Discovery of Tankyrase scaffolding inhibitor specifically targeting the ARC4 peptide binding domain

Discovery of Tankyrase scaffolding inhibitor specifically targeting the ARC4 peptide binding domain | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Discovery of Tankyrase scaffolding inhibitor specifically targeting the ARC4 peptide binding domain View ORCID Profile Chiara Bosetti , View ORCID Profile Albert Galera-Prat , View ORCID Profile Sven T. Sowa , Alexandra Gade , Cláudia Braga , Shoshy A. Brinch , View ORCID Profile Faranak Nami , Johan Pääkkönen , Veeti Pulju , View ORCID Profile Jo Waaler , View ORCID Profile Mads H. Clausen , View ORCID Profile Lari Lehtiö doi: https://doi.org/10.1101/2025.03.31.646301 Chiara Bosetti 1 Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu , Oulu, Finland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Chiara Bosetti Albert Galera-Prat 1 Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu , Oulu, Finland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Albert Galera-Prat Sven T. Sowa 1 Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu , Oulu, Finland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sven T. Sowa Alexandra Gade 2 Norwegian Centre for Molecular Biosciences and Medicine, University of Oslo , Oslo, Norway Find this author on Google Scholar Find this author on PubMed Search for this author on this site Cláudia Braga 3 Department of Chemistry, Technical University of Denmark , Kgs. Lyngby, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shoshy A. Brinch 4 Department of Immunology, Oslo University Hospital , Oslo, Norway 5 Hybrid Technology Hub-Centre of Excellence, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo , Oslo, Norway Find this author on Google Scholar Find this author on PubMed Search for this author on this site Faranak Nami 3 Department of Chemistry, Technical University of Denmark , Kgs. Lyngby, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Faranak Nami Johan Pääkkönen 1 Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu , Oulu, Finland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Veeti Pulju 1 Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu , Oulu, Finland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jo Waaler 4 Department of Immunology, Oslo University Hospital , Oslo, Norway 5 Hybrid Technology Hub-Centre of Excellence, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo , Oslo, Norway Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jo Waaler Mads H. Clausen 3 Department of Chemistry, Technical University of Denmark , Kgs. Lyngby, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mads H. Clausen Lari Lehtiö 1 Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu , Oulu, Finland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lari Lehtiö For correspondence: lari.lehtio{at}oulu.fi Abstract Full Text Info/History Metrics Supplementary material Preview PDF Summary In the past, development of tankyrase inhibitors has focused on the ADP-ribosyltransferase domain. Targeting tankyrases ability to interact with protein substrates through their ARC domains represents an alternative strategy to be explored as a therapeutic approach against specific protein-protein interactions. In this paper, we employed a FRET-based assay to identify ARC4-binding compounds by screening the EU-OPENSCREEN Pilot and Commercials Diversity libraries. We discovered an effective series of compounds with the same scaffold and through chemical synthesis we obtained the compound S8 ( ARCher-142 ), which binds selectively to ARC4 with potency of 8 µM. NMR analysis and X-ray crystallography allowed us to identify the binding site in ARC4 and to rationalize the observed selectivity. Despite binding exclusively to ARC4, the inhibitor can attenuate the WNT/β-catenin signaling pathway in cells. Our work demonstrates that targeting single ARC domains is possible, offering an inhibition approach tailored to tankyrase ARC4 inhibition. Significance Tankyrases impact a variety of cellular processes by binding proteins through their ARC domains and the inhibition of these scaffolding functions represents an alternative therapeutic approach to catalytic inhibitors. With a FRET-based high-throughput screening of the EU-OPENSCREEN Pilot and Commercials Diversity libraries we discovered a pyrrolone-based scaffold that is interestingly selective towards ARC4, despite the high conservation of the ARC binding site. Our synthesized compound S8 ( ARCher-142 ) displays an 8 µM potency for TNKS2 ARC4. With NMR and X-ray crystallography we demonstrate that S8 ( ARCher-142 ) competes with the peptide optimized for binding and extends to a unique hydrophobic sub-pocket of ARC4. The compound attenuates the WNT/β-catenin signaling pathway in cells and interestingly offers the possibility to target specific protein-protein interactions mediated by ARC4, paving the way for the development of a pyrrolone-based class of tankyrase scaffolding inhibitors. Introduction Ankyrin repeat (ANK) domains are ubiquitous protein modules designed to mediate protein-protein interactions. They are characterized by an ANK motif (30–34 residues), which is tandemly repeated and which structurally organizes into a helix-loop-helix architecture. 1 , 2 Among the human enzyme family of Diphtheria toxin-like ADP-ribosyltransferases (ARTDs), tankyrases (paralogs TNKS1 and TNKS2) are the only members containing ANK domains, also defined as ankyrin repeat cluster (ARC) domains. Both tankyrases have five ARCs (ARC1/2/3/4/5), each of them characterized by five repetitions of the ANK motif. 3 Once bound to the ARC domains, the protein partners can undergo poly-ADP-ribosylation (PARylation), a reaction that consists in the covalent attachment of ADP-ribose moieties from the substrate β-nicotinamide adenine dinucleotide (NAD + ) into an elongated PAR chain. Tankyrases perform the PARylation reaction through their C-terminal catalytic domain (ART), whose function is possible only upon polymerization of multiple tankyrase molecules through their sterile alpha motif (SAM) domains ( Figure 1 ). 4 – 7 Targets that are subjected to this post-translational modification are subsequently ubiquitylated by E3 ubiquitin-protein ligase RNF146 and directed to proteasomal degradation. 8 – 12 Download figure Open in new tab Figure 1. Domain organization of tankyrases and TNKS2 ARC4 peptide binding pocket. Tankyrases contain a C-terminal catalytic domain (ART), a SAM domain that mediates polymerization and five ARC domains responsible for protein-protein interactions. TNKS1 has an additional N-terminal histidine, proline and serine-rich region (HPS). The crystal structure of TNKS2 ARC4 in complex with the TBM of 3BP2 (PDB: 3TWR 3 ) allowed to identify different regions of the binding pocket: the “arginine cradle” (residues in cyan), the “central patch” (blue), the “aromatic glycine sandwich” (magenta) and residues that offer additional contacts with the peptide (pink). The bound 3BP2 peptide is yellow. Human tankyrase ARCs are highly similar and display a strong conservation throughout evolution that is particularly evident in the pocket for interaction with protein partners. 3 , 13 This pocket can bind proteins provided with a specific 6–8 residue pattern defined as tankyrase-binding motif (TBM). The consensus rules of the TBM have been defined (R-X-X-[small hydrophobic or G]-[D/E/I/P]-G-[no P]-[D/E]) and indicate that arginine at position 1 and glycine at position 6 are essential for the binding to take place. 3 Crystal structures of ARCs in complex with binding TBMs provided insights into the anatomy of the pocket ( Figure 1 ). 3 , 14 – 18 Notably, the mandatory arginine at position 1 of the TBM fits into an “arginine cradle”, while glycine at position 6 is accommodated into an “aromatic glycine sandwich”, which encompasses two tyrosine residues that form a gate crossed by the peptide. The central residues of the binding peptide occupy the “central patch”, and residues of the ARC domain can also provide additional interactions with the terminal residues of the TBM. Interestingly, ARC domains show different aptitudes for protein binding. ARC3 has no binding capacity and this behavior is reflected by its scarce conservation in comparison to other ARCs, 3 , 13 leading to the hypothesis that ARC3 may play a structural role. 3 , 14 , 16 ARC2 and ARC5 display stronger affinity towards TBMs in comparison to ARC1 and ARC4, where ARC1 is the weakest binder. 3 Collectively, the five ARCs act as a multivalent platform that can adapt to binding partners. 16 The ability to interact with and modify a variety of proteins allows tankyrases to orchestrate a vast range of cellular processes. 19 – 21 By binding and PARylating paralogs AXIN1/2, tankyrases modulate the WNT/β-catenin pathway, 22 a cascade of signaling events that affects processes like cell proliferation and differentiation. 23 Deregulation of the WNT/β-catenin pathway contributes to the development of several diseases, 24 encompassing diverse types of cancer 25 and tissue fibrosis. 26 Therefore, the regulating action of tankyrases on WNT/β-catenin signaling events can be potentially exploited as therapeutic strategy to re-tune the pathway 22 , 27 and, in the past years, a wide range of catalytic inhibitors has been developed for this purpose. 28 Inhibition of tankyrases has also proven to suppress YAP oncogenic activity 29 – 31 and to potentiate cell sensitivity to DNA-damaging drugs by targeting MERIT40 PARylation. 