Off-target interaction of the amyloid PET imaging tracer PiB with acetylcholinesterase

in silico studies; computational analysis; Alzheimer’s disease; molecular dynamics; molecular docking; amyloid, tau * To whom correspondence should be addressed: Dr. Alberto Granzotto Center for Advanced Studies and Technology – CAST Department of Neuroscience, Imaging, and Clinical Sciences University G. d’ Annunzio of Chieti-Pescara, Via L. Polacchi, 11, 66100, Chieti (CH), Italy Email: [email protected] .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint

Pittsburgh compound B (PiB) is a widely used Positron Emission Tomography (PET) tracer for detecting amyloid-β (Aβ) deposits in Alzheimer’s disease (AD). While PiB is assumed to bind selectively to Aβ, emerging evidence suggests off-target interactions that may complicate PET signal interpretation. Here, we report that PiB can interact with acetylcholinesterase (AchE), a key enzyme in the cholinergic system. Similarity screening identified the AchE ligand thioflavin T (ThT) as the top structural analog of PiB. Docking studies and molecular dynamics simulations showed that PiB stably binds the peripheral anionic site (PAS) of AchE, with binding energies comparable to ThT and clinically relevant AchE inhibitors. In vitro fluorescence-based assays confirmed this interaction and suggest an involvement of the PAS. These findings indicate a stable off-target interaction between PiB and AChE with implications for interpreting PiB-PET signals in AD, particularly in regions with altered AchE expression or under AchE inhibitor therapy. .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint

Positron emission tomography (PET)-based biomarkers are largely employed in research and clinical settings for disease diagnosis and monitoring, patient stratification, or as an efficacy outcome of interventions [1]. In Alzheimer’s disease (AD), PET tracers have been developed to quantitatively detect changes in the accumulation of key pathological markers, like cortical amyloid-β (Aβ) deposits, hyperphosphorylated tau (p-tau) protein buildup, and neurodegeneration [2,3]. Alterations in these biomarkers mirror disease progression and are the “gold standard” for diagnosing AD and for the early detection of people at-risk of developing the condition [4,5]. The shift from a clinical- to a biomarker- based definition of AD is also at the basis of the “ATN research framework”, a biological definition of the disease (i.e., 'A' – amyloid, ‘T′ – tau, and ‘N′ – neurodegeneration) aimed at offering a quantifiable and unbiased staging of AD [4]. The approach is relevant since the pathological alterations of AD can occur and are detectable long before the onset of cognitive and behavioral symptoms [6,7]. Early identification of individuals in the very early stages of the condition represents a transformative step for the effective development and targeted implementation of disease-modifying interventions. Alterations of Aβ levels are widely recognized as one of the earliest molecular changes that can foreshadow the onset of AD pathology, although the specific contribution of Aβ to disease pathogenesis is debated [8,9]. Quantitative assessment of Aβ is either performed in biological fluids like liquor and plasma, where decreases in Aβ abundance reflect the cerebral deposition of the peptide, or by PET- based imaging, where specific radioligands are employed to detect the presence of fibrillar Aβ (fAβ) aggregates in the brain. Several Aβ radiotracers have been developed since the early 2000s with Pittsburgh compound B (11C-PiB), a thioflavin T (ThT) analog, being the first of this class of imaging agents. The short half-life of 11C-PiB led to the development of fluorine-18 derivatives more suitable for clinical applications, like 18F-flutemetamol or the trans-stilbene-based compounds 18F-florbetapir and 18F- florbetaben. Nevertheless, 11C-PiB is still broadly adopted in clinical research settings. Although these radioligands are widely employed for the diagnosis of AD and for monitoring target engagement of Aβ-targeting interventions, doubts have been cast on their specificity and sensitivity [10–13]. Previous studies demonstrated that 2-aryl-6-hydroxybenzothiazole-based tracers can effectively bind to off-target molecules, like sulfotransferases, that likely contribute to PET signals unrelated to the overall Aβ load [10,12]. However, it is unclear whether this class of Aβ radioligands has additional off-target effects. This study aims to investigate PiB binding characteristics at the molecular level and, by employing unbiased in silico screening, docking calculations, molecular dynamics (MD) simulations, and in vitro assay, we surveyed for potential novel binding partners unrelated to Aβ pathology.

