Activity-Based Proteomics Discovery of Deubiquitinating Enzyme Inhibitors with Immunomodulatory Activity

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L. Jones, Simeon D. Draganov, Sofia Schönbauer Huamán, and 27 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9106698/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Deubiquitinases (DUBs) play key roles in human pathologies, including cancer, infectious, autoimmune, and neurodegenerative diseases. The progress of potent and selective active site-targeting DUB inhibitors into clinical trials has demonstrated the therapeutic opportunities of such molecules; however, the shared catalytic geometry among DUB family members poses a challenge for achieving inhibitor selectivity. We describe a high-throughput, DUB-focused, activity-based proteomics workflow to identify new types of cysteine-reactive DUB inhibitors. We demonstrate the potential of αβ,α′β′-diepoxyketones (DEKs) and ubiquitin-derived covalently reacting alkynes as the first selective, cell-active inhibitors of USP47, OTUD7B (Cezanne), and USP5. These DUBs are reported to have critical roles in inflammasome formation, hypoxia, and viral replication, respectively. The identified inhibitors phenocopy these findings, demonstrating the tractability of these DUBs as immunotherapeutic targets. Biological sciences/Biochemistry Biological sciences/Computational biology and bioinformatics Biological sciences/Drug discovery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Post-translational (poly)-ubiquitination regulates key cellular processes, including protein and organelle homeostasis, through the interplay of ubiquitinating and deubiquitinating molecular machineries ( 1 – 4 ). Deubiquitinases (DUBs) are mostly nucleophilic cysteine enzymes which cleave ubiquitin (Ub) from biomolecule substrates, mainly proteins, modulating their fate ( 5 , 6 ). Consequently, dysregulated DUB functions have been linked to diseases ( 1 ). DUBs cleave after the conserved C-terminal LRGG motif of ubiquitin within their catalytic cleft. Despite sharing a broadly conserved active-site architecture, individual DUBs discriminate among distinct polyubiquitin chain linkages and ubiquitinated substrates through differences in ubiquitin-binding interfaces and allosteric regulatory elements. This combination of conserved catalytic geometry and context-dependent substrate recognition complicates the development of selective active-site-directed DUB inhibitors ( 7 ). Nonetheless, both active-site targeting and allosteric small-molecule inhibitors of USP1, USP28, and USP30 have been developed, and progressed into clinical trials for treatment of advanced solid tumours ( 8 , 9 ), relapsed/refractory acute myeloid leukemia (R/R AML)( 10 ), chronic kidney disease ( 11 ), and Parkinson’s disease ( 3 , 12 ). DUB activity-based protein profiling (ABPP) was established to profile DUB activities in complex systems. DUB ABPP employs activity-based probes (ABPs) that react with the nucleophilic cysteine of DUBs, ( 13 – 16 ) including haemagglutinin (HA)-tagged ubiquitin-propargylamide (HA-Ub-PA) which bears an alkyne warhead for irreversible covalent reaction with active-site cysteines. Coupled with affinity purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS), ABPP enables the analysis of endogenous DUB-ABP complexes. Unlike recombinant enzyme assays, ABPP supports the identification of potent and selective inhibitors in a cellular environment, including those for challenging targets, ( 17 , 18 ) whilst also providing information on cell permeability and stability ( 19 – 21 ). Historically, the scope of ABPP has been limited by sensitivity and throughput because of labor-intensive workflows involving ABP labeling, purification, sample preparation, MS analysis, and data processing. Our group and others, with the aim of expanding ABPP’s utility for DUB inhibitor discovery, have automated the established methodology to enable analysis of ~ 100 samples per day (ABPP-HT) ( 22 ) and implemented data-independent acquisition LC-MS/MS (DIA), enabling deeper and more comprehensive DUBome profiling (ABPP-HT*) ( 23 ). Additional improvements aimed at enhancing sensitivity were tested and include using diverse Ub probe approaches, high-pH pre-fractionation ( 24 ), and isobaric labelling (Fig S1 A-C ) ( 17 ). Here, we advance the ABPP-HT* platform into a broadly accessible and scalable workflow, enhancing its sensitivity, flexibility, and ease of use while establishing a fully functional on-bench (non-automated) format. As a proof of concept, we applied the platform to the discovery of unconventional cysteine-reactive and selective DUB inhibitors αβ,α′β′-diepoxyketones (DEKs) that selectively engage the active sites of USP47/USP7 and OTUD7B. The platform also enabled the identification and optimization of a ubiquitin-derived inhibitor class that uses an alkyne as a latent electrophile for covalent engagement, yielding chemical probes with selective cellular inhibition of USP5. These inhibitors performed robustly in functional assays and are useful tools for assigning DUB-dependent phenotypes in cells. Consistent with reported biology, the USP47/USP7, OTUD7B, and USP5 inhibitors modulated pathways linked to innate immunity, hypoxic signaling, and viral infection by suppressing inflammasome activation, stabilizing HIF-1α, and relieving inhibition of type I interferon signaling, respectively. Results We modified our reported ABPP-HT* methodology ( 22 , 23 ) by adapting the immunoprecipitation step to either a multi-well magnetic rack for on-bench processing or a Thermo KingFisher Apex purification system, demonstrating the workflow’s flexibility (Fig S1 D ). Compared to using an automated liquid handling platform (Assay MAP from Agilent), the new method manifested comparable or improved enrichment of cysteine-reactive DUBs (Fig. S1 E-I ). The improved and user-friendly “on-bench” ABPP-HT* set-up was then used to identify novel DUB inhibitors from a set of unconventional cysteine-reactive small-molecules (Fig. 1 A ). Four complementary classes of small-molecules were investigated in the screen: (i) αβ,α′β′-diepoxyketones (DEKs), which are reported to covalently react with the nucleophilic cysteine residues of bacterial and viral enzymes ( 25 ), (ii) γ-lactams which are substantially less reactive than DEKs, but can covalently react with Cys145 of the SARS-CoV-2 main protease ( 26 ), (iii) Michael acceptors, which are a common motif in covalently reacting small-molecule drugs, and (iv) Ub-derived alkyne and nitrile inhibitors that may bind at the active site (Table S1 -3) (Fig. 1 B ); note that classes (i), (ii), and (iv) are relatively uncommon in established DUB inhibitor libraries. The latter class of inhibitors was designed based on the Ub C-terminal LRGG motif ( 27 ), replacing the C-terminal glycine by 2-aminoacetonitrile or propargyl amide (PA) to enable reversible or irreversible covalent reaction with the DUB active site cysteine thiol, respectively; note that the arginine residue at the P3-equivalent position was substituted for a lysine group to facilitate chemical modification. This design strategy was informed by the observations that Ub-PA ( 16 , 28 – 30 ) and nitrile as well as alkyne-containing substrate-derived small-molecules are potent and selective active site-targeting inhibitors of nucleophilic cysteine enzymes ( 31 – 36 ). A total of 41 small-molecules were thus incubated with MCF-7 breast cancer cell lysates in a 96 well plate in technical duplicates. FT827, a well-characterized USP7 inhibitor ( 37 ), was included as a reference inhibitor; Negative and positive controls (absence or presence of HA-Ub-PA) were included on the plate. The results reveal that 38 DUBs were consistently enriched > 5-fold across replicates using the ABPP-HT* workflow, with an average coefficient of variation (CV) of 16%, confirming robustness (Fig. S2 A ). Importantly, we confirmed potent and selective inhibition of USP7 by FT827, ( 37 ) further validating this assay. Eight compounds significantly inhibited at least one DUB (> 20% intensity loss, p USP7), Compound 6 (OTUD7B), Compound 7 (USP30/USP47/VCPIP1), Compound 25 (USP5), and Compounds 26 and 32 (USP33) (Fig. 1 D ). Notably, USP5-selective Compound 25 is a diastereomer of the inactive Compound 27 , highlighting the workflow’s ability to resolve isomer-specific effects (Fig. S2 B , S2 C ). Hit Validation and Dose-Response Analysis Western blot analysis supported inhibition of USP47/USP7 by Compound 3 , OTUD7B by Compound 6 , and USP5 by Compound 25 (Fig. 2 A , 2 B ). DUB inhibition calculated from western blot densitometric analysis was concentration-dependent (Fig. 2 C ) and IC₅₀ values revealed the preference of Compound 3 for USP47 (8.8 µM) over USP7 (36 µM) (Fig. 2 D ), which was consistent with screening data. The apparent preference of Compound 3 for USP47 over USP7 inhibition was remarkable given that reported USP47 inhibitors typically also inhibit USP7 with similar or lower potency ( 38 , 39 ), likely reflecting the close evolutionary relationship between human USP47 and USP7 (their catalytic domains share ~ 40–45% sequence identity, depending on the alignment boundaries used) ( 40 , 41 ). Compounds 26 and 32 appeared to be false positive hits and could not be validated (Fig. S2 D ), while Compound 7 induced protein cross-linking, likely due to its bifunctional epoxide structure (Fig. S2 E ) ( 25 ). To assess binding stability, Compounds 3 , 6 , and 25 were pre-incubated with MCF-7 lysates for 30 min before addition of HA-Ub-PA and incubation for 5, 15, or 45 min. Efficient prevention of HA-Ub-PA binding was observed across all timepoints (Fig. S2 F–H ), suggesting irreversible binding of Compounds 3 , 6 , and 25 at the DUB active sites. The increased throughput of the ABPP-HT* workflow enabled systematic evaluation of the selectivity of Compounds 3 , 6 , and 25 across multiple concentrations and replicates (Fig. 2 E-G ). The dose-dependent inhibition curves and IC 50 values for the three compounds were comparable to those generated by immunoblotting (Fig. S3 A-D ) ( 22 ). The results reveal that Compound 3 exclusively inhibited USP47 and USP7 up to 50 µM, with USP40 and USP48 inhibition being observed at higher concentrations (Fig. 2 E , 2 H ). Compound 6 was selective for OTUD7B up to 100 µM but also inhibited USP16 and USP28 at the highest concentration (250 µM) (Fig. 2 F , 2 I ). Note that Compounds 3 and 6 are reported to inhibit the mycobacterial L,D-transpeptidase Ldt Mt2 and the SARS-CoV-2 main protease ( 25 ), implying that they may also react with additional nucleophilic cysteine enzymes other than DUBs. By contrast to Compounds 3 and 6 , Compound 25 was highly selective, only inhibiting USP5 across the tested concentration range (Fig. 2 G , 3 J ). Molecular Basis for Selective Inhibition of USP47 by Compound 3 Given the preference of Compound 3 for USP47 over USP7 inhibition, we investigated its mode of action. The results of in vitro activity assays using recombinant DUBs and a fluorogenic ubiquitin-rhodamine (Ub-Rho) substrate revealed that Compound 3 was ~ 10-fold more potent in inhibiting USP47 than USP7 after 30 min preincubation (IC₅₀: 0.48 vs. 4.3 µM; Fig. 3 A , Fig. S4 A-D ). Time-dependency experiments indicated covalent or slow tight-binding inhibition for USP47 (Fig. 3 B ), while USP7 inhibition was weaker and time-independent (Fig. 3 C ). For both enzymes, progress curves in the presence of Compound 3 were non-linear, indicating slow initial binding (Fig. S4 E , S4 F ). Molecular docking studies were initially performed to investigate the preferred reaction of Compound 3 with USP47. In the absence of a human USP47 Protein Data Bank (PDB) structure, the structure of human USP7 in complex with the covalent inhibitor FT827 (PDB: 5NGF) was used as the template to generate a homology model of human USP47 with an appropriate conformational state for covalent inhibitor docking studies. Compound 3 most likely forms a covalent adduct with the catalytic cysteine of human USP47 (C197) via 1,2-carbonyl attack and retro-aldol fragmentation based on previously reported work (Fig. S4 G ) ( 25 ). The docking studies also suggest that Compound 3 covalently binds at the thumb-palm cleft (Fig. S4 H ) and sterically blocks Ub binding (Fig. S4 I ), analogous to the covalent USP7 inhibitor FT827 (PDB: 5NGF; Fig. S4 J ). The binding site is flanked by 14 residues including R402, the side chain of which is predicted to form a hydrogen bond with the epoxide moiety of Compound 3 (Fig. 3 D ). In human USP7, the substitution of R402 with an asparagine residue (N418), which cannot interact with Compound 3 , may rationalize the apparent selectivity of Compound 3 for USP47 inhibition (Fig. 3 E ). USP47 apoform (PDB: 8ITN) and Ub-bound (PDB: 8ITP) structures exist for the closely related catalytic domain of C. elegans USP47 (44% sequence identity). Comparison of the human USP47-Compound 3 model with the apo C. elegans USP47 structure suggests that Compound 3 binding disrupts the hydrogen bond between R402 and H271 that stabilizes the switching loop in its catalytically incompetent “in” conformation. Disruption of this interaction is predicted to favor the active “out” conformation of the switching loop, compatible with activation peptide binding, as described for USP7 (Fig. S4 O , S4 P ) ( 42 , 43 ). Hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies were performed to investigate binding of Compound 3 to USP47 and USP7 (Fig. 3 F , 3 G , and Fig. S4 K , S4 L ). The conformational shifts observed by HDX-MS indicate a switching loop rearrangement in USP47 (Fig. S4 M ) and potential engagement of the activation peptide (Fig. 3 H ) ( 43 ). Notably, HDX-MS analysis with USP7 showed no such distal conformational changes, indicating that Compound 3 does not promote an equivalent switching loop reorganization or activation peptide binding in USP7 (Fig. 3 I and Fig. S4 N ), further rationalizing its selectivity for USP47. Inhibition of USP47 with Compound 3 Further Supports its Roles in Inflammasome Activation ABP assays in MCF-7 cells revealed that Compound 3 is cell permeable with USP47 inhibition occurring in a concentration dependent manner (Fig. 4 A ); cytotoxicity was observed at 250 µM, indicative of off-target effects, in line with the reported reactivity of DEKs with other enzymes. To explore whether Compound 3 could serve as a useful research tool probe, we investigated its ability to replicate reports describing a role of both USP47 and USP7 in the activation of the NLRP3 inflammasome by regulating NLRP3 ubiquitination ( 40 ). Human monocyte (THP-1) cells were treated with lipopolysaccharide (LPS) followed by a 45 min or 2 h nigericin incubation to activate the NLRP3 inflammasome. Under these conditions, treatment with Compound 3 inhibited inflammasome activation, as evidenced by reduced Caspase-1 activity (Fig. 4 B , Fig. S5 A-C ). To ensure Compound 3 did not reduce Caspase-1 activity via direct inhibition, the upstream formation of the NLRP3 inflammasome was also examined: THP-1 treatment with Compound 3 , in combination with LPS and Nigericin, blocked the oligomerisation of apoptosis-associated speck-like protein containing a CARD (ASC), and prevented the cleavage of Caspase-1, IL-1β, and gasdermin D (GSDMD) (Fig. 4 C ). While a decrease in NLRP3 levels was observed, the treatment of cells with Compound 3 resulted in appearance of a high molecular weight NLRP3 smear, consistent with ubiquitination (Fig. 4 C ). While further investigation is required to fully characterize this smear, it may be attributable to the ubiquitination of NLRP3, as previously proposed ( 40 ). No decrease in THP-1 cell viability was detected when incubating with up to 100 µM Compound 3 , when the LPS treatment was followed by 45 min nigericin treatment (Fig. S5 D). In contrast, 2 h of nigericin exposure induced toxicity at Compound 3 concentrations of 30 µM and higher. (Fig. 4 D ), further signifying additional targets beyond DUBs at these high concentrations ( 25 ). In contrast, and consistent with an on-target reduction in inflammasome activation, lower concentrations of Compound 3 increased THP-1 viability upon LPS and a 4 h nigericin treatment, indicating reduced inflammasome-mediated pyroptosis (Fig. 4 D ). Taken together, the data demonstrates that Compound 3 can reproduce phenotypes previously seen by USP47/7 gene knock-out (KO) and inhibition, through the prevention of NLRP3 inflammasome formation (Fig. 4 E ). Inhibition of OTUD7B with Compound 6 Further Supports its Roles in Hypoxic Signaling and Associated SARS-CoV-2 Repression Orthogonal in vitro assays using the recombinant catalytic domain of OTUD7B and a ubiquitin–rhodamine substrate demonstrated that Compound 6 inhibits OTUD7B with an IC₅₀ of 1.9 µM (95% confidence interval, 1.8–2.1 µM) (Fig. 5 A ). Intact protein mass spectrometry revealed that incubation of the catalytic domain of OTUD7B with Compound 6 (molecular weight: 326.35 Da) for 1 min results in a + 326 Da mass shift, consistent with covalent reaction of OTUD7B with Compound 6 in a 1:1 stoichiometry. After 15 min, the formation of multiple species derived from the + 326 Da adduct were observed with a + 190 Da adduct dominating. The + 190 Da adduct likely arises from elimination of ortho -methoxybenzaldehyde from the initially formed + 326 Da intermediate, consistent with the crystallographically validated mechanism by which nucleophilic cysteine enzymes react with DEKs (Fig. 5 B , 5 C ) ( 25 ). Compound 6 was active and cell permeable in MCF-7 cells (Fig. 5 D ), enabling subsequent evaluation of the phenotypic consequences of OTUD7B inhibition. OTUD7B has been shown to stabilize hypoxia-inducible factor 1α (HIF-1α) under hypoxic conditions in a ubiquitin-dependent manner, counteracting the E3 ubiquitin ligase activity of the von Hippel–Lindau tumor suppressor (VHL) ( 44 ). Consistent with these findings, treatment of Calu-3 cells with Compound 6 under hypoxic conditions reduced HIF-1α levels in a dose-dependent manner (Fig. 5 E ). Furthermore, in renal carcinoma cells lacking active VHL (RCC4-VA)( 45 ), we observed that treatment with Compound 6 had no impact on HIF-1α protein levels. When wild-type VHL (RCC4-VHL) expression was restored, however, a dose-dependent reduction in hypoxic HIF-1α protein levels were observed (Fig. 5 F ). In Chronic Myeloid Leukemia (CML)-derived HAP1 wild-type (WT) cells, treatment with Compound 6 also resulted in a dose-dependent reduction in HIF-1α protein levels. Importantly, HIF-1α levels were markedly reduced in HAP1 OTUD7B KO cells compared to WT, consistent with published findings ( 44 ), and treatment of KO cells with Compound 6 did not further reduce HIF-1α, supporting an on-target mechanism at the tested concentrations (Fig. 5 G ). Previous studies have shown that SARS-CoV-2 replication is reduced under hypoxic conditions, attributable to increased HIF-1α protein levels ( 46 ). Consistent with these findings, we observed decreased viral replication in Calu-3 cells under hypoxia compared with normoxia. Notably, treatment with Compound 6 significantly attenuated the antiviral effect of hypoxia (Fig. 5 H ). These results are consistent with reduced HIF-1α protein levels following OTUD7B inhibition. The ability of Compound 6 to phenocopy OTUD7B knockout supports on-target engagement and provides further evidence that OTUD7B catalytic activity directly contributes to HIF-1α stability under hypoxic conditions. Moreover, we provide the first functional validation of OTUD7B inhibition in the regulation of SARS-CoV-2 replication under hypoxia (Fig. 5 I ). Inhibition of USP5 Demonstrates Its Essential Role in SARS-CoV-2 Replication The active sites of DUBs and deISGylases, cellular enzymes cleaving the ubiquitin-like protein ISG15 (interferon-stimulated gene 15), are structurally very similar. Most deISGylating enzymes are cross-reactive DUBs, catalysing removal of both Ub and ISG15, with the notable exception of USP18 ( 41 , 47 – 49 ). Given USP5’s dual Ub/ISG15 specificity, we evaluated the effect of Compound 25 on deISGylases by immunoblotting, using a bio-ISG15-PA ABP. The results reveal that the extent of USP5 inhibition by Compound 25 is comparable using the the bio-ISG15-PA and Ub-PA ABPs, and that Compound 25 did not cross-react with USP18 or any of the other detected deISGylases in the tested concentration range (up to 125 µM), demonstrating the high selectivity of Compound 25 for USP5 inhibition (Fig. 6 A ). Interestingly, ABP assays in MCF-7 cells revealed that Compound 25 was inactive in intact cells, limiting its applicability (Fig. 6 B ). To optimize Compound 25 for cellular activity, we modified the alkyne and amine protecting groups. Substituting the terminal alkyne reduced potency (Fig. 6 C ; Table S4 ; synthesis detailed in SI), supporting that Compound 25 covalently reacts with the USP5 nucleophilic cysteine via its alkyne group which serves as a latent electrophile. By contrast, altering amine-protecting groups at the N-terminus (R 1 ) and/or the N ε -lysine amino group (R 2 ), we generated the active and cell permeable derivatives Compounds 25a and 25b bearing trifluoromethyl groups at R1 and trifluoroacetyl ( 25a ) or tert -butyloxycarbonyl (Boc; 25b ) at R2, respectively (Fig. 6 C , 6 D ). Both 25a and 25b retained the potency and selectivity for USP5 inhibition observed in lysates (Fig. S6 A ), offering promising leads for functional studies and possibly therapeutic development. To study the binding and selectivity of Compound 25 and its derivatives to USP5, we generated five biotinylated derivatives with different linker strategies, including biotinylation via the N-terminal amino group or the N ε -lysine amino group (Table S5 ). All five biotinylated versions of Compound 25 bound to endogenous USP5 in MCF-7 lysates. Binding was confirmed by streptavidin western-blotting, with a strong band at the expected molecular weight of USP5 (~ 95 kDa; Fig. 6 E , S7 B, S7D ). By contrast, no signal was observed for another USP, USP42, at ~ 150 kDa. Interestingly, despite comparable binding to USP5, derivatives bio-b-25a and bio-a-25b were less active than the other three biotinylated derivatives, as shown by Ub-PA probe competition assays, at two different concentrations (Fig. S7 A , S7 B ). Selective binding of these Compound 25 derivatives to the catalytic cysteine of USP5 was confirmed by competition experiments using free Compound 25 and N-ethylmaleimide ( NEM ), a broad-spectrum cysteine-reactive alkylating agent. In both cases, competition resulted in loss of the USP5 band at ~ 95 kDa, consistent with covalent engagement of the nucleophilic cysteine. (Fig. S7 C , 6 E , S7 D ). To functionally validate Compound 25b as an on-target inhibitor of USP5 we investigated its effect on the role of USP5 in viral replication. USP5 has been linked to IFN-I production by regulating the ubiquitination of different innate immune factors such as RIG-I and IRF3 ( 50 , 51 ). In agreement with these observations, when IFN-I production was induced by synthetic RNA Poly I:C, there was a significant amplification of IFN-I production in HAP1 cells co-treated with Compound 25b (Fig. 6 F ). In line with the observed elevated IFN-I levels, Compound 25b exhibited a dose-dependent reduction in viral replication against SARS-CoV-2 in infected Calu-3 cells. In comparison to Remdesivir, a guanosine nucleoside analogue which targets the viral replicase complex and is in clinical use as a broad-spectrum antiviral medication ( 52 ), Compound 25b showed a ~ 10-fold increase in potency (Fig. 6 G ) and did not show any significant cellular toxicity at concentrations as high as 200 µM in HAP1 cells (Fig. S8 A ). The increased IFN-I production and prevention of viral replication by Compound 25b both validates it as a tool compound for investigating roles of USP5 in signaling, and provides evidence of USP5’s potential as a therapeutic target (Fig. 6 H ). Discussion As interest in DUBs as drug targets grows ( 6 , 53 , 54 ), so does the need for improved discovery strategies. High-throughput screening (HTS) has enabled rapid evaluation of large compound libraries ( 37 , 55 ), yet it remains constrained by high cost, low hit rates, and reliance on recombinant proteins that may not fully capture native regulation and complex formation. Consequently, many HTS-derived hits require substantial optimization and secondary validation before demonstrating cellular activity. To overcome these limitations, computational approaches and activity-based proteomics have emerged as complementary strategies. AI-enabled virtual screening platforms such as AtomNet and Boltz-2 prioritize compounds in silico , even in the absence of prior ligand data ( 56 ). In parallel, chemoproteomic profiling of reactive cysteines has enabled more directed targeting of cysteine-containing proteins, including DUBs ( 57 – 60 ). Recent advances in DUB-focused ABPP