32 Tankyrases also regulate telomere length, 33 – 35 apoptosis, 36 mitotic spindle assembly, 37 – 39 vesicular transport 38 , 40 and glucose metabolism. 40 – 43 In the past, research efforts have focused on the discovery of catalytic inhibitors. Although there are reports of dose-limiting intestinal toxicity in mice, 44 – 46 optimized lead shows no substantial toxicity and a clear therapeutic window. 47 The development of scaffolding-targeting compounds can provide new routes to explore tankyrase inhibition, with the possibility to impact distinct biological activities. While inhibitors of SAM-mediated multimerization have not been reported yet, macrocyclized peptides and some ARC inhibitors have already been developed. 48 – 51 In this study, we screened the EU-OPENSCREEN Pilot Library and Commercials Diversity collection of compounds by employing a fluorescence resonance energy transfer (FRET) based assay designed for the discovery of novel compounds targeting scaffolding functions of tankyrases. 52 We aimed at the identification of compounds that target the TBM pocket by inhibiting the interaction between TNKS2 ARC4 and the peptide optimized for ARC binding (REAGDGEE). 3 The same libraries were tested in a counter screen for inhibition of SAM domain dimer formation to identify non-specific compounds, while simultaneously assessing possible hit compounds that would interfere with the SAM-SAM interaction. We identified a common scaffold in the best hits and defined their binding site to TNKS2 ARC4 with NMR titration experiments. Subsequently, analogous compounds were acquired commercially or synthesized and analyzed for the study of structure-activity relationships (SARs). One synthesized analogue S8 displayed increased potency, and we solved its co-crystal structure with TNKS2 ARC4. We reveal that the compound occupies the TBM pocket and it extends towards a region that is unique of TNKS2 ARC4. Profiling of the scaffold with ARC domains of TNKS1 and TNKS2 explained the compound selectivity towards TNKS1/2 ARC4. In addition, S8 is active in cells as it attenuates the WNT/β-catenin signaling pathway. These findings indicate that it is possible to develop inhibitors tailored towards the inhibition of specific ARC-mediated interactions and affecting potentially distinct cellular pathways. Overall, this study provides novel insights into the inhibition of tankyrase scaffolding functions and opens the route to the development of a new class of tankyrase inhibitors. Results Discovery of TNKS2 ARC4 binders through FRET-based high-throughput screening To discover novel compounds targeting the TBM pocket in ARC domains, we performed a high-throughput screening based on a FRET assay designed for the purpose. 52 In the assay, we employed human recombinant TNKS2 ARC4 fused with mCerulean (CFP) and the 8-residues peptide optimized for the binding of ARCs (REAGDGEE) fused with mCitrine (YFP). 3 The interaction is evaluated by calculating the ratiometric FRET (rFRET), which is the ratio of the fluorescent intensity of the acceptor at the emission wavelength to the fluorescent intensity of the donor at emission wavelength. The control indicating total inhibition of the interaction (positive control) is represented by the same FRET pair in presence of 1 M guanidine hydrochloride. At the same time, we employed the FRET assay developed for the discovery of compounds inhibiting SAM-SAM interaction to screen the same libraries and to identify overlapping, hence non-selective, hits. We first tested the compounds of the EU-OPENSCREEN Pilot Library, which encompasses 4928 compounds including bioactive compounds and representative members of the Commercials Diversity library. This pilot screen was used to set a threshold of 20% activity for hit determination. The screening resulted in the identification of 50 primary hits (1% hit rate), active at a concentration of 30 µM, which were later analyzed with DSF assay. As the assay performed well, the larger EU-OPENSCREEN Commercials Diversity collection of compounds was screened. A total of 96076 compounds were screened, identifying 772 primary hits (0.8% hit rate). Out of 772 primary hits, 12 were found to overlap with hits of the counter screen for the SAM-SAM interaction, so they did not undergo further validation. As a result, 760 hits were subjected to validation through potency measurements, where compounds were studied at different concentrations ranging from 0.5 µM to 100 µM. A primary hit was considered validated when the calculated IC 50 value fell in the 0.5–100 µM range and when its activity change was at least 25%. Out of 760 compounds, 114 were validated and were further tested with DSF assay. Complete data of the screening is available at the European Chemical Biology Database (ECBD). 53 , 54 Pyrrolone-based compounds inhibit and are selective towards TNKS1/2 ARC4 We used DSF assay to validate the binding of the primary hits to TNKS2 ARC4. A total of 50 primary hits from the Pilot Library and 114 compounds from the Commercials Diversity Library were tested ( Figure S1, S2 ). Two compounds from the Pilot Library were found to mildly stabilize TNKS2 ARC4 with ΔTm equal to 0.45 °C and 0.41 °C ( Figure S1 ). Validation of primary hits from the Commercials Diversity collection led to the identification of nine molecules that share a common pyrrolone-based scaffold and that remarkably stabilized the protein up to 2.25 °C ( Figure S2 ). Given the notable stabilization of the pyrrolone-based compounds from the Commercials Diversity collection, we decided to focus on their common scaffold for compound optimization. Pyrrolone is indeed a versatile moiety that is largely employed for the design of compounds with a variety of biological effects. 55 The nine primary hits were acquired from commercial suppliers, they underwent quality control, additional DSF validation and determination of potency through FRET-based IC 50 measurements ( Table 1 ). All the tested compounds include a benzodioxane moiety (R 1 ), a phenyl group with substitutions in the para position (R 2 ), and an R 3 group with an aliphatic character and a tertiary amine. All the compounds studied significantly stabilized TNKS2 ARC4, with ΔTm values ranging from 1.40 °C to 4.58 °C. In addition, all molecules, except for 7 , have IC 50 values below 100 µM. Among the nine molecules, 1 performed the best in terms of stabilization of TNKS2 ARC4 and potency. Direct comparison of 1 with 2 indicates that a polar hydrogen bond donor group (-OH) in the para position of the phenyl at R 2 is preferred in comparison to a fluorine substituent. In the same position, the presence of a hydrophobic methyl group ( 3, 4 ), other halogen substituents ( 5 ) and alkoxy groups ( 6 – 9 ) is also tolerated, even if not optimal. Focusing on the R 3 group, all the nine primary hits have a common 3-carbon chain and comparisons between 3 – 4 , and 8 – 9 , show that the morpholine-based moiety performs slightly worse than the dimethylamino-based substituent. View this table: View inline View popup Table 1. The nine primary hit compounds from the Commercials Diversity collection share a common pyrrolone scaffold and profiling of compounds 1 , 4 , 6 against TNKS1/2 ARC domains reveals selectivity of the scaffold towards TNKS1/2 ARC4 domains. FRET-based potency measurements are indicated as IC 50 (μM) and pIC 50 ± SEM, with number of repetitions n=3 unless indicated otherwise (n=1 for measurements with IC 50 >1000 μM). Thermal stabilization (ΔTm ± SD) is also reported (n=4). Profiling of 1 against a panel of ARC domains from TNKS1 and TNKS2 interestingly indicates that the compound inhibits the interaction of the optimized peptide only with TNKS1/2 ARC4 and not with other ARCs ( Table 1 ). Profiling of other primary hits 4 and 6 also shows selectivity towards TNKS1/2 ARC4, confirming the selectivity of the scaffold for ARC4 inhibition ( Table 1 ). Pyrrolone-based compounds bind to a common site in TNKS2 ARC4 binding pocket To identify the binding site of the compounds and the reason behind the observed selectivity, we performed 1 H- 15 N HSQC NMR titration experiments with 15 N-labelled TNKS2 ARC4 and compounds 1 , 4 and 6 . Compounds appear to bind with intermediate exchange rate to a common binding site ( Figure 2 ). This site overlaps with the TBM pocket and intriguingly extends to a sub-pocket with a hydrophobic character ( Figure 3A - B ). The bottom of this sub-pocket is indeed decorated with a leucine and a phenylalanine residue (L497, F532) that may provide the correct hydrophobic environment to accommodate the aliphatic R 3 group of the compounds, while the tertiary amine at R 3 may extend towards other residues that surround the sub-pocket. Notably, residue F532 is characterized by a significantly high chemical shift perturbation (CFP), possibly due to the vicinity of the pyrrolone core, which is likely located at the center of the identified binding area. The alignment of the sequences of ARC domains indicates that phenylalanine at this position is conserved only in TNKS1/2 ARC4 and TNKS1/2 ARC1, where other ARCs contain leucine instead ( Figure 3C ). It is necessary to highlight that ARC1 domains show an overall lower conservation of the residues lining the binding pocket and are weaker peptide binders in comparison to ARC2/4/5. This is mainly due to the presence of phenylalanine residues instead of the two tyrosines at the “aromatic glycine sandwich” present in other ARCs. Since the “aromatic glycine sandwich” and neighboring residues form the binding pocket of the analyzed compounds, the