Reagents and chemicals PiB was purchased from TargetMol. Acetylcholinesterase from Electrophorus electricus (eeAchE), Catalase from bovine liver and all the other chemicals were from Sigma-Aldrich. .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint Library screening Similarity screening for the PiB amyloid PET tracer was performed with the SwissSimilarity 2021 Web Tool (http://www.swisssimilarity.ch/ [14,15]) on August 3, 2024. The search was limited to ligands present in the Protein Data Bank (LigandExpo; 19500 compounds) using a consensus 2D/3D screening using a score based on both FP2 Tanimoto coefficient and Electroshape-5D Manhattan distance [14]. Screening scores and SMILES notations were downloaded for further analysis. Molecular modeling All the molecules investigated in this study were downloaded as three-dimensional conformer .sdf files from PubChem [16]. The Energy Minimization Experiment function, using YASARA AutoSMILES for automatic force field parameter assignment, was used to optimize the 3D structure before docking. Docking calculations were performed using VINA default docking parameters as implemented in the YASARA suite, as previously described [17]. Briefly, the crystal structure of the target protein was downloaded from the PDB (PDB ID: pdb_00004ey7), a cell encompassing all atoms extending 5 Å from the surface of the structure of the ligand was generated, and the crystallized ligand was removed. Global ligand docking was performed using VINA using the default parameters and further refined with VINA Local Search [18]. The molecular dynamics simulations of the acetylcholinesterase complexes were run with the same YASARA suite [19] by employing the macro md_runfast. A cuboid periodic simulation cell extending 20 Å from the protein surface was set and filled with water (density: 0.997 g/mL). The setup included an optimization of the hydrogen bonding network [20] to increase the solute stability, and a pKa prediction to fine-tune the protonation states of protein residues at pH 7.4 [21]. NaCl ions were added at a physiological concentration of 0.9%. After steepest descent and simulated annealing minimizations to remove clashes, the simulation was run for 300 nanoseconds using the AMBER14 force field [22] for the solute, GAFF2 [23] and AM1BCC [24] for ligands and TIP3P for water. The cutoff was 8 Å for Van der Waals forces [25], no cutoff was applied to electrostatic forces (using the Particle Mesh Ewald algorithm) [26]. The equations of motions were integrated with a multiple timestep of 2.5 fs for bonded interactions and 5.0 fs for non-bonded interactions at a temperature of 298K and a pressure of 1 atm (NPT ensemble) using algorithms described previously [27]. MD conformations were recorded every 250 ps. The energies of binding and the MD trajectory have been calculated using the md_analyzebindenergy macro implemented in the YASARA suite employing the MM/PBSA method as previously described [28]. Ligand movement root mean square displacement (RMSD) was calculated with the YASARA md_analyze function after superposing on the receptor. PiB spectra A 20 µM PiB (0.8 % DMSO final concentration) solution was prepared in a 100 mM potassium phosphate buffer solution (KPi, pH 7.4). Absorbance spectrum was measured by employing a PerkinElmer Lambda 35 spectrophotometer (Range: 200 – 900 nm; slit: 2nm; resolution: 1 nm; speed: 240 nm/min). Fluorescence emission spectrum was measured with a BioTek Synergy H1 plate reader (Ex λ: 350 nm; Em range: 380 – 700 nm; resolution: 1 nm; gain: 60 a.u.). Turbidity assay .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint The turbidity assay was performed as previously described [29]. In brief, the absorbance of increasing concentrations of PiB (1.56 µM to 1600 µM) was measured at 405 nm using a PerkinElmer SPECTRAmax 190 microplate reader. Absorbance readings from the buffer alone (KPi containing 0.8% DMSO) served as reference. The purpose of the assay was to determine the highest PiB concentration that does not result in precipitation of the compound. Fluorescence-based interaction assay A fluorescence-based binding assay was performed to assess the potential interaction between PiB and eeAChE. PiB (25 µM final concentration; dissolved in 100mM KPi, 0.1 % DMSO) was incubated in vitro either in the presence or absence of 2 µg of eeAChE in a total volume of 50 µL. Incubations were carried out for 5 hours at 30 °C under gentle agitation (1100 rpm). Following incubation, each mixture was filtered using Amicon Ultra-0.5 centrifugal filters with a 30 kDa molecular weight cutoff (Millipore), centrifuged at 14,000 × g for 10 minutes at room temperature to separate unbound PiB from AChE- bound PiB. The retentate, containing eeAChE and any bound PiB, was recovered and transferred to a black walled 96-well plate for fluorescence measurement. Fluorescence was measured using a BioTek Synergy H1 plate reader with excitation at 350 nm and emission at 440 nm. The retentate was subsequently spotted onto a nitrocellulose membrane, stained with Ponceau S, and imaged to assess protein recovery. Propidium iodide displacement assay To evaluate the interaction between PiB and the PAS of eeAChE, we employed a propidium iodide (PI) displacement assay. A total of 25 U of eeAChE were incubated overnight with PI (1 µM), either alone or in the presence of PiB (20 µM, 0.8% DMSO). A parallel experiment using donepezil (20 µM, 0.8% DMSO) served as a positive control. PI fluorescence was measured using a BioTek Synergy H1 plate reader with excitation at 535 nm and emission at 630 nm. Background fluorescence from PI alone was subtracted from all readings. Data were then normalized as Fx/Fvehicle, where Fx represents the PI fluorescence for each condition, and Fvehicle is the PI fluorescence in the presence of eeAChE and 0.8% DMSO. Statistical analysis Microsoft Excel (Microsoft) and OriginPro (OriginLab) were employed for statistical analysis and data plotting. Data in Fig. 2 are represented as mean ± 1 standard error of the mean (s.e.m.); data points represent individual experiments. Exact P values are reported for each relevant comparison. The number of replicates and the statistical test used are provided in the figure legends.