have produced high-throughput-ready platforms, including our ABPP-HT* workflow ( 17 , 22 , 61 ). ABPP-HT* combines sensitivity, cost-efficiency, and flexibility, operating in both manual and automated formats while maintaining compatibility with diverse probe chemistries and enzyme classes. Here, we demonstrate a proof-of-concept application of the ABPP-HT* platform for DUB inhibitor discovery and characterization. Screening a curated electrophile-containing library, including αβ,α′β′-diepoxyketones (DEKs), γ-lactams, and ubiquitin-derived peptide inhibitors specifically designed for this study, yielded a hit rate approximately tenfold higher than traditional DUB HTS approaches. Following validation, we identified selective inhibitors of USP47, OTUD7B, and USP5 and further characterized their potency, permeability, selectivity, and functional consequences. These findings underscore the efficiency of endogenous chemoproteomic screening for rapid identification of cell-active DUB inhibitors. USP47 represents an emerging therapeutic target ( 62 – 64 ), yet previously reported inhibitors display comparable potency toward its close homolog USP7 ( 38 , 39 ), consistent with their substantial sequence similarity ( 40 , 41 ). We identified Compound 3 as a first-in-class inhibitor exhibiting preference for USP47 over USP7. Orthogonal kinetic analyses and HDX-MS-guided structural modeling revealed molecular determinants underlying this selectivity. Importantly, Compound 3 recapitulated prior findings that USP47/USP7 inhibition prevents inflammasome activation ( 40 ), providing functional validation of selective USP47 targeting. Although the roles of OTUD7B in disease are still being defined ( 65 ) accumulating evidence supports its therapeutic potential ( 44 , 66 – 68 ), and recent reports describe emerging OTUD7B-directed ligands ( 61 , 69 ). We identified Compound 6 as a potent, selective, and cell-permeable OTUD7B inhibitor that reduced HIF-1α protein levels under hypoxia, phenocopying OTUD7B knockdown ( 44 ). Given that hypoxia restricts SARS-CoV-2 infection in a HIF-1α-dependent manner ( 46 , 70 ), Compound 6 correspondingly rescued viral replication under hypoxic conditions ( 70 ). These findings establish pharmacological inhibition of OTUD7B as a tool to interrogate hypoxia-driven cellular responses. Compound 25 displayed high selectivity for USP5 over related DUBs such as USP16 and USP22 ( 71 ). USP5 hydrolyzes free polyubiquitin chains via a zinc finger ubiquitin-binding domain (ZnF UBP) that recognizes the C-terminal diglycine motif of ubiquitin ( 72 ). Although USP5 has been implicated in cancer, pain, and inflammatory disorders ( 73 ), existing inhibitors remain limited in selectivity, scope, and cellular validation (Fig. S10 ) ( 74 , 75 ). In contrast to prior inhibitors designed to engage the ZnF UBP carboxylate-binding pocket ( 74 , 76 ), our ubiquitin-derived inhibitors substitute the C-terminal glycine with a propargyl amide group, inspired by the covalent reactivity of Ub-PA probes. Remarkably, these relatively simple substrate-inspired molecules achieved high selectivity, highlighting subtle but functionally significant differences in active-site architecture across DUBs. Mechanistically, Compound 25 likely modifies the catalytic cysteine of USP5 via its alkyne warhead, supported by structural precedent (PDB: 3IHP) ( 77 ), related USP-Ub-PA complexes, and reduced activity upon steric modification (Table S4 ). USP5’s unique domain architecture, including a disulfide linkage between C793 and C195 in the USP5-Ub complex, may constrain blocking loop flexibility and promote favorable compound engagement (Fig S8 B ). The use of a latent electrophile may further enhance selectivity through differential binding kinetics across DUBs ( 78 – 81 ). The clinical success of electrophilic alkynes such as propiolamides in Bruton’s tyrosine kinase inhibitors ( 82 , 83 ), supports the translational potential of this strategy. Structure-activity optimization yielded the fluorinated, cell-permeable analogues 25a and 25b that retained cellular activity and were suitable for functional studies. Biotinylated derivatives confirmed covalent engagement and proteome-wide selectivity for USP5. Notably, Compound 25b enhanced type I interferon production and exhibited strong antiviral activity, extending established USP5 functions. The combined results manifest the potential of high-throughput ABPP workflows for identifying DUB inhibitors and streamlining the development of potent and, at least, partially selective compounds for use as biochemical tools in functional assignment and mechanistic studies. Importantly, Compounds 3 and 6 , as well as Compounds 25a and 25b , are novel classes of active site-targeting covalently reacting DUB inhibitors that show promise as chemical probes and scaffolds for drug development. By eliciting pathway-relevant phenotypes in innate immunity, hypoxic signaling, and viral infection models, these inhibitors further reinforce the therapeutic potential of DUBs. Methods Small-molecule synthesis αβ,α′β′-Diepoxyketones (DEKs) ( Compounds 1–8 ) ( 25 ) and γ-lactams ( Compounds 9–23 ) ( 26 ) reported. The synthesis of Ub-derived small-molecules ( 25 – 31 and 25a-k ) is described in the Supplementary Information (Fig. S11 and S12). The structures of Compounds 1–31 are in the Supplementary Information (Table S1 -3), the structures of Compounds 25a-k are in the Table S4 . The synthesis and structures of biotinylated Compound 25 derivates are described in the Supplementary Information (Fig. S13 and Table S5 ). Cell culture MCF-7 cells were cultured in high glucose DMEM supplemented with 10% (v/v) fetal bovine serum (FBS) and L-glutamine (2 mM). Cells were maintained at 37°C, 5% CO 2 . MCF-7 cells were washed with phosphate buffered saline (PBS) and collected in PBS by scraping and centrifugation at 200 xg for 10 min. For data shown in Fig. S1 A-C, MCF-7 cells were collected by the addition of TrypLE followed by media and centrifuged at 1600 rpm for 5 min with pellets being resuspended in PBS (3 cycles). HAP1 cells were cultured in IMDM media (#21980065) supplemented with 10% FBS (v/v), and maintained at 37°C, 5% CO 2 . ~3×10 6 cells were seeded per 10 cm plate in 10 mL media overnight. After seeding, 4 mL of media were aspirated from each plate, and recombinant human interferon-alpha, IFN-α2 (α2b; PBL Assay Science; Cat. No. 11105-1), was added to the remaining media (1000 units/mL final concentration). IFN-α treatment was performed for 24 h (to induce USP18), media from each plate was then aspirated and cells were washed once with PBS (5 mL per plate). Cells were subsequently stored at − 80°C until lysis. THP-1 cells were cultured in RPMI 1640 supplemented with 10% (v/v) FBS, 0.05 mM β-mercaptoethanol, GlutaMAX Supplement, and Penicillin-Streptomycin. Cells were maintained at 37°C 5% CO 2 . RCC4-VHL and VA cells were cultured in DMEM media supplemented with 10% fetal bovine serum (FBS), GlutaMAX, and Penicillin-Streptomycin ( 45 ). Calu-3 cells were maintained in Advanced DMEM media, 10% FCS, L-glutamine, and Penicillin-Streptomycin. High-throughput ABPP workflows Sample preparation for Agilent Bravo ABPP-HT* The data presented here on the Agilent Bravo ABPP immunoprecipitation is from published work,( 22 ) and is included for comparison. Sample preparation for “on bench” ABPP-HT* high-throughput screen and compound concentration-dependence validations HA-Ub-PA was synthesised as reported ( 22 , 24 ). MCF-7 cells were washed with PBS, scraped in PBS and centrifuged at 200 x g for 10 min. Pellets were then resuspended in lysis buffer (50 mM Tris, 5 mM MgCl 2 , 0.5 mM EDTA, 250 mM sucrose, 1 mM dithiothreitol (DTT), pH 7.5), and lysed using glass beads (2:1 lysate to beads ratio) with vortexing (10 cycles of 30 s vortexing, followed by 2 min incubation on ice). The MCF-7 lysate was then clarified at 600 x g for 10 min at 4°C. Lysate protein concentration was determined by BCA, and 250 µg was aliquoted per well of a 96 well plate at a concentration of ~ 4.7 mg/mL. The compound library was diluted to 1 mM DMSO stock solutions in a 96 well plate and multichannel-pipetted with mixing into the lysate plate to a final compound concentration of 25 µM in duplicate for the screen. For the concentration-dependence validations, the indicated inhibitor concentrations were added in duplicate. Additional control wells (4 x negative, 4 x positive for the screen, 3 x negative and 3 x positive for the concentration-dependence validations) were treated with DMSO. The plate was then incubated at 37°C for 1 h on a thermomixer without shaking. A total of 2 µg of HA-Ub-PA was multichannel-pipetted with mixing into to the positive control wells and all inhibitor-treated wells, with an equivalent volume of buffer added to the negative control wells. The lysates were then incubated with the HA-Ub-PA at 37°C for 45 min on a thermomixer without shaking. The final reaction volume with the MCF-7 lysate, inhibitor/DMSO and HA-Ub-PA/buffer was 60 µL. Reactions were quenched by the addition of SDS and NP-40 (IGEPAL CA-630) to final concentrations of 0.4% (v/v) and 0.5% (v/v), respectively. Reactions were then diluted by addition of 200 µL NP40 buffer (50 mM Tris, 0.5% NP-40 (IGEPAL CA-630), 150 mM NaCl, 20 mM MgCl 2 ·6 H 2 O, pH 7.4). 75 µL of anti-HA magnetic beads were aliquoted into a separate 96 well plate and washed 5x with 200 µL of NP40 buffer (50 mM Tris Base, 0.5% NP-40 (IGEPAL CA-630), 150 mM NaCl, 20 mM MgCl 2 ·6 H 2 O, pH 7.4), using a magnetic rack for bead separation and multichannel pipetting with cut tips for mixing. Reaction mixtures were then added to the washed magnetic beads, mixed by multichannel-pipetting, and incubated in a thermomixer overnight at 4°C with gentle agitation (350 rpm). The beads were then washed 5x with 200 µL of NP40 buffer, as before. Proteins were eluted from the beads and reduced by addition of 100 µL of 2 x Laemmli buffer supplemented with 20 mM DTT, and incubated for 10 min at 90°C with agitation (350 rpm) on a thermomixer. Eluates were then removed from the beads using a magnetic rack and transferred to a fresh 96 well plate. Eluates were alkylated by the addition of iodoacetamide (IAA) to a final concentration of 40 mM. Proteins were then digested using trypsin and cleaned up using a 96 well S-trap plate according to the manufacturer’s instructions ( 84 ), dried down in a speedvac and resuspended in 0.1% (v/v) aqueous formic acid. Sample preparation for the Thermo KingFisher Apex ABPP-HT* The bio-ISG15-PA probe was synthesised as previously described ( 85 ). Cell lysis, subsequent labelling with HA-Ub-PA or bio-ISG15-PA probes, immunoprecipitation and protein enrichment, and processing of samples for analysis by western blot and LC-MS/MS was performed as described ( 86 ). Briefly, HAP1 cells were treated and prepared as described in the subsection ‘ Cell culture’ . Cells were resuspended in lysis buffer (50 mM Tris, 5 mM MgCl 2 , 0.5 mM EDTA, 2.5% (v/v) glycerol, 1 mM dithiothreitol (DTT), pH 7.5), and lysed using glass beads (2:1 ratio lysate to beads) with vortexing (10 x 30 s vortexing, followed by 1 min incubation on ice). The HAP1 lysates were then centrifuged (600 x g for 5 min at 4°C; ~80% of the supernatant was transferred into tubes, then the remaining ~ 20% of supernatant was centrifuged at 14 000 x g for 10 min at 4°C, and then the two fractions from each spin were combined). The protein concentration in the lysates was estimated using the BCA assay kit (#23227), and 270 µg were aliquoted per well into a 96-deep well plate. The total volume in each well was made up to 143 µL for samples to be labelled with HA-Ub-PA, or 147 µL for samples to be labelled with bio-ISG15-PA, with lysis buffer. Negative and probe-only positive controls were treated with 1.5 µL of DMSO in triplicates. The plate was then incubated at RT for 1 h without shaking. 5.4 µL of HA-Ub-PA or 2.35 µL of bio-ISG15-PA (volumes determined from probe titration assays for protein labelling) were transferred into the respective wells using a multichannel pipette, with an equivalent volume of buffer added to the corresponding negative control wells, and incubated at 37°C for 45 min without shaking. The final reaction volume with the HAP1 lysate, inhibitor or DMSO, and HA-Ub-PA or bio-ISG15-PA or buffer was 150 µL. Reactions were quenched by addition of SDS and NP-40 (IGEPAL CA-630) to final concentrations of 0.4% (v/v) and 0.5% (v/v), respectively, from a master mixture. The master mixture was prepared immediately before quenching by mixing a 5% (v/v) SDS stock with a 10% (v/v) NP-40 stock in a ratio of 1.6:1 and adding 21.5 µL of the mixture to each well. 20 µL of each reaction mixture was kept as ‘input’ samples (to evaluate efficiency of probe labelling), and the remaining ~ 150 µL of reaction volume was diluted by addition of 250 µL NP-40 buffer (50 mM Tris, 0.5% (v/v) NP-40 (IGEPAL CA-630), 150 mM NaCl, 20 mM MgCl 2 , pH 7.4) to a total volume of 400 µL. For immunoprecipitation/pull-down steps, 50 µL of anti-HA magnetic beads or 37.5 µL of streptavidin magnetic Sepharose beads were used per sample. The appropriate total amount of anti-HA magnetic beads or streptavidin magnetic Sepharose beads were split into 450 µL aliquots, washed with 4 × 1.6 mL of NP-40 buffer (50 mM Tris, 0.5% NP-40 (IGEPAL CA-630), 150 mM NaCl, 20 mM MgCl 2 , pH 7.4) using a magnetic rack for bead separation, and transferred into the respective reaction wells using a multichannel pipette with cut tips. The 96-deep well plate was then incubated for 4 h at 4°C on the Thermo KingFisher Apex system, with the mixing speed set to ‘medium’. The beads were then washed with 4 × 500 µL NP-40 buffer, as described above, and proteins were eluted from the beads twice with 2× Laemmli buffer supplemented with 50 mM DTT (2 × 55 µL, 2 × 5 min at 95°C with the mixing speed set to ‘slow’). Beads were removed from the eluates by programming the system to capture and release the beads into an empty 96-deep well plate. Eluates were reduced with DTT to a final concentration of 10 mM (15 min, RT), then alkylated by the addition of iodoacetamide (IAA) to a final concentration of 20 mM (10 min, RT). Proteins were then digested using trypsin and cleaned up using a 96-well S-trap plate according to the manufacturer’s instructions(84, dried down in a speedvac and resuspended in 0.1% (v/v) aqueous formic acid. LC-MS/MS timsTOF for “on bench” ABPP-HT* high-throughput screen and compound concentration-dependence validations Peptide samples were loaded onto Evotips (Evosep) desalting columns according to the manufacturer’s instructions, then chromatographically separated on an 8 cm x 150 µm analytical column with bead size 1.5 µm (EV1109, Evosep), using an Evosep One liquid chromatography (LC) system with the 100 samples per day (spd) standard method. Peptides were eluted onto a TimsTOF Pro mass spectrometer (Bruker) operated in diaPASEF mode using 8 diaPASEF scans per TIMS-MS scan. The ion mobility range was set to 0.85–1.3 Vs/cm 2 . Each mass window isolated was 25 m/z wide, ranging from 475–1000 m/z with an ion mobility-dependent collision energy that increased linearly from 20 eV to 59 eV between 0.6–1.6 Vs/cm 2 . LC-MS/MS for timsTOF Agilent Bravo ABPP-HT* The previously reported timsTOF data collected after immunoprecipitation using the Agilent robot sample prep methodology is identical to that detailed above ( 23 ), other than a different ion mobility range (0.6–1.6 Vs/cm 2 ), and mass range (400–1000 m/z). LC-MS/MS for timsTOF for Thermo KingFisher Apex ABPP-HT* Peptides were analysed by nanoLC-MS/MS using an Evosep One LC system coupled with a timsTOF HT (Bruker) equipped with an 8 cm x 150 µm, 1.5 µm analytical column (Evosep). 200 ng peptides were separated using the Evosep 60SPD workflow (Analytical solvents A: 0.1% FA and B: acetonitrile plus 0.1% FA). Column was held at 40°C. Data were acquired in data-independent acquisition PASEF mode with the following settings: m/z range from 100 m/z to 1700 m/z, ion mobility range from 1/K0 = 1.30 to 0.85 Vs/cm 2 using equal ion accumulation and ramp times in the dual TIMS analyser of 100 ms each. Each cycle consisted of 8 PASEF ramps covering 21 mass steps each with 25 Da windows each with 2/3 non-overlapping ion mobility windows covering the 475 to 1000 m/z range and 0.85 and 1.26 Vs/cm 2 ion mobility range. The collision energy was lowered as a function of increasing ion mobility from 59 eV at 1/K0 = 1.6 Vs/cm 2 to 20 eV at 1/K0 = 0.6 Vs/cm 2 . Agilent Bravo vs “on bench” ABPP-HT* comparison LC-MS/MS data analysis Data was searched in DIA-NN 1.8.1 with search settings left as default and match between runs enabled. Data was searched using a Uniprot Homo sapiens database containing isoforms (42,469 entries, retrieved on 15/08/2023). N = 3 for HA-Ub-PA positive controls, N = 1 for negative controls with no HA-Ub-PA present. The ‘report.pg_matrix’ output was used for analysis. The volcano plot at Fig. S1 G (N = 3 HA-Ub-PA positive and negative controls) was generated from a two-sample student’s t-test in Perseus (Version 1.6.15). Thermo KingFisher Apex ABPP-HT* workflow LC-MS/MS data analysis Data was searched in DIA-NN 1.8.1 with search settings left as default and match between runs enabled. Data was searched using a Uniprot Homo sapiens database containing isoforms (20412 entries, retrieved 13/12/2023). The ‘report.pg_matrix’ output was used (identical number of DUBs were identified in the ‘report.unique_genes_matrix’ output). DUBs were filtered to select hits > 1.5-fold enriched in the positive HA-Ub-PA control vs the negative control without HA-Ub-PA. Volcano plots were generated/calculated using GraphPad Prism (Version 10.2.3). ABPP-HT* screen and concentration-dependence data analysis Data was searched in DIA-NN 1.8.1 with search settings left as default and match between runs enabled. Data was searched using a Uniprot Homo sapiens database, not containing isoforms (20,416 entries, retrieved on 15/02/2023). The ‘report.unique_genes_matrix’ output was used to ensure inhibitor identification unique to specific DUBs. DUBs were filtered to remove those that were not > 5-fold enriched in the positive HA-Ub-PA control vs the negative control without HA-Ub-PA. For the concentration-dependent data the following DUBs were discounted due to their unreliable detection across replicates, likely attributable to their low intensities: OTUD1, USP42, OTUD5, USP1, USP30, USP33, USP37, USP43 and OTUB2. Intensities were normalized as a percentage of the positive HA-Ub-PA control and for the screen multiple two-tailed unpaired (homoscedastic) T-tests were carried out (Microsoft Excel) to identify DUBs with significantly different intensities relative to the positive control. The resultant data were then filtered to select hits with a > 20% average reduction in DUB activity. The concentration-dependence ABPP outputs were processed using CurveCurator (Version 0.3.0),(87 with the alpha asymptote set to 5%. In-depth ABPP workflows Sample preparation Cell lysis and DUB Activity-Based Enrichment: Cells were lysed in lysis buffer (50 mM Tris, pH 7.5, 5 mM MgCl 2 , 0.5 mM EDTA, 1 mM DTT, 250 mM sucrose) containing acid-washed glass beads (Sigma Aldrich #G4649). Glass beads made up approximately one third of the total lysis buffer volume. Cell lysates were vortexed and centrifuged (14K x g, 4°C, 25 min) to separate the pellet, glass beads, nuclei, and membranes from supernatant. The supernatant was transferred to Eppendorf tubes and the pellets were discarded. Proteins extracted from ~ 1·10 7 cells were used as starting materials. When profiling a DUB inhibitor, PR-619 (Sigma-Aldrich # 662141) was first incubated and mixed at 1,000 rpm at 37°C for 1 h. 25 µg of a DUB probe (HA-Ahx-Ahx-Ub-PA (UbiQ-078), HA-Ahx-Ahx-Ub-VME (UbiQ-035), HA-Ahx-Ahx-Ub-VS (UbiQ-178)) or probe mixtures were added to each of the samples and mixed at 1,000 rpm at 37°C for 1 h. The reaction was quenched by the addition of SDS to 0.4% (v/v) and NP-40 to 0.5% (v/v). Lysate mixtures were then diluted to 1 mL, 0.5 mg/mL with NP-40 lysis buffer (pH 7.4, 50 mM Tris, 0.5% (v/v) NP-40, 150 mM NaCl, 20 mM MgCl 2 ). 200 µL of anti-HA-Agarose (Pierce # 26182) slurry, previously washed three times with NP-40 lysis buffer, was added to the samples and incubated on a rotator overnight at 4°C. After a first centrifugation step (2,000 x g , 4°C, 1 min), beads were washed four times with 500 µL of NP-40 lysis buffer. Protein complexes were eluted by boiling beads in 250 µL SDS Laemmli 2X sample buffer. Methanol-chloroform precipitation was performed prior to protease digestion. For each sample, a 4x volume of methanol was added and vortexed for 5 s. A 1x volume of chloroform was added to the samples, which were then vortexed for 5 s. A 3x volume of water was added, and samples were vortexed for 5 s. Samples were then centrifuged for 10 min at 10,000 rpm. The aqueous and organic phases were removed to leave only the protein pellet. The pellets were subsequently washed with 4x volume of methanol and centrifuged for 10 min at 10,000 rpm. The supernatant was discarded, and pellets were re-dissolved in 100 µL of water. Protein precipitation was repeated for a second time to ensure sufficient removal of detergent. Sample digestion and TMT labelling were performed using the IST-NHS 96x kit (PreOmics # P.O.00030) according to the manufacturer’s instructions. Briefly, 100 µL lysis buffer was added to each sample and heated to 95°C while mixing at 1,000 rpm for 10 min. Lyophilised digestion enzymes were dissolved in 100 µL water before being added to the samples. The samples were digested at 37°C for 2 h. For samples to be analysed by a Label-Free quantitation MS method, digestion was stopped, and the sample was transferred to the cartridge in a centrifuge at 2000 g for 1 min before proceeding the washing steps with wash solvent 1 and 2 provided with the kit. For the remaining samples, 200 µg TMTpro 18 Plex reagents (ThermoFischer Scientific # A52045) were added to each of the samples and incubated for 1 h while shaking at 1000 rpm. A total of 10 µL of 5% (v/v) aqueous hydroxylamine was added to each of the samples. A total of 100 µL of stop reagent was also added to the samples before transferring to cartridge for washing with washing solvent 1 and 2 provided with the kit. Samples were eluted with 250 µL of 50% (v/v) acetonitrile in 0.01% (v/v) aqueous formic acid. Samples labelled with TMT reagents were combined, dried and subjected to high pH fractionation using HPLC (Agilent). Peptides were loaded into a XBridge C18 column 4.6 x 250 mm (Waters # PN186003117) in buffer A (H2O, pH 10) and eluted with increasing buffer B (90% (v/v) aqueous acetonitrile, pH 10) at a 0.50 mL/min flowrate over a 96 min gradient. Eluted samples were collected every minute and concatenated to give a total of 24 fractions. Desalting was performed by solid phase extraction (SPE) employing reversed-phase C18 cartridges (Sep-Pak C18 light). LC-MS/MS methodology For LFQ Data Dependent Acquisition (DDA) and Data independent Acquisition (DIA) workflows, peptides were analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using an Ultimate 3000 UHPLC coupled to an Orbitrap Fusion Lumos Mass Spectrometer (both from ThermoFisher). Tryptic peptides were separated on an EASY-spray PepMap column (2 µm, 75 µm x 50cm; ThermoScientific) using a 60 min linear gradient from 2 to 35% buffer B (5% DMSO, 100% acetonitrile, 0.1% TFA) with a 250 nL/min flow and analysed on the Orbitrap Fusion Lumos (Thermo Scientific). Data were acquired in DDA and DIA as ( 19 , 86 , 87 ). In both cases the advance peak detection (ADP) was enabled. For DDA, survey scans were acquired in the Orbitrap at 120 k resolution over a m/z range 400–1500, AGC target of 4e5 and S-lens RF of 30. Fragment ion spectra (MS/MS) were obtained in the Ion trap (rapid scan mode) with a Quad isolation window of 1.6, 40% AGC target and a maximum injection time of 35 ms, with HCD activation and 28% collision energy. For DIA, MS1 scans were acquired in the Orbitrap with 120k resolution (m/z 350–1650), an AGC target of 5 × 10⁵ and a maximum injection time of 20ms. The full MS events were followed by 40 DIA scan windows per cycle (with variable isolation width) covering the m/z range from 350–1650. The MS/MS scans were acquired in the orbitrap at 30k resolution and normalised HCD set up at 30%. For TMT labelled samples, mass spectrometric analyses were carried out with a Proxeon Easy Nano-LC fitted with a PepMap C18 column 75 µm x 50 cm (Thermos Scientific #PN ES903) and connected to a Lumos Orbitrap mass spectrometer (Thermo Scientific). Peptides were eluted at 450 nL flowrate during 120 min gradient. MS data was acquired with SPS M3 approach. Each full orbitrap scan (R=60K, m/z scan range: 385–1350, AGC target: 100k, max. injection time: 100 ms) was acquired and followed with dependent IT MS/MS scans (isolation window: 0.5, CID collision