described absence of tyrosines may explain the lack of binding of the compounds to ARC1 domains. Additional differences between TNKS1/2 ARC4 and other ARC domains in the binding pocket of the compounds are provided by the loop lining the sub-pocket (R520 to T528). Specifically, positions occupied by residues I522 and E523 in TNKS2 ARC4 (L673 and E674 in TNKS1 ARC4) vary among other ARCs ( Figure 3C ). Download figure Open in new tab Figure 2. Analysis of NMR titration experiments. CSPs of TNKS2 ARC4 induced by binding of compounds 1 ( A ), 4 ( B ) and 6 ( C ). Each plot is accompanied by the structure of the titrated compound and the crystal structure of TNKS2 ARC4 bound to the TBM of 3BP2 (PDB: 3TWR 3 ). On the surface of the protein, residues significantly perturbed are indicated in orange (2σ < CSP ≤ 1σ) and red (CSP ≥ 2σ). The TBM peptide of 3BP2 is shown (blue) to highlight the overlapping between its binding site and the binding site of the compounds. To identify significant differences in the binding sites of 1 , 4 and 6 , the Euclidean distances between the residues peaks at saturation have been calculated (ΔCSP) and plotted ( D , E , F ). This approach highlights peaks that underwent different changes in the environment, including peaks that covered different distances and peaks that shifted to different locations. Relevant residues are indicated and colored in orange (ΔCSP ≥ 0.1 ppm) and red (ΔCSP ≥ 0.2 ppm). ( D ) Plot indicating ΔCSPs induced by compounds 1 and 4 . ( E ) Plot indicating ΔCSPs induced by compounds 1 and 6 . ( F ) Plot indicating ΔCSPs induced by compounds 4 and 6 . In all plots, residues that could not be assigned (R525, G535, A584) are shown with * (color magenta). Proline residues spots on the plots are left empty. Download figure Open in new tab Figure 3. Identification and characterization of the compounds binding site in TNKS2 ARC4. ( A ) Crystal structure of TNKS2 ARC4 (PDB: 3TWR 3 ) with bound TBM of 3BP2 (blue). The surface of the protein is colored in a gradient from white to red according to the Kyte-Doolittle hydrophobicity scale, where red indicates residues with high hydrophobic score. The hydrophobic pocket lined by L497 and F532 is indicated with a dotted line. ( B ) Crystal structure of TNKS2 ARC4 (PDB: 3TWR 3 ) with residues that were significantly perturbed upon compound titration shown as sticks. Residues that likely form the shared binding site of the compounds, that is residues with CSP ≥ 1 σ in all titrations, are shown in magenta. Residues Q495 and H531 appear to undergo significant environmental changes with compounds 4 and 6 only and are colored orange. Residues that display different behavior with one compound titration only are shown in yellow. Residue E498 has significantly high CSP induced only by 1 , while residue L612 moves to a different location with 1 . Residue K557 appears instead to be perturbed significantly only upon compound 6 titration. Residue L497 is shown in green, and it forms the floor of the hydrophobic pocket (yellow halo) with H532. ( C ) Sequence alignment of TNKS1/2 ARCs. Residues are highlighted with a blue color gradient according to the sequence identity. Residues that appear to be perturbed upon compound binding are indicated with arrows in the same color scheme as in ( B ). TNKS1/2 ARC3 sequences are excluded from the alignment since they are reported not to bind TBMs of protein partners. Residues that were not assigned in NMR titrations are indicated with *. The NMR titration experiments reveal that 1 , 4 and 6 bind TNKS2 ARC4 in the same fashion and only some residues appear to have different roles in the binding sites. Residues with significantly different behavior were identified by calculating the Euclidean distances between the peak positions at compound saturation (ΔCSP) ( Figure 2D - F ). The compounds differ only in their R 2 and R 3 groups and share the core and the benzodioxane moiety (R 1 ). Compounds 4 and 6 share a morpholine-based R 3 group, which provides a heterocyclic ring in comparison to the R 3 group of 1 . All three compounds have a three-carbon aliphatic chain in the R 3 group that can be accommodated in the hydrophobic sub-pocket. Consequently, the presence of the morpholine moiety may explain the involvement of residue Q495 with 4 and 6 , while E498 is affected only by 1 binding, indicating a possible ion-pair interaction with the positively charged tertiary amine. In addition, residue H531 displays significantly higher CSPs in 4 and 6 titrations, while its CSP in titration with 1 appears to be below the calculated average. Analogue 6 has a bulkier methoxy substitution on the para position of the phenyl ring at R 2 and the high CSP of K557 may indicate the substituent pointing in the direction of the residue. Other minor differences in CSPs involve E523 and Q526, which are more perturbed in the presence of 1 . In addition, residue L612 has comparable CSPs in the titrations with the different compounds, but its peak appears to shift to a different location with 1 only. Synthesis of pyrrolone-based analogues To explore the potential of the pyrrolone scaffold, analogues of 1 with different R 1 , R 2 and R 3 substituents ( Table S1 , compounds S1 – S12 ) were synthesized using a two-step procedure, as depicted in Scheme 1 . Aryl methyl ketones 1a – c were treated with a dialkyl oxalate under basic conditions, yielding 2,4-diketoesters 2a – c via a crossed Claisen condensation. 56 Pyrrolone analogues were synthesized using commercially available substituted benzaldehydes, substituted primary amines and 2,4-diketoesters 2a – c , through a one pot three-component reaction, 57 , 58 yielding the desired products in relatively low yields (3–22%). In addition to pyrrolone ring cyclization, an undesired competing side reaction was observed, in which 2,4-diketoesters underwent aminolysis with primary amines. Compound S9 was synthesized by reducing the commercially available para -nitro analogue with zinc dust and ammonium chloride by heating a H₂O/EtOH solution to 80 °C. 1 H NMR spectra of the synthesized analogues show characteristic singlet signals for the CH proton at the 5-position at 5.2–5.4 ppm ( Figure S3 - S31 ). Additionally, the CH 2 protons from the alkyl chain substituent at R 3 , positioned close to the chiral center are diastereotopic and exhibit distinct chemical shifts. Download figure Open in new tab Scheme 1. General synthetic route for the synthesis of pyrrolone analogues. Reagents and conditions: i) dialkyl oxalate, NaH, THF, Δ; ii) R 2 CHO, R 3 NH 2 , THF, EtOH/AcOH or AcOH/NH 4 Ac, rt or Δ. SAR investigation A SAR investigation was conducted with purchased and synthesized analogues of 1 through FRET-based potency measurements and DSF assay with TNKS2 ARC4 ( Table 2 , Table S2 ). All the compounds commercially acquired underwent quality control. Modifications at R 1 indicate that the presence of a double ring system with at least one oxygen is essential for maintaining high inhibitory activity and stabilization of the protein ( 10 IC 50 31 µM, 11 / S6 / S7 IC 50 > 500 µM). Diverse variations of the substituents on the phenyl ring at R 2 appear to be tolerated, including substituents in the meta position and large alkoxy substituents as seen in Table 1 ( 8 IC 50 69 µM, 9 IC 50 81 µM). Notably, the methoxy group in the meta position and the dimethylamino group in the para position moderately improve potency and allow a stabilization of the protein ΔTm > 4 °C ( 12 IC 50 11 µM, 19 IC 50 10 µM). Changes to the length of the aliphatic chain at R 3 greatly impacts both potency and protein stabilization. Comparison of compounds 13 (IC 50 24 µM) with 14 (IC 50 139 µM) and 16 (IC 50 31 µM) with 17 (IC 50 274 µM) indicates indeed that a 3-carbon aliphatic chain is superior to a 2-carbon chain. At the same time, a longer 4-carbon chain has a negative impact on the measured values ( S12 IC 50 74 µM). The diethylamino-based substituent at R 3 ( 13 IC 50 24 µM) has slightly improved potency and a greater TNKS2 ARC4 stabilization in comparison to the dimethylamino-based substituent ( 16 IC 50 31 µM). In addition, at R 3 the piperazine ( S1 IC 50 250 µM), guanidine ( S4 IC 50 142 µM), methoxypropyl ( 15 IC 50 368 µM), morpholine ( 18 IC 50 85 µM, 23 IC 50 411 µM), pyridyl ( 22 IC 50 325 µM) and phenyl ( 25 IC 50 56 µM, 26 IC 50 30 µM) groups decrease potency towards TNKS2 ARC4. On the contrary, compound S8 , characterized by a fully aliphatic R 3 substituent, is the best tested analogue with an IC 50 value of 8 µM and a 1.9-fold improved potency in comparison to 1 . Compounds 25 and 26 further indicate that the presence of tertiary amine at R 3 is not necessary to achieve binding. Profiling of S8 against TNKS1/2 ARCs indicates that the analogue maintains selectivity towards TNKS1/2 ARC4 ( Table 2 ). View this table: View inline View popup Table 2. Structures of 1 and its derivatives for investigation of SARs and profiling of compound S8 against TNKS1/2 ARCs. FRET-based potency measurements are indicated as IC 50 (μM) and pIC 50 ± SEM, with number of repetitions n=3 unless indicated otherwise (n=1 for measurements with IC 50 >1000 μM unless differently indicated). Thermal stabilization (ΔTm ± SD) is indicated (n=4). The full list of all analyzed compounds is available in Table S2 . Crystal structure of S8 in complex with TNKS2 ARC4 reveals its binding mode To study the binding mode of the scaffold, we co-crystallized the compound displaying the best potency ( S8 ) with TNKS2 ARC4 ( Figure 4 ) (PDB: 9QFC). The crystal structure