To identify potential off-target partners of Aβ PET tracers, we performed an unbiased screening of biologically relevant molecules that show structural similarity with PiB by employing the SwissSimilarity 2021 Web Tool. Our analysis returned the score of 400 molecules (Fig. 1A and Supplementary Table 1) with ThT being the top-scoring molecule (score 0.996). More importantly, ThT .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint was identified as the molecule is a ligand for acetylcholinesterase (AchE) in the PDB (PDB ID: pdb_00002j3q). To test the hypothesis that PiB interacts with AchE, we performed docking studies within the AchE pocket using the crystal structure of the human AchE (PDB ID: pdb_00004ey7). AchE has two binding sites: the catalytic site and the peripheral anionic site (PAS; [30,31]). We focused on the latter, located at the entrance of the catalytic gorge, since it mediates the interaction of AchE with ThT [32–34]. Analysis of docking results shows that PiB has binding energy comparable with that of ThT (8.603 and 8.771 kcal/mol, respectively; Fig. 1B). Binding energy of clinically approved AchE inhibitors (donepezil, galantamine, and rivastigmine) was calculated for comparison (Fig. 1B). Fig. 1C and D show the 2D and 3D poses and the interaction of PiB with the amino acid residues in the AchE PAS. PiB forms a π–π-sulfur interaction with residue Phe297 along with several hydrophobic, alkyl, and Van der Waals interactions with residues Trp86, His447, Tyr337, Phe338, Tyr72, Trp286, Phe295, Tyr341, and Tyr124 (Fig. 1C, D). We further investigated the PiB-AchE complex by performing a 300 ns molecular dynamics (MD) simulation. Analysis of the energy of binding shows that PiB maintains a high and stable binding energy throughout the simulation (Fig. 1E). The stability of the PiB-AchE complex is also supported by the RMSD analysis of the ligand movement after superimposing the molecule on the enzyme structure (Fig. 1F). After a stabilization phase, the ligand remains within the AchE PAS. The compound exhibits only a few modest, sharp fluctuations that return to baseline levels during the simulation (Fig. 1F). We attribute the stability of the complex to the sulfur interaction, along with the dense network of hydrophobic interactions that keep PiB within the AchE PAS. .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint Figure 1. Identification and in silico characterization of AchE as a potential target of PiB. (A) The plot illustrates the similarity score of each compound screened with the SwissSimilarity 2021 Web Tool. (B) The histogram depicts the binding energy calculation of the listed ligands after docking on AchE. (C - D) Two-dimensional (C) and three-dimensional (D) docking poses and interactions of PiB in the AchE PAS. The dashed yellow line indicates π–π-sulfur interaction; dashed pink lines indicate π–π-alkyl interactions; magenta lines indicate π–π and T-shaped interactions; green residues show van der Waals interactions. (E - F) Time course of energy of binding (E) and root mean squared displacement (RMSD; F) for the PiB- AchE complex over a 300 ns MD simulation. To further evaluate PiB binding to the PAS of eeAChE, we performed PI displacement, an assay used for probing the interaction of candidate drugs with the PAS of AchE [35]. The binding of PI to the PAS increases dye fluorescence. Meanwhile its displacement by PAS-interacting compounds leads to signal reduction [36]. In the presence of 20 µM PiB, PI fluorescence was reduced by approximately 15% compared to control conditions (Fig. 2F). Although this difference did not reach statistical significance (P .