energy (%): 35, AGC target: 30k ions, max. injection time: 175 ms) for a 2.7s cycle time. SPS MS3 scans were acquired with the following parameters (R: 50k, isolation window = 1.5, AGC target: 120k, HCD collision energy (%): 55, max. injection time: 100 ms). LC-MS/MS data analysis Single shot LFQ Data was searched using a Uniprot Homo sapiens database containing isoforms (42,469 entries, retrieved on 15/08/2023). Software versions: DIA-NN 1.8.1, Fragpipe 22.0, MaxQuant/MaxDIA 2.6.1.0. All search settings were left as default, with match between runs enabled and cysteine carbamidomethylation included as a fixed modification. Each search was 3 x HA-Ub-* and 3 x HA-Ub-*+ 50 µM PR619. The PA warhead alone was searched separately from the PA/VME/VS cocktail of warheads. The intensities output ‘report.pg_matrix’ was used from DIA-NN, the LFQ intensities from ‘combined_protein’ output was used from Fragpipe, and the LFQ intensities from the ‘protein groups’ was used from Maxquant for both DDA and DIA searches. LC-MS/MS data analysis TMT fractions MS data processing was performed with Proteome Discoverer Version 2.4.0.305 (Thermo Scientific) with UniProtKB Swiss-Prot (TaxID = 9606, Homo sapiens ) fasta. Peptide identifications were carried out using the Sequest™ search engine with the following parameters: precursor tolerance: 10 ppm, fragment tolerance = 0.6 Da, max. missed cleavage sites: 3, dynamic modifications: M oxidation and N/Q deamination, fixed modifications: PreOMics cysteine modification (+ 113.084 Da) and TMTpro (+ 304.207 N-terminus and K ABP western blot compound validations ABPP-HT* screen hit validations For positive hit validations by western blot the sample preparation detailed for the “on bench” ABPP-HT* were used with the concentrations of compounds indicated in figures. Following compound incubation and reaction with HA-Ub-PA, the reaction was quenched directly with Laemmli buffer supplemented with 100 mM DTT, boiled, and analysed by western blot. IC 50 values were extracted by quantifying the HA-Ub-PA labelled band by densitometry (Image studio lite) normalised to the β-actin signal and subsequently normalising the signal of the DUB activity to the negative and positive HA-Ub-PA controls. Concentration-dependencies were fit to Y = 100/(1 + 10^((LogIC 50 -X)*HillSlope)) in Graphpad Prism (Version 10.1.1). Compound vs HA-Ub-PA time dependence competition studies Samples were processed as for the ABP western blot compound concentration-dependence validations, with the HA-Ub-PA incubated for the indicated time. ABP compound cellular permeability studies MCF-7 cells were cultured in 6-well plates to 90% confluence, with a media change to 1 mL prior to DMSO/compound treatment at the indicated concentration for 4 h. Cells were then washed 3 x with PBS, and collected by scraping in PBS and centrifuged (200 x g for 10 min). Pellets were then processed as outlined above in the ABPP screen sample preparation section, with 0.4 µg of HA-Ub-PA being used per 50 µg of lysate protein. The mixture was incubated for 45 min at 37 ˚°C, before being quenched by addition of Laemmli buffer supplemented with 100 mM of DTT; the mixture was then analysed by western blotting. Compound 25 selectivity with biotin-ISG15-PA ABP For Compound 25 selectivity with biotin-ISG15-PA the sample preparation detailed for the Thermo KingFisher Apex ABPP-HT* was used with the concentrations of compounds indicated in figures. For each compound, stock solutions (100×) were prepared at a concentration of 0.46, 1.39, 4.16 or 12.5 mM in DMSO in a 96-well plate, and 1.5 µL of each stock were transferred into the respective wells using a multichannel pipette (resulting in a final concentration of 4.6, 13.9, 41.6 or 125 µM). Negative and probe-only positive controls were treated with 1.5 µL of DMSO. The plate was then incubated at RT for 1 h without shaking. Following compound incubation 2.35 µL of bio-ISG15-PA was transferred into the respective wells using a multichannel pipette, with an equivalent volume of buffer added to the corresponding negative control wells and incubated at 37°C for 45 min without shaking. Following compound incubation and reaction with HA-Ub-PA, the reaction was quenched directly with Laemmli buffer supplemented with 50 mM DTT, boiled, and analysed by western blot. Biotinylated USP5 inhibitors HA-Ub-PA ABP For HA-Ub-PA ABP assays MCF-7 cell lysates were lysed as detailed for the Thermo KingFisher Apex ABPP-HT* methodology and incubated with biotinylated Compound 25 derivative inhibitors at either 150 µM or 250 µM) for 1 h at room temperature. Following the initial incubation, 0.5 µl of HA-Ub-PA was added and incubated at 37°C for 45 min. Reactions were quenched with Laemmli buffer and DTT (50 mM), boiled and analyzed by western blot with immunoblotting for streptavidin, USP5, USP42, and GAPDH. DMSO-treated samples and reactions lacking HA-Ub-PA ABP were included as vehicle controls. Reactions with non-biotinylated USP5 inhibitor (Compound 25b) at 150 µM was included as a negative control. Immunoblotting against USP5, USP42 and GAPDH were also included as controls, positive, negative, and loading, respectively. Competition assay with non-biotinylated inhibitor (Compound 25) and N-Ethylmaleimide (NEM) For non-biotinylated Compound 25 and NEM competition assays MCF-7 cell lysates were lysed as detailed for the Thermo KingFisher Apex ABPP-HT* methodology and pre-incubated with either non-biotinylated inhibitor (Compound 25) at 250 µM or NEM at 30 mM at room temperature for 1 h. Following the initial incubation, biotinylated Compound 25 derivative inhibitors at 150 µM or DMSO control were added and incubated at room temperature for 1 h. Reactions were quenched with Laemmli buffer and DTT (50 mM), boiled and analyzed by western blot with immunoblotting for streptavidin, USP5, USP42, and GAPDH. DMSO-treated samples lacking biotinylated USP5 inhibitors and/or competitors were included as vehicle controls. Immunoblotting against USP5, USP42 and GAPDH were also included as controls, positive, negative, and loading, respectively. OTUD7B Protein expression and purification OTUD7B (129–438) cloned into pOPIN E vector was expressed in Rosetta2 (DE3) E. coli cells grown in 2xTY medium supplemented with 100 µg/mL carbenicillin and induced with 400 µM Isopropyl-β-D-thiogalactoside (IPTG) at 18°C overnight. E.coli pellets were lysed in 20 mM Tris pH 8.5, 500 mM NaCl, 20 mM Imidazole, 2 mM β-mercaptoethanol supplemented with lysozyme, DNase, 2 µM pepstatin, 4 µM leupeptin and 1 mM PMSF. Clarified lysates were applied to a HisTrap HP column (Cytiva) and eluted in 20 mM Tris pH 8.5, 500 mM NaCl, 500 mM Imidazole, 2 mM β-mercaptoethanol. Further purification was achieved by anion exchange chromatography (Resource Q, Cytiva) followed by size exclusion chromatography (Superdex 75, Cytiva) in 20 mM HEPES pH 7.5, 200 mM NaCl, 4 mM DTT. Concentrated protein was flash frozen and stored in single-use aliquots at -75°C. Enzyme kinetics Fluorescence intensity measurements were used to monitor the cleavage of a ubiquitin-rhodamine substrate. All activity assays were performed in black 384-well plates in assay buffer (20 mM Tris, pH 8.0, 150 mM potassium glutamate, 0.1 mM TCEP, 0.03% Bovine Gamma Globulin) with a final assay volume of 20 µL. USP7, 1 nM (USP7 (1-1102), DU15644, MRC Protein Phosphorylation and Ubiquitylation Unit) or USP47, 10 nM (USP47 (N-terminal His), DU15682, MRC Protein Phosphorylation and Ubiquitylation Unit) was added and preincubated with Compound 3 for 30 min. OTUD7B (OTU domain) was added and preincubated with Compound 6 for 30 min. 180 nM ubiquitin-rhodamine 110 (Ubiquigent for USP47/7, Bio-Techne for OTUD7B) was added to initiate the reaction and the fluorescence intensity was recorded for 30 min on a PherastarFSX (BMG Labtech) with an Ex 485 /Em 520 optic module. Initial rates were plotted against compound concentration to determine IC 50 . To investigate the time-dependence of the potency of Compound 3 , activity assays were repeated without a preincubation step. HDX-MS Sample Preparation Hydrogen Deuterium eXchange Mass Spectrometry (HDX-MS) experiments were performed on the same stock of recombinant USP7 and USP47 protein constructs as those used in the in vitro enzyme kinetics experiments. USP7 was provided at a concentration of 25.3 µM and USP47 at 16.5 µM, respectively. Both proteins were prepared in 50 mM HEPES, pH 7.5, 10% glycerol, 150 mM NaCl, 1 mM DTT. The initial stock concentration of Compound 3 was 30 mM in DMSO. In solution HDX-MS was performed by preparing a volume-to-volume mixture at a molar ratio of 1:2 of protein: Compound 3 ( i.e ., the holo-state). Samples were diluted to achieve a final concentration of 16 pmol on column per reaction for USP7 and 21 pmol on column per reaction for USP47. Equivalent control samples ( i.e ., the apo-state) were prepared where DMSO was supplemented in place of the compound. Data Acquisition HDX-MS was performed as( 88 ). Briefly, an identical labeling buffer was prepared as that of the protein stocks except in deuterium oxide D 2 O (99+ %D, Cambridge Isotope Laboratories, Tewksbury, MA) and with the 10% glycerol was removed (a final composition of 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM DTT). The pH of the labeling buffer was measured and corrected to pD (pD = pH+0.4). The quenching buffer comprises 2M Guanidine HCl, 100 mM citric acid, pH 2.3 in H 2 O. Proteins +/- Compound 3 were preincubated for 30 min at room temperature to allow complexes to form. Samples were then diluted with the labeling buffer in a 1:20 ratio, achieving an excess D 2 O concentration of 95%. Several labeling time points were sampled at 20 ᵒC, more specifically, at 30, 600, and 3600 s. All analyses were acquired in triplicate. Non-deuterated controls were prepared in an identical fashion, with H 2 O in place of D 2 O. At the end of each labeling time point, samples were quenched upon addition of quench buffer (1:1 ratio), resulting in a final pH of 2.5. Samples were subsequently digested with a dual pepsin/proteaseXIII column (2.1 x 3.0 mm; NovaBioAssays, MA) at 8 ᵒC for 3 min. Peptides were trapped on a 1.0 mm x 5.0 mm, 5.0 µm trap cartridge (Thermo Scientific™ Acclaim PepMap100) for desalting. The flow rate was maintained at 150 µL/min. Peptides were then separated on a Thermo Scientific™ Hypersil Gold™, 50 x 1 mm, 1.9 um, C18 column increasing hydrophobicity achieved by a linear gradient of 10% to 40% Buffer B (A: water, 0.1% FA; B: ACN, 0.1% FA). The flow rate was maintained at 40 µL/min. A protease wash (2 M guanidine, 100 mM citric acid, pH 2.3 in H 2 O) was performed for each run to limit carry-over. To minimize back-exchange, the quenching, trapping, and separation steps were performed at 1.5ᵒC. Labeling, quenching, and online digestion steps were performed with the aid of an automated HDX robot (Trajan Scientific and Medical). Sample preparation was managed in Chronos (version 5.4.1). All samples were acquired in MS1 mode on Thermo Scientific™ Orbitrap Exploris™ 480 Hybrid™ mass spectrometer. Data Analysis Before each HDX-MS experiment, an unspecific digested peptide database was created in BioPharma Finder (version 5.2) for non-deuterated USP7 and USP47 proteins through a data-dependent and targeted HCD-MS 2 acquisition regime ( 88 ). Labeling data were processed and manually curated in HDExaminer version 3.4.2 (Trajan Scientific and Medical). The charge state with the highest quality spectra for all replicates for each peptide across all HDX-MS labeling times was used in the final analysis. The significant differences observed at each residue were used to map HDX-MS consensus effects (based on overlapping peptides) onto the AlphaFold model. Caspase-1 assay PMA-differentiated THP-1 cells were treated as indicated, and the caspase-1 activity in the culture medium (supernatant) was measured using the Caspase-Glo 1 Inflammasome Assay (Promega) according to the manufacturer’s instruction. Briefly, the Caspase-Glo 1 Reagent was prepared by resuspending the Z-WEHD substrate in Caspase-Glo 1 buffer and addition of MG132 inhibitor to a final concentration of 60 µM. Cell culture medium (50 µL) was then added to the Caspase-Glo 1 Reagent (50 µL) in a 96-well plate and the resultant mixture was incubated at room temperature for 1 h. Luminescence was measured using a FLUOstar Omega multi-mode microplate reader (BMG LABTECH) according to the manufacturer's instructions. MTS cell viability assay Cell viability was assessed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS, Promega). The One Solution Reagent (20 µL) was added to each well of a 96-well plate containing 100 µL of sample in culture medium. Following incubation at 37°C for 1 h, absorbance was measured at the wavelength of 490 nm using a FLUOstar Omega multi-mode microplate reader (BMG LABTECH). Cell viability was calculated according to the manufacturer's instruction. ASC speck oligomer cross-linking ASC oligomer chemical cross-linking was performed as previously described ( 89 ). Briefly, PMA-differentiated THP-1 cells were primed with LPS (1 µg/mL) for 4 h, followed by stimulation with nigericin (10 µM) for 45 min and indicated concentrations of Compound 3 . Cells were lysed on ice in Buffer A (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 320 mM sucrose, and protease inhibitors), and homogenised using a 21-gauge needle. Lysates were centrifuged at 300 × g for 8 min at 4°C. The supernatants were collected, mixed 1:1 with CHAPS buffer (20 mM HEPES, pH 7.5, 5 mM MgCl₂, 0.5 mM EGTA, 0.1% CHAPS, and protease inhibitors), and centrifuged at 2,400 × g for 8 min to pellet crude inflammasome complexes. Pellets were washed twice with ice-cold PBS, resuspended in CHAPS buffer, and cross-linked with 2 mM DSS (prepared in DMSO) at 37°C for 20 min. The reaction was quenched with NuPAGE™ LDS Sample Buffer (4X) and subjected to western blot analysis. Alignment and structure prediction of homo sapiens and Pan troglodytes USP47 For kinetic and HDX MS studies of Compound 3 , full-length Homo Sapiens USP7 and full-length Pan troglodytes isoform 2 were used. Sequences of full-length Homo sapiens canonical USP47 and full-length USP47 from Pan troglodytes isoform 2 were aligned using T-coffee multiple sequence alignment with differences reported in Fig. S9 A ( 90 ). The sequences, including the His-tag of the recombinant Pan troglodytes isoform 2, were then used to predict the structures using AlphaFold 3 ( 91 ), with the highest-ranking structures being superposed using PyMOL (version 2.5.0) with an RMSD of 2.511 (Fig. S9 B and S9 C ). OTUD7B Intact MS Binding of OTUD7B to compound 6 was detected by Liquid Chromatography Mass Spectrometry (LCMS) using an Agilent 1290 infinity II LC system connected to an Agilent 6550 accurate mass iFunnel quadrupole time of flight (QTOF) mass spectrometer. OTUD7B was incubated with compound 6 in 50 mM HEPES pH 7.5 for 1.0 and 15.0 min and 1.0 µL of sample was injected and loaded onto a ProSwift RP-4H monolithic phenyl reverse phase , 1.0 x 50 mm, HPLC column (Thermo). Solvent A consisted of LCMS grade water containing formic acid (0.1% v/v) and solvent B consisted of acetonitrile containing formic acid (0.1% v/v). OTUD7B (OTU domain) and Compound 6 were separated using a step wise gradient (0 min – 95.0% solvent A, 1.0 min − 80% solvent A, 9.0 min − 45% solvent A, 10 min − 0% solvent A, 11 min − 0% solvent A, 12.0 min – 95.0% solvent A), followed by a 3.0-min post-run with solvent A. All flow rates were 0.2 mL/min. The mass spectrometer was operated in positive ion mode with a drying gas temperature (225°C), drying gas flow rate (13 L/min), nebulizer pressure (20 psi), sheath gas temperature (350°C), sheath gas flow rate (12 L/min), capillary voltage (4000 V), nozzle voltage (1500 V), fragmentor voltage (365 V). All acquired data were analysed using Agilent MassHunter Qualitative Analysis (Version B.07.00) software. HIF-1α hypoxia conditions and Covid-19 infection assay SARS-CoV-2 Australia/VIC01/2020 virus and cells were provided by the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia at P1 and passaged twice in Vero/hSLAM cells (Cat#04091501) - obtained from the European Collection of Cell Cultures (ECACC), UK. Virus infectivity was determined by plaque assay on Vero-TMPRSS2 cells as previously reported ( 92 ). Calu-3 cells were infected with the above strain of SARS-CoV-2 at a MOI of 0.01 for 2 h. Viral inocula were removed, cells washed three times in PBS and maintained in growth media until harvest. Cell lines were maintained at 37°C and 5% CO 2 in a standard culture incubator and exposed to hypoxia using an atmosphere-regulated workstation set to 37°C, 5% CO 2 :1%–5% O 2 :balance N 2 (InvivO 2 200, Baker-Ruskinn Technologies). For quantification of viral RNA, total cellular RNA was extracted using the RNeasy kit (Qiagen) according to manufacturer’s instructions. Equal amounts of RNA, as determined by Nanodrop analysis, were used in a one-step RT-qPCR using the Takyon-One Step RT probe mastermix (Eurogentec) and run on a Roche Light Cycler 96. For quantification of viral copy numbers, qPCR runs contained serial dilutions of viral RNA standards. Total SARS-CoV-2 RNA was quantified using: 2019-nCoV_N1-F: 5’-GAC CCC AAA ATC AGC GAA AT-3’, 2019-nCoV_N1-R: 5’-TCT GGT TAC TGC CAG TTG AA TCT G-3’, 2019nCoV_N1-Probe: 5’-FAM-ACC CCG CAT TAC GTT TGG ACC-BHQ1-3’. For assaying the antiviral response against Compound 25b , Calu-3 cells were infected with a SARS-CoV-2 reporter virus containing a mNeonGreen fluorescent reporter as previously described at a MOI of 0.1 ( 93 ). Infected cells were treated with serial dilutions of the compound for 24 h followed by fixation in 4% PFA. Cells were stained with DAPI and mNeonGreen fluorescence was quantified by Clariostar. Fluorescence data was normalised to the DAPI signal and plotted as relative to the DMSO control. Type I interferon production Type I interferon production was performed as previously described ( 94 ), using HEK293 cells transduced with a pGreenFire-ISRE reporter. In brief, HAP1 cells were seeded at 5,000 cells per well in a 96-well plate in DMEM containing 10% FBS and 2 mM glutamine. After attachment, cells were treated with Compound 25b for 1 h, followed by poly(I:C) (0.5mg/mL) for 48 h. HAP1 media was then collected, centrifuged, and transferred to HEK293 IFN reporter cells, which were seeded at 25,000 cells per well in a 96-well plate and allowed to attach. Recombinant human IFNα2 (PBL assay science) was used for standards. After 24 h, the One-Glo Luciferase Assay System (Promega) was used to quantify luciferase expression following manufacturer’s instructions. Cell growth and proliferation assays A total of 5,000 cells were seeded into either 6-well or 12-well plates and imaged using an IncuCyte Zoom Imager (Sartorius) for live-cell imaging. Imaging was performed at the specified time points using the phase-contrast channel, as previously described ( 85 ). Cell growth was assessed by measuring the percentage confluence. Molecular docking A model for human USP47 catalytic domain (UniProt: Q96K76), which is representative of a covalently bound inhibitor conformational state, was generated using the structure of human USP7 in complex with the covalent inhibitor, FT827 (PDB: 5NGF; Turnbull et al ., 2017), as the template in SwissModel ( 95 ). Coordinates for Compound 3 were generated using ChemDraw Prime 23.0.1.10 and molecular docking was performed using CovDock ( 96 ) implemented in Maestro version 13.8.135 (Schrödinger Release 2024-3: Maestro, Schrödinger, LLC, New York, NY, 2024). The best docking pose for Compound 3 corresponded to a docking score of -5.704. Antibodies The antibodies used for immunoblotting in this study are listed in Table 1. Table 1 List of antibodies for immunoblotting used in this study. Reagent Brand Catalogue Number Notes USP5 Proteintech 15158-1-AP USP7 ENZO BML-PW0540-0100 USP18 (D4E7) Cell Signaling Technology 4813 USP42 Santa Cruz sc-390604 USP30 Atlas antibodies HPA016952 USP33 Proteintech 20445-1-AP USP47 Abcam ab72143 OTUD7B Cell Signaling Technology 14817 VCPIP1 Cell Signaling Technology 88153 HA (12CA5) Roche 11666606001 CFL1 Cell Signaling Technology 5175 NLRP3(D4D8T) Cell Signaling Technology 15101 Caspase-1(D7F10) Cell Signaling Technology 3866 ASC(B-3) Santa Cruz sc-514414 IL-1b(D3U3E) Cell Signaling Technology 12703 Cleaved-IL-1b (Asp116) (D3A3Z) Cell Signaling Technology 83186 GSDMD Novus Biologicals NBP2-33422 HIF-1α BD Biosciences 610959 β-actin Sigma Aldrich A5441 Used in all figures except for Fig. 5A β-actin (8H10D10) Cell Signaling Technology 3700 Used in Fig. 5A IRDye 680RD Streptavidin Li-Cor 926-68079 GAPDH Cell Signaling Technology 97166 Declarations Data availability The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD064209. The mass spectrometry HDX data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD064060. Acknowledgments The A.P.-F. and B.M.K. labs were supported by the Chinese Academy of Medical Sciences Innovation Fund for Medical Science, China (grant numbers: 2018-I2M-2-002 and 2024-I2M-2-001-1) and by Pfizer Inc. The A.P.-F. lab was also supported by CRUK, Ono Pharma, and Boehringer Ingelheim. H.B.L.J. was supported by a Bristol-Myers Squibb fellowship. We thank Prof. Jan Rehwinkel for generously providing the type I interferon reporter cells and Prof Nicole Zitzmann’s lab for generously providing the Calu-3 cells. Author Contributions: H.B.L.J., S.D., L.B., and A.P.-F. designed research; H.B.L.J., S.D., S.S.H., P.A.C.W., C.N., Z.L., J.D., J.L., E.M., A.B., A.C., A.P., L.W., S.S., J.W.H., M.E.R., S.F., A.T., D.O.B., E.S., and A.P.T. performed research; C.J.E and P.R.E. contributed new reagents/analytic tools; I.V., E.W.T., D.O.B., G.M.W., B.M.K., C.J.S., L.B., A.P.-F. provided supervision; H.B.L.J., L.B., and A.P.-F. wrote the paper, and D.O.B., S.D., S.S.H., A.P.T., C.J.S., P.R.E., and B.M.K. reviewed and corrected the manuscript. Competing Interests: The authors declare that they have no competing interests affecting the contents of this article. References N. J. Schauer, R. S. Magin, X. Liu, L. M. Doherty, S. J. Buhrlage, Advances in Discovering Deubiquitinating Enzyme (DUB) Inhibitors. J. Med. Chem. 63, 2731–2750 (2020). T. S. Z. Fang, et al. , Knockout or inhibition of USP30 protects dopaminergic neurons in a Parkinson’s disease mouse model. Nature Communications 2023 14:1 14, 1–16 (2023). 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Supplementary Files JonesDraganovetalSupplementaryInformationDrugDiscovery2026.pdf JonesDraganovetalSupplementaryDataS4HDXSummaryTableforFigure4KLandS3FGDrugDiscovery2026.xlsx JonesDraganovetalSupplementaryDataS9DataforFigureS1GDrugDiscovery2026.xlsx JonesDraganovetalSupplemetaryDataS7DataforFigureS1EDrugDiscovery2026.xlsx JonesDraganovetalSupplementaryDataS5HDXUptakeSummaryTableforFigure4KLandSFGDrugDiscovery2026.xlsx JonesDraganovetalSupplementaryDataS3DataforFigure2EJandS3DrugDiscovery2026.xlsx JonesDraganovetalSupplementaryDataS1UncroppedBlotsDrugDiscovery2026.pdf JonesDraganovetalSupplementaryDataS2DataforFigure1CDDrugDiscovery2026.xlsx JonesDraganovetalSupplementaryDataS8DataforFigureS1FS1HS1IDrugDiscovery2026.xlsx JonesDraganovetalSupplementaryDataS6DataforFigureS1AS1BS1CDrugDiscovery2026.xlsx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 29 Apr, 2026 Reviews received at journal 28 Apr, 2026 Reviews received at journal 19 Apr, 2026 Reviewers agreed at journal 09 Apr, 2026 Reviewers agreed at journal 08 Apr, 2026 Reviewers invited by journal 07 Apr, 2026 Editor assigned by journal 18 Mar, 2026 Submission checks completed at journal 16 Mar, 2026 First submitted to journal 12 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9106698","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":620384022,"identity":"4c6c4c9d-62ec-401b-87b3-b608736ae8db","order_by":0,"name":"Hannah B. L. 