confirms that the compound binds to the TBM pocket and interestingly extends towards the hydrophobic sub-pocket, as indicated by our NMR titration experiments. The hydrophobic sub-pocket accommodates the 4-ethylhexan-1-yl substituent (R 3 ) of the compound. Specifically, R 3 forms hydrophobic contacts with F532 and L497, packing against the nonpolar component of R494, E498 and K501 side chains. The pyrrolone core is located between F532 and Y536 and it forms hydrogen bonds with K501 and Y536 side chains. In this way, the hydroxybenzene substituent (R 2 ) packs against R525 and is directed towards the solvent, with the formation of a hydrogen bond with a water molecule. Below R 2 , the carbonyl group that connects the pyrrolone to the benzodioxane moiety (R 1 ) forms an hydrogen bond with S527. This group also forms a hydrogen bond with a water molecule, which bridges a hydrogen bond interaction with D521. The residue F532 forms a π-π stacking interaction with the R 1 benzene ring and directs R 1 between Y536 and Y569, the tyrosine gate encasing the irreplaceable glycine of a TBM. In this way, the benzodioxane moiety is cozily surrounded by three aromatic ring side chains (F532, Y536, Y569). In addition, one of the oxygens of the dioxane ring could form a weak hydrogen bond with the hydroxyl group of Y536 (average distance 3.3 Å). The placement of the R 1 moiety in the aromatic glycine sandwich explains the ability of the compound to inhibit the binding of the optimized peptide in the FRET assay, since the paramount glycine cannot be placed between the two tyrosine residues in the presence of the compound ( Figure 4C ). Download figure Open in new tab Figure 4. Crystal structure TNKS2 ARC4 in complex with S8. Panels ( A ) and ( B ) show two different perspectives of compound S8 (green) in complex with TNKS2 ARC4 (blue) (PDB 9QFC). The mobile loop that packs against the compound is shown in orange. The sigma-A weighted 2Fo-Fc electron density map is grey and contoured at 1.0 σ. Hydrogen bonds are represented with black dashed lines. ( C ) Superimposed crystal structures of TNKS2 ARC4 in complex with 3BP2 TBM (PDB: 3TWR 3 , protein in color sand and TBM in yellow) and TNKS2 ARC4 in complex with S8 (blue with mobile loop in orange). It is evident that the mobile loop, and especially I522, E523, G524, move to accommodate S8 . The 3BP2 peptide is shown as cartoon and it overlaps with the compound in the binding pocket. The crystal structure confirms the binding mode hypothesized with the our NMR experiments. Particularly evident is the perturbation of the loop formed by residues R520 to T528, against which the compound packs. Structural superimposition of our crystal structure with the crystal structure of TNKS2 ARC4 bound to the TBM of 3BP2 (PDB: 3TWR 3 ) shows indeed that the loop moves to make space for the binding of S8 ( Figure 4C ). The major displacement is observed for I522, whose α-carbon moves 2.3 Å, E523 (α-carbon movement of 2.6 Å) and G524 (α-carbon movement of 2.4 Å). This loop faces directly the R 2 substituent of the compound, and its flexibility may explain the reason behind the tolerance towards different R 2 moieties highlighted by our SAR analysis. S8 affects the WNT/β-catenin signaling pathway in cells To test the effect of S8 on the WNT/β-catenin pathway in cellular context, we employed a luciferase-based WNT/β-catenin reporter assay with the use of SuperTOP-Luciferase/Renilla HEK-293 cells. 59 In the assay, the canonical WNT/β-catenin signaling events are activated by the presence of WNT3a in the cellular medium, leading to the binding of β-catenin to TCF/LEF transcription factors and to transcription of downstream genes of the WNT pathway. At the same time, firefly luciferase expression is increased, because it is under the control of a promoter with 7 TCF binding sites. 60 Incubation of HEK293 cells with S8 results in an attenuation of the WNT/β-catenin pathway with IC 50 value of 45 ± 11 µM (n=3). This is in line with previous studies in which inhibition of ARCs leads to suppression of the WNT/β-catenin pathway. 48 – 50 Discussion In this stufy, we employed a FRET-based high-throughput screening of the EU-OPENSCREEN Pilot and Commercials Diversity libraries (101004 compounds screened in total) to discover a new pyrrolone-based scaffold that specifically targets TNKS2 ARC4. The screening results are disclosed and available for consultation on ECBD. 53 , 54 NMR analysis of the best hit compound 1 and its analogues 4 and 6 revealed a common binding site in TNKS2 ARC4. This binding site overlaps with the binding pocket of TBMs and extends towards a hydrophobic sub-pocket lined by a leucine residue (L497) and a phenylalanine (F532). The co-crystal structure of the analogue S8 with TNKS2 ARC4 reveals that this site is indeed occupied by the aliphatic R 3 substituent. SAR investigation indicates that a 3-carbon chain is ideal to achieve optimal potency, while longer or shorter aliphatic chains are detrimental. The positive charge provided by the tertiary amine present at R 3 of most compounds is beneficial, as it could form an ion-pair interaction with E498, but it is not necessary to achieve high potency. The co-crystal structure also highlights the importance of the benzodioxane moiety (R 1 ), as confirmed by the SAR investigation. Notably, F532 guides the double ring system inside the tyrosine gate (the “aromatic glycine sandwich”) of the peptide binding pocket, inhibiting the interaction of the optimized peptide. The phenylalanine residue at this position is present only in ARC4 and ARC1 domains, where ARC1 domains are devoid of the tyrosine gate and cannot form the hydrogen bond that instead anchors the pyrrolone core to ARC4, as evident from the crystal structure. Several substituents on the benzene ring at R 2 are tolerated, since this moiety packs against a flexible loop, variable across ARCs, and is directed towards the solvent. Compound S8 is the best analogue from our SARs analysis: it maintains selectivity towards TNKS1/2 ARC4 and it displays a potency of 8 μM against TNKS2 ARC4 with optimal protein stabilization in the DSF assay. The compound attenuates the WNT/β-catenin signaling in cells, indicating that the compound can enter the cellular environment and affect the tankyrase-modulated signaling pathway despite only affecting one ARC. At present, tankyrase partners able to bind exclusively to ARC4 have not been discovered, but ARC-selective inhibitors such as S8 can provide the tools to identify specific proteins and relative pathways controlled by different ARCs. The development of ARC inhibitors further allows the investigation of the interplay between tankyrase scaffolding and catalytic functions, and it may offer an alternative therapeutic approach to catalytic inhibition. Materials and Methods Cloning Cloning was performed as previously described. 52 The method employed to clone the expression constructs into vectors was the one-step sequence– and ligation-independent cloning (SLIC). 61 The employed expression vectors (pNIC-CFP, pNIC-YFP, pNIC-MBP) originated from the pNIC28-Bsa4 plasmid (26103, Addgene). Specifically, mCerulean (CFP, 29726, Addgene plasmid), mCitrine (YFP, 29724, Addgene plasmid) and E. coli maltose-binding protein (Uniprot P0AEX9, residues 27-392) sequences were inserted between the 6xHis-tag and the TEV protease site of the pNIC28-Bsa4 plasmid. Purified PCR products of the constructs were mixed with linearized plasmids (100 ng) in 1:3 molar ratio in presence of T4 polymerase (M0203L, New England Biolabs). The mix was then kept at room temperature for 2.5 minutes and incubated on ice for 10 minutes. After incubation, the reactions were transformed into NEB® 5-alpha Competent E. coli (High Efficiency) cells (C2987H, New England BioLabs) according to the high efficiency transformation protocol provided by the manufacturer. 62 , 63 Colonies were grown overnight at 37 °C on LB agar with 50 μg/mL kanamycin (KAN0025, Formedium Ltd.) and 5% sucrose for sacB counter-selection. 64 TNKS2 SAM mutants (Y920A and E897K) were obtained through DpnI mediated site-directed mutagenesis. 52 , 65 The sequences of the inserted regions were verified through sequencing. Protein expression Expression of YFP and CFP fused proteins was performed as described before. 52 Plasmids were transformed into BL21(DE3) Competent E. coli cells (C2527H, New England BioLabs) according to the manufacturer’s instructions. 66 Colonies were grown overnight at 37 °C on LB agar supplemented with kanamycin (50 μg/mL) and 5% sucrose. The day after, single colonies were selected for overnight growth of pre-cultures in Terrific Broth (TB, TRB0102, Formedium Ltd.) media with 50 μg/mL kanamycin. Pre-cultures were then added to TB autoinduction media including trace elements (AIMTB0210, Formedium Ltd) with 8 g/L glycerol and 50 μg/ml kanamycin, or LB media with the same antibiotic (for 15 N TNKS2 ARC4). The cultures were grown at 37 °C in shaking conditions until OD 600 = 1. At this point, the procedure for the expression of the isotopically labelled 15 N TNKS2 ARC4 required additional steps, including washing the cells with M9 medium without labelled nitrogen sources, centrifugation (5000 x g), resuspension of the pellet in M9 medium with 15 N source and incubation to allow recovery of cell growth. 