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint = 0.10), the data suggest a potential trend toward PI displacement. The reduction increased to ≈ 78% in the presence of the high-affinity, PAS-binding AchE inhibitor donepezil (20 µM; Fig. 2F) [30,37]. Together, these findings support the formation of a stable PiB–eeAchE complex in vitro and are consistent with the possibility that PiB interacts with the PAS of the enzyme. Figure 2. Experimental validation of the formation of the PiB-eeAchE complex. (A) Absorption (dashed line) and emission (solid line) normalized spectra of PiB in KPi buffer (pH 7.4). (B) The bar graph depicts normalized fluorescence of PiB following incubation of the compound with or without eeAchE (2 µg) and size-exclusion filtration (PiB n = 6 and PiB + eeAchE n = 5 independent experiments). (C) Ponceau S staining of the retentate was spotted onto a nitrocellulose membrane to assess protein recovery. (D) The bar graph depicts normalized fluorescence of PiB following incubation of the compound with or without Catalase (2 µg) and size-exclusion filtration (PiB n = 4 and PiB + Catalase n = 4 independent experiments). (E) Ponceau S staining of the retentate was spotted onto a nitrocellulose membrane to assess protein recovery. (F) The bar graph depicts normalized fluorescence of PI following incubation with eeAchE in the presence of vehicle (Control; 0.8% DMSO), PiB (20 µM, 0.8% DMSO), or Donepezil (20 µM, 0.8% DMSO). Note the ≈ 15% signal reduction in the presence of PiB. In B and D, the comparison of mean values was assessed by the Mann-Whitney U Test. In F, mean values were compared by one-way ANOVA followed by Tukey's post-hoc test. .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint

In this study, we provide computational and experimental evidence that the amyloid PET tracer PiB can bind AchE, suggesting a previously unrecognized off-target interaction. Our findings extend prior observations that ThT-based compounds may interact with non-amyloid targets and raise important questions on the specificity of PiB and related PET tracers used in AD research and diagnostics [32]. Our in silico similarity screening identified ThT – a known AchE ligand – as the compound most structurally related to PiB among biologically relevant molecules in the PDB database, pointing to a potential interaction between PiB and AchE. We further tested this hypothesis using molecular docking and MD simulations. Docking results showed that PiB binds the PAS of AchE with binding energy comparable to ThT and within the range of clinically relevant AchE inhibitors. PiB establishes π–sulfur and hydrophobic interactions with residues located in the PAS and near the gorge of the active site, like Phe297, Trp286, Tyr337, and His447. These residues were found to be key for the interaction with AchE- targeting drugs [30]. MD simulations further confirmed the persistence of these interactions, indicating a stable and energetically favorable complex. We validated the computational predictions with a binding assay that exploits the intrinsic fluorescence properties of PiB, supporting the formation of a stable PiB–AchE complex in vitro and suggesting that the interaction occurs at the PAS of the enzyme. It is important to notice that a more thorough investigation of the interaction between PiB and AchE was hampered by both the physicochemical properties of PiB and the sensitivity of AchE to PiB-compatible solvents. We found that in conditions suitable for the AchE enzymatic assay, PiB began to precipitate at concentrations above 25 µM (Supplementary Fig. 1B). The use of alternative solvents or surfactants was unsuccessful (unpublished observations). Moreover, higher concentrations of DMSO were shown to substantially impair AchE activity [38]. In a further attempt to directly examine PiB-PAS interaction, we also tested an in vitro competition assay in the presence of donepezil [30,37]. However, preliminary control experiments showed a substantial spectral overlap between PiB and donepezil (Supplementary Fig. 1B), making fluorescence-based comparisons difficult to interpret and prone to bias. While these technical constraints limit our ability to perform orthogonal or competitive binding assays, they do not undermine the core observation that PiB interacts with eeAchE, as suggested in our fluorescence-based filtration assay and PI displacement. The identification of AChE as a potential off-target of PiB has several implications. First, it imposes the need to carefully interpret PET signals in brain regions where AchE is abundantly expressed, particularly in early-stage or atypical AD presentations, where amyloid deposition may not be the