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C.","lastName":"Wing","suffix":""},{"id":620384043,"identity":"057e4301-13c3-4483-8a20-5525b17d6f7e","order_by":4,"name":"Chuong Nguyen","email":"","orcid":"","institution":"Pfizer (United States)","correspondingAuthor":false,"prefix":"","firstName":"Chuong","middleName":"","lastName":"Nguyen","suffix":""},{"id":620384049,"identity":"d84e7cc0-6eae-43d9-bc88-ae23c79df32c","order_by":5,"name":"Zhu Liang","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Zhu","middleName":"","lastName":"Liang","suffix":""},{"id":620384053,"identity":"ac9a7fe5-13ed-40e2-94c7-bf3806a6f9ae","order_by":6,"name":"Julia Dörner","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Dörner","suffix":""},{"id":620384057,"identity":"75548a52-f750-4719-b06e-e09ade48b3ba","order_by":7,"name":"Jasper Lithgow","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Jasper","middleName":"","lastName":"Lithgow","suffix":""},{"id":620384060,"identity":"d30389a1-3bfc-4253-822f-b7c8504d736c","order_by":8,"name":"Emma Murphy","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Emma","middleName":"","lastName":"Murphy","suffix":""},{"id":620384063,"identity":"636d2700-d292-42aa-9e19-ba41c1b45335","order_by":9,"name":"Alice Beard","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Alice","middleName":"","lastName":"Beard","suffix":""},{"id":620384064,"identity":"261e4d45-f2c1-478b-a104-f156606e328b","order_by":10,"name":"Angela Chen","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Angela","middleName":"","lastName":"Chen","suffix":""},{"id":620384065,"identity":"897d74bf-eba8-4192-b42e-5bbe210132fe","order_by":11,"name":"Andrea Pierangelini","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Pierangelini","suffix":""},{"id":620384066,"identity":"4feb1442-d289-43c4-945f-d75d505e3d9a","order_by":12,"name":"Jack W. Houghton","email":"","orcid":"","institution":"Imperial College London","correspondingAuthor":false,"prefix":"","firstName":"Jack","middleName":"W.","lastName":"Houghton","suffix":""},{"id":620384067,"identity":"bd5693df-3d88-4834-90b3-70f330cbbb57","order_by":13,"name":"Mackenzie E. Robert","email":"","orcid":"","institution":"Pfizer (United States)","correspondingAuthor":false,"prefix":"","firstName":"Mackenzie","middleName":"E.","lastName":"Robert","suffix":""},{"id":620384068,"identity":"075779c7-af23-4515-9af7-43ef5d8bf733","order_by":14,"name":"Sarah Flannery","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Flannery","suffix":""},{"id":620384071,"identity":"702e4e30-e76c-4b7f-a394-38ab31cfe32a","order_by":15,"name":"Cara J. 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Schofield","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Christopher","middleName":"J.","lastName":"Schofield","suffix":""},{"id":620384096,"identity":"6d7e21ec-2a8d-4949-a224-3f6ffaeeab97","order_by":27,"name":"Benedikt M. Kessler","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Benedikt","middleName":"M.","lastName":"Kessler","suffix":""},{"id":620384099,"identity":"bb452ee4-3121-46e6-ba8f-e074885d5396","order_by":28,"name":"Lennart Brewitz","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Lennart","middleName":"","lastName":"Brewitz","suffix":""},{"id":620384103,"identity":"104cba75-2208-45e6-8723-6f25f81d053b","order_by":29,"name":"Adán Pinto-Fernández","email":"data:image/png;base64,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","orcid":"","institution":"University of Oxford","correspondingAuthor":true,"prefix":"","firstName":"Adán","middleName":"","lastName":"Pinto-Fernández","suffix":""}],"badges":[],"createdAt":"2026-03-12 15:53:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9106698/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9106698/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106960369,"identity":"6fe8da44-85ed-4756-adb0-6e187d8db1ea","added_by":"auto","created_at":"2026-04-15 09:20:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":491057,"visible":true,"origin":"","legend":"\u003cp\u003eDUB inhibitor screen using activity proteomics and a cysteine-centric compound library. (A) ABPP-HT* DUB inhibitor screen workflow. Created in BioRender. Jones, H. (2025) \u003ca href=\"https://biorender.com/yl1zds3\"\u003e(License\u003c/a\u003e\u003cu\u003e link to be inserted upon publication)\u003c/u\u003e. (B) Design rationale for DUB inhibitor chemotypes: ubiquitin peptide-alkyne inhibitors exploit native ubiquitin recognition, whereas αβ,α′β′-diepoxyketones (DEKs) and γ-lactams probe non-canonical cysteine reactivity and active-site accessibility independent of ubiquitin binding. (C) Heatmap of DUBs identified from ABPP-HT* screen, performed using MCF-7 lysates. DUB activity in the presence of 25 µM of \u003cstrong\u003eCompounds 1-41\u003c/strong\u003e (\u003cstrong\u003eCompound 42\u003c/strong\u003e = established USP7 inhibitor FT827) as a % of positive control activity (n = 2, controls n = 4). Cells outlined with black squares indicate \u0026gt; 20 % inhibition (p \u0026lt; 0.05 - two tailed unpaired t-test, (\u003cstrong\u003eCompound 6\u003c/strong\u003e OTUD7B p= 0.066)), with significant inhibition from \u003cstrong\u003eCompounds 4\u003c/strong\u003e and \u003cstrong\u003e27 \u003c/strong\u003enot highlighted due to lack of selectivity. (D) Significant hits from ABPP-HT* screen taken forward for validations, showing reduction in activity relative to other DUBs.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9106698/v1/5759fe7e25156a19c7e8fbbe.png"},{"id":106830935,"identity":"3bc1621e-2d4c-4bfa-b715-45379f339969","added_by":"auto","created_at":"2026-04-13 23:40:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":647344,"visible":true,"origin":"","legend":"\u003cp\u003eHit Validation and Dose-Response Analysis.\u003cstrong\u003e \u003c/strong\u003e(A) Compound structures of positive hits. (B) HA-Ub-PA ABP validations in MCF-7 cell lysates showing concentration-dependent inhibition of DUBs by positive hits identified from ABPP-HT* screen. (C) Densitometric quantitation from western blots of positive hits (n = 2) showing log inhibitor concentration \u003cem\u003evs \u003c/em\u003enormalised DUB activity, fit to Y=100/(1+10^((LogIC\u003csub\u003e50\u003c/sub\u003e-X)*HillSlope)) (error bars = SD). (D)\u003cstrong\u003e \u003c/strong\u003eIC\u003csub\u003e50\u003c/sub\u003e values extracted from fits in C, (CI= Confidence interval).\u0026nbsp; (E-G) ABPP-HT* DUB activity with concentration-dependence of Compounds 3 (E), 6 (F) and 25 (G) (n = 2, controls n = 3). (H-J) Curve curator volcano plots (87), showing Log\u003csub\u003e2\u003c/sub\u003e Fold change decrease in DUB intensity with 250 µM of Compound 3 (H), 6 (I), and 25 (J) Colour is reflective of pEC50 values across concentration-dependence shown in E-G.\u0026nbsp;\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9106698/v1/e74835fcc787579deccdc439.png"},{"id":106960297,"identity":"f6d9b83e-35d6-46be-97f0-0e0345b758ed","added_by":"auto","created_at":"2026-04-15 09:19:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":700105,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular Basis for Selective Inhibition of USP47 by Compound 3.\u003cstrong\u003e \u003c/strong\u003e(A) Inhibition of purified USP47 and USP7 determined from Ub-Rho cleavage after a 30 min pre-incubation with varying concentrations of \u003cstrong\u003eCompound 3\u003c/strong\u003e. IC\u003csub\u003e50\u003c/sub\u003e curves fit to:\u0026nbsp; Y=Bottom + (Top-Bottom)/(1+10^((LogIC\u003csub\u003e50\u003c/sub\u003e-X)*HillSlope)) (Error bars = SD). (B) Inhibition of USP47 with \u003cstrong\u003eCompound 3\u003c/strong\u003e without preincubation, determined from Ub-Rho cleavage. Each curve represents inhibition data at an individual incubation time from 18 to 1782 s. (C) Inhibition of USP7 with \u003cstrong\u003eCompound 3\u003c/strong\u003e without pre-incubation, determined from Ub-Rho cleavage. Each curve represents inhibition data at an individual incubation time from 18 to 1782 s.\u003cstrong\u003e \u003c/strong\u003e(D) Modelled structure of human USP47 in complex with \u003cstrong\u003eCompound 3\u003c/strong\u003e, shown as a stick representation with carbon atoms coloured green. Residues within 5 Å of the \u003cstrong\u003eCompound 3\u003c/strong\u003e-C197 adduct are highlighted, and the key hydrogen-bonding interaction between the epoxide moiety of \u003cstrong\u003eCompound 3\u003c/strong\u003e and the side chain of R402 is represented as a dotted line. Figure prepared using PyMOL (The PyMOL Molecular Graphics System, version 2.5.8; Schrödinger, LLC). (E) Superposition of the modelled structure of human USP47 (grey) in complex with \u003cstrong\u003eCompound 3\u003c/strong\u003e (green carbon atoms) on the X-ray structure of human USP7 (cyan) in complex with the covalent inhibitor, FT827 (yellow carbon atoms; PDB: 5NGF). C* denotes the catalytic cysteine (corresponding to C197 in human USP47 and C223 in human USP7). \u003cstrong\u003eCompound 3\u003c/strong\u003e is predicted to bind within the thumb-palm cleft in a similar fashion to FT827, with FT827 extending out further towards the fingers subdomain. Sequence differences are labelled for human USP47 and the corresponding residues are labelled and underlined for human USP7. R402 in USP47 is predicted to hydrogen bond directly to \u003cstrong\u003eCompound 3\u003c/strong\u003e (represented as a dotted line). R402 in USP47 corresponds to N418 (disordered side chain in PDB: 5NGF) in USP7 with a shorter side chain that cannot interact with the compound, which may explain the higher potency of \u003cstrong\u003eCompound 3\u003c/strong\u003e for USP47. (F-G) Residual ΔHDX plots showing differences in deuterium uptake between holo- and apo- states for USP47 (F) and USP7 (G), across all indicated peptide fragments and time points (30 sec to 1 h).\u0026nbsp; These zoomed-in views highlight the peptides surrounding only those regions of the full-length protein (residues 116-323 of USP47 (F) and 45-347 of USP7 (G)). These regions also encompass the catalytic cysteine residue of each DUB.\u0026nbsp; Positive ΔHDX values indicate increased solvent exposure or flexibility in the presence of \u003cstrong\u003eCompound 3\u003c/strong\u003e, whilst negative values suggest protection or structural stabilisation. A grey shaded area marks the significance threshold for ΔHDX applied in each study. The green boxes indicate those peptides which encompass the catalytic cysteine. Due to the disappearance of these peptides in the holo-state of USP47, these peptides could not be visualised on the plot. This is further evidence of the covalent attachment of \u003cstrong\u003eCompound 3\u003c/strong\u003e to the catalytic cysteine in USP47. (H) Modelled structure of human USP47 catalytic domain (grey) in complex with \u003cstrong\u003eCompound 3\u003c/strong\u003e (carbon atoms in green) highlighting regions identified in the HDX-MS analysis coloured red (mean perturbation over 60 min of 5–20%) and magenta (mean perturbation over 60 min of \u0026lt;60%). The 19-residue C-terminal activation peptide of human USP7 (PDB: 5JTV; corresponding to residues 1084 to 1102) is shown in gold with key residues labelled and underlined. The activation peptide of USP47 (corresponding to residues 1345 to 1362) (42) is proposed to play an important role in activation and regulation analogous to the activation peptide of USP7, which maintains the switching loop (SL; especially W285 and H294) in the active “out” conformation (43). W285 and H294 in human USP7 correspond to W262 and H271 in human USP47. Regions identified in the USP47 HDX-MS analysis correlate with a conformational shift in the SL and potential engagement of the activation peptide upon \u003cstrong\u003eCompound 3\u003c/strong\u003e binding.\u003cstrong\u003e \u003c/strong\u003e(I) Human USP7 catalytic domain (cyan; from PDB: 5NGF) with the superimposed modelled position of \u003cstrong\u003eCompound 3\u003c/strong\u003e in human USP47 (green carbons) highlighting regions identified in the HDX-MS analysis in red (mean perturbation over 60 min of 5–20%) and magenta (mean perturbation over 60 min of \u0026lt;60%). The 19-residue C-terminal activation peptide of human USP7 (PDB: 5JTV; corresponding to residues 1084 to 1102) is in gold with key residues labelled and underlined. Analysis of human USP7 HDX-MS data reveals that the region of highest confidence directly flanks the proposed compound binding site and differences are not observed in structural elements implicated in activation peptide binding, suggesting that \u003cstrong\u003eCompound 3\u003c/strong\u003e does not trigger an equivalent shift in SL to the active “out” conformation and subsequent binding of the activation peptide as predicted for USP47.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9106698/v1/19fe1fe66d8044d9458d3760.png"},{"id":106961325,"identity":"0b525a5c-6279-44cb-9fee-70a1cb72b5f8","added_by":"auto","created_at":"2026-04-15 09:25:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":566738,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of USP47 with Compound 3 further supports its roles in inflammasome activation. (A) Cell permeability concentration-dependence of \u003cstrong\u003eCompound 3 \u003c/strong\u003eafter incubation with MCF-7 cells for 4 h, lysis and subsequent labelling with HA-Ub-PA. Red = lysate control (B) Caspase-1 activity in the media (supernatant) of PMA-differentiated THP-1 cells treated with indicated concentrations of\u003cstrong\u003e Compound 3\u003c/strong\u003e, determined using Caspase-Glo 1 luminescence assay. \u003cstrong\u003e\u0026nbsp;Compound 3\u003c/strong\u003eincubation for 4 h, ± 1 µg/mL LPS incubation for 4 h and 10 µM Nigericin incubation for 45 min (error bars = SD). One-way ANOVA was used. ***P \u0026lt; 0.001. (C) Immunoblots of NLRP3, Caspase-1 (Casp-1), and gasdermin d (GSDMD) from whole cell lysates of PMA-differentiated THP-1 cells treated with indicated concentrations of \u003cstrong\u003eCompound 3\u003c/strong\u003e. Immunoblot of ASC oligomerisation of the NP40-insoluble fraction of PMA-differentiated THP-1 cells after treatment with the indicated concentrations of\u003cstrong\u003e Compound 3\u003c/strong\u003eand DSS cross-linking. \u003cstrong\u003e\u0026nbsp;Compound 3\u003c/strong\u003eincubation for 4 h, ± 1 µg/mL LPS incubation for 4 h and 10 µM Nigericin incubation for 45 min. (D) MTS cell viability of THP-1 cells treated with indicated concentrations of \u003cstrong\u003eCompound 3\u003c/strong\u003e ± LPS or LPS/Nigericin. Compound treatment for 4 h, 1 µg/mL LPS treatment for 4 h, 10 µM Nigericin treatment for 2 h (error bars = SD). Red dashed boxes indicate cell death. Two-way ANOVA was used, discounting data in red dashed boxes, with Tukey multiple comparisons correction. *P\u0026lt; 0.0332, ** P\u0026lt; 0.0021, *** P\u0026lt;0.0002. (E) Formation of the NLRP3 inflammasome complex upon stimulation with bacterial products such as LPS and nigericin, with USP47 / USP7 deubiquitinating NLRP3, and the ubiquitination of NLRP3 upon USP47 / USP7 inhibition with \u003cstrong\u003eCompound 3 \u003c/strong\u003epreventing the formation of the NLRP3 inflammasome. Created in BioRender. Jones, H. (2025) (License link to be inserted upon publication).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9106698/v1/372b82d1a92e6056313199f5.png"},{"id":106961697,"identity":"7cad0807-8693-4d8a-bda0-b28e1ef3796e","added_by":"auto","created_at":"2026-04-15 09:26:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":602721,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of OTUD7B with Compound 6 further supports its roles in\u0026nbsp;hypoxic signaling and associated SARS-CoV-2 repression. (A) Inhibition of purified OTU domain of OTUD7B determined from Ub-Rho cleavage after a 30 min pre-incubation with varying concentrations of \u003cstrong\u003eCompound 6\u003c/strong\u003e. IC\u003csub\u003e50\u003c/sub\u003e curves fit to:\u0026nbsp; Y=100/(1+10^((LogIC50-X)*HillSlope)) (Error bars = SD, N=3). (B) LC-MS (liquid chromatography-mass spectrometry) analysis of the reaction of OTUD7B (top spectrum) following 1 min (central spectrum) and 15 min (bottom spectrum) incubation with \u003cstrong\u003eCompound 6\u003c/strong\u003e. (C) Mechanism for the covalent reaction of OTUD7B with \u003cstrong\u003eCompound 6\u003c/strong\u003e. (D) Cell permeability concentration-dependence of \u003cstrong\u003eCompound 6 \u003c/strong\u003eafter incubation with MCF-7 cells for 4 h, lysis and subsequent labelling with HA-Ub-PA. Red = Lysate control (E) Immunoblot of HIF-1α in Calu-3 cells incubated in 18% or 1% O\u003csub\u003e2\u003c/sub\u003e for 24 h followed by treatment with increasing doses of \u003cstrong\u003eCompound 6\u003c/strong\u003e for 1h. (F) Expression of HIF-1α in RCC4-VA (Mutant VHL) or –VHL (WT VHL) cells treated with increasing doses of \u003cstrong\u003eCompound 6\u003c/strong\u003e. Lysates from hypoxic RCC4-VHL cells treated with\u003cstrong\u003e Compound 6\u003c/strong\u003e were also performed to induce HIF-1α expression. (G) HAP1-WT and OTUD7B KO cells were incubated in 18% or 1% O\u003csub\u003e2\u003c/sub\u003e for 24 h followed by treatment with indicated doses of\u003cstrong\u003e Compound 6\u003c/strong\u003e for 1 h. HIF-1α expression was then determined by immunoblot in WT and KO cells. (H) Calu-3 cells were infected with SARS-CoV-2 (VIC02/20) at a multiplicity of infection (MOI) of 0.01 for 1h. The inoculum was removed and cells incubated at 18% or 1% O\u003csub\u003e2\u003c/sub\u003e for 24 h treated with or without 26.6 µM of\u003cstrong\u003e Compound 6\u003c/strong\u003e. Viral replication was quantified by qPCR of intracellular viral RNA and expressed as copies µg-1 of total cellular RNA. Data is representative of three biological replicates and data presented as mean -/+ S.D. Significance from Mann-Whitney test, * = p \u0026lt; 0.0332. (I) Under hypoxic conditions, HIF1α expression is increased, leading to reduced ACE2 receptor expression and subsequent reduced SARS-CoV-2 viral entry and replication (46). Inhibition of OTUD7B by \u003cstrong\u003eCompound 6\u003c/strong\u003e leads to increased ubiquitination, and subsequently decreased levels, of HIF1α under hypoxic conditions. This results in increased ACE2 receptor expression, allowing for increased SARS-CoV-2 viral entry and replication. Created in BioRender. Jones, H. (2025) \u003ca href=\"https://BioRender.com/yl1zds3\"\u003e(License\u003c/a\u003e\u003cu\u003e link to be inserted upon publication)\u003c/u\u003e.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9106698/v1/2eeadea0ad770e7d45f50704.png"},{"id":106960970,"identity":"cd664e8c-9c6e-4c0b-b792-01e33c8bba6b","added_by":"auto","created_at":"2026-04-15 09:23:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":556656,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of USP5 with \u003cstrong\u003eCompound 25\u003c/strong\u003e demonstrates its essential role in SARS-CoV-2 replication. (A) \u003cstrong\u003eCompound 25\u003c/strong\u003e concentration dependence bio-ISG15-PA ABP with USP5 and USP18 in lysates from HAP1 cells treated with 1000 U/mL of human interferon α 2 (α2b) for 24 h. (B) Cell permeability concentration-dependence of \u003cstrong\u003eCompound\u003c/strong\u003e \u003cstrong\u003e25 \u003c/strong\u003eafter incubation with MCF-7 cells for 4 h, lysis and subsequent labelling with HA-Ub-PA or ISG15-PA. Red = Lysate control. (C) Varying the R1 and R2 groups of \u003cstrong\u003eCompound 25\u003c/strong\u003e enhances cellular permeability. (D) Cellular permeability concentration-dependence of USP5 inhibition with \u003cstrong\u003eCompounds 25a\u003c/strong\u003e and \u003cstrong\u003e25b \u003c/strong\u003eafter incubation with MCF-7 cells for 4 h, lysis and subsequence labelling with HA-Ub-PA. (E) MCF-7 lysates were incubated with DMSO or 250 µM of \u003cstrong\u003eCompound 25\u003c/strong\u003e for 1 h., followed by incubation with 150 µM of biotinylated Compound 25 derivatives for 1 h.\u0026nbsp; (F) Interferon I (IFN-I) production by HAP1 cells treated with \u003cstrong\u003eCompound 25b\u003c/strong\u003e/DMSO for 1 hour, followed by poly(I:C) (0.5 µg/mL) for 48 h. HEK293 IFN reporter cells were incubated with HAP1 media for 24 h, and luminescence determined with the One-Glo luciferase assay system. Data is representative of three replicates with data presented as mean -/+ SEM. Significance from 2-way ANOVA, with Tukey’s multiple comparisons test, * = p \u0026lt; 0.0332 (G) Calu-3 cells were infected with SARS-CoV-2-mNeonGreen reporter virus at a MOI of 0.1 and treated with a range of doses of the USP5 inhibitor \u003cstrong\u003eCompound 25b\u003c/strong\u003e or remdesivir (97). Data is representative of three biological replicates and data presented as mean -/+ SEM. Dose response curves were determined using a non-linear regression analysis to calculate IC\u003csub\u003e50\u003c/sub\u003e values for each compound. (H) Upon SARS-CoV-2 infection, ubiquitinated IRF3 is activated leading to antiviral signalling, thanks to upregulation of IFN-I production. USP5 deubiquitinates IRF3(50), and upon SARS-CoV-2 infection, inhibition of USP5 with \u003cstrong\u003eCompound 25b \u003c/strong\u003eleads to an increase in IRF3 ubiquitination, resulting in an upregulation of IRF-3-mediated IFN-I production, and subsequent reduced viral replication. \u0026nbsp;Created in BioRender. Jones, H. (2025) \u003ca href=\"https://BioRender.com/yl1zds3\"\u003e(License\u003c/a\u003e\u003cu\u003e link to be inserted upon publication)\u003c/u\u003e.\u0026nbsp;\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9106698/v1/3c6e83e05a41a3a33bb6179c.png"},{"id":106963229,"identity":"10c6aa88-4bf7-4228-be77-2308e3fcac31","added_by":"auto","created_at":"2026-04-15 09:43:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6254001,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9106698/v1/131f5b90-18d7-4cb3-8cfb-ce118637ae9a.pdf"},{"id":106830930,"identity":"8b1db4fb-ed11-4384-a067-0a50deb9b4e4","added_by":"auto","created_at":"2026-04-13 23:40:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":6222271,"visible":true,"origin":"","legend":"","description":"","filename":"JonesDraganovetalSupplementaryInformationDrugDiscovery2026.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9106698/v1/f12ee658158edb6b8c4afa18.pdf"},{"id":106830931,"identity":"1018ad25-f5d9-4be8-b412-789233af1cfb","added_by":"auto","created_at":"2026-04-13 23:40:59","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":384007,"visible":true,"origin":"","legend":"","description":"","filename":"JonesDraganovetalSupplementaryDataS4HDXSummaryTableforFigure4KLandS3FGDrugDiscovery2026.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9106698/v1/8bfa1718fc964015a78134f4.xlsx"},{"id":106830933,"identity":"5e9d2868-af5d-44e0-9e55-dfb316763cde","added_by":"auto","created_at":"2026-04-13 23:40:59","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":347890,"visible":true,"origin":"","legend":"","description":"","filename":"JonesDraganovetalSupplementaryDataS9DataforFigureS1GDrugDiscovery2026.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9106698/v1/4f00d38cc86131b79ffd7323.xlsx"},{"id":106960365,"identity":"f91a7d23-e484-4afa-bd7c-50703aa4a09f","added_by":"auto","created_at":"2026-04-15 09:20:33","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":320796,"visible":true,"origin":"","legend":"","description":"","filename":"JonesDraganovetalSupplemetaryDataS7DataforFigureS1EDrugDiscovery2026.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9106698/v1/8e58bee802cd7f25e020a5d0.xlsx"},{"id":106961372,"identity":"68c1dfb3-0fbc-45e5-8a65-06f322e882ce","added_by":"auto","created_at":"2026-04-15 09:25:18","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":468660,"visible":true,"origin":"","legend":"","description":"","filename":"JonesDraganovetalSupplementaryDataS5HDXUptakeSummaryTableforFigure4KLandSFGDrugDiscovery2026.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9106698/v1/6d9d193cba5bce9b7cab1845.xlsx"},{"id":106830938,"identity":"03452137-6303-460b-9a6a-08592c1cd2ac","added_by":"auto","created_at":"2026-04-13 23:40:59","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":798821,"visible":true,"origin":"","legend":"","description":"","filename":"JonesDraganovetalSupplementaryDataS3DataforFigure2EJandS3DrugDiscovery2026.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9106698/v1/3a9a4a947e2fd6966868a428.xlsx"},{"id":106961038,"identity":"f07795ca-c09c-4734-a05c-404278a85a83","added_by":"auto","created_at":"2026-04-15 09:24:02","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1127350,"visible":true,"origin":"","legend":"","description":"","filename":"JonesDraganovetalSupplementaryDataS1UncroppedBlotsDrugDiscovery2026.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9106698/v1/57d50b5d7e64c1fec2c127a7.pdf"},{"id":106960453,"identity":"fb91d8c6-a833-4148-81fd-b47af87565d5","added_by":"auto","created_at":"2026-04-15 09:21:12","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":2233126,"visible":true,"origin":"","legend":"","description":"","filename":"JonesDraganovetalSupplementaryDataS2DataforFigure1CDDrugDiscovery2026.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9106698/v1/08af068509aa7c93e4bb9b26.xlsx"},{"id":106830940,"identity":"0f30294e-9f39-4b8c-aa58-a60e0db6b515","added_by":"auto","created_at":"2026-04-13 23:40:59","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":3139392,"visible":true,"origin":"","legend":"","description":"","filename":"JonesDraganovetalSupplementaryDataS8DataforFigureS1FS1HS1IDrugDiscovery2026.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9106698/v1/6f1db47ef5197bdaf10bad04.xlsx"},{"id":106830942,"identity":"7c79d708-efce-4735-b1e5-4f36ebf8db80","added_by":"auto","created_at":"2026-04-13 23:40:59","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":8400141,"visible":true,"origin":"","legend":"","description":"","filename":"JonesDraganovetalSupplementaryDataS6DataforFigureS1AS1BS1CDrugDiscovery2026.