67 Expression of 15 N TNKS2 ARC4 was induced by addition of 1 mM isopropyl β-D-thiogalactopyranoside (IPTG, IPTG025, Formedium Ltd.). After cell growth, the temperature of the cell cultures was set to 18 °C (17 °C for 15 N TNKS2 ARC4) for overnight incubation. The morning after, cells were harvested by centrifugation at 4200 × g for 30 minutes at 4°C, and they were resuspended in lysis buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 10% volume/volume glycerol, 0.5 mM TCEP) in the presence of proteinase k inhibitor (Pefabloc® SC, 11585916001, Roche) and DNase I (10104159001, Roche). Resuspended cells were frozen in liquid nitrogen and stored at –20 °C until purification. Protein Purification Purification of YFP and CFP fused proteins was carried out as previously described. 52 Cells were thawed, lysed by sonication, centrifugated at 16000 x g for 30 minutes and filtered through a 0.45 µm filter. All the proteins underwent a first step of purification through immobilized metal affinity chromatography (IMAC) on Ni 2+ loaded HiTrap Chelating HP column (17040901, Cytiva). MBP-tagged TNKS2 ARC4 was purified with an additional step of MBP-based affinity chromatography by using MBP-Trap HP column (28918779, Cytiva). TNKS2 ARC4 and 15 N TNKS2 ARC4 (MBP-tagged) were dialyzed against size exclusion chromatography (SEC) buffer (30 mM HEPES pH 7.5, 350 mM NaCl, 10% volume/volume glycerol, 0.5 mM TCEP) in presence of 6xHis-tagged TEV protease (1:30 molar ratio, 20 hours, 4 °C) to cleave MBP. TNKS2 ARC4 and 15 N TNKS2 ARC4 were also purified through reverse-MBP chromatography and reverse-IMAC to remove TEV and MBP contaminants. All proteins were further purified through SEC using a HiLoad 16/600 Superdex 75 pg (28989333, Cytiva) and SEC buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 0.5 mM TCEP for TNKS2 ARC4 purification). Fractions containing pure 15 N TNKS2 ARC4 were pooled together, concentrated and buffer exchanged to the final buffer (20 mM NaPhosphate pH 7.0, 50 mM NaCl). All proteins were aliquoted, frozen in liquid nitrogen and stored at –70 °C. Mass spectrometry analysis of 15 N TNKS2 ARC4 indicated a labelling efficiency of 92.6%. FRET-based high-throughput screening and dose response validation The FRET assay for discovery of chemical probes targeting ARC and SAM domains of tankyrases was developed in a previous study. 52 The high-throughput screening of the EU-OPENSCREEN Pilot and Commercials Diversity libraries was performed at the Norwegian Centre for Molecular Biosciences and Medicine (NCMBM, formerly NCMM). For the primary screen, assay-ready plates containing library compound sufficient to reach a screening concentration of 30 µM were prepared on black shallow-well 384-microwell plates (Greiner bio-one small volume, high base, non-binding microplate black, 784900 or Fisherbrand™ 384-Well ShallowWell Polypropylene Microplates, 12-566-617, Fisher Scientific) using 30 nL of 10 mM DMSO compound stocks dispensed with an Echo 550 liquid handler (Labcyte). Each screening plate also contained DMSO controls at equivalent volumes to compounds for positive, negative, and assay buffer blank controls, 16 wells per control type. Stock solutions of CFP-TNKS2 ARC4 and YFP-REAGDGEE or CFP-SAM(E897K) and YFP-SAM(Y920A) were combined and diluted in assay buffer (10 mM BTP pH 7, 3% weight/volume PEG 20000, 0.01% volume/volume Triton, 0.5 mM TCEP) to final concentrations of 100 nM and 200 nM or 150 and 300 nM, respectively. The positive control was prepared by addition of guanidine hydrochloride to the above assay buffer to a final concentration of 1M. A volume of 10 µL of these solutions was added to each well of the assay-ready plates using a BioTek MultiFlo FX dispenser (Agilent) equipped with 5 uL dispensing cassettes for the peristaltic pumps (screening solution and control) and a syringe pump (assay buffer blank). After liquid handling, plates were incubated at RT for 30 minutes before reading fluorescence using a BioTek Synergy Neo2 plate reader (Agilent) with Gen5 software (version 3.02) at four wavelength combinations – 410/477, 410/527, 430/477, 430/527 – each with an excitation bandwidth of 20 nm and an emission bandwidth of 10 nm; a gain setting of 135; a “Normal” read speed with 10 msec delay after plate movement, 50 measurements per data point, low lamp energy, and a “Standard” dynamic range (0 to 99,999); and a read height of 4.5 mm. Analysis proceeded using a custom workflow created in KNIME version 4.3.4. All plates had to pass a quality filter of Z’ ≥ 0.5 and SSMD ≤ –5. These quality metrics were calculated using the means and standard deviations of the control groups on each plate, as follows: Z’=1-(3×(σ_Positive+σ_Negative))/(|μ_Positive – μ_Negative|), SSMD=(μ_Positive – μ_Negative)/√(〖 σ_Positive〗^2+〖σ_Negative〗^2). The buffer blank was subtracted from each emission measurement and the rFRET was calculated by dividing the fluorescence intensity at 527 nm by the fluorescence intensity at 477 nm for both excitation at 410 nm and 430 nm to give r410 and r430, respectively. These values were then normalized on a plate-wise basis, setting the response of the positive control (screening solution with 1M guanidine hydrochloride and DMSO) to 100% and the response of the negative control (screening solution with DMSO) to 0%. Both donor emission and the r410 value were used to filter fluorescently interfering compounds. Any compounds with donor emission falling outside of 150-50% of the mean of donor emission for the controls on the same plate were flagged as interfering. Any compounds where r410 had a percent difference from r430 greater than 50%, calculated as 100%*(|r430-r410|/0.5*(r430+r410)), were also flagged as interfering. In the early screen this filter was originally a percentage point difference between normalized r430 and r410, excluding compounds where |r410%-r430%| > 50% – changing the filter led to a small amount of previously excluded compounds being reclassified as hits, but these were not rescreened. For the dose response validation, assay-ready plates containing library compound sufficient to reach concentrations of 0.5, 1.25, 2.5, 5, 10, 25, 50, and 100 µM were prepared on black shallow-well 384-microwell plates (Greiner bio-one small volume, high base, non-binding microplate black, 784900 or Fisherbrand™ 384-Well ShallowWell Polypropylene Microplates, 12-566-617, Fisher Scientific) using 10 mM DMSO compound stocks dispensed with an Echo 550 (Labcyte). Each compound was tested at these concentrations in duplicate. Compound-containing wells were backfilled with DMSO to a total volume of 100 nL, and on each plate 16 wells for each of the positive, negative, and assay blank controls also received 100 nL DMSO. Screening solutions were added to the assay ready plates and fluorescence read as detailed above for the primary screen. Dose response data were filtered to remove compounds displaying fluorescence interference and normalized as detailed above. Compounds that had at least four doses which passed the fluorescence filtering were further analyzed for dose response. Within KNIME, we used a custom R script calling the function drm from the package ‘drc’ with fct=LL.4 to fit the normalized data to a four-parameter log-logistic curve. This function returned the curve upper limit, lower limit, slope and IC 50 as parameters. From these parameters and the curve function, derived responses at the maximum and minimum doses used in the curve fit were used to calculate an activity change by subtraction of the latter from the former. For each compound, if the activity change was ≥ 25% and if the IC 50 value fell within the tested concentrations that passed the interference filters, then that compound was considered a validated hit. FRET-based assay IC 50 measurements To measure the potency of compounds against ARC inhibitors, CFP-TNKS1/2 ARCs (100 nM) and YFP-REAGDGEE (200 nM) were mixed with different concentrations of the compounds (100 µM to 3 nM) in FRET buffer (10 mM Bis-Tris Propane pH 7, 3% weight/volume PEG 20000, 0.01% volume/volume Triton X-100, 0.5 mM TCEP). The control for absence of interaction was represented by the FRET pair in absence of compound, while the control for total inhibition was the FRET pair in FRET Inhibiting buffer (10 mM Bis-Tris Propane pH 7, 3% weight/volume PEG 20000, 0.01% volume/volume Triton X-100, 0.5 mM TCEP, 1 M guanidine hydrochloride). The controls were set to be 2 log units apart from the lowest and highest concentrations of compounds tested, respectively. Compounds were dispensed using Echo 650T liquid handler (Labcyte), while protein mixtures and buffer were dispensed using a Formulatrix Mantis liquid dispenser. Each condition was studied with at least four replicates in 384-well plates (Fisherbrand™ 384-Well ShallowWell Polypropylene Microplates, 12-566-617, Fisher Scientific) with a final volume of 10 µl per well. Measurements were carried out by employing 430 nm excitation wavelength and emission wavelengths of 477 nm and 527 nm. Fluorescence readings were performed with the use of a Tecan Spark multimode microplate reader. The blank was subtracted from each emission measurement and the rFRET was calculated by dividing the fluorescence intensity at 527 nm by the fluorescence intensity at 477 nm. Potency values were calculated with GraphPad Prism 10 using a nonlinear regression analysis (sigmoidal dose–response fitting with variable slope). Differential scanning fluorimetry assay This assay was performed by incubating 5 μM TNKS2 ARC4 with 100 μM of compound in buffer (10 mM Bis-Tris-Propane pH 7.0, 3% weight/volume PEG 20000, 0.01% volume/volume Triton X-100, 0.5 mM TCEP) with the presence 5X SYPRO™ Orange dye (S6650, Invitrogen). Three controls were employed: TNKS2 ARC4 with REAGDGEE peptide (100 μM) as positive control; TNKS2 ARC4 with 1% DMSO, which is the same amount of DMSO present when incubating the protein with 100 μM compound (stored in DMSO); TNKS2 ARC4, to measure the melting temperature of the protein only. Each condition was studied in four replicates of 20 μL and measured in a sealed Multiplate™ 96-Well PCR plate (MLL9601, Bio-Rad) with the use of a CFX96 Real-Time PCR Detection System (Bio-Rad). During the experiment, the temperature was gradually raised from 20°C to 95°C (1°C increase per minute) with measurements performed at intervals of 1 minute. After the experiment, data were normalized and analyzed with GraphPad Prism 10 by using a nonlinear regression analysis (Boltzmann sigmoid equation). NMR titration experiments and analysis of NMR data All NMR data for the human TNKS2 ARC4 domain were measured using the Bruker Avance III HD NMR spectrometer, operating at 850 MHz of 1 H frequency and equipped with a 5 mm 1 H/ 13 C/ 15 N triple-resonance TCI cryoprobe. Mapping of the inhibitor binding site on the TNKS2 ARC4 domain was carried out by measuring two-dimensional 1 H, 15 N correlation spectra ( 15 N-HSQC) of 0.2 mM 15 N-labeled TNKS2 ARC4 in 20 mM sodium phosphate buffer pH 7, 5% DMSO, 50 mM NaCl without inhibitor and in the presence 0.25, 0.5, 1, 2, 4, 8 and 16 molar excess of unlabelled compounds 4 and 6 . For compound 1 , the inhibitor: protein ratios of 1:4, 1:2, 1:1, 2:1, 4:1 and 12:1 were used. Given that ligand stocks were dissolved in DMSO, the titration series were prepared in such a way that at each titration point the NMR sample contained 0.2 mM ARC domain and the desired molar ratio of inhibitor in 5% DMSO to eliminate chemical shift perturbations stemming from DMSO-buffer mixture. The 15 N-HSQC data were collected at 20 °C using 128 and 1024 complex points in 15 N (F 1 ) and 1 H (F 2 ) dimensions, respectively. This corresponds to acquisition time of 48 and 75 ms in F 1 and F 2 dimensions, respectively. Spectra were collected using 4-64 scans per FID with the repetition rate of 1.1 seconds. Resolution in F 1 dimension was improved by linear prediction and zero-filled 4x prior to Fourier transform. Spectra were processed using Topspin 3.5 pl7 software package (Bruker). NMR data were analyzed with CcpNmr Analysis Version 3 software. 68 Full backbone and partial side chain assignment of the human protein TNKS2 ARC4 has been previously reported by Zaleska et al. 69 (BMRB Entry 27747). This assignment allowed us to identify backbone peaks, which were assigned manually in all spectra of the titration experiments. Chemical shift perturbations were calculated for each residue backbone signals as Euclidean distances, with weighting of 15 N signals by a factor of 0.14, between the positions at compound saturation and in the control spectrum (DMSO only). Residues that displayed CSPs higher than 1σ or 2σ (σ being the standard deviation for all residues CSPs) were relevant for the determination of the compounds binding sites. 70 To identify differences between the binding sites of the compounds, Euclidean distances between the residues peaks at saturation (ΔCSP) were calculated with weighting of 15 N signals by a factor of 0.14. ΔCSP values higher than 0.1 or 2 ppm were considered important for differential binding of the compounds. Co-crystallization, data collection, processing and refinement TNKS2 ARC4 domain (residues 487-649, 8 mg/mL) was incubated with 2 mM S8 in ice for 1 hour. Protein crystals were grown at 4°C by mixing the protein and compound mixture in a 3:1 ratio (protein: precipitant solution) with 0.1 M phosphate/citrate buffer pH 4.2, 40% volume/volume PEG 300. The crystallization was carried out with the sitting-drop vapor diffusion method in SWISSCI 96-well 3-Lens crystallization plate. Long needles of protein crystals were fully grown after five days. The crystals were cryo-protected by soaking into precipitant solution added with 20% volume/volume glycerol and then frozen in liquid nitrogen. Data were collected at Diamond (beamline I24). Diffraction data were processed using XDS package. 71 Molecular replacement was performed with PHASER 72 by using TNKS2 ARC4 structure (PDB: 3TWR 3 ) as model. Protein model building was carried out with Coot, while REFMAC5 and Phenix were employed for refinement of the crystal structure. 73 – 75 In the crystal structure, each copy of TNKS2 ARC4 is bound to two S8 molecules, one in the pocket for binding TBMs ( S enantiomer) and one at crystal contacts ( R enantiomer). Data collection and refinement statistics for the crystal structure is provided in Table S3 . Cell Culture The human cell line HEK-293 (CRL-1573, from embryo’s kidney) was obtained from the American Type Culture Collection (ATCC). HEK 293 cells were cultured in high-glucose DMEM (Dulbecco′s Modified Eagle′s Medium, D6429, Sigma-Aldrich). The medium contained 10% Fetal Bovine Serum (FBS, 10270106, Gibco) and 1% Penicillin-Streptomycin (P4333, Sigma-Aldrich). Cells were cultured at 37 °C in a humidified cell incubator with 5% CO 2 . The cell culture was kept below 20 passages (∼10 weeks) and routinely monitored (upon thawing and monthly) for Mycoplasma infections with MycoAlert® Mycoplasma Detection Kit (Lonza). The cell line was authenticated by short tandem repeat profiling to confirm identity (Eurofins). Transfection of HEK-293 cells A stable HEK-293 cell line containing the SuperTOP-Flash plasmid (ST-Luc HEK-293) provided with a 7×TCF binding sites promoter was kindly provided by Dr. Vladimir Korinek. To generate SuperTOP-Luciferase/Renilla HEK-293 cells (ST-Luc/Ren HEK-293), the pRL-TK (Renilla, E2241, Promega) cassette was subcloned into pPUR (Promega), creating the pRL-TK-puro construct. The linearized pRL-TK-puro was transfected into ST-Luc HEK-293 cells using FuGENE® 6 Transfection Reagent (E2691, Promega) before selection with 2.5 μg/mL Puromycin (P9620, Sigma-Aldrich). Wnt3a-Conditioned Medium (Wnt3a-CM) The Wnt3a-expressing cells were purchased from ATCC (male mouse, CRL-2647) and maintained according to the supplier’s recommendations, under the same conditions as HEK-293 cells. Wnt3a-CM was collected following ATCC’s instructions. 76 WNT/β-catenin signaling pathway reporter cell assay A total of 40000 ST-Luc/Renilla HEK-293 cells were seeded in 96-well plates coated with poly-L-lysine (sc-286689, Santa Cruz Biotechnology). The day after, the cells were incubated for a 24 hours treatment with increasing concentrations of S8 compound in 50% Wnt3a-CM. After compound exposure, the cells were lysed, and firefly luciferase and Renilla activities were measured using a GloMax® Multi Detection System (E7031, Promega) with the Dual-Glo Luciferase Assay System (E1980, Promega). Chemistry: General methods Commercially available reagents were used without further purification and all solvents were of HPLC grade quality. Anhydrous THF and DMF were obtained using PureSolve MD-7 solvent purification system. All reactions were monitored by thin-layer chromatography (TLC) using Merck aluminum sheets precoated with 0.25 mm silica gel, 60 F 254 plates and/or by reversed-phase ultra-high performance liquid chromatography mass spectrometry (RP-UPLC-MS). Analytical RP-UPLC-MS (ESI) analysis was performed on a S2 Waters AQUITY RP-UPLC system equipped with a diode array detector using an Thermo Accucore C18 column (d 2.6 μm, 2.1 x 50 mm; column temp: 50 °C; flow: 1.0 mL/min). Eluents A (H 2 O + 0.1% formic acid) and B (ACN + 0.1% formic acid) were used in a linear gradient (5% B to 100% B) in 2.4 min and then held for 0.1 min at 100% B (total run time: 2.6 min). The LC system was coupled to a SQD mass spectrometer. Flash column chromatography was performed using Merck Geduran® Si 60 (40–63 µm) silica gel. All new compounds were characterized by 1 H NMR, 13 C NMR, HRMS (ESI) and melting point. Preparative HPLC analyses were performed using a Shimadzu LC-20 HPLC system comprised of an LC-20AP pump, an SPD-M20A PDA array detector and a SIL-10AP autosampler (injection volume: 3500 µL). Chromatographic separations were achieved on a Shimadzu column (C18, 10 µm, 20 x 250 mm) placed in a CTO-20AC column oven. Eluents A (H 2 O with 0.1% formic acid) and B (acetonitrile with 0.1% formic acid) were used in a linear gradient (5% B to 60% B) in 23 min, up to 100% B in 2 min, held until 27 min and then column was re-equilibrated at 5% B for 3 min (total run time: 30 min), at a flow rate of 20 mL/min. NMR spectra were acquired at 298 K using either a 400 MHz Bruker AVANCE III HD spectrometer equipped with a Prodigy CryoProbe, a 600 MHz Bruker AVANCE III spectrometer equipped with a Bruker BBFO SmartProbe or an 800 MHz Bruker AVANCE III HD spectrometer equipped with a TCI CryoProbe; chemical shifts (δ) are reported in parts per million (ppm) relative to tetramethylsilane (TMS) as internal standard and coupling constants ( J ) are expressed in hertz (Hz). Melting points were obtained using a Stuart SMP30 melting point apparatus and are uncorrected. HRMS was performed using an Agilent Infinity II 1290 UHPLC system (Agilent Technologies, Santa Clara, CA, USA) for injection directly into the MS. About 90% of the flow was diverted off using a static splitter. MS detection was performed in positive mode on an Agilent 6545 QTOF MS equipped with Agilent Dual Jet Stream electrospray ion source with a drying gas temperature of 200 °C, gas flow of 8 L/min, nebulizer pressure of 35 psi, sheath gas temperature of 200 °C, sheath gas flow of 11 L/min, a fragmentor value of 175 V, skimmer voltage of 65 V and octapole 1 frequency of 750 Vpp. The mass spectra were acquired in the 100–3000 m/z mass range at a rate of 1 spectrum per second, collecting 5890 transients per spectrum. Constant reference mass correction was applied using all the masses defined in the instrument supplier-provided solution (ESI-L Low Concentration Tuning Mix from Agilent Technologies, prod. no. G1969-85000) delivered from the instrument’s reference mass bottle directly to the secondary nebulizer of the ion source. The data was analyzed using the qualitative analysis of Mass Hunter B.07.00. Purity of compounds as determined by HPLC-MS, 1 H and 13 C NMR ( Figure S3 - S32 ) was higher than 95%. General procedure for the synthesis of 2,4-diketoesters 2a–c Download figure Open in new tab 2,4-diketo esters 2a–c were synthesized via a Claisen condensation reaction between an aryl methyl ketone and a dialkyl oxalate, by modifying a previously published method. 