unique contributor to the tracer uptake. Second, given that AchE expression and activity can change in the aging brain and neurodegenerative conditions beyond AD [39,40]. The off-target binding of PiB to AchE could contribute to false positives or elevated baseline signals in specific populations. Third, the PiB signal could be influenced by the use of AchE inhibitors that act by binding the PAS of the enzyme, leading to false negative results. Moreover, the high lipophilicity of PiB could also explain the elevated retention of the tracer in lipid-enriched white matter regions [41,42]. Our findings align with previous reports of off-target binding for other radiotracers used in AD, for whom interactions with enzymes like monoamine oxidases and sulfotransferases have been reported .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint [10,11,43]. In addition, our similarity virtual screening does not rule out the existence of additional PiB binding partners. However, to our knowledge, this is the first study to indicate an interaction between PiB and AchE at both the computational and experimental levels. While the functional consequences of this binding remain to be explored, such interactions may alter AchE activity or affect PiB signals in vivo. Further studies using radiolabeled PiB and AchE inhibitors in vivo are warranted to confirm whether this interaction occurs under pathophysiological conditions and contributes to PET signals. In conclusion, these results underscore the importance of integrative approaches combining computational modeling with biochemical validation to uncover and assess the biological relevance of such interactions. Acknowledgments A.G. is supported by the European Union - Next Generation EU, Mission 4 Component 1, CUP: D53D23019280001. AI-assisted technology (ChatGPT 4o) has been used in the writing process to improve the readability and language of the manuscript.

[1] Nasrallah I, Dubroff J (2013) An overview of PET neuroimaging. Semin Nucl Med 43, 449–461. [2] Burkett BJ, Babcock JC, Lowe VJ, Graff-Radford J, Subramaniam RM, Johnson DR (2022) PET Imaging of Dementia: Update 2022. Clin Nucl Med 47, 763–773. [3] Wang J, Jin C, Zhou J, Zhou R, Tian M, Lee HJ, Zhang H (2023) PET molecular imaging for pathophysiological visualization in Alzheimer’s disease. Eur J Nucl Med Mol Imaging 50, 765–783. [4] Jack CR, Bennett DA, Blennow K, Carrillo MC, Feldman HH, Frisoni GB, Hampel H, Jagust WJ, Johnson KA, Knopman DS, Petersen RC, Scheltens P , Sperling RA, Dubois B (2016) A/T/N: An unbiased descriptive classification scheme for Alzheimer disease biomarkers. Neurology 87, 539– 547. [5] Rabinovici GD, Knopman DS, Arbizu J, Benzinger TLS, Donohoe KJ, Hansson O, Herscovitch P , Kuo PH, Lingler JH, Minoshima S, Murray ME, Price JC, Salloway SP , Weber CJ, Carrillo MC, Johnson KA (2025) Updated Appropriate Use Criteria for Amyloid and Tau PET: A Report from the Alzheimer’s Association and Society for Nuclear Medicine and Molecular Imaging Workgroup. J Nucl Med. [6] Aisen PS, Cummings J, Jack CR, Morris JC, Sperling R, Frölich L, Jones RW, Dowsett SA, Matthews BR, Raskin J, Scheltens P , Dubois B (2017) On the path to 2025: understanding the Alzheimer’s disease continuum. Alzheimers Res Ther 9, 60. [7] Frisoni GB, Altomare D, Thal DR, Ribaldi F, van der Kant R, Ossenkoppele R, Blennow K, Cummings J, van Duijn C, Nilsson PM, Dietrich P-Y , Scheltens P , Dubois B (2022) The probabilistic model of Alzheimer disease: the amyloid hypothesis revised. Nat Rev Neurosci 23, 53–66. [8] Kepp KP , Robakis NK, Høilund-Carlsen PF, Sensi SL, Vissel B (2023) The amyloid cascade hypothesis: an updated critical review. Brain awad159. [9] Granzotto A, Sensi SL (2024) Once upon a time, the Amyloid Cascade Hypothesis. Ageing Res Rev 93, 102161. .