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9106698/v1/c042b7832b474f2fd9d93eb3.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Activity-Based Proteomics Discovery of Deubiquitinating Enzyme Inhibitors with Immunomodulatory Activity","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePost-translational (poly)-ubiquitination regulates key cellular processes, including protein and organelle homeostasis, through the interplay of ubiquitinating and deubiquitinating molecular machineries (\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Deubiquitinases (DUBs) are mostly nucleophilic cysteine enzymes which cleave ubiquitin (Ub) from biomolecule substrates, mainly proteins, modulating their fate (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Consequently, dysregulated DUB functions have been linked to diseases (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDUBs cleave after the conserved C-terminal LRGG motif of ubiquitin within their catalytic cleft. Despite sharing a broadly conserved active-site architecture, individual DUBs discriminate among distinct polyubiquitin chain linkages and ubiquitinated substrates through differences in ubiquitin-binding interfaces and allosteric regulatory elements. This combination of conserved catalytic geometry and context-dependent substrate recognition complicates the development of selective active-site-directed DUB inhibitors (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Nonetheless, both active-site targeting and allosteric small-molecule inhibitors of USP1, USP28, and USP30 have been developed, and progressed into clinical trials for treatment of advanced solid tumours (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), relapsed/refractory acute myeloid leukemia (R/R AML)(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), chronic kidney disease (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), and Parkinson\u0026rsquo;s disease (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDUB activity-based protein profiling (ABPP) was established to profile DUB activities in complex systems. DUB ABPP employs activity-based probes (ABPs) that react with the nucleophilic cysteine of DUBs, (\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) including haemagglutinin (HA)-tagged ubiquitin-propargylamide (HA-Ub-PA) which bears an alkyne warhead for irreversible covalent reaction with active-site cysteines. Coupled with affinity purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS), ABPP enables the analysis of endogenous DUB-ABP complexes. Unlike recombinant enzyme assays, ABPP supports the identification of potent and selective inhibitors in a cellular environment, including those for challenging targets, (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) whilst also providing information on cell permeability and stability (\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHistorically, the scope of ABPP has been limited by sensitivity and throughput because of labor-intensive workflows involving ABP labeling, purification, sample preparation, MS analysis, and data processing. Our group and others, with the aim of expanding ABPP\u0026rsquo;s utility for DUB inhibitor discovery, have automated the established methodology to enable analysis of ~\u0026thinsp;100 samples per day (ABPP-HT) (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) and implemented data-independent acquisition LC-MS/MS (DIA), enabling deeper and more comprehensive DUBome profiling (ABPP-HT*) (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Additional improvements aimed at enhancing sensitivity were tested and include using diverse Ub probe approaches, high-pH pre-fractionation (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), and isobaric labelling (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003cem\u003eA-C\u003c/em\u003e) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHere, we advance the ABPP-HT* platform into a broadly accessible and scalable workflow, enhancing its sensitivity, flexibility, and ease of use while establishing a fully functional on-bench (non-automated) format. As a proof of concept, we applied the platform to the discovery of unconventional cysteine-reactive and selective DUB inhibitors αβ,α\u0026prime;β\u0026prime;-diepoxyketones (DEKs) that selectively engage the active sites of USP47/USP7 and OTUD7B. The platform also enabled the identification and optimization of a ubiquitin-derived inhibitor class that uses an alkyne as a latent electrophile for covalent engagement, yielding chemical probes with selective cellular inhibition of USP5. These inhibitors performed robustly in functional assays and are useful tools for assigning DUB-dependent phenotypes in cells. Consistent with reported biology, the USP47/USP7, OTUD7B, and USP5 inhibitors modulated pathways linked to innate immunity, hypoxic signaling, and viral infection by suppressing inflammasome activation, stabilizing HIF-1α, and relieving inhibition of type I interferon signaling, respectively.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eWe modified our reported ABPP-HT* methodology (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) by adapting the immunoprecipitation step to either a multi-well magnetic rack for on-bench processing or a Thermo KingFisher Apex purification system, demonstrating the workflow\u0026rsquo;s flexibility (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003cem\u003eD\u003c/em\u003e). Compared to using an automated liquid handling platform (Assay MAP from Agilent), the new method manifested comparable or improved enrichment of cysteine-reactive DUBs (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003cem\u003eE-I\u003c/em\u003e). The improved and user-friendly \u0026ldquo;on-bench\u0026rdquo; ABPP-HT* set-up was then used to identify novel DUB inhibitors from a set of unconventional cysteine-reactive small-molecules (Fig.\u0026nbsp;1\u003cem\u003eA\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eFour complementary classes of small-molecules were investigated in the screen: (i) αβ,α\u0026prime;β\u0026prime;-diepoxyketones (DEKs), which are reported to covalently react with the nucleophilic cysteine residues of bacterial and viral enzymes (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), (ii) γ-lactams which are substantially less reactive than DEKs, but can covalently react with Cys145 of the SARS-CoV-2 main protease (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), (iii) Michael acceptors, which are a common motif in covalently reacting small-molecule drugs, and (iv) Ub-derived alkyne and nitrile inhibitors that may bind at the active site (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-3) (Fig.\u0026nbsp;1\u003cem\u003eB\u003c/em\u003e); note that classes (i), (ii), and (iv) are relatively uncommon in established DUB inhibitor libraries. The latter class of inhibitors was designed based on the Ub C-terminal LRGG motif (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), replacing the C-terminal glycine by 2-aminoacetonitrile or propargyl amide (PA) to enable reversible or irreversible covalent reaction with the DUB active site cysteine thiol, respectively; note that the arginine residue at the P3-equivalent position was substituted for a lysine group to facilitate chemical modification. This design strategy was informed by the observations that Ub-PA (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) and nitrile as well as alkyne-containing substrate-derived small-molecules are potent and selective active site-targeting inhibitors of nucleophilic cysteine enzymes (\u003cspan additionalcitationids=\"CR32 CR33 CR34 CR35\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA total of 41 small-molecules were thus incubated with MCF-7 breast cancer cell lysates in a 96 well plate in technical duplicates. FT827, a well-characterized USP7 inhibitor (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), was included as a reference inhibitor; Negative and positive controls (absence or presence of HA-Ub-PA) were included on the plate. The results reveal that 38 DUBs were consistently enriched\u0026thinsp;\u0026gt;\u0026thinsp;5-fold across replicates using the ABPP-HT* workflow, with an average coefficient of variation (CV) of 16%, confirming robustness (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003cem\u003eA\u003c/em\u003e). Importantly, we confirmed potent and selective inhibition of USP7 by FT827, (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) further validating this assay. Eight compounds significantly inhibited at least one DUB (\u0026gt;\u0026thinsp;20% intensity loss, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;1\u003cem\u003eC\u003c/em\u003e). Of these, \u003cb\u003eCompounds 4\u003c/b\u003e and \u003cb\u003e27\u003c/b\u003e were excluded due to a lack of selectivity. Selective, or partially selective, hits included: \u003cb\u003eCompound 3\u003c/b\u003e (USP47\u0026thinsp;\u0026gt;\u0026thinsp;USP7), \u003cb\u003eCompound 6\u003c/b\u003e (OTUD7B), \u003cb\u003eCompound 7\u003c/b\u003e (USP30/USP47/VCPIP1), \u003cb\u003eCompound 25\u003c/b\u003e (USP5), and \u003cb\u003eCompounds 26\u003c/b\u003e and \u003cb\u003e32\u003c/b\u003e (USP33) (Fig.\u0026nbsp;1\u003cem\u003eD\u003c/em\u003e). Notably, USP5-selective \u003cb\u003eCompound 25\u003c/b\u003e is a diastereomer of the inactive \u003cb\u003eCompound 27\u003c/b\u003e, highlighting the workflow\u0026rsquo;s ability to resolve isomer-specific effects (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003cem\u003eB\u003c/em\u003e, S2\u003cem\u003eC\u003c/em\u003e).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHit Validation and Dose-Response Analysis\u003c/h2\u003e \u003cp\u003eWestern blot analysis supported inhibition of USP47/USP7 by \u003cb\u003eCompound 3\u003c/b\u003e, OTUD7B by \u003cb\u003eCompound 6\u003c/b\u003e, and USP5 by \u003cb\u003eCompound 25\u003c/b\u003e (Fig.\u0026nbsp;2\u003cem\u003eA\u003c/em\u003e, 2\u003cem\u003eB\u003c/em\u003e). DUB inhibition calculated from western blot densitometric analysis was concentration-dependent (Fig.\u0026nbsp;2\u003cem\u003eC\u003c/em\u003e) and IC₅₀ values revealed the preference of \u003cb\u003eCompound 3\u003c/b\u003e for USP47 (8.8 \u0026micro;M) over USP7 (36 \u0026micro;M) (Fig.\u0026nbsp;2\u003cem\u003eD\u003c/em\u003e), which was consistent with screening data. The apparent preference of \u003cb\u003eCompound 3\u003c/b\u003e for USP47 over USP7 inhibition was remarkable given that reported USP47 inhibitors typically also inhibit USP7 with similar or lower potency (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), likely reflecting the close evolutionary relationship between human USP47 and USP7 (their catalytic domains share\u0026thinsp;~\u0026thinsp;40\u0026ndash;45% sequence identity, depending on the alignment boundaries used) (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCompounds 26\u003c/b\u003e and \u003cb\u003e32\u003c/b\u003e appeared to be false positive hits and could not be validated (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003cem\u003eD\u003c/em\u003e), while \u003cb\u003eCompound 7\u003c/b\u003e induced protein cross-linking, likely due to its bifunctional epoxide structure (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003cem\u003eE\u003c/em\u003e) (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). To assess binding stability, \u003cb\u003eCompounds 3\u003c/b\u003e, \u003cb\u003e6\u003c/b\u003e, and \u003cb\u003e25\u003c/b\u003e were pre-incubated with MCF-7 lysates for 30 min before addition of HA-Ub-PA and incubation for 5, 15, or 45 min. Efficient prevention of HA-Ub-PA binding was observed across all timepoints (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003cem\u003eF\u0026ndash;H\u003c/em\u003e), suggesting irreversible binding of \u003cb\u003eCompounds 3\u003c/b\u003e, \u003cb\u003e6\u003c/b\u003e, and \u003cb\u003e25\u003c/b\u003e at the DUB active sites.\u003c/p\u003e \u003cp\u003eThe increased throughput of the ABPP-HT* workflow enabled systematic evaluation of the selectivity of \u003cb\u003eCompounds 3\u003c/b\u003e, \u003cb\u003e6\u003c/b\u003e, and \u003cb\u003e25\u003c/b\u003e across multiple concentrations and replicates (Fig.\u0026nbsp;2\u003cem\u003eE-G\u003c/em\u003e). The dose-dependent inhibition curves and IC\u003csub\u003e50\u003c/sub\u003e values for the three compounds were comparable to those generated by immunoblotting (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003cem\u003eA-D\u003c/em\u003e) (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). The results reveal that \u003cb\u003eCompound 3\u003c/b\u003e exclusively inhibited USP47 and USP7 up to 50 \u0026micro;M, with USP40 and USP48 inhibition being observed at higher concentrations (Fig.\u0026nbsp;2\u003cem\u003eE\u003c/em\u003e, 2\u003cem\u003eH\u003c/em\u003e). \u003cb\u003eCompound 6\u003c/b\u003e was selective for OTUD7B up to 100 \u0026micro;M but also inhibited USP16 and USP28 at the highest concentration (250 \u0026micro;M) (Fig.\u0026nbsp;2\u003cem\u003eF\u003c/em\u003e, 2\u003cem\u003eI\u003c/em\u003e). Note that \u003cb\u003eCompounds 3\u003c/b\u003e and \u003cb\u003e6\u003c/b\u003e are reported to inhibit the mycobacterial L,D-transpeptidase Ldt\u003csub\u003eMt2\u003c/sub\u003e and the SARS-CoV-2 main protease (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), implying that they may also react with additional nucleophilic cysteine enzymes other than DUBs. By contrast to \u003cb\u003eCompounds 3\u003c/b\u003e and \u003cb\u003e6\u003c/b\u003e, \u003cb\u003eCompound 25\u003c/b\u003e was highly selective, only inhibiting USP5 across the tested concentration range (Fig.\u0026nbsp;2\u003cem\u003eG\u003c/em\u003e, 3\u003cem\u003eJ\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular Basis for Selective Inhibition of USP47 by Compound 3\u003c/h3\u003e\n\u003cp\u003eGiven the preference of \u003cb\u003eCompound 3\u003c/b\u003e for USP47 over USP7 inhibition, we investigated its mode of action. The results of \u003cem\u003ein vitro\u003c/em\u003e activity assays using recombinant DUBs and a fluorogenic ubiquitin-rhodamine (Ub-Rho) substrate revealed that \u003cb\u003eCompound 3\u003c/b\u003e was ~\u0026thinsp;10-fold more potent in inhibiting USP47 than USP7 after 30 min preincubation (IC₅₀: 0.48 vs. 4.3 \u0026micro;M; Fig.\u0026nbsp;3\u003cem\u003eA\u003c/em\u003e, Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003cem\u003eA-D\u003c/em\u003e). Time-dependency experiments indicated covalent or slow tight-binding inhibition for USP47 (Fig.\u0026nbsp;3\u003cem\u003eB\u003c/em\u003e), while USP7 inhibition was weaker and time-independent (Fig.\u0026nbsp;3\u003cem\u003eC\u003c/em\u003e). For both enzymes, progress curves in the presence of \u003cb\u003eCompound 3\u003c/b\u003e were non-linear, indicating slow initial binding (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003cem\u003eE\u003c/em\u003e, S4\u003cem\u003eF\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eMolecular docking studies were initially performed to investigate the preferred reaction of \u003cb\u003eCompound 3\u003c/b\u003e with USP47. In the absence of a human USP47 Protein Data Bank (PDB) structure, the structure of human USP7 in complex with the covalent inhibitor FT827 (PDB: 5NGF) was used as the template to generate a homology model of human USP47 with an appropriate conformational state for covalent inhibitor docking studies. \u003cb\u003eCompound 3\u003c/b\u003e most likely forms a covalent adduct with the catalytic cysteine of human USP47 (C197) via 1,2-carbonyl attack and retro-aldol fragmentation based on previously reported work (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003cem\u003eG\u003c/em\u003e) (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe docking studies also suggest that \u003cb\u003eCompound 3\u003c/b\u003e covalently binds at the thumb-palm cleft (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003cem\u003eH\u003c/em\u003e) and sterically blocks Ub binding (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003cem\u003eI\u003c/em\u003e), analogous to the covalent USP7 inhibitor FT827 (PDB: 5NGF; Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003cem\u003eJ\u003c/em\u003e). The binding site is flanked by 14 residues including R402, the side chain of which is predicted to form a hydrogen bond with the epoxide moiety of \u003cb\u003eCompound 3\u003c/b\u003e (Fig.\u0026nbsp;3\u003cem\u003eD\u003c/em\u003e). In human USP7, the substitution of R402 with an asparagine residue (N418), which cannot interact with \u003cb\u003eCompound 3\u003c/b\u003e, may rationalize the apparent selectivity of \u003cb\u003eCompound 3\u003c/b\u003e for USP47 inhibition (Fig.\u0026nbsp;3\u003cem\u003eE\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eUSP47 apoform (PDB: 8ITN) and Ub-bound (PDB: 8ITP) structures exist for the closely related catalytic domain of \u003cem\u003eC. elegans\u003c/em\u003e USP47 (44% sequence identity). Comparison of the human USP47-Compound 3 model with the apo \u003cem\u003eC. elegans\u003c/em\u003e USP47 structure suggests that Compound 3 binding disrupts the hydrogen bond between R402 and H271 that stabilizes the switching loop in its catalytically incompetent \u0026ldquo;in\u0026rdquo; conformation. Disruption of this interaction is predicted to favor the active \u0026ldquo;out\u0026rdquo; conformation of the switching loop, compatible with activation peptide binding, as described for USP7 (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003cem\u003eO\u003c/em\u003e, S4\u003cem\u003eP\u003c/em\u003e) (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHydrogen-deuterium exchange mass spectrometry (HDX-MS) studies were performed to investigate binding of \u003cb\u003eCompound 3\u003c/b\u003e to USP47 and USP7 (Fig.\u0026nbsp;3\u003cem\u003eF\u003c/em\u003e, 3\u003cem\u003eG\u003c/em\u003e, and Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003cem\u003eK\u003c/em\u003e, S4\u003cem\u003eL\u003c/em\u003e). The conformational shifts observed by HDX-MS indicate a switching loop rearrangement in USP47 (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003cem\u003eM\u003c/em\u003e) and potential engagement of the activation peptide (Fig.\u0026nbsp;3\u003cem\u003eH\u003c/em\u003e) (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Notably, HDX-MS analysis with USP7 showed no such distal conformational changes, indicating that \u003cb\u003eCompound 3\u003c/b\u003e does not promote an equivalent switching loop reorganization or activation peptide binding in USP7 (Fig.\u0026nbsp;3\u003cem\u003eI\u003c/em\u003e and Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003cem\u003eN\u003c/em\u003e), further rationalizing its selectivity for USP47.\u003c/p\u003e\n\u003ch3\u003eInhibition of USP47 with Compound 3 Further Supports its Roles in Inflammasome Activation\u003c/h3\u003e\n\u003cp\u003eABP assays in MCF-7 cells revealed that \u003cb\u003eCompound 3\u003c/b\u003e is cell permeable with USP47 inhibition occurring in a concentration dependent manner (Fig.\u0026nbsp;4\u003cem\u003eA\u003c/em\u003e); cytotoxicity was observed at 250 \u0026micro;M, indicative of off-target effects, in line with the reported reactivity of DEKs with other enzymes. To explore whether \u003cb\u003eCompound 3\u003c/b\u003e could serve as a useful research tool probe, we investigated its ability to replicate reports describing a role of both USP47 and USP7 in the activation of the NLRP3 inflammasome by regulating NLRP3 ubiquitination (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHuman monocyte (THP-1) cells were treated with lipopolysaccharide (LPS) followed by a 45 min or 2 h nigericin incubation to activate the NLRP3 inflammasome. Under these conditions, treatment with \u003cb\u003eCompound 3\u003c/b\u003e inhibited inflammasome activation, as evidenced by reduced Caspase-1 activity (Fig.\u0026nbsp;4\u003cem\u003eB\u003c/em\u003e, Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e\u003cem\u003eA-C\u003c/em\u003e). To ensure \u003cb\u003eCompound 3\u003c/b\u003e did not reduce Caspase-1 activity via direct inhibition, the upstream formation of the NLRP3 inflammasome was also examined: THP-1 treatment with \u003cb\u003eCompound 3\u003c/b\u003e, in combination with LPS and Nigericin, blocked the oligomerisation of apoptosis-associated speck-like protein containing a CARD (ASC), and prevented the cleavage of Caspase-1, IL-1β, and gasdermin D (GSDMD) (Fig.\u0026nbsp;4\u003cem\u003eC\u003c/em\u003e). While a decrease in NLRP3 levels was observed, the treatment of cells with \u003cb\u003eCompound 3\u003c/b\u003e resulted in appearance of a high molecular weight NLRP3 smear, consistent with ubiquitination (Fig.\u0026nbsp;4\u003cem\u003eC\u003c/em\u003e). While further investigation is required to fully characterize this smear, it may be attributable to the ubiquitination of NLRP3, as previously proposed (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNo decrease in THP-1 cell viability was detected when incubating with up to 100 \u0026micro;M \u003cb\u003eCompound 3\u003c/b\u003e, when the LPS treatment was followed by 45 min nigericin treatment (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eD). In contrast, 2 h of nigericin exposure induced toxicity at \u003cb\u003eCompound 3\u003c/b\u003e concentrations of 30 \u0026micro;M and higher. (Fig.\u0026nbsp;4\u003cem\u003eD\u003c/em\u003e), further signifying additional targets beyond DUBs at these high concentrations (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In contrast, and consistent with an on-target reduction in inflammasome activation, lower concentrations of \u003cb\u003eCompound 3\u003c/b\u003e increased THP-1 viability upon LPS and a 4 h nigericin treatment, indicating reduced inflammasome-mediated pyroptosis (Fig.\u0026nbsp;4\u003cem\u003eD\u003c/em\u003e). Taken together, the data demonstrates that \u003cb\u003eCompound 3\u003c/b\u003e can reproduce phenotypes previously seen by USP47/7 gene knock-out (KO) and inhibition, through the prevention of NLRP3 inflammasome formation (Fig.\u0026nbsp;4\u003cem\u003eE\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eInhibition of OTUD7B with Compound 6 Further Supports its Roles in Hypoxic Signaling and Associated SARS-CoV-2 Repression\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOrthogonal \u003cem\u003ein vitro\u003c/em\u003e assays using the recombinant catalytic domain of OTUD7B and a ubiquitin\u0026ndash;rhodamine substrate demonstrated that Compound 6 inhibits OTUD7B with an IC₅₀ of 1.9 \u0026micro;M (95% confidence interval, 1.8\u0026ndash;2.1 \u0026micro;M) (Fig.\u0026nbsp;5\u003cem\u003eA\u003c/em\u003e). Intact protein mass spectrometry revealed that incubation of the catalytic domain of OTUD7B with \u003cb\u003eCompound 6\u003c/b\u003e (molecular weight: 326.35 Da) for 1 min results in a\u0026thinsp;+\u0026thinsp;326 Da mass shift, consistent with covalent reaction of OTUD7B with \u003cb\u003eCompound 6\u003c/b\u003e in a 1:1 stoichiometry. After 15 min, the formation of multiple species derived from the +\u0026thinsp;326 Da adduct were observed with a\u0026thinsp;+\u0026thinsp;190 Da adduct dominating. The +\u0026thinsp;190 Da adduct likely arises from elimination of \u003cem\u003eortho\u003c/em\u003e-methoxybenzaldehyde from the initially formed\u0026thinsp;+\u0026thinsp;326 Da intermediate, consistent with the crystallographically validated mechanism by which nucleophilic cysteine enzymes react with DEKs (Fig.\u0026nbsp;5\u003cem\u003eB\u003c/em\u003e, 5\u003cem\u003eC\u003c/em\u003e) (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCompound 6\u003c/b\u003e was active and cell permeable in MCF-7 cells (Fig.\u0026nbsp;5\u003cem\u003eD\u003c/em\u003e), enabling subsequent evaluation of the phenotypic consequences of OTUD7B inhibition. OTUD7B has been shown to stabilize hypoxia-inducible factor 1α (HIF-1α) under hypoxic conditions in a ubiquitin-dependent manner, counteracting the E3 ubiquitin ligase activity of the von Hippel\u0026ndash;Lindau tumor suppressor (VHL) (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Consistent with these findings, treatment of Calu-3 cells with Compound 6 under hypoxic conditions reduced HIF-1α levels in a dose-dependent manner (Fig.\u0026nbsp;5\u003cem\u003eE\u003c/em\u003e). Furthermore, in renal carcinoma cells lacking active VHL (RCC4-VA)(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), we observed that treatment with \u003cb\u003eCompound 6\u003c/b\u003e had no impact on HIF-1α protein levels. When wild-type VHL (RCC4-VHL) expression was restored, however, a dose-dependent reduction in hypoxic HIF-1α protein levels were observed (Fig.