56 Aryl ketone (5.6 mmol, 1.0 equiv.) and dialkyl oxalate (9.5 mmol, 1.7 equiv.) were dissolved in 3 mL of anhydrous THF. This solution was added dropwise to a stirred suspension of 60% sodium hydride (11.2 mmol, 2.0 equiv.) in 6 mL of anhydrous THF at 0 °C. Upon completion of the addition, the ice bath was removed, and the reaction mixture was heated to reflux for 3 h. THF was then removed under reduced pressure and 10 mL of iced water, 6 mL of EtOAc and 560 µL of conc. H 2 SO 4 were added to the residue. The mixture was stirred and transferred to a separatory funnel. Organic and aqueous phases were separated, and the aqueous layer was extracted with EtOAc (3 x 20 mL). The combined organic phases were washed with brine, dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. 2,4-diketo esters were purified by flash column chromatography (gradient elution, 20–70% EtOAc / n -heptane) yielding the pure intermediates. 1 H and 13 C NMR spectra were in good agreement with the experimental data reported in the literature. 77 – 79 General procedure for the synthesis of pyrrolones Method A: Adapted from a previously published method 57 . To a stirred solution of the appropriate aldehyde (0.323 mmol, 1.0 equiv.) in dry THF (1 mL) under N 2 , 3-(piperazin-1-yl)propan-1-amine (0.323 mmol, 1.0 equiv.) was added dropwise. After 15 min, a solution of the 2,4-diketo ester 2a (0.323 mmol, 1.0 equiv.) in THF (2 mL) was added and the mixture was stirred overnight at room temperature. The solvent was removed under vacuum and the resulting crude product was purified by preparative RP-HPLC. Method B: Adapted from a previously published method 58 . To a solution of anhydrous sodium acetate (0.288 mmol, 1.0 equiv.) in glacial acetic acid (2 mL) were added the 2,4-diketo ester 2a (0.288 mmol, 1.0 equiv.), the appropriate aldehyde and N -(3-aminopropyl)guanidine hydrochloride (0.288 mmol, 1.0 equiv.). The mixture was heated to reflux to ensure complete dissolution of the starting materials and then, it was allowed to cool down to room temperature and stirred overnight. Toluene was added to the mixture and the solvent was removed under reduced pressure. The resulting crude product was purified by preparative RP-HPLC. Method C: To a stirred solution of the appropriate aldehyde (0.390 mmol, 1.0 equiv.) in EtOH (2 mL) and catalytic glacial acetic acid (≈ 5 drops), the appropriate amine (0.390 mmol, 1.0 equiv.) was added dropwise. After 10 min, a suspension of the 2,4-diketo ester (0.390 mmol, 1.0 equiv.) in EtOH (2 mL) was added. The mixture was heated to reflux to ensure complete dissolution of the starting materials and then, it was allowed to cool down to room temperature and stirred overnight. Then, toluene was added to the mixture and the solvent was removed under vacuum. The resulting crude product was purified by preparative RP-HPLC. Compound S1 Download figure Open in new tab Method A was followed using 3-methoxybenzaldehyde, 3-(piperazin-1-yl)propan-1-amine and 2,4-diketo ester 2a . The crude product was purified by preparative RP-HPLC (H 2 O + 0.1% HCOOH / ACN + 0.1% HCOOH) using linear gradient elution (5–60% ACN (0.1% HCOOH), to afford S1 as a yellow solid (Yield: 18%); m.p.: 139–141 °C; 1 H NMR (800 MHz, (CD 3 ) 2 SO) δ 8.19 (s, 1H), 7.50 (d, J = 2.0 Hz, 1H), 7.35 (dd, J = 8.3, 2.0 Hz, 1H), 7.16 (t, J = 7.9 Hz, 1H), 6.84–6.89 (m, 2H), 6.77–6.73 (m, 1H), 6.72 (d, J = 8.4 Hz, 1H), 5.17 (s, 1H), 4.27–4.16 (m, 4H), 3.69 (s, 3H), 3.54–3.47 (m, 1H), 3.00 (bs, 4H), 2.73–2.63 (m, 1H), 2.42 (bs, 4H), 2.25–2.20 (m, 1H), 2.20–2.16 (m, 1H), 1.58–1.52 (m, 1H), 1.50–1.44 (m, 1H); 13 C NMR (200 MHz, (CD 3 ) 2 SO) δ 183.28, 170.33, 165.34, 163.54, 158.89, 144.82, 143.50, 141.77, 134.45, 128.82, 122.34, 119.69, 118.31, 115.23, 113.68, 111.75, 64.26, 63.87, 60.73, 55.00, 54.87, 49.55, 43.03, 38.19, 24.45; HRMS (ESI): m/z [M + H] + calcd for C 27 H 32 N 3 O 6 : 494.2286; found: 494.2285. Compound S2 Download figure Open in new tab Compound S2 was synthesized following Method A using 3-hydroxybenzaldehyde, 3-(piperazin-1-yl)propan-1-amine and 2,4-diketo ester 2a . The crude product was purified by preparative RP-HPLC (H 2 O + 0.1% HCOOH / ACN + 0.1% HCOOH) using linear gradient elution (5–60% ACN (0.1% HCOOH), to afford S2 as a yellow solid (Yield: 11%); m.p.: 195–197 °C; 1 H NMR (600 MHz, (CD 3 ) 2 SO) δ 8.18 (s, 1H), 7.44 (dd, J = 15.4, 2.0 Hz, 1H), 7.32 (td, J = 8.3, 2.0 Hz, 1H), 7.07–7.02 (m, 1H), 6.78–6.73 (m, 1H), 6.69 (d, J = 7.5 Hz, 1H), 6.65 (s, 1H), 6.57 (dd, J = 7.9, 2.6 Hz, 1H), 5.17 (s, 1H), 4.24–4.16 (m, 4H), 3.54–3.50 (m, 1H), 3.00 (bs, 4H), 2.70–2.62 (m, 1H), 2.44 (bs, 4H), 2.29–2.18 (m, 2H), 1.68–1.53 (m, 1H), 1.53–1.42 (m, 1H); 13 C NMR (150 MHz, (CD 3 ) 2 SO) δ 184.31, 169.56, 163.56, 160.77, 157.16, 145.42, 142.16, 142.00, 133.85, 128.89, 122.50, 118.55, 118.45, 115.59, 114.28, 114.20, 64.38, 63.94, 60.87, 54.90, 49.34, 42.98, 38.28, 24.46; HRMS (ESI): m/z [M + H] + calcd for C 26 H 30 N 3 O 6 : 480.2129; found: 480.2128. Compound S3 Download figure Open in new tab Compound S3 was synthesized following Method B using benzaldehyde, N-(3-aminopropyl)guanidine hydrochloride and 2,4-diketo ester 2a . The crude product was purified by preparative RP-HPLC (H 2 O + 0.1% HCOOH / ACN + 0.1% HCOOH) using linear gradient elution (5–60% ACN (0.1% HCOOH), to afford S3 as a pale yellow solid (Yield: 10%); m.p.: 237–239 °C; 1 H NMR (800 MHz, (CD 3 ) 2 SO) δ 7.94 (s, 1H), 7.51–7.15 (m, 7H), 6.69 (d, J = 8.4 Hz, 1H), 5.20 (s, 1H), 4.30–4.12 (m, 4H), 3.55–3.44 (m, 1H), 3.04–2.99 (m, 2H), 2.62–2.56 (m, 1H), 1.67–1.51 (m, 2H); 13 C NMR (200 MHz, (CD 3 ) 2 SO) δ 182.94, 170.93, 165.94, 163.53, 156.80, 144.66, 141.72, 134.62, 127.78, 127.53, 126.75, 122.32, 118.19, 115.13, 111.59, 64.23, 63.86, 60.91, 38.34, 37.58, 27.01; HRMS (ESI): m/z [M + H] + calcd for C 23 H 25 N 4 O 5 : 437.1819; found: 437.1827. Compound S4 Download figure Open in new tab Compound S4 was synthesized following Method B using 3-methoxybenzaldehyde, N-(3-aminopropyl)guanidine hydrochloride and 2,4-diketo ester 2a . The crude product was purified by preparative RP-HPLC (H 2 O + 0.1% HCOOH / ACN + 0.1% HCOOH) using linear gradient elution (5–60% ACN (0.1% HCOOH), to afford S4 as a pale orange solid (Yield: 11%); m.p.: 197–199 °C; 1 H NMR (400 MHz, (CD 3 OD) δ 8.25 (s, 2H), 7.32–7.27 (m, 2H), 7.21 (t, J = 7.9 Hz, 1H), 6.89–6.84 (m, 2H), 6.82–6.76 (m, 2H), 5.40 (s, 1H), 4.30–4.18 (m, 4H), 3.74 (s, 3H), 3.66–3.57 (m, 1H), 3.18–3.02 (m, 2H), 3.00–2.91 (m, 1H), 1.74–1.61 (m, 2H); 13 C NMR (100 MHz, (CD 3 OD) δ 189.63, 170.40, 166.62, 161.37, 158.59, 148.61, 144.31, 140.60, 133.78, 130.75, 124.12, 120.97, 119.54, 118.11, 117.53, 114.67, 114.62, 65.94, 65.42, 63.26, 55.69, 39.82, 39.39, 28.44; HRMS (ESI): m/z [M + H] + calcd for C 24 H 27 N 4 O 6 : 467.1925; found: 467.1933. Compound S5 Download figure Open in new tab Compound S5 was synthesized following Method B using 3-hydroxybenzaldehyde, N -(3-aminopropyl)guanidine hydrochloride and 2,4-diketo ester 2a . The crude product was purified by preparative RP-HPLC (H 2 O + 0.1% HCOOH / ACN + 0.1% HCOOH) using linear gradient elution (5–60% ACN (0.1% HCOOH), to afford SCALL0122 as a pale orange solid (Yield: 17%); m.p.: 187-189 °C; 1 H NMR (400 MHz, CD 3 OD) δ 8.27 (s, 2H), 7.31–7.29 (m, 2H), 7.11 (t, J = 7.8 Hz, 1H), 6.81–6.77 (m, 2H), 6.73–6.64 (m, 2H), 5.37 (s, 1H), 4.30–4.20 (m, 4H), 3.63–3.59 (m, 1H), 3.13–3.08 (m, 2H), 3.00–2.96 (m, 1H), 1.69–1.65 (m, 2H); 13 C NMR (100 MHz, CD 3 OD) δ 189.65, 170.85, 167.44, 167.42, 158.82, 158.59, 148.40, 144.25, 140.92, 134.06, 130.62, 124.08, 120.23, 119.57, 117.44, 116.20, 115.37, 65.93, 65.42, 63.18, 39.83, 39.34, 28.43; HRMS (ESI): m/z [M + H] + calcd for C 23 H 25 N 4 O 6 : 453.1769; found: 453.1778. Compound S6 Download figure Open in new tab Compound S6 was synthesized following Method C using 4-hydroxybenzaldehyde, N,N- diethyl-3-diaminopropane and 2,4-diketo ester 2b . The crude product was purified by preparative RP-HPLC (H 2 O + 0.1% HCOOH / ACN + 0.1% HCOOH) using linear gradient elution (5–60% ACN (0.1% HCOOH), to afford S6 as a pale yellow solid (Yield: 22%); m.p.: 213–214 °C; 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 9.26 (s, 1H), 8.40 (s, 1H), 7.90 (dd, J = 18.2, 7.7 Hz, 2H), 7.77 (q, J = 8.4 Hz, 2H), 7.58–7.41 (m, 2H), 7.14 (d, J = 8.0 Hz, 2H), 6.68 (d, J = 7.9 Hz, 2H), 5.23 (s, 1H), 3.46–3.39 (m, 1H), 3.01–2.89 (m, 4H), 2.87–2.75 (m, 3H), 1.84–1.58 (m, 2H), 1.05 (t, J = 7.3 Hz, 6H); 13 C NMR (100 MHz, (CD 3 ) 2 SO) δ 185.08, 179.74, 169.84, 156.53, 138.42, 133.79, 132.04, 130.31, 128.89, 128.82, 128.65, 127.35, 126.63, 126.60, 126.27, 126.05, 125.72, 114.81, 60.52, 49.04, 45.84, 