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint [10] Cole GB, Keum G, Liu J, Small GW, Satyamurthy N, Kepe V, Barrio JR (2010) Specific estrogen sulfotransferase (SULT1E1) substrates and molecular imaging probe candidates. Proc Natl Acad Sci U S A 107, 6222–6227. [11] Surmak AJ, Wong K-P , Cole GB, Hirata K, Aabedi AA, Mirfendereski O, Mirfendereski P , Yu AS, Huang S-C, Ringman JM, Liebeskind DS, Barrio JR (2020) Probing Estrogen Sulfotransferase- Mediated Inflammation with [11C]-PiB in the Living Human Brain. J Alzheimers Dis JAD 73, 1023– 1033. [12] Høilund-Carlsen PF, Alavi A, Castellani RJ, Neve RL, Perry G, Revheim M-E, Barrio JR (2024) Alzheimer’s Amyloid Hypothesis and Antibody Therapy: Melting Glaciers? Int J Mol Sci 25, 3892. [13] Høilund-Carlsen PF, Alavi A, Barrio JR (2024) PET/CT/MRI in Clinical Trials of Alzheimer’s Disease. J Alzheimer’s Dis 101, S579–S601. [14] Zoete V, Daina A, Bovigny C, Michielin O (2016) SwissSimilarity: A Web Tool for Low to Ultra High Throughput Ligand-Based Virtual Screening. J Chem Inf Model 56, 1399–1404. [15] Bragina ME, Daina A, Perez MAS, Michielin O, Zoete V (2022) The SwissSimilarity 2021 Web Tool: Novel Chemical Libraries and Additional Methods for an Enhanced Ligand-Based Virtual Screening Experience. Int J Mol Sci 23, 811. [16] Kim S, Chen J, Cheng T, Gindulyte A, He J, He S, Li Q, Shoemaker BA, Thiessen PA, Yu B, Zaslavsky L, Zhang J, Bolton EE (2023) PubChem 2023 update. Nucleic Acids Res 51, D1373–D1380. [17] Floresta G, Granzotto A, Patamia V, Arillotta D, Papanti GD, Guirguis A, Corkery JM, Martinotti G, Sensi SL, Schifano F (2025) Xylazine as an emerging new psychoactive substance; focuses on both 5-HT7 and κ-opioid receptors’ molecular interactions and isosteric replacement. Arch Pharm (Weinheim) 358, e2500041. [18] Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J Comput Chem 31, 455. [19] Krieger E, Vriend G (2014) YASARA View - molecular graphics for all devices - from smartphones to workstations. Bioinforma Oxf Engl 30, 2981–2982. [20] Krieger E, Dunbrack RL, Hooft RWW, Krieger B (2012) Assignment of protonation states in proteins and ligands: combining pKa prediction with hydrogen bonding network optimization. Methods Mol Biol Clifton NJ 819, 405–421. [21] Krieger E, Nielsen JE, Spronk CAEM, Vriend G (2006) Fast empirical pKa prediction by Ewald summation. J Mol Graph Model 25, 481–486. [22] Maier JA, Martinez C, Kasavajhala K, Wickstrom L, Hauser KE, Simmerling C (2015) ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J Chem Theory Comput 11, 3696–3713. [23] Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA (2004) Development and testing of a general amber force field. J Comput Chem 25, 1157–1174. [24] Jakalian A, Jack DB, Bayly CI (2002) Fast, efficient generation of high-quality atomic charges. AM1- BCC model: II. Parameterization and validation. J Comput Chem 23, 1623–1641. [25] Hornak V, Abel R, Okur A, Strockbine B, Roitberg A, Simmerling C (2006) Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins 65, 712–725. [26] Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG (1995) A smooth particle mesh Ewald method. J Chem Phys 103, 8577–8593. [27] Krieger E, Vriend G (2015) New ways to boost molecular dynamics simulations. J Comput Chem 36, 996–1007. [28] Floresta G, Patamia V, Mazzeo PP , Lombardo GM, Pistarà V, Bacchi A, Rescifina A, Punzo F (2024) Structural, morphological, and modeling studies of N-(benzoyloxy)benzamide as a specific inhibitor of Type II inosine monophosphate dehydrogenase. J Mol Struct 1303, 137588. .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint [29] Granzotto A, Bolognin S, Scancar J, Milacic R, Zatta P (2011) Beta-amyloid toxicity increases with hydrophobicity in the presence of metal ions. Monatshefte Chem 142, 421–430. [30] Cheung J, Rudolph MJ, Burshteyn F, Cassidy MS, Gary EN, Love J, Franklin MC, Height JJ (2012) Structures of Human Acetylcholinesterase in Complex with Pharmacologically Important Ligands. J Med Chem 55, 10282–10286. [31] Dvir H, Silman I, Harel M, Rosenberry TL, Sussman JL (2010) Acetylcholinesterase: From 3D Structure to Function. Chem Biol Interact 187, 10–22. [32] Auletta JT , Johnson JL, Rosenberry TL (2010) Molecular basis of inhibition of substrate hydrolysis by a ligand bound to the peripheral site of acetylcholinesterase. Chem Biol Interact 187, 135–141. [33] Harel M, Sonoda LK, Silman I, Sussman JL, Rosenberry TL (2008) Crystal Structure of Thioflavin T Bound to the Peripheral Site of Torpedo californica Acetylcholinesterase Reveals