\u0026nbsp;5\u003cem\u003eF\u003c/em\u003e). In Chronic Myeloid Leukemia (CML)-derived HAP1 wild-type (WT) cells, treatment with \u003cb\u003eCompound 6\u003c/b\u003e also resulted in a dose-dependent reduction in HIF-1α protein levels. Importantly, HIF-1α levels were markedly reduced in HAP1 OTUD7B KO cells compared to WT, consistent with published findings (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e), and treatment of KO cells with \u003cb\u003eCompound 6\u003c/b\u003e did not further reduce HIF-1α, supporting an on-target mechanism at the tested concentrations (Fig.\u0026nbsp;5\u003cem\u003eG\u003c/em\u003e).\u003c/p\u003e \u003cp\u003ePrevious studies have shown that SARS-CoV-2 replication is reduced under hypoxic conditions, attributable to increased HIF-1α protein levels (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Consistent with these findings, we observed decreased viral replication in Calu-3 cells under hypoxia compared with normoxia. Notably, treatment with \u003cb\u003eCompound 6\u003c/b\u003e significantly attenuated the antiviral effect of hypoxia (Fig.\u0026nbsp;5\u003cem\u003eH\u003c/em\u003e). These results are consistent with reduced HIF-1α protein levels following OTUD7B inhibition. The ability of \u003cb\u003eCompound 6\u003c/b\u003e to phenocopy OTUD7B knockout supports on-target engagement and provides further evidence that OTUD7B catalytic activity directly contributes to HIF-1α stability under hypoxic conditions. Moreover, we provide the first functional validation of OTUD7B inhibition in the regulation of SARS-CoV-2 replication under hypoxia (Fig.\u0026nbsp;5\u003cem\u003eI\u003c/em\u003e).\u003c/p\u003e\n\u003ch3\u003eInhibition of USP5 Demonstrates Its Essential Role in SARS-CoV-2 Replication\u003c/h3\u003e\n\u003cp\u003eThe active sites of DUBs and deISGylases, cellular enzymes cleaving the ubiquitin-like protein ISG15 (interferon-stimulated gene 15), are structurally very similar. Most deISGylating enzymes are cross-reactive DUBs, catalysing removal of both Ub and ISG15, with the notable exception of USP18 (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Given USP5\u0026rsquo;s dual Ub/ISG15 specificity, we evaluated the effect of \u003cb\u003eCompound 25\u003c/b\u003e on deISGylases by immunoblotting, using a bio-ISG15-PA ABP. The results reveal that the extent of USP5 inhibition by \u003cb\u003eCompound 25\u003c/b\u003e is comparable using the the bio-ISG15-PA and Ub-PA ABPs, and that \u003cb\u003eCompound 25\u003c/b\u003e did not cross-react with USP18 or any of the other detected deISGylases in the tested concentration range (up to 125 \u0026micro;M), demonstrating the high selectivity of \u003cb\u003eCompound 25\u003c/b\u003e for USP5 inhibition (Fig.\u0026nbsp;6\u003cem\u003eA\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eInterestingly, ABP assays in MCF-7 cells revealed that \u003cb\u003eCompound 25\u003c/b\u003e was inactive in intact cells, limiting its applicability (Fig.\u0026nbsp;6\u003cem\u003eB\u003c/em\u003e). To optimize \u003cb\u003eCompound 25\u003c/b\u003e for cellular activity, we modified the alkyne and amine protecting groups. Substituting the terminal alkyne reduced potency (Fig.\u0026nbsp;6\u003cem\u003eC\u003c/em\u003e; Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e; synthesis detailed in SI), supporting that \u003cb\u003eCompound 25\u003c/b\u003e covalently reacts with the USP5 nucleophilic cysteine via its alkyne group which serves as a latent electrophile. By contrast, altering amine-protecting groups at the N-terminus (R\u003csub\u003e1\u003c/sub\u003e) and/or the \u003cem\u003eN\u003c/em\u003e\u003csup\u003eε\u003c/sup\u003e-lysine amino group (R\u003csub\u003e2\u003c/sub\u003e), we generated the active and cell permeable derivatives \u003cb\u003eCompounds 25a\u003c/b\u003e and \u003cb\u003e25b\u003c/b\u003e bearing trifluoromethyl groups at R1 and trifluoroacetyl (\u003cb\u003e25a\u003c/b\u003e) or \u003cem\u003etert\u003c/em\u003e-butyloxycarbonyl (Boc; \u003cb\u003e25b\u003c/b\u003e) at R2, respectively (Fig.\u0026nbsp;6\u003cem\u003eC\u003c/em\u003e, 6\u003cem\u003eD\u003c/em\u003e). Both \u003cb\u003e25a\u003c/b\u003e and \u003cb\u003e25b\u003c/b\u003e retained the potency and selectivity for USP5 inhibition observed in lysates (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e\u003cem\u003eA\u003c/em\u003e), offering promising leads for functional studies and possibly therapeutic development.\u003c/p\u003e \u003cp\u003eTo study the binding and selectivity of \u003cb\u003eCompound 25 and its derivatives\u003c/b\u003e to USP5, we generated five biotinylated derivatives with different linker strategies, including biotinylation via the N-terminal amino group or the \u003cem\u003eN\u003c/em\u003e\u003csup\u003eε\u003c/sup\u003e-lysine amino group (Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). All five biotinylated versions of \u003cb\u003eCompound 25\u003c/b\u003e bound to endogenous USP5 in MCF-7 lysates. Binding was confirmed by streptavidin western-blotting, with a strong band at the expected molecular weight of USP5 (~\u0026thinsp;95 kDa; Fig.\u0026nbsp;6\u003cem\u003eE\u003c/em\u003e, S7\u003cem\u003eB, S7D\u003c/em\u003e). By contrast, no signal was observed for another USP, USP42, at ~\u0026thinsp;150 kDa. Interestingly, despite comparable binding to USP5, derivatives \u003cb\u003ebio-b-25a\u003c/b\u003e and \u003cb\u003ebio-a-25b\u003c/b\u003e were less active than the other three biotinylated derivatives, as shown by Ub-PA probe competition assays, at two different concentrations (Fig. \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e\u003cem\u003eA\u003c/em\u003e, S7\u003cem\u003eB\u003c/em\u003e). Selective binding of these \u003cb\u003eCompound 25\u003c/b\u003e derivatives to the catalytic cysteine of USP5 was confirmed by competition experiments using free \u003cb\u003eCompound 25\u003c/b\u003e and N-ethylmaleimide (\u003cb\u003eNEM\u003c/b\u003e), a broad-spectrum cysteine-reactive alkylating agent. In both cases, competition resulted in loss of the USP5 band at ~\u0026thinsp;95 kDa, consistent with covalent engagement of the nucleophilic cysteine. (Fig. \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e\u003cem\u003eC\u003c/em\u003e, 6\u003cem\u003eE\u003c/em\u003e, S7\u003cem\u003eD\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eTo functionally validate \u003cb\u003eCompound 25b\u003c/b\u003e as an on-target inhibitor of USP5 we investigated its effect on the role of USP5 in viral replication. USP5 has been linked to IFN-I production by regulating the ubiquitination of different innate immune factors such as RIG-I and IRF3 (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). In agreement with these observations, when IFN-I production was induced by synthetic RNA Poly I:C, there was a significant amplification of IFN-I production in HAP1 cells co-treated with \u003cb\u003eCompound 25b\u003c/b\u003e (Fig.\u0026nbsp;6\u003cem\u003eF\u003c/em\u003e). In line with the observed elevated IFN-I levels, \u003cb\u003eCompound 25b\u003c/b\u003e exhibited a dose-dependent reduction in viral replication against SARS-CoV-2 in infected Calu-3 cells. In comparison to Remdesivir, a guanosine nucleoside analogue which targets the viral replicase complex and is in clinical use as a broad-spectrum antiviral medication (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e), \u003cb\u003eCompound 25b\u003c/b\u003e showed a\u0026thinsp;~\u0026thinsp;10-fold increase in potency (Fig.\u0026nbsp;6\u003cem\u003eG\u003c/em\u003e) and did not show any significant cellular toxicity at concentrations as high as 200 \u0026micro;M in HAP1 cells (Fig. \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e\u003cem\u003eA\u003c/em\u003e). The increased IFN-I production and prevention of viral replication by \u003cb\u003eCompound 25b\u003c/b\u003e both validates it as a tool compound for investigating roles of USP5 in signaling, and provides evidence of USP5\u0026rsquo;s potential as a therapeutic target (Fig.\u0026nbsp;6\u003cem\u003eH\u003c/em\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs interest in DUBs as drug targets grows (\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e), so does the need for improved discovery strategies. High-throughput screening (HTS) has enabled rapid evaluation of large compound libraries (\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e), yet it remains constrained by high cost, low hit rates, and reliance on recombinant proteins that may not fully capture native regulation and complex formation. Consequently, many HTS-derived hits require substantial optimization and secondary validation before demonstrating cellular activity.\u003c/p\u003e \u003cp\u003eTo overcome these limitations, computational approaches and activity-based proteomics have emerged as complementary strategies. AI-enabled virtual screening platforms such as AtomNet and Boltz-2 prioritize compounds \u003cem\u003ein silico\u003c/em\u003e, even in the absence of prior ligand data (\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e). In parallel, chemoproteomic profiling of reactive cysteines has enabled more directed targeting of cysteine-containing proteins, including DUBs (\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e). Recent advances in DUB-focused ABPP have produced high-throughput-ready platforms, including our ABPP-HT* workflow (\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e). ABPP-HT* combines sensitivity, cost-efficiency, and flexibility, operating in both manual and automated formats while maintaining compatibility with diverse probe chemistries and enzyme classes.\u003c/p\u003e \u003cp\u003eHere, we demonstrate a proof-of-concept application of the ABPP-HT* platform for DUB inhibitor discovery and characterization. Screening a curated electrophile-containing library, including αβ,α′β′-diepoxyketones (DEKs), γ-lactams, and ubiquitin-derived peptide inhibitors specifically designed for this study, yielded a hit rate approximately tenfold higher than traditional DUB HTS approaches. Following validation, we identified selective inhibitors of USP47, OTUD7B, and USP5 and further characterized their potency, permeability, selectivity, and functional consequences. These findings underscore the efficiency of endogenous chemoproteomic screening for rapid identification of cell-active DUB inhibitors.\u003c/p\u003e \u003cp\u003eUSP47 represents an emerging therapeutic target (\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e), yet previously reported inhibitors display comparable potency toward its close homolog USP7 (\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e), consistent with their substantial sequence similarity (\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e). We identified \u003cb\u003eCompound 3\u003c/b\u003e as a first-in-class inhibitor exhibiting preference for USP47 over USP7. Orthogonal kinetic analyses and HDX-MS-guided structural modeling revealed molecular determinants underlying this selectivity. Importantly, \u003cb\u003eCompound 3\u003c/b\u003e recapitulated prior findings that USP47/USP7 inhibition prevents inflammasome activation (\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e), providing functional validation of selective USP47 targeting.\u003c/p\u003e \u003cp\u003eAlthough the roles of OTUD7B in disease are still being defined (\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e) accumulating evidence supports its therapeutic potential (\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e), and recent reports describe emerging OTUD7B-directed ligands (\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e). We identified \u003cb\u003eCompound 6\u003c/b\u003e as a potent, selective, and cell-permeable OTUD7B inhibitor that reduced HIF-1α protein levels under hypoxia, phenocopying OTUD7B knockdown (\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e). Given that hypoxia restricts SARS-CoV-2 infection in a HIF-1α-dependent manner (\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e), \u003cb\u003eCompound 6\u003c/b\u003e correspondingly rescued viral replication under hypoxic conditions (\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e). These findings establish pharmacological inhibition of OTUD7B as a tool to interrogate hypoxia-driven cellular responses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCompound 25\u003c/b\u003e displayed high selectivity for USP5 over related DUBs such as USP16 and USP22 (\u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e). USP5 hydrolyzes free polyubiquitin chains via a zinc finger ubiquitin-binding domain (ZnF UBP) that recognizes the C-terminal diglycine motif of ubiquitin (\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e). Although USP5 has been implicated in cancer, pain, and inflammatory disorders (\u003cspan class=\"CitationRef\"\u003e73\u003c/span\u003e), existing inhibitors remain limited in selectivity, scope, and cellular validation (Fig. \u003cspan class=\"InternalRef\"\u003eS10\u003c/span\u003e) (\u003cspan class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e75\u003c/span\u003e). In contrast to prior inhibitors designed to engage the ZnF UBP carboxylate-binding pocket (\u003cspan class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e76\u003c/span\u003e), our ubiquitin-derived inhibitors substitute the C-terminal glycine with a propargyl amide group, inspired by the covalent reactivity of Ub-PA probes. Remarkably, these relatively simple substrate-inspired molecules achieved high selectivity, highlighting subtle but functionally significant differences in active-site architecture across DUBs.\u003c/p\u003e \u003cp\u003eMechanistically, \u003cb\u003eCompound 25\u003c/b\u003e likely modifies the catalytic cysteine of USP5 via its alkyne warhead, supported by structural precedent (PDB: 3IHP) (\u003cspan class=\"CitationRef\"\u003e77\u003c/span\u003e), related USP-Ub-PA complexes, and reduced activity upon steric modification (Table \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e). USP5’s unique domain architecture, including a disulfide linkage between C793 and C195 in the USP5-Ub complex, may constrain blocking loop flexibility and promote favorable compound engagement (Fig \u003cspan class=\"InternalRef\"\u003eS8\u003c/span\u003e\u003cem\u003eB\u003c/em\u003e). The use of a latent electrophile may further enhance selectivity through differential binding kinetics across DUBs (\u003cspan class=\"CitationRef\"\u003e78\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e81\u003c/span\u003e). The clinical success of electrophilic alkynes such as propiolamides in Bruton’s tyrosine kinase inhibitors (\u003cspan class=\"CitationRef\"\u003e82\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e83\u003c/span\u003e), supports the translational potential of this strategy.\u003c/p\u003e \u003cp\u003eStructure-activity optimization yielded the fluorinated, cell-permeable analogues \u003cb\u003e25a\u003c/b\u003e and \u003cb\u003e25b\u003c/b\u003e that retained cellular activity and were suitable for functional studies. Biotinylated derivatives confirmed covalent engagement and proteome-wide selectivity for USP5. Notably, \u003cb\u003eCompound 25b\u003c/b\u003e enhanced type I interferon production and exhibited strong antiviral activity, extending established USP5 functions.\u003c/p\u003e \u003cp\u003eThe combined results manifest the potential of high-throughput ABPP workflows for identifying DUB inhibitors and streamlining the development of potent and, at least, partially selective compounds for use as biochemical tools in functional assignment and mechanistic studies. Importantly, \u003cb\u003eCompounds 3\u003c/b\u003e and \u003cb\u003e6\u003c/b\u003e, as well as \u003cb\u003eCompounds 25a\u003c/b\u003e and \u003cb\u003e25b\u003c/b\u003e, are novel classes of active site-targeting covalently reacting DUB inhibitors that show promise as chemical probes and scaffolds for drug development. By eliciting pathway-relevant phenotypes in innate immunity, hypoxic signaling, and viral infection models, these inhibitors further reinforce the therapeutic potential of DUBs.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Methods","content":"\u003ch2\u003eSmall-molecule synthesis\u003c/h2\u003e\u003cp\u003eαβ,α′β′-Diepoxyketones (DEKs) (\u003cb\u003eCompounds 1–8\u003c/b\u003e) (\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e) and γ-lactams (\u003cb\u003eCompounds 9–23\u003c/b\u003e) (\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e) reported. The synthesis of Ub-derived small-molecules (\u003cb\u003e25\u003c/b\u003e–\u003cb\u003e31 and 25a-k\u003c/b\u003e) is described in the Supplementary Information (Fig. S11 and S12). The structures of \u003cb\u003eCompounds 1–31\u003c/b\u003e are in the Supplementary Information (Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e-3), the structures of \u003cb\u003eCompounds 25a-k\u003c/b\u003e are in the Table \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e. The synthesis and structures of biotinylated \u003cb\u003eCompound 25 derivates\u003c/b\u003e are described in the Supplementary Information (Fig. S13 and Table \u003cspan class=\"InternalRef\"\u003eS5\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eMCF-7 cells were cultured in high glucose DMEM supplemented with 10% (v/v) fetal bovine serum (FBS) and L-glutamine (2 mM). Cells were maintained at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e. MCF-7 cells were washed with phosphate buffered saline (PBS) and collected in PBS by scraping and centrifugation at 200 \u003cem\u003exg\u003c/em\u003e for 10 min. For data shown in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-C, MCF-7 cells were collected by the addition of TrypLE followed by media and centrifuged at 1600 rpm for 5 min with pellets being resuspended in PBS (3 cycles). HAP1 cells were cultured in IMDM media (#21980065) supplemented with 10% FBS (v/v), and maintained at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e. ~3\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells were seeded per 10 cm plate in 10 mL media overnight. After seeding, 4 mL of media were aspirated from each plate, and recombinant human interferon-alpha, IFN-α2 (α2b; PBL Assay Science; Cat. No. 11105-1), was added to the remaining media (1000 units/mL final concentration). IFN-α treatment was performed for 24 h (to induce USP18), media from each plate was then aspirated and cells were washed once with PBS (5 mL per plate). Cells were subsequently stored at \u0026minus;\u0026thinsp;80\u0026deg;C until lysis. THP-1 cells were cultured in RPMI 1640 supplemented with 10% (v/v) FBS, 0.05 mM β-mercaptoethanol, GlutaMAX Supplement, and Penicillin-Streptomycin. Cells were maintained at 37\u0026deg;C 5% CO\u003csub\u003e2\u003c/sub\u003e. RCC4-VHL and VA cells were cultured in DMEM media supplemented with 10% fetal bovine serum (FBS), GlutaMAX, and Penicillin-Streptomycin (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Calu-3 cells were maintained in Advanced DMEM media, 10% FCS, L-glutamine, and Penicillin-Streptomycin.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHigh-throughput ABPP workflows\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eSample preparation for Agilent Bravo ABPP-HT*\u003c/h2\u003e \u003cp\u003eThe data presented here on the Agilent Bravo ABPP immunoprecipitation is from published work,(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) and is included for comparison.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSample preparation for \u0026ldquo;on bench\u0026rdquo; ABPP-HT* high-throughput screen and compound concentration-dependence validations\u003c/h2\u003e \u003cp\u003eHA-Ub-PA was synthesised as reported (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). MCF-7 cells were washed with PBS, scraped in PBS and centrifuged at 200 x \u003cem\u003eg\u003c/em\u003e for 10 min. Pellets were then resuspended in lysis buffer (50 mM Tris, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.5 mM EDTA, 250 mM sucrose, 1 mM dithiothreitol (DTT), pH 7.5), and lysed using glass beads (2:1 lysate to beads ratio) with vortexing (10 cycles of 30 s vortexing, followed by 2 min incubation on ice). The MCF-7 lysate was then clarified at 600 \u003cem\u003ex g\u003c/em\u003e for 10 min at 4\u0026deg;C. Lysate protein concentration was determined by BCA, and 250 \u0026micro;g was aliquoted per well of a 96 well plate at a concentration of ~\u0026thinsp;4.7 mg/mL.\u003c/p\u003e \u003cp\u003eThe compound library was diluted to 1 mM DMSO stock solutions in a 96 well plate and multichannel-pipetted with mixing into the lysate plate to a final compound concentration of 25 \u0026micro;M in duplicate for the screen. For the concentration-dependence validations, the indicated inhibitor concentrations were added in duplicate. Additional control wells (4 x negative, 4 x positive for the screen, 3 x negative and 3 x positive for the concentration-dependence validations) were treated with DMSO. The plate was then incubated at 37\u0026deg;C for 1 h on a thermomixer without shaking.\u003c/p\u003e \u003cp\u003eA total of 2 \u0026micro;g of HA-Ub-PA was multichannel-pipetted with mixing into to the positive control wells and all inhibitor-treated wells, with an equivalent volume of buffer added to the negative control wells. The lysates were then incubated with the HA-Ub-PA at 37\u0026deg;C for 45 min on a thermomixer without shaking. The final reaction volume with the MCF-7 lysate, inhibitor/DMSO and HA-Ub-PA/buffer was 60 \u0026micro;L. Reactions were quenched by the addition of SDS and NP-40 (IGEPAL CA-630) to final concentrations of 0.4% (v/v) and 0.5% (v/v), respectively. Reactions were then diluted by addition of 200 \u0026micro;L NP40 buffer (50 mM Tris, 0.5% NP-40 (IGEPAL CA-630), 150 mM NaCl, 20 mM MgCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6 H\u003csub\u003e2\u003c/sub\u003eO, pH 7.4).\u003c/p\u003e \u003cp\u003e75 \u0026micro;L of anti-HA magnetic beads were aliquoted into a separate 96 well plate and washed 5x with 200 \u0026micro;L of NP40 buffer (50 mM Tris Base, 0.5% NP-40 (IGEPAL CA-630), 150 mM NaCl, 20 mM MgCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6 H\u003csub\u003e2\u003c/sub\u003eO, pH 7.4), using a magnetic rack for bead separation and multichannel pipetting with cut tips for mixing. Reaction mixtures were then added to the washed magnetic beads, mixed by multichannel-pipetting, and incubated in a thermomixer overnight at 4\u0026deg;C with gentle agitation (350 rpm). The beads were then washed 5x with 200 \u0026micro;L of NP40 buffer, as before.\u003c/p\u003e \u003cp\u003eProteins were eluted from the beads and reduced by addition of 100 \u0026micro;L of 2 x Laemmli buffer supplemented with 20 mM DTT, and incubated for 10 min at 90\u0026deg;C with agitation (350 rpm) on a thermomixer. Eluates were then removed from the beads using a magnetic rack and transferred to a fresh 96 well plate. Eluates were alkylated by the addition of iodoacetamide (IAA) to a final concentration of 40 mM. Proteins were then digested using trypsin and cleaned up using a 96 well S-trap plate according to the manufacturer\u0026rsquo;s instructions (\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e), dried down in a speedvac and resuspended in 0.1% (v/v) aqueous formic acid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSample preparation for the Thermo KingFisher Apex ABPP-HT*\u003c/h2\u003e \u003cp\u003eThe bio-ISG15-PA probe was synthesised as previously described (\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e). Cell lysis, subsequent labelling with HA-Ub-PA or bio-ISG15-PA probes, immunoprecipitation and protein enrichment, and processing of samples for analysis by western blot and LC-MS/MS was performed as described (\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e). Briefly, HAP1 cells were treated and prepared as described in the subsection \u0026lsquo;\u003cem\u003eCell culture\u0026rsquo;\u003c/em\u003e. Cells were resuspended in lysis buffer (50 mM Tris, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.5 mM EDTA, 2.5% (v/v) glycerol, 1 mM dithiothreitol (DTT), pH 7.5), and lysed using glass beads (2:1 ratio lysate to beads) with vortexing (10 x 30 s vortexing, followed by 1 min incubation on ice). The HAP1 lysates were then centrifuged (600 x \u003cem\u003eg\u003c/em\u003e for 5 min at 4\u0026deg;C; ~80% of the supernatant was transferred into tubes, then the remaining\u0026thinsp;~\u0026thinsp;20% of supernatant was centrifuged at 14 000 x \u003cem\u003eg\u003c/em\u003e for 10 min at 4\u0026deg;C, and then the two fractions from each spin were combined). The protein concentration in the lysates was estimated using the BCA assay kit (#23227), and 270 \u0026micro;g were aliquoted per well into a 96-deep well plate. The total volume in each well was made up to 143 \u0026micro;L for samples to be labelled with HA-Ub-PA, or 147 \u0026micro;L for samples to be labelled with bio-ISG15-PA, with lysis buffer.\u003c/p\u003e \u003cp\u003eNegative and probe-only positive controls were treated with 1.5 \u0026micro;L of DMSO in triplicates. The plate was then incubated at RT for 1 h without shaking.