37.81, 21.78, 8.70; HRMS (ESI): m/z [M + H] + calcd for C 28 H 31 N 2 O 4 : 459.2278; found: 459.2285. Compound S7 Download figure Open in new tab Compound S7 was synthesized following Method C using 4-hydroxybenzaldehyde, N,N- diethyl-3-diaminopropane and 2,4-diketo ester 2c . The crude product was purified by preparative RP-HPLC (H 2 O + 0.1% HCOOH / ACN + 0.1% HCOOH) using linear gradient elution (5–60% ACN (0.1% HCOOH), to afford S7 as a pale yellow solid (Yield: 12%); m.p.: 243–245 °C; 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 9.24 (s, 1H), 7.59 (bs, 2H), 7.06 (d, J = 8.0 Hz, 2H), 6.86 (d, J = 8.7 Hz, 1H), 6.65 (d, J = 8.0 Hz, 2H), 5.17 (s, 1H), 3.76 (s, 1H), 3.73 (s, 1H), 3.47–3.43 (m, 1H), 2.91 (q, J = 7.3 Hz, 4H), 2.79–2.70 (m, 3H), 1.69–1.64 (m, 2H), 1.09 (t, J = 7.1 Hz, 6H); 13 C NMR (100 MHz, (CD 3 ) 2 SO) δ 184.25, 169.75, 156.47, 150.65, 147.27, 133.15, 130.84, 128.62, 122.83, 114.74, 112.71, 109.90, 60.69, 56.02, 55.48, 55.35, 49.05, 45.83, 37.83, 22.09, 18.57, 9.00; HRMS (ESI): m/z [M + H] + calcd for C 26 H 33 N 2 O 6 : 469.2333; found: 469.2342. Compound S8 Download figure Open in new tab Compound S8 was synthesized following Method C using 4-hydroxybenzaldehyde, 4-ethylhexan-1-amine and 2,4-diketo ester 2a . The crude product was purified by preparative RP-HPLC (H 2 O + 0.1% HCOOH / ACN + 0.1% HCOOH) using linear gradient elution (5–60% ACN (0.1% HCOOH), to afford S8 as a beige solid (Yield: 3%); m.p.: 203–205 °C; 1 H NMR (400 MHz, CD 3 OD) δ 7.33 (dd, J = 8.5, 2.1 Hz, 1H), 7.28 (d, J = 2.0 Hz, 1H), 7.06 (d, J = 8.5 Hz, 2H), 6.84 (d, J = 8.4 Hz, 1H), 6.71 (d, J = 8.5 Hz, 2H), 5.44 (s, 1H), 4.32–4.20 (m, 4H), 3.61–3.46 (m, 1H), 2.93–2.79 (m, 1H), 1.55–1.44 (m, 1H), 1.41–1.30 (m, 1H), 1.28–1.21 (m, 4H), 1.20–1.08 (m, 3H), 0.82 (td, J = 7.4, 1.4 Hz, 6H); 13 C NMR (100 MHz, CD 3 OD) δ 190.04, 166.90, 159.01, 153.55, 149.73, 144.63, 132.27, 130.12, 126.93, 124.46, 121.53, 119.59, 117.92, 116.63, 66.04, 65.40, 63.12, 42.34, 41.25, 30.77, 26.41, 26.29, 11.22, 11.13; HRMS (ESI): m/z [M + H] + calcd for C 27 H 32 NO 6 : 466.2224; found: 466.2232. Compound S9 Download figure Open in new tab Compound S9 was synthesized following a modified version of a previously reported procedure 80 . To a solution of the commercially available 1-(3-(diethylamino)propyl)-4-(2,3-dihydrobenzo[b][1,4]dioxine-6-carbonyl)-3-hydroxy-5-(4-nitrophenyl)-1h-pyrrol-2(5h)-one (0.121 mmol, 1equiv.) in EtOH/H 2 O (1 mL/320 µL) were added ammonium chloride (0.158 equiv., 1.3 equiv.) and zinc powder (0.968, 8 equiv.). The mixture was stirred and heated to 80°C for 1h30. After cooling to room temperature, solvent was removed under reduced pressure, the residue was dispersed in MeOH and zinc powder was removed by filtration. The crude product was then purified by preparative RP-HPLC (H 2 O + 0.1% HCOOH / ACN + 0.1% HCOOH) using linear gradient elution (5–60% ACN (0.1% HCOOH), to afford S9 as a yellow solid (Yield: 12%); m.p.: 193–196 °C; 1 H NMR (800 MHz, (CD 3 ) 2 SO) δ 8.20 (s, 1H), 7.46 (s, 1H), 7.33 (dd, J = 8.3, 2.0 Hz, 1H), 6.86 (d, J = 8.2 Hz, 2H), 6.70 (d, J = 8.4 Hz, 1H), 6.42 (d, J = 8.5 Hz, 2H), 5.32 (t, J = 4.8 Hz, NH 2 ), 5.03 (s, 1H), 4.31–4.17 (m, 4H), 3.41–3.39 (m, 1H), 2.65–2.60 (m, 1H), 2.43–2.34 (m, 2H), 2.04–1.93 (m, 1H), 1.60–1.54 (m, 1H), 1.49–1.41 (m, 1H), 1.30–1.20 (m, 3H), 0.94 (t, J = 7.2 Hz, 6H); 13 C NMR (200 MHz, (CD 3 ) 2 SO) δ 174.65, 147.90, 145.00, 142.21, 135.34, 130.07, 128.68, 128.46, 122.74, 118.80, 115.67, 113.86, 64.66, 64.34, 60.90, 50.06, 46.51, 38.35, 35.65, 29.11, 27.13, 25.67, 24.82, 11.34; HRMS (ESI): m/z [M + H] + calcd for C 26 H 32 N 3 O 5 : 466.2336; found: 466.2344. Compound S10 Download figure Open in new tab Compound S10 was synthesized following Method C using benzaldehyde, N,N- diethyl-3-diaminopropane and 2,4-diketo ester 2c . The crude product was purified by preparative RP-HPLC (H 2 O + 0.1% HCOOH / ACN + 0.1% HCOOH) using linear gradient elution (5–60% ACN (0.1% HCOOH), to afford S10 as a pale yellow solid (Yield: 17%); m.p.: 203–205 °C; 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 7.67–7.56 (m, 2H), 7.30–7.24 (m, 4H), 7.23–7.15 (m, 1H), 6.85 (d, J = 8.8 Hz, 1H), 5.27 (s, 1H), 3.76 (s, 3H), 3.73 (s, 3H), 3.48–3.41 (m, 1H), 2.95 (q, J = 7.2 Hz 4H), 2.87–2.77 (m, 2H), 2.75–2.63 (m, 1H), 1.80–1.60 (m, 2H), 1.10 (t, J = 7.2 Hz, 6H); 13 C NMR (100 MHz, (CD 3 ) 2 SO) δ 184.12, 170.17, 163.61, 150.63, 147.25, 141.17, 133.11, 127.97, 127.64, 127.03, 122.79, 112.88, 112.74, 109.88, 61.19, 55.48, 55.35, 49.00, 45.84, 38.01, 21.85, 8.81; HRMS (ESI): m/z [M + H] + calcd for C 26 H 33 N 2 O 5 : 453.2384; found: 453.2392. Compound S11 Download figure Open in new tab Compound S11 was synthesized following Method C using 3-hydroxybenzaldehyde, N,N- diethyl-3-diaminopropane and 2,4-diketo ester 2c . The crude product was purified by preparative RP-HPLC (H 2 O + 0.1% HCOOH / ACN + 0.1% HCOOH) using linear gradient elution (5–60 % ACN (0.1% HCOOH), to afford S11 as a yellow solid (Yield: 15%); m.p.: 196–198 °C; 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 9.30 (s, 1H), 7.66–7.57 (m, 2H), 7.06 (t, J = 7.8 Hz, 1H), 6.86 (d, J = 8.2 Hz, 1H), 6.74 (d, J = 7.9 Hz, 1H), 6.71–6.66 (m, 1H), 6.59 (dd, J = 8.0, 2.4 Hz, 1H), 5.18 (s, 1H), 3.76 (s, 3H), 3.73 (s, 3H), 3.47–3.41 (m, 1H), 2.95 (q, J = 7.2 Hz, 4H), 2.88–2.80 (m, 2H), 2.79–2.71 (m, 1H), 1.79–1.58 (m, 2H), 1.11 (t, J = 7.2 Hz, 6H); 13 C NMR (100 MHz, (CD 3 ) 2 SO) δ 184.60, 70.66, 164.98, 157.39, 150.86, 147.51, 143.00, 133.48, 129.24, 123.12, 119.11, 114.51, 114.37, 113.02, 112.76, 110.22, 61.38, 55.79, 55.67, 49.23, 46.38, 38.24, 22.23, 9.01; HRMS (ESI): m/z [M + H] + calcd for C 26 H 33 N 2 O 6 : 469.2333; found: 469.2340. Compound S12 Download figure Open in new tab Compound S12 was synthesized following Method C using 4-hydroxybenzaldehyde, N,N -diethylbutane-1,4-diamine and 2,4-diketo ester 2a . The crude product was purified by preparative RP-HPLC (H 2 O + 0.1% HCOOH / ACN + 0.1% HCOOH) using linear gradient elution (5–60% ACN (0.1% HCOOH), to afford S12 as a yellow solid (Yield: 7%); m.p.: 124–126 °C; 1 H NMR (400 MHz, CD 3 OD) δ 8.27 (s, 1H), 7.36–7.26 (m, 2H), 7.11 (d, J = 8.2 Hz, 2H), 6.84 – 6.77 (m, 1H), 6.71 (d, J = 8.2 Hz, 2H), 5.37 (s, 1H), 4.29 – 4.20 (m, 4H), 3.66 – 3.53 (m, 1H), 3.12 (q, J = 7.2 Hz, 4H), 3.03 (t, J = 7.5 Hz, 2H), 2.98–2.89 (m, 1H), 1.64–1.42 (m, 4H), 1.25 (t, J = 7.2 Hz, 6H); 13 C NMR (100 MHz, CD 3 OD) δ 189.67, 169.76, 166.83, 158.66, 148.71, 144.33, 133.68, 130.12, 129.14, 124.14, 119.63, 118.50, 117.59, 116.45, 65.97, 65.44, 62.65, 52.54, 48.13, 40.87, 26.15, 22.24, 9.03; HRMS (ESI): m/z [M + H] + calcd for C 27 H 33 N 2 O 6 : 481.2333; found: 481.2341. Data availability The coordinates and structure factors of the crystal structure are deposited to PDB with ID 9QFC. Raw diffraction data and NMR data will be available at www.fairdata.fi ( https://doi.org/10.23729/0dfe3539-a7b9-4a60-ad72-a698576de760 ). Author contributions Conceptualization, Ch.B., S.T.S., L.L.; methodology, Ch.B.; investigation, Ch.B., A.G.P., A.G., Cl.B., S.A.B., F.N., V.P.; data curation, Ch.B., A.G.P., A.G., J.P.; formal analysis, Ch.B., A.G.P., A.G., Cl.B., S.A.B., J.P., V.P.; writing-original draft, Ch.B.; writing-review & editing, Ch.B., A.G.P., S.T.S., A.G., Cl.B., S.A.B., F.N., J.P., V.P., J.W., M.H.C., L.L.; visualization, Ch.B.; validation, Ch.B.; funding acquisition, L.L.; resources, L.L; supervision, L.L., J.W., M.H.C. Declaration of interests The authors declare no competing interests. Funding This work was funded by Jane and Aatos Erkko foundation, and by the Biocenter Oulu spearhead project. This project received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 823893 (EU-OPENSCREEN-DRIVE). S.A.B. and J.W. were supported by the South-Eastern Norway Regional Health Authority (grant no. 2021035 and 2025031) and the Research council of Norway (grant no. 354458). F.N and M.H.C. acknowledge funding for the DTU Screening Core from the Novo Nordisk Foundation (NNF19OC0055818) and the Carlsberg Foundation (CF19-0072). Cl.B. and M.H.C. acknowledge support by the EU project Fragment-Screen, grant agreement ID: 101094131. Supplemental information Document S1 . Figure S1-S32 and Table S1-S3. Acknowledgements The use of the facilities and expertise of the Biocenter Oulu Structural Biology core facility (a member of Biocenter Finland, Instruct-ERIC Centre Finland and FINStruct), Proteomics and Protein Analysis core facility (a member of Biocenter Finland) and Biocenter Oulu sequencing center are gratefully acknowledged. We gratefully acknowledge the staff at the Chemical Biology Platform at the Norwegian Centre for Molecular Biosciences and Medicine (formerly NCMM), especially Eirin Solberg, for performing the high-throughput screen. This work benefited from access to the Instruct-ERIC Centre Finland NMR facility at the University of Helsinki. We greatly thank Perttu Permi and Tuomas Niemi-Aro for the help with NMR data collection. Financial support was provided by Instruct-ERIC (PID 23611). We are grateful to local contacts at Diamond for providing assistance in using beamline I24. Footnotes ↵ 6 Lead contact References 1. ↵ Sedgwick SG , Smerdon SJ . The ankyrin repeat: a diversity of interactions on a common structural framework . Trends in Biochemical Sciences . 1999 ; 24 ( 8 ): 311 – 316 . doi: 10.1016/S0968-0004(99)01426-7 OpenUrl CrossRef PubMed Web of Science 2. ↵ Li J , Mahajan A , Tsai MD . Ankyrin Repeat: A Unique Motif Mediating Protein−Protein Interactions † . 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