How Thioflavin T Acts as a Sensitive Fluorescent Reporter of Ligand Binding to the Acylation Site. J Am Chem Soc 130, 7856–7861. [34] Sulatskaya AI, Rychkov GN, Sulatsky MI, Rodina NP , Kuznetsova IM, Turoverov KK (2018) Thioflavin T Interaction with Acetylcholinesterase: New Evidence of 1:1 Binding Stoichiometry Obtained with Samples Prepared by Equilibrium Microdialysis. ACS Chem Neurosci 9, 1793–1801. [35] Alarcón-Enos J, Muñoz-Núñez ,Evelyn, Gutiérrez ,Margarita, Quiroz-Carreño ,Soledad, Pastene- Navarrete ,Edgar, and Céspedes Acuña C (2022) Dyhidro-β-agarofurans natural and synthetic as acetylcholinesterase and COX inhibitors: interaction with the peripheral anionic site (AChE-PAS), and anti-inflammatory potentials. J Enzyme Inhib Med Chem 37, 1845–1856. [36] Singh YP , Shankar G, Jahan S, Singh G, Kumar N, Barik A, Upadhyay P , Singh L, Kamble K, Singh GK, Tiwari S, Garg P , Gupta S, Modi G (2021) Further SAR studies on natural template based neuroprotective molecules for the treatment of Alzheimer’s disease. Bioorg Med Chem 46, 116385. [37] Sugimoto H, Yamanishi Y , Iimura Y , Kawakami Y (2000) Donepezil hydrochloride (E2020) and other acetylcholinesterase inhibitors. Curr Med Chem 7, 303–339. [38] David B, Schneider P , Schäfer P , Pietruszka J, Gohlke H (2021) Discovery of new acetylcholinesterase inhibitors for Alzheimer’s disease: virtual screening and in vitro characterisation. J Enzyme Inhib Med Chem 36, 491–496. [39] García-Ayllón M-S, Small DH, Avila J, Saez-Valero J (2011) Revisiting the Role of Acetylcholinesterase in Alzheimer’s Disease: Cross-Talk with P-tau and β-Amyloid. Front Mol Neurosci 4,. [40] Campanari M-L, Navarrete F, Ginsberg SD, Manzanares J, Sáez-Valero J, García-Ayllón M-S (2016) Increased Expression of Readthrough Acetylcholinesterase Variants in the Brains of Alzheimer’s Disease Patients. J Alzheimer’s Dis 53, 831–841. [41] Glodzik L, Rusinek H, Li J, Zhou C, Tsui W, Mosconi L, Li Y , Osorio R, Williams S, Randall C, Spector N, McHugh P , Murray J, Pirraglia E, Vallabhajolusa S, de Leon M (2015) Reduced retention of Pittsburgh compound B in white matter lesions. Eur J Nucl Med Mol Imaging 42, 97–102. [42] Klunk WE, Engler H, Nordberg A, Wang Y , Blomqvist G, Holt DP , Bergstr??m M, Savitcheva I, Huang GF, Estrada S, Aus??n B, Debnath ML, Barletta J, Price JC, Sandell J, Lopresti BJ, Wall A, Koivisto P , Antoni G, Mathis CA, L??ngstr??m B (2004) Imaging Brain Amyloid in Alzheimer’s Disease with Pittsburgh Compound-B. Ann Neurol 55, 306–319. [43] Murugan NA, Chiotis K, Rodriguez-Vieitez E, Lemoine L, Ågren H, Nordberg A (2019) Cross- interaction of tau PET tracers with monoamine oxidase B: evidence from in silico modelling and in vivo imaging. Eur J Nucl Med Mol Imaging 46, 1369–1382. .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint Supplementary Information Supplementary Figure 1. Limited PiB solubility and spectral properties of PiB and donepezil complicate fluorescence- and enzymatic-based assays. (A) The scatter plot depicts absorbance of PiB measured across increasing concentrations (1.56 – 1600 µM) and reveals precipitation above 25 µM, limiting its use in assays requiring higher concentrations. (B) Normalized absorbance spectra of PiB (blue) and donepezil (orange) highlight substantial spectral overlap which complicates interpretation of fluorescence-based competition assays involving both compounds. Data are representative of at least two independent experiments. a.u., arbitrary units. .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint Supplementary table 1 Ligand PDB ID Similarity Score SMILES TFL 0.996 CN(C)C1=CC=C(C=C1)C1=[N+](C)C2=CC=C(O)C=C2S1 GK1 0.767 CC1=CC=C(O)C=C1NC1=CC=NC2=CC(=CC=C12)C1=CSC(C=O)=N1 EVW 0.715 NC1=NC2=CC=C(C=C2S1)C(=O)NC(C1=CC=CC=C1)C1=CC=CC=C1 UUL 0.672 OC1=CC=C(NC2=NC(=CS2)C2=CC=C(Cl)C=C2)C=C1 JMT 0.616 OC1=CC=C2NC=C(CN3CCN(CC3)C3=NC4=CC=CC=C4S3)C2=C1 09H 0.606 O=C(NC1=CC=CC=C1N1CCNCC1)C1=CSC(=N1)C1=CC=C2OCCC2=C1 JMW 0.594 OC1=CC=C2NC=C(CN3CCN(CC3)C3=NC4=CC(Cl)=CC=C4S3)C2=C1 EWK 0.585 NC1=NC2=CC=C(SCC3=CC=C(C=C3)C(=O)NCC3=CC=CC=C3)C=C2S1 EWT 0.574 NC1=NC2=CC=C(C=C2S1)C(=O)NCC1=CC(Cl)=C(Cl)C=C1 2WJ 0.565 CC(=O)NC1=NC2=CC=C(C=C2S1)C1=CC=CN=C1 