\u003c/p\u003e \u003cp\u003e5.4 \u0026micro;L of HA-Ub-PA or 2.35 \u0026micro;L of bio-ISG15-PA (volumes determined from probe titration assays for protein labelling) were transferred into the respective wells using a multichannel pipette, with an equivalent volume of buffer added to the corresponding negative control wells, and incubated at 37\u0026deg;C for 45 min without shaking. The final reaction volume with the HAP1 lysate, inhibitor or DMSO, and HA-Ub-PA or bio-ISG15-PA or buffer was 150 \u0026micro;L. Reactions were quenched by addition of SDS and NP-40 (IGEPAL CA-630) to final concentrations of 0.4% (v/v) and 0.5% (v/v), respectively, from a master mixture. The master mixture was prepared immediately before quenching by mixing a 5% (v/v) SDS stock with a 10% (v/v) NP-40 stock in a ratio of 1.6:1 and adding 21.5 \u0026micro;L of the mixture to each well. 20 \u0026micro;L of each reaction mixture was kept as \u0026lsquo;input\u0026rsquo; samples (to evaluate efficiency of probe labelling), and the remaining\u0026thinsp;~\u0026thinsp;150 \u0026micro;L of reaction volume was diluted by addition of 250 \u0026micro;L NP-40 buffer (50 mM Tris, 0.5% (v/v) NP-40 (IGEPAL CA-630), 150 mM NaCl, 20 mM MgCl\u003csub\u003e2\u003c/sub\u003e, pH 7.4) to a total volume of 400 \u0026micro;L.\u003c/p\u003e \u003cp\u003eFor immunoprecipitation/pull-down steps, 50 \u0026micro;L of anti-HA magnetic beads or 37.5 \u0026micro;L of streptavidin magnetic Sepharose beads were used per sample. The appropriate total amount of anti-HA magnetic beads or streptavidin magnetic Sepharose beads were split into 450 \u0026micro;L aliquots, washed with 4 \u0026times; 1.6 mL of NP-40 buffer (50 mM Tris, 0.5% NP-40 (IGEPAL CA-630), 150 mM NaCl, 20 mM MgCl\u003csub\u003e2\u003c/sub\u003e, pH 7.4) using a magnetic rack for bead separation, and transferred into the respective reaction wells using a multichannel pipette with cut tips. The 96-deep well plate was then incubated for 4 h at 4\u0026deg;C on the Thermo KingFisher Apex system, with the mixing speed set to \u0026lsquo;medium\u0026rsquo;. The beads were then washed with 4 \u0026times; 500 \u0026micro;L NP-40 buffer, as described above, and proteins were eluted from the beads twice with 2\u0026times; Laemmli buffer supplemented with 50 mM DTT (2 \u0026times; 55 \u0026micro;L, 2 \u0026times; 5 min at 95\u0026deg;C with the mixing speed set to \u0026lsquo;slow\u0026rsquo;). Beads were removed from the eluates by programming the system to capture and release the beads into an empty 96-deep well plate. Eluates were reduced with DTT to a final concentration of 10 mM (15 min, RT), then alkylated by the addition of iodoacetamide (IAA) to a final concentration of 20 mM (10 min, RT). Proteins were then digested using trypsin and cleaned up using a 96-well S-trap plate according to the manufacturer\u0026rsquo;s instructions(84, dried down in a speedvac and resuspended in 0.1% (v/v) aqueous formic acid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS/MS timsTOF for \u0026ldquo;on bench\u0026rdquo; ABPP-HT* high-throughput screen and compound concentration-dependence validations\u003c/h2\u003e \u003cp\u003ePeptide samples were loaded onto Evotips (Evosep) desalting columns according to the manufacturer\u0026rsquo;s instructions, then chromatographically separated on an 8 cm x 150 \u0026micro;m analytical column with bead size 1.5 \u0026micro;m (EV1109, Evosep), using an Evosep One liquid chromatography (LC) system with the 100 samples per day (spd) standard method. Peptides were eluted onto a TimsTOF Pro mass spectrometer (Bruker) operated in diaPASEF mode using 8 diaPASEF scans per TIMS-MS scan. The ion mobility range was set to 0.85\u0026ndash;1.3 Vs/cm\u003csup\u003e2\u003c/sup\u003e. Each mass window isolated was 25 m/z wide, ranging from 475\u0026ndash;1000 m/z with an ion mobility-dependent collision energy that increased linearly from 20 eV to 59 eV between 0.6\u0026ndash;1.6 Vs/cm\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS/MS for timsTOF Agilent Bravo ABPP-HT*\u003c/h2\u003e \u003cp\u003eThe previously reported timsTOF data collected after immunoprecipitation using the Agilent robot sample prep methodology is identical to that detailed above (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), other than a different ion mobility range (0.6\u0026ndash;1.6 Vs/cm\u003csup\u003e2\u003c/sup\u003e), and mass range (400\u0026ndash;1000 m/z).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS/MS for timsTOF for Thermo KingFisher Apex ABPP-HT*\u003c/h2\u003e \u003cp\u003ePeptides were analysed by nanoLC-MS/MS using an Evosep One LC system coupled with a timsTOF HT (Bruker) equipped with an 8 cm x 150 \u0026micro;m, 1.5 \u0026micro;m analytical column (Evosep). 200 ng peptides were separated using the Evosep 60SPD workflow (Analytical solvents A: 0.1% FA and B: acetonitrile plus 0.1% FA). Column was held at 40\u0026deg;C. Data were acquired in data-independent acquisition PASEF mode with the following settings: m/z range from 100 m/z to 1700 m/z, ion mobility range from 1/K0\u0026thinsp;=\u0026thinsp;1.30 to 0.85 Vs/cm\u003csup\u003e2\u003c/sup\u003e using equal ion accumulation and ramp times in the dual TIMS analyser of 100 ms each. Each cycle consisted of 8 PASEF ramps covering 21 mass steps each with 25 Da windows each with 2/3 non-overlapping ion mobility windows covering the 475 to 1000 m/z range and 0.85 and 1.26 Vs/cm\u003csup\u003e2\u003c/sup\u003e ion mobility range. The collision energy was lowered as a function of increasing ion mobility from 59 eV at 1/K0\u0026thinsp;=\u0026thinsp;1.6 Vs/cm\u003csup\u003e2\u003c/sup\u003e to 20 eV at 1/K0\u0026thinsp;=\u0026thinsp;0.6 Vs/cm\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAgilent Bravo\u003c/b\u003e \u003cb\u003evs\u003c/b\u003e \u003cb\u003e\u0026ldquo;on bench\u0026rdquo; ABPP-HT* comparison LC-MS/MS data analysis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eData was searched in DIA-NN 1.8.1 with search settings left as default and match between runs enabled. Data was searched using a Uniprot \u003cem\u003eHomo sapiens\u003c/em\u003e database containing isoforms (42,469 entries, retrieved on 15/08/2023). N\u0026thinsp;=\u0026thinsp;3 for HA-Ub-PA positive controls, N\u0026thinsp;=\u0026thinsp;1 for negative controls with no HA-Ub-PA present. The \u0026lsquo;report.pg_matrix\u0026rsquo; output was used for analysis. The volcano plot at Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG (N\u0026thinsp;=\u0026thinsp;3 HA-Ub-PA positive and negative controls) was generated from a two-sample student\u0026rsquo;s t-test in Perseus (Version 1.6.15).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThermo KingFisher Apex ABPP-HT* workflow LC-MS/MS data analysis\u003c/h2\u003e \u003cp\u003eData was searched in DIA-NN 1.8.1 with search settings left as default and match between runs enabled. Data was searched using a Uniprot \u003cem\u003eHomo sapiens\u003c/em\u003e database containing isoforms (20412 entries, retrieved 13/12/2023). The \u0026lsquo;report.pg_matrix\u0026rsquo; output was used (identical number of DUBs were identified in the \u0026lsquo;report.unique_genes_matrix\u0026rsquo; output). DUBs were filtered to select hits\u0026thinsp;\u0026gt;\u0026thinsp;1.5-fold enriched in the positive HA-Ub-PA control \u003cem\u003evs\u003c/em\u003e the negative control without HA-Ub-PA. Volcano plots were generated/calculated using GraphPad Prism (Version 10.2.3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eABPP-HT* screen and concentration-dependence data analysis\u003c/h2\u003e \u003cp\u003eData was searched in DIA-NN 1.8.1 with search settings left as default and match between runs enabled. Data was searched using a Uniprot \u003cem\u003eHomo sapiens\u003c/em\u003e database, not containing isoforms (20,416 entries, retrieved on 15/02/2023). The \u0026lsquo;report.unique_genes_matrix\u0026rsquo; output was used to ensure inhibitor identification unique to specific DUBs. DUBs were filtered to remove those that were not \u0026gt;\u0026thinsp;5-fold enriched in the positive HA-Ub-PA control \u003cem\u003evs\u003c/em\u003e the negative control without HA-Ub-PA. For the concentration-dependent data the following DUBs were discounted due to their unreliable detection across replicates, likely attributable to their low intensities: OTUD1, USP42, OTUD5, USP1, USP30, USP33, USP37, USP43 and OTUB2. Intensities were normalized as a percentage of the positive HA-Ub-PA control and for the screen multiple two-tailed unpaired (homoscedastic) T-tests were carried out (Microsoft Excel) to identify DUBs with significantly different intensities relative to the positive control. The resultant data were then filtered to select hits with a\u0026thinsp;\u0026gt;\u0026thinsp;20% average reduction in DUB activity. The concentration-dependence ABPP outputs were processed using CurveCurator (Version 0.3.0),(87 with the alpha asymptote set to 5%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eIn-depth ABPP workflows\u003c/h2\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003eSample preparation\u003c/h2\u003e \u003cp\u003eCell lysis and DUB Activity-Based Enrichment: Cells were lysed in lysis buffer (50 mM Tris, pH 7.5, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.5 mM EDTA, 1 mM DTT, 250 mM sucrose) containing acid-washed glass beads (Sigma Aldrich #G4649). Glass beads made up approximately one third of the total lysis buffer volume. Cell lysates were vortexed and centrifuged (14K x g, 4\u0026deg;C, 25 min) to separate the pellet, glass beads, nuclei, and membranes from supernatant. The supernatant was transferred to Eppendorf tubes and the pellets were discarded.\u003c/p\u003e \u003cp\u003eProteins extracted from ~\u0026thinsp;1\u0026middot;10\u003csup\u003e7\u003c/sup\u003e cells were used as starting materials. When profiling a DUB inhibitor, PR-619 (Sigma-Aldrich # 662141) was first incubated and mixed at 1,000 rpm at 37\u0026deg;C for 1 h. 25 \u0026micro;g of a DUB probe (HA-Ahx-Ahx-Ub-PA (UbiQ-078), HA-Ahx-Ahx-Ub-VME (UbiQ-035), HA-Ahx-Ahx-Ub-VS (UbiQ-178)) or probe mixtures were added to each of the samples and mixed at 1,000 rpm at 37\u0026deg;C for 1 h. The reaction was quenched by the addition of SDS to 0.4% (v/v) and NP-40 to 0.5% (v/v). Lysate mixtures were then diluted to 1 mL, 0.5 mg/mL with NP-40 lysis buffer (pH 7.4, 50 mM Tris, 0.5% (v/v) NP-40, 150 mM NaCl, 20 mM MgCl\u003csub\u003e2\u003c/sub\u003e). 200 \u0026micro;L of anti-HA-Agarose (Pierce # 26182) slurry, previously washed three times with NP-40 lysis buffer, was added to the samples and incubated on a rotator overnight at 4\u0026deg;C. After a first centrifugation step (2,000 \u003cem\u003ex g\u003c/em\u003e, 4\u0026deg;C, 1 min), beads were washed four times with 500 \u0026micro;L of NP-40 lysis buffer. Protein complexes were eluted by boiling beads in 250 \u0026micro;L SDS Laemmli 2X sample buffer.\u003c/p\u003e \u003cp\u003eMethanol-chloroform precipitation was performed prior to protease digestion. For each sample, a 4x volume of methanol was added and vortexed for 5 s. A 1x volume of chloroform was added to the samples, which were then vortexed for 5 s. A 3x volume of water was added, and samples were vortexed for 5 s. Samples were then centrifuged for 10 min at 10,000 rpm. The aqueous and organic phases were removed to leave only the protein pellet. The pellets were subsequently washed with 4x volume of methanol and centrifuged for 10 min at 10,000 rpm. The supernatant was discarded, and pellets were re-dissolved in 100 \u0026micro;L of water. Protein precipitation was repeated for a second time to ensure sufficient removal of detergent.\u003c/p\u003e \u003cp\u003eSample digestion and TMT labelling were performed using the IST-NHS 96x kit (PreOmics # P.O.00030) according to the manufacturer\u0026rsquo;s instructions. Briefly, 100 \u0026micro;L lysis buffer was added to each sample and heated to 95\u0026deg;C while mixing at 1,000 rpm for 10 min. Lyophilised digestion enzymes were dissolved in 100 \u0026micro;L water before being added to the samples. The samples were digested at 37\u0026deg;C for 2 h. For samples to be analysed by a Label-Free quantitation MS method, digestion was stopped, and the sample was transferred to the cartridge in a centrifuge at 2000 g for 1 min before proceeding the washing steps with wash solvent 1 and 2 provided with the kit. For the remaining samples, 200 \u0026micro;g TMTpro 18 Plex reagents (ThermoFischer Scientific # A52045) were added to each of the samples and incubated for 1 h while shaking at 1000 rpm. A total of 10 \u0026micro;L of 5% (v/v) aqueous hydroxylamine was added to each of the samples. A total of 100 \u0026micro;L of stop reagent was also added to the samples before transferring to cartridge for washing with washing solvent 1 and 2 provided with the kit. Samples were eluted with 250 \u0026micro;L of 50% (v/v) acetonitrile in 0.01% (v/v) aqueous formic acid. Samples labelled with TMT reagents were combined, dried and subjected to high pH fractionation using HPLC (Agilent). Peptides were loaded into a XBridge C18 column 4.6 x 250 mm (Waters # PN186003117) in buffer A (H2O, pH 10) and eluted with increasing buffer B (90% (v/v) aqueous acetonitrile, pH 10) at a 0.50 mL/min flowrate over a 96 min gradient. Eluted samples were collected every minute and concatenated to give a total of 24 fractions. Desalting was performed by solid phase extraction (SPE) employing reversed-phase C18 cartridges (Sep-Pak C18 light).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS/MS methodology\u003c/h2\u003e \u003cp\u003eFor LFQ Data Dependent Acquisition (DDA) and Data independent Acquisition (DIA) workflows, peptides were analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using an Ultimate 3000 UHPLC coupled to an Orbitrap Fusion Lumos Mass Spectrometer (both from ThermoFisher). Tryptic peptides were separated on an EASY-spray PepMap column (2 \u0026micro;m, 75 \u0026micro;m x 50cm; ThermoScientific) using a 60 min linear gradient from 2 to 35% buffer B (5% DMSO, 100% acetonitrile, 0.1% TFA) with a 250 nL/min flow and analysed on the Orbitrap Fusion Lumos (Thermo Scientific). Data were acquired in DDA and DIA as (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e). In both cases the advance peak detection (ADP) was enabled. For DDA, survey scans were acquired in the Orbitrap at 120 k resolution over a m/z range 400\u0026ndash;1500, AGC target of 4e5 and S-lens RF of 30. Fragment ion spectra (MS/MS) were obtained in the Ion trap (rapid scan mode) with a Quad isolation window of 1.6, 40% AGC target and a maximum injection time of 35 ms, with HCD activation and 28% collision energy. For DIA, MS1 scans were acquired in the Orbitrap with 120k resolution (m/z 350\u0026ndash;1650), an AGC target of 5 \u0026times; 10⁵ and a maximum injection time of 20ms. The full MS events were followed by 40 DIA scan windows per cycle (with variable isolation width) covering the m/z range from 350\u0026ndash;1650. The MS/MS scans were acquired in the orbitrap at 30k resolution and normalised HCD set up at 30%.\u003c/p\u003e \u003cp\u003eFor TMT labelled samples, mass spectrometric analyses were carried out with a Proxeon Easy Nano-LC fitted with a PepMap C18 column 75 \u0026micro;m x 50 cm (Thermos Scientific #PN ES903) and connected to a Lumos Orbitrap mass spectrometer (Thermo Scientific). Peptides were eluted at 450 nL flowrate during 120 min gradient. MS data was acquired with SPS M3 approach. Each full orbitrap scan (R=60K, m/z scan range: 385\u0026ndash;1350, AGC target: 100k, max. injection time: 100 ms) was acquired and followed with dependent IT MS/MS scans (isolation window: 0.5, CID collision energy (%): 35, AGC target: 30k ions, max. injection time: 175 ms) for a 2.7s cycle time. SPS MS3 scans were acquired with the following parameters (R: 50k, isolation window\u0026thinsp;=\u0026thinsp;1.5, AGC target: 120k, HCD collision energy (%): 55, max. injection time: 100 ms).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eLC-MS/MS data analysis Single shot LFQ\u003c/h2\u003e \u003cp\u003eData was searched using a Uniprot \u003cem\u003eHomo sapiens\u003c/em\u003e database containing isoforms (42,469 entries, retrieved on 15/08/2023). Software versions: DIA-NN 1.8.1, Fragpipe 22.0, MaxQuant/MaxDIA 2.6.1.0. All search settings were left as default, with match between runs enabled and cysteine carbamidomethylation included as a fixed modification. Each search was 3 x HA-Ub-* and 3 x HA-Ub-*+ 50 \u0026micro;M PR619. The PA warhead alone was searched separately from the PA/VME/VS cocktail of warheads. The intensities output \u0026lsquo;report.pg_matrix\u0026rsquo; was used from DIA-NN, the LFQ intensities from \u0026lsquo;combined_protein\u0026rsquo; output was used from Fragpipe, and the LFQ intensities from the \u0026lsquo;protein groups\u0026rsquo; was used from Maxquant for both DDA and DIA searches.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS/MS data analysis TMT fractions\u003c/h2\u003e \u003cp\u003eMS data processing was performed with Proteome Discoverer Version 2.4.0.305 (Thermo Scientific) with UniProtKB Swiss-Prot (TaxID\u0026thinsp;=\u0026thinsp;9606, \u003cem\u003eHomo sapiens\u003c/em\u003e) fasta. Peptide identifications were carried out using the Sequest\u0026trade; search engine with the following parameters: precursor tolerance: 10 ppm, fragment tolerance\u0026thinsp;=\u0026thinsp;0.6 Da, max. missed cleavage sites: 3, dynamic modifications: M oxidation and N/Q deamination, fixed modifications: PreOMics cysteine modification (+\u0026thinsp;113.084 Da) and TMTpro (+\u0026thinsp;304.207 N-terminus and K ABP western blot compound validations\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eABPP-HT* screen hit validations\u003c/h2\u003e \u003cp\u003eFor positive hit validations by western blot the sample preparation detailed for the \u0026ldquo;on bench\u0026rdquo; ABPP-HT* were used with the concentrations of compounds indicated in figures. Following compound incubation and reaction with HA-Ub-PA, the reaction was quenched directly with Laemmli buffer supplemented with 100 mM DTT, boiled, and analysed by western blot. IC\u003csub\u003e50\u003c/sub\u003e values were extracted by quantifying the HA-Ub-PA labelled band by densitometry (Image studio lite) normalised to the β-actin signal and subsequently normalising the signal of the DUB activity to the negative and positive HA-Ub-PA controls. Concentration-dependencies were fit to Y\u0026thinsp;=\u0026thinsp;100/(1\u0026thinsp;+\u0026thinsp;10^((LogIC\u003csub\u003e50\u003c/sub\u003e-X)*HillSlope)) in Graphpad Prism (Version 10.1.1).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eCompound\u003c/span\u003e \u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003evs\u003c/span\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eHA-Ub-PA time dependence competition studies\u003c/span\u003e\u003c/p\u003e \u003cp\u003eSamples were processed as for the ABP western blot compound concentration-dependence validations, with the HA-Ub-PA incubated for the indicated time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eABP compound cellular permeability studies\u003c/h2\u003e \u003cp\u003eMCF-7 cells were cultured in 6-well plates to 90% confluence, with a media change to 1 mL prior to DMSO/compound treatment at the indicated concentration for 4 h. Cells were then washed 3 x with PBS, and collected by scraping in PBS and centrifuged (200 x \u003cem\u003eg\u003c/em\u003e for 10 min). Pellets were then processed as outlined above in the ABPP screen sample preparation section, with 0.4 \u0026micro;g of HA-Ub-PA being used per 50 \u0026micro;g of lysate protein. The mixture was incubated for 45 min at 37 ˚\u0026deg;C, before being quenched by addition of Laemmli buffer supplemented with 100 mM of DTT; the mixture was then analysed by western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eCompound 25 selectivity with biotin-ISG15-PA ABP\u003c/h2\u003e \u003cp\u003eFor Compound 25 selectivity with biotin-ISG15-PA the sample preparation detailed for the Thermo KingFisher Apex ABPP-HT* was used with the concentrations of compounds indicated in figures. For each compound, stock solutions (100\u0026times;) were prepared at a concentration of 0.46, 1.39, 4.16 or 12.5 mM in DMSO in a 96-well plate, and 1.5 \u0026micro;L of each stock were transferred into the respective wells using a multichannel pipette (resulting in a final concentration of 4.6, 13.9, 41.6 or 125 \u0026micro;M). Negative and probe-only positive controls were treated with 1.5 \u0026micro;L of DMSO. The plate was then incubated at RT for 1 h without shaking. Following compound incubation 2.35 \u0026micro;L of bio-ISG15-PA was transferred into the respective wells using a multichannel pipette, with an equivalent volume of buffer added to the corresponding negative control wells and incubated at 37\u0026deg;C for 45 min without shaking. Following compound incubation and reaction with HA-Ub-PA, the reaction was quenched directly with Laemmli buffer supplemented with 50 mM DTT, boiled, and analysed by western blot.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eBiotinylated USP5 inhibitors HA-Ub-PA ABP\u003c/h2\u003e \u003cp\u003eFor HA-Ub-PA ABP assays MCF-7 cell lysates were lysed as detailed for the Thermo KingFisher Apex ABPP-HT* methodology and incubated with biotinylated Compound 25 derivative inhibitors at either 150 \u0026micro;M or 250 \u0026micro;M) for 1 h at room temperature. Following the initial incubation, 0.5 \u0026micro;l of HA-Ub-PA was added and incubated at 37\u0026deg;C for 45 min. Reactions were quenched with Laemmli buffer and DTT (50 mM), boiled and analyzed by western blot with immunoblotting for streptavidin, USP5, USP42, and GAPDH. DMSO-treated samples and reactions lacking HA-Ub-PA ABP were included as vehicle controls. Reactions with non-biotinylated USP5 inhibitor (Compound 25b) at 150 \u0026micro;M was included as a negative control. Immunoblotting against USP5, USP42 and GAPDH were also included as controls, positive, negative, and loading, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eCompetition assay with non-biotinylated inhibitor (Compound 25) and N-Ethylmaleimide (NEM)\u003c/h2\u003e \u003cp\u003eFor non-biotinylated Compound 25 and NEM competition assays MCF-7 cell lysates were lysed as detailed for the Thermo KingFisher Apex ABPP-HT* methodology and pre-incubated with either non-biotinylated inhibitor (Compound 25) at 250 \u0026micro;M or NEM at 30 mM at room temperature for 1 h. Following the initial incubation, biotinylated Compound 25 derivative inhibitors at 150 \u0026micro;M or DMSO control were added and incubated at room temperature for 1 h. Reactions were quenched with Laemmli buffer and DTT (50 mM), boiled and analyzed by western blot with immunoblotting for streptavidin, USP5, USP42, and GAPDH. DMSO-treated samples lacking biotinylated USP5 inhibitors and/or competitors were included as vehicle controls. Immunoblotting against USP5, USP42 and GAPDH were also included as controls, positive, negative, and loading, respectively.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOTUD7B Protein expression and purification\u003c/h3\u003e\n\u003cp\u003eOTUD7B (129\u0026ndash;438) cloned into pOPIN E vector was expressed in Rosetta2 (DE3) \u003cem\u003eE. coli\u003c/em\u003e cells grown in 2xTY medium supplemented with 100 \u0026micro;g/mL carbenicillin and induced with 400 \u0026micro;M Isopropyl-β-D-thiogalactoside (IPTG) at 18\u0026deg;C overnight. \u003cem\u003eE.coli\u003c/em\u003e pellets were lysed in 20 mM Tris pH 8.5, 500 mM NaCl, 20 mM Imidazole, 2 mM β-mercaptoethanol supplemented with lysozyme, DNase, 2 \u0026micro;M pepstatin, 4 \u0026micro;M leupeptin and 1 mM PMSF. Clarified lysates were applied to a HisTrap HP column (Cytiva) and eluted in 20 mM Tris pH 8.5, 500 mM NaCl, 500 mM Imidazole, 2 mM β-mercaptoethanol. Further purification was achieved by anion exchange chromatography (Resource Q, Cytiva) followed by size exclusion chromatography (Superdex 75, Cytiva) in 20 mM HEPES pH 7.5, 200 mM NaCl, 4 mM DTT. Concentrated protein was flash frozen and stored in single-use aliquots at -75\u0026deg;C.\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme kinetics\u003c/h2\u003e \u003cp\u003eFluorescence intensity measurements were used to monitor the cleavage of a ubiquitin-rhodamine substrate. All activity assays were performed in black 384-well plates in assay buffer (20 mM Tris, pH 8.0, 150 mM potassium glutamate, 0.1 mM TCEP, 0.03% Bovine Gamma Globulin) with a final assay volume of 20 \u0026micro;L. USP7, 1 nM (USP7 (1-1102), DU15644, MRC Protein Phosphorylation and Ubiquitylation Unit) or USP47, 10 nM (USP47 (N-terminal His), DU15682, MRC Protein Phosphorylation and Ubiquitylation Unit) was added and preincubated with \u003cb\u003eCompound 3\u003c/b\u003e for 30 min. OTUD7B (OTU domain) was added and preincubated with Compound 6 for 30 min. 180 nM ubiquitin-rhodamine 110 (Ubiquigent for USP47/7, Bio-Techne for OTUD7B) was added to initiate the reaction and the fluorescence intensity was recorded for 30 min on a PherastarFSX (BMG Labtech) with an Ex\u003csub\u003e485\u003c/sub\u003e/Em\u003csub\u003e520\u003c/sub\u003e optic module. Initial rates were plotted against compound concentration to determine IC\u003csub\u003e50\u003c/sub\u003e. To investigate the time-dependence of the potency of \u003cb\u003eCompound 3\u003c/b\u003e, activity assays were repeated without a preincubation step.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eHDX-MS\u003c/h2\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eSample Preparation\u003c/h2\u003e \u003cp\u003eHydrogen Deuterium eXchange Mass Spectrometry (HDX-MS) experiments were performed on the same stock of recombinant USP7 and USP47 protein constructs as those used in the \u003cem\u003ein vitro\u003c/em\u003e enzyme kinetics experiments. USP7 was provided at a concentration of 25.3 \u0026micro;M and USP47 at 16.5 \u0026micro;M, respectively. Both proteins were prepared in 50 mM HEPES, pH 7.5, 10% glycerol, 150 mM NaCl, 1 mM DTT. The initial stock concentration of \u003cb\u003eCompound 3\u003c/b\u003e was 30 mM in DMSO. In solution HDX-MS was performed by preparing a volume-to-volume mixture at a molar ratio of 1:2 of protein:\u003cb\u003eCompound 3\u003c/b\u003e (\u003cem\u003ei.e\u003c/em\u003e., the holo-state). Samples were diluted to achieve a final concentration of 16 pmol on column per reaction for USP7 and 21 pmol on column per reaction for USP47. Equivalent control samples (\u003cem\u003ei.e\u003c/em\u003e., the apo-state) were prepared where DMSO was supplemented in place of the compound.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eData Acquisition\u003c/h2\u003e \u003cp\u003eHDX-MS was performed as(\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e). Briefly, an identical labeling buffer was prepared as that of the protein stocks except in deuterium oxide D\u003csub\u003e2\u003c/sub\u003eO (99+ %D, Cambridge Isotope Laboratories, Tewksbury, MA) and with the 10% glycerol was removed (a final composition of 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM DTT). The pH of the labeling buffer was measured and corrected to pD (pD\u0026thinsp;=\u0026thinsp;pH+0.4). The quenching buffer comprises 2M Guanidine HCl, 100 mM citric acid, pH 2.3 in H\u003csub\u003e2\u003c/sub\u003eO. Proteins +/- \u003cb\u003eCompound 3\u003c/b\u003e were preincubated for 30 min at room temperature to allow complexes to form. Samples were then diluted with the labeling buffer in a 1:20 ratio, achieving an excess D\u003csub\u003e2\u003c/sub\u003eO concentration of 95%. Several labeling time points were sampled at 20 ᵒC, more specifically, at 30, 600, and 3600 s. All analyses were acquired in triplicate. Non-deuterated controls were prepared in an identical fashion, with H\u003csub\u003e2\u003c/sub\u003eO in place of D\u003csub\u003e2\u003c/sub\u003eO. At the end of each labeling time point, samples were quenched upon addition of quench buffer (1:1 ratio), resulting in a final pH of 2.5. Samples were subsequently digested with a dual pepsin/proteaseXIII column (2.1 x 3.0 mm; NovaBioAssays, MA) at 8 ᵒC for 3 min. Peptides were trapped on a 1.0 mm x 5.0 mm, 5.0 \u0026micro;m trap cartridge (Thermo Scientific\u0026trade; Acclaim PepMap100) for desalting. The flow rate was maintained at 150 \u0026micro;L/min. Peptides were then separated on a Thermo Scientific\u0026trade; Hypersil Gold\u0026trade;, 50 x 1 mm, 1.9 um, C18 column increasing hydrophobicity achieved by a linear gradient of 10% to 40% Buffer B (A: water, 0.1% FA; B: ACN, 0.1% FA). The flow rate was maintained at 40 \u0026micro;L/min. A protease wash (2 M guanidine, 100 mM citric acid, pH 2.3 in H\u003csub\u003e2\u003c/sub\u003eO) was performed for each run to limit carry-over. To minimize back-exchange, the quenching, trapping, and separation steps were performed at 1.5ᵒC. Labeling, quenching, and online digestion steps were performed with the aid of an automated HDX robot (Trajan Scientific and Medical). Sample preparation was managed in Chronos (version 5.4.1). All samples were acquired in MS1 mode on Thermo Scientific\u0026trade; Orbitrap Exploris\u0026trade; 480 Hybrid\u0026trade; mass spectrometer.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec35\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eBefore each HDX-MS experiment, an unspecific digested peptide database was created in BioPharma Finder (version 5.2) for non-deuterated USP7 and USP47 proteins through a data-dependent and targeted HCD-MS\u003csup\u003e2\u003c/sup\u003e acquisition regime (\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e). Labeling data were processed and manually curated in HDExaminer version 3.4.2 (Trajan Scientific and Medical). The charge state with the highest quality spectra for all replicates for each peptide across all HDX-MS labeling times was used in the final analysis. The significant differences observed at each residue were used to map HDX-MS consensus effects (based on overlapping peptides) onto the AlphaFold model.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCaspase-1 assay\u003c/h3\u003e\n\u003cp\u003ePMA-differentiated THP-1 cells were treated as indicated, and the caspase-1 activity in the culture medium (supernatant) was measured using the Caspase-Glo 1 Inflammasome Assay (Promega) according to the manufacturer\u0026rsquo;s instruction. Briefly, the Caspase-Glo 1 Reagent was prepared by resuspending the Z-WEHD substrate in Caspase-Glo 1 buffer and addition of MG132 inhibitor to a final concentration of 60 \u0026micro;M. Cell culture medium (50 \u0026micro;L) was then added to the Caspase-Glo 1 Reagent (50 \u0026micro;L) in a 96-well plate and the resultant mixture was incubated at room temperature for 1 h. Luminescence was measured using a FLUOstar Omega multi-mode microplate reader (BMG LABTECH) according to the manufacturer's instructions.\u003c/p\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003eMTS cell viability assay\u003c/h2\u003e \u003cp\u003eCell viability was assessed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS, Promega). The One Solution Reagent (20 \u0026micro;L) was added to each well of a 96-well plate containing 100 \u0026micro;L of sample in culture medium. Following incubation at 37\u0026deg;C for 1 h, absorbance was measured at the wavelength of 490 nm using a FLUOstar Omega multi-mode microplate reader (BMG LABTECH). Cell viability was calculated according to the manufacturer's instruction.\u003c/p\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003ch2\u003eASC speck oligomer cross-linking\u003c/h2\u003e \u003cp\u003eASC oligomer chemical cross-linking was performed as previously described (\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e). Briefly, PMA-differentiated THP-1 cells were primed with LPS (1 \u0026micro;g/mL) for 4 h, followed by stimulation with nigericin (10 \u0026micro;M) for 45 min and indicated concentrations of \u003cb\u003eCompound 3\u003c/b\u003e. Cells were lysed on ice in Buffer A (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 320 mM sucrose, and protease inhibitors), and homogenised using a 21-gauge needle. Lysates were centrifuged at 300 \u0026times; g for 8 min at 4\u0026deg;C. The supernatants were collected, mixed 1:1 with CHAPS buffer (20 mM HEPES, pH 7.5, 5 mM MgCl₂, 0.5 mM EGTA, 0.1% CHAPS, and protease inhibitors), and centrifuged at 2,400 \u0026times; g for 8 min to pellet crude inflammasome complexes. Pellets were washed twice with ice-cold PBS, resuspended in CHAPS buffer, and cross-linked with 2 mM DSS (prepared in DMSO) at 37\u0026deg;C for 20 min. The reaction was quenched with NuPAGE\u0026trade; LDS Sample Buffer (4X) and subjected to western blot analysis.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAlignment and structure prediction of\u003c/span\u003e \u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003ehomo sapiens\u003c/span\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eand Pan\u003c/span\u003e \u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003etroglodytes\u003c/span\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eUSP47\u003c/span\u003e\u003c/p\u003e \u003cp\u003eFor kinetic and HDX MS studies of \u003cb\u003eCompound 3\u003c/b\u003e, full-length \u003cem\u003eHomo Sapiens\u003c/em\u003e USP7 and full-length \u003cem\u003ePan troglodytes\u003c/em\u003e isoform 2 were used. Sequences of full-length \u003cem\u003eHomo sapiens\u003c/em\u003e canonical USP47 and full-length USP47 from \u003cem\u003ePan troglodytes\u003c/em\u003e isoform 2 were aligned using T-coffee multiple sequence alignment with differences reported in Fig. \u003cspan refid=\"MOESM9\" class=\"InternalRef\"\u003eS9\u003c/span\u003e\u003cem\u003eA\u003c/em\u003e (\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e). The sequences, including the His-tag of the recombinant \u003cem\u003ePan troglodytes\u003c/em\u003e isoform 2, were then used to predict the structures using AlphaFold 3 (\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e), with the highest-ranking structures being superposed using PyMOL (version 2.5.0) with an RMSD of 2.511 (Fig. \u003cspan refid=\"MOESM9\" class=\"InternalRef\"\u003eS9\u003c/span\u003e\u003cem\u003eB\u003c/em\u003e and S9\u003cem\u003eC\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section2\"\u003e \u003ch2\u003eOTUD7B Intact MS\u003c/h2\u003e \u003cp\u003eBinding of OTUD7B to compound 6 was detected by Liquid Chromatography Mass Spectrometry (LCMS) using an Agilent 1290 infinity II LC system connected to an Agilent 6550 accurate mass iFunnel quadrupole time of flight (QTOF) mass spectrometer. OTUD7B was incubated with compound 6 in 50 mM HEPES pH 7.5 for 1.0 and 15.0 min and 1.0 \u0026micro;L of sample was injected and loaded onto a \u003cem\u003eProSwift RP-4H monolithic phenyl reverse phase\u003c/em\u003e, 1.0 x 50 mm, HPLC column (Thermo). Solvent A consisted of LCMS grade water containing formic acid (0.1% v/v) and solvent B consisted of acetonitrile containing formic acid (0.1% v/v). OTUD7B (OTU domain) and \u003cb\u003eCompound 6\u003c/b\u003e were separated using a step wise gradient (0 min \u0026ndash; 95.0% solvent A, 1.0 min\u0026thinsp;\u0026minus;\u0026thinsp;80% solvent A, 9.0 min\u0026thinsp;\u0026minus;\u0026thinsp;45% solvent A, 10 min\u0026thinsp;\u0026minus;\u0026thinsp;0% solvent A, 11 min\u0026thinsp;\u0026minus;\u0026thinsp;0% solvent A, 12.0 min \u0026ndash; 95.0% solvent A), followed by a 3.0-min post-run with solvent A. All flow rates were 0.2 mL/min. The mass spectrometer was operated in positive ion mode with a drying gas temperature (225\u0026deg;C), drying gas flow rate (13 L/min), nebulizer pressure (20 psi), sheath gas temperature (350\u0026deg;C), sheath gas flow rate (12 L/min), capillary voltage (4000 V), nozzle voltage (1500 V), fragmentor voltage (365 V). All acquired data were analysed using Agilent MassHunter Qualitative Analysis (Version B.07.00) software.\u003c/p\u003e \u003cdiv id=\"Sec40\" class=\"Section3\"\u003e \u003ch2\u003eHIF-1α hypoxia conditions and Covid-19 infection assay\u003c/h2\u003e \u003cp\u003eSARS-CoV-2 Australia/VIC01/2020 virus and cells were provided by the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia at P1 and passaged twice in Vero/hSLAM cells (Cat#04091501) - obtained from the European Collection of Cell Cultures (ECACC), UK. Virus infectivity was determined by plaque assay on Vero-TMPRSS2 cells as previously reported (\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e). Calu-3 cells were infected with the above strain of SARS-CoV-2 at a MOI of 0.01 for 2 h. Viral inocula were removed, cells washed three times in PBS and maintained in growth media until harvest. Cell lines were maintained at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e in a standard culture incubator and exposed to hypoxia using an atmosphere-regulated workstation set to 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e:1%\u0026ndash;5% O\u003csub\u003e2\u003c/sub\u003e:balance N\u003csub\u003e2\u003c/sub\u003e (InvivO\u003csub\u003e2\u003c/sub\u003e 200, Baker-Ruskinn Technologies). For quantification of viral RNA, total cellular RNA was extracted using the RNeasy kit (Qiagen) according to manufacturer\u0026rsquo;s instructions. Equal amounts of RNA, as determined by Nanodrop analysis, were used in a one-step RT-qPCR using the Takyon-One Step RT probe mastermix (Eurogentec) and run on a Roche Light Cycler 96. For quantification of viral copy numbers, qPCR runs contained serial dilutions of viral RNA standards. Total SARS-CoV-2 RNA was quantified using: 2019-nCoV_N1-F: 5\u0026rsquo;-GAC CCC AAA ATC AGC GAA AT-3\u0026rsquo;, 2019-nCoV_N1-R: 5\u0026rsquo;-TCT GGT TAC TGC CAG TTG AA TCT G-3\u0026rsquo;, 2019nCoV_N1-Probe: 5\u0026rsquo;-FAM-ACC CCG CAT TAC GTT TGG ACC-BHQ1-3\u0026rsquo;. For assaying the antiviral response against \u003cb\u003eCompound 25b\u003c/b\u003e, Calu-3 cells were infected with a SARS-CoV-2 reporter virus containing a mNeonGreen fluorescent reporter as previously described at a MOI of 0.1 (\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e). Infected cells were treated with serial dilutions of the compound for 24 h followed by fixation in 4% PFA. Cells were stained with DAPI and mNeonGreen fluorescence was quantified by Clariostar. Fluorescence data was normalised to the DAPI signal and plotted as relative to the DMSO control.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eType I interferon production\u003c/span\u003e \u003c/p\u003e \u003cp\u003eType I interferon production was performed as previously described (\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e), using HEK293 cells transduced with a pGreenFire-ISRE reporter. In brief, HAP1 cells were seeded at 5,000 cells per well in a 96-well plate in DMEM containing 10% FBS and 2 mM glutamine. After attachment, cells were treated with \u003cb\u003eCompound 25b\u003c/b\u003e for 1 h, followed by poly(I:C) (0.5mg/mL) for 48 h. HAP1 media was then collected, centrifuged, and transferred to HEK293 IFN reporter cells, which were seeded at 25,000 cells per well in a 96-well plate and allowed to attach. Recombinant human IFNα2 (PBL assay science) was used for standards. After 24 h, the One-Glo Luciferase Assay System (Promega) was used to quantify luciferase expression following manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eCell growth and proliferation assays\u003c/span\u003e \u003c/p\u003e \u003cp\u003eA total of 5,000 cells were seeded into either 6-well or 12-well plates and imaged using an IncuCyte Zoom Imager (Sartorius) for live-cell imaging. Imaging was performed at the specified time points using the phase-contrast channel, as previously described (\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e). Cell growth was assessed by measuring the percentage confluence.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eMolecular docking\u003c/span\u003e \u003c/p\u003e \u003cp\u003eA model for human USP47 catalytic domain (UniProt: Q96K76), which is representative of a covalently bound inhibitor conformational state, was generated using the structure of human USP7 in complex with the covalent inhibitor, FT827 (PDB: 5NGF; Turnbull \u003cem\u003eet al\u003c/em\u003e., 2017), as the template in SwissModel (\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e). Coordinates for \u003cb\u003eCompound 3\u003c/b\u003e were generated using ChemDraw Prime 23.0.1.10 and molecular docking was performed using CovDock (\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e) implemented in Maestro version 13.8.135 (Schr\u0026ouml;dinger Release 2024-3: Maestro, Schr\u0026ouml;dinger, LLC, New York, NY, 2024). The best docking pose for \u003cb\u003eCompound 3\u003c/b\u003e corresponded to a docking score of -5.704.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAntibodies\u003c/span\u003e \u003c/p\u003e \u003cp\u003eThe antibodies used for immunoblotting in this study are listed in Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of antibodies for immunoblotting used in this study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e Reagent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrand\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCatalogue Number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNotes\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUSP5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15158-1-AP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUSP7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eENZO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBML-PW0540-0100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUSP18 (D4E7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4813\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUSP42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSanta Cruz\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003esc-390604\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUSP30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAtlas antibodies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHPA016952\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUSP33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20445-1-AP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUSP47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eab72143\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOTUD7B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14817\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVCPIP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e88153\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHA (12CA5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRoche\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11666606001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCFL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5175\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNLRP3(D4D8T)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaspase-1(D7F10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3866\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASC(B-3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSanta Cruz\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003esc-514414\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-1b(D3U3E)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12703\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCleaved-IL-1b (Asp116) (D3A3Z)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e83186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGSDMD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNovus Biologicals\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNBP2-33422\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHIF-1α\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBD Biosciences\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e610959\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSigma Aldrich\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA5441\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUsed in all figures except for Fig.\u0026nbsp;5A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-actin (8H10D10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUsed in Fig.\u0026nbsp;5A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIRDye 680RD Streptavidin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLi-Cor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e926-68079\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAPDH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e97166\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD064209. The mass spectrometry HDX data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD064060. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe A.P.-F. and B.M.K. labs were supported by the Chinese Academy of Medical Sciences Innovation Fund for Medical Science, China (grant numbers: 2018-I2M-2-002 and 2024-I2M-2-001-1) and by Pfizer Inc. The A.P.-F. lab was also supported by CRUK, Ono Pharma, and Boehringer Ingelheim. H.B.L.J. was supported by a Bristol-Myers Squibb fellowship. We thank Prof. Jan Rehwinkel for generously providing the type I interferon reporter cells and Prof Nicole Zitzmann\u0026rsquo;s lab for generously providing the Calu-3 cells.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions: \u003c/strong\u003eH.B.L.J., S.D., L.B., and A.P.-F. designed research; H.B.L.J., S.D., S.S.H., P.A.C.W., C.N., Z.L., J.D., J.L., E.M., A.B., A.C., A.P., L.W., S.S., J.W.H., M.E.R., S.F., A.T., D.O.B., E.S., and A.P.T. performed research; C.J.E and P.R.E. contributed new reagents/analytic tools; I.V., E.W.T., D.O.B., G.M.W., B.M.K., C.J.S., L.B., A.P.-F. provided supervision; H.B.L.J., L.B., and A.P.-F. wrote the paper, and D.O.B., S.D., S.S.H., A.P.T., C.J.S., P.R.E., and B.M.K. reviewed and corrected the manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting Interests: \u003c/strong\u003eThe authors declare that they have no competing interests affecting the contents of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eN. 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Erdmann, \u003cem\u003eet al.\u003c/em\u003e, A Novel Toolkit of SARS-CoV-2 Sub-Genomic Replicons for Efficient Antiviral Screening. \u003cem\u003eViruses\u003c/em\u003e 17, 597 (2025).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-drug-discovery","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Drug Discovery](https://www.nature.com/npjdrugdiscov/)","snPcode":"44386","submissionUrl":"https://submission.springernature.com/new-submission/44386/3","title":"npj Drug Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9106698/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9106698/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDeubiquitinases (DUBs) play key roles in human pathologies, including cancer, infectious, autoimmune, and neurodegenerative diseases. The progress of potent and selective active site-targeting DUB inhibitors into clinical trials has demonstrated the therapeutic opportunities of such molecules; however, the shared catalytic geometry among DUB family members poses a challenge for achieving inhibitor selectivity. We describe a high-throughput, DUB-focused, activity-based proteomics workflow to identify new types of cysteine-reactive DUB inhibitors. We demonstrate the potential of αβ,α′β′-diepoxyketones (DEKs) and ubiquitin-derived covalently reacting alkynes as the first selective, cell-active inhibitors of USP47, OTUD7B (Cezanne), and USP5. These DUBs are reported to have critical roles in inflammasome formation, hypoxia, and viral replication, respectively. The identified inhibitors phenocopy these findings, demonstrating the tractability of these DUBs as immunotherapeutic targets.\u003c/p\u003e","manuscriptTitle":"Activity-Based Proteomics Discovery of Deubiquitinating Enzyme Inhibitors with Immunomodulatory Activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-13 23:40:40","doi":"10.21203/rs.3.rs-9106698/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-29T10:41:59+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-29T03:26:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-19T20:32:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"322819144092442582994739580352281098085","date":"2026-04-09T13:54:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"102649916531829739163990105430939959337","date":"2026-04-08T12:03:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-07T12:49:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-18T12:27:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-16T12:05:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Drug Discovery","date":"2026-03-12T15:45:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-drug-discovery","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Drug Discovery](https://www.nature.com/npjdrugdiscov/)","snPcode":"44386","submissionUrl":"https://submission.springernature.com/new-submission/44386/3","title":"npj Drug Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"19ce7684-d2be-4d23-b322-948857d05bc0","owner":[],"postedDate":"April 13th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-04-29T10:41:59+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-29T03:26:00+00:00","index":22,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":66023200,"name":"Biological sciences/Biochemistry"},{"id":66023201,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":66023202,"name":"Biological sciences/Drug discovery"}],"tags":[],"updatedAt":"2026-04-29T10:55:01+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-13 23:40:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9106698","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9106698","identity":"rs-9106698","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}