AQE 0.563 C1CC[C@@]2(CCCN(C2)C2=C3C(NC=C3C3=NC=CS3)=NC=C2)NC1 94U 0.522 CCN(CC)C1=CC=C(NC(=O)C2=CC3=C(N2)N=CS3)C=C1 G4A 0.514 CN1\\C(OC2=CC=CC=C12)=C\\C1=[N+](CCCS(O)(=O)=O)C2=CC=CC=C2S1 3TI 0.51 OC1=CC=C(C=C1)\\N=C\\C1=C2C=CC=CC2=CC=C1O N0E 0.505 OC1=CC=C(NC(=O)CCC2=CC=CC=C2)C=C1 2JR 0.5 C1CCN(C1)C1(CCCCC1)C1=CN=C(S1)C1=CC=C2NC=CC2=C1 EV8 0.498 COC(=O)C1CCN(CC1)C(=O)C1=CC=C(CNC(=O)C2=CC=C3N=C(N)SC3=C2)C=C1 CP9 0.497 CC1=NC2=CN=CC=C2N1C1=CC=C(CN2C(=O)SC3=CC=CC=C23)C=C1 X1H 0.492 COC1=CC=C(C=C1)C(=O)C1=C(SC2=CC(O)=CC=C12)C1=CC=C(O)C=C1 B4K 0.484 CC(=O)NC1=C2C=CC(=NC2=NN1)C1=CC=C(O)C(O)=C1 P2X 0.48 CC(C)N1N=C(C2=CC3=CC(O)=CC=C3N2)C2=C(N)N=CN=C12 RU5 0.475 NC(=O)C1=CC=C2NC(=NC2=C1)C1=CC=C(OC2=CC=C(Cl)C=C2)C=C1 3F4 0.474 OC1=CC=C(C=C1)C1=NC(=O)C2=CC=CC=C2N1 MKY 0.472 CCOC(=O)CN1\\C(SC2=CC(O)=CC=C12)=N\\C(N)=N OFI 0.471 CCCC(=O)NC1=NNC2=CC(=CC=C12)C1=CC=C(O)C=C1 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint NU3 0.466 NC(=O)C1=C2N=C(NC2=CC=C1)C1=CC=C(O)C=C1 LNJ 0.462 COC1=CC=C(CC2=CC=C(C=C2)C2=CSC(N)=N2)C=C1 C2J 0.442 OC1=CC=C(C=C1)C1=CC=C2C(NN=C2NC(=O)C2CC2)=C1 57X 0.44 CCCN1C2=NNC(C3=CN=C(S3)C3=CC=CN=C3)=C2C=CC1=O 972 0.419 CC(C)COC1=CC=CC(C2=NC3=CC(C(N)=N)=C(Cl)C=C3N2)=C1O JV5 0.418 CC1=CC=CC=C1OCC(=O)NC1=CC2=NNN=C2C=C1 917 0.417 CC(=O)NC[C@H]1CN(C(=O)O1)C1=CC=C(C=C1)C1=CN=CS1 2RE 0.416 OC1=CC=C(C=C1)C1=NC(=C(N1)C1=CC=NC=C1)C1=CC=C(F)C=C1 0HD 0.416 O=C(NCCC1=CC=CC=C1)NC1=CC2=CNN=C2C=C1 656 0.413 CC(C)COC1=CC=CC(C2=NC3=CC(=CC=C3N2)C(N)=N)=C1O 3U6 0.411 NC1=NN=C(S1)C1=CC=C2NC=C(C2=C1)C1=NC(NC2CCCC2)=CC=C1 D58 0.408 C[C@H]1NCCC[C@@H]1NC1=C2C=C(SC2=C(C=N1)C(N)=O)C1=CC=CC=C1 EK7 0.404 CN(C)C1=C2C(CCC3=C2N=C(NC2=CC=CC(O)=C2)N=C3)=C(S1)C#N 3TX 0.4 OC1=CC=C(C=C1)N1C=C(N=N1)C1=NC2=CC=CC=C2C=C1 3J7 0.4 CC(C)(N)CNC1=C2C=CN=CC2=NC(=N1)C1=CC2=CNN=C2C=C1 41Z 0.394 CC1=NC2=C(C=CC=C2C(NCC2=C(C)C=CC=C2C)=C1)C(N)=O 97K 0.393 O=C1N=C(NC2=CC=CC=C12)C1=CC2=CNN=C2C=C1 I0D 0.379 CN1CCC2=C(C1)C=CC=C2NC1=C(Cl)C(=O)N(C)N=C1 79X 0.379 COC1=CC2=C(C=C1OC)C1=CC3=CC(O)=CC=C3N1C2=O 4GM 0.379 NC(=O)C1=CC=C(NCC2=CC=CC=C2O)N=C1 IOK 0.377 C[C@H](CCC1=CC=C(O)C=C1)NC(=O)CC1=C(NC2=CC=CC=C12)C1=CC=CC=C1 3T9 0.376 COC1=CC(=CC=C1O)C1=NC2=NNC(=C2C=C1)C1=CC=CC=C1 655 0.374 NC(=N)C1=CC=C2NC(=NC2=C1)C1=C(O)C(OC2CCCC2)=CC=C1 WTF 0.373 CCS(=O)(=O)C1=CC=CC=C1C(=O)N1CCN(C[C@@H]1C)C1=NC2=CC=C(F)C=C2S1 WAM 0.37 COC1=CC(\\C=C\\C2=[N+](C)C3=CC=CC=C3C(=C2)C(N)=O)=CC=C1O C70 0.368 NC(=O)C1=C2SC(=CC2=C(N[C@H]2CCCNC2)N=N1)C1=CC=C(Cl)C=C1 NUW 0.367 CNC(=O)C1=CC=C2N(CC3CCN(CC3)C(C)=O)C(=NC2=C1)C1=CC(C)=C(O)C(C)=C1 4KK 0.365 COC1=CC=CC(CC(=O)NC2=NC(=CS2)C2=CC=NC=C2)=C1 824 0.365 OC1=CC2=C(NC3=C2C2=C(C(=O)NC2=O)C(=C3)C2=CC=CC=C2)C=C1 950 0.364 CC(C)COC1=CC=CC(C2=NC3=CC(F)=C(C=C3N2)C(N)=N)=C1O .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint 879 0.364 NC1=NC(CN2C(=CC=C2C2=CC=CC=C2Cl)C2=CC=C(OC3=CN=CN=C3)C=C2)=CC=C1 133 0.364 CC(C)COC1=C(O)C(=CC=C1)C1=NC2=CC(F)=C(C=C2N1)C(N)=N G4E 0.362 CC1=CC=CC(NC2=NNC(=N2)C2=CC=C(OC3=CC=NC=C3)C=C2)=C1C CJH 0.361 CCOC1=CC=CC(=C1)C1=CC=C(NC(=O)C(C#N)C(C)=O)C=C1 6QJ 0.356 CC1=CC(=CC=C1O)C1=CC=CC(=N1)C(=O)C1=CC=C(F)C(O)=C1 9ET 0.354 CC(=O)OC[C@]1(C)OC2=C(C=C1)C1=C(C=C2C)C2=C(N1)C=C(O)C=C2 YVQ 0.353 N1C=C(C=N1)C1=CN2C(C=N1)=NC=C2C1=CNC2=CC=CC=C12 Q4A 0.352 COC1=CC2=NC(=NC(NC3CCN(CC4=CC=CC=C4)CC3)=C2C=C1OC)N1CCCN(C)CC1 0VN 0.35 CC(C)(C)C1=CC=C(NC2=NC3=CC(=CC=C3N2)C#N)C=C1 QP8 0.347 CC(C)(C)OC(=O)N1CCN(CC1)C1=CC(=NN=C1N)C1=CC=CC=C1O 859 0.347 NC(=O)C1=CC=CC=C1NC1=CC=NC(NC2=CC=CC(O)=C2)=N1 6W3 0.346 CN1C(=CC2=C1C=CS2)C(=O)NC1=CC=CC=C1COC1=CC=C(OC2CCN(C)CC2)C=C1 C72 0.345 NC(=O)C1=C2SC(=CC2=C(N[C@H]2CCCNC2)N=C1)C1=CC=C(Cl)C=C1 28C 0.345 CC1=NN2C=NN=C2C(NCCC2=CC=C(O)C=C2)=C1 U81 0.343 BrC1=CC2=C(OCC[C@H]2NCCCNC2=CC(=O)C3=C(N2)C=CS3)C(Br)=C1 A3F 0.343 COC1=CC(=CC(OC)=C1OC)C1=CC(=CN=C1N)C1=CC=CC(O)=C1 6H2 0.343 OC1=CC=C(C=C1O)C1=CN2C=CC=CC2=N1 2YX 0.341 NC1=NC(=O)C2=CC3=C(NC(NCCC4=CC=C(C=C4)C#N)=N3)C=C2N1 ZZF 0.34 CC1=CC=C(OC2=CC=NC(NC3=CC=C(C=C3)S(N)(=O)=O)=C2)C(C)=N1 MCV 0.338 COC1=CC=C(OC)C(CCC2=CSC3=NC(N)=NC(N)=C23)=C1 8UN 0.338 C[C@@H](C1CCCCC1)N1C2=CC=C(C=C2N=C1C1=CC2=C(OCO2)C=C1Br)C(=O)NC1=CC=C(C=C1)C#N PKJ 0.337 CC1=CC=C(C=C1)C1=CSC2=NN=C(SCC(=O)NC3=CC=C4OCOC4=C3)N12 O1Q 0.332 CC1=CC=CC(=C1)N1N=CC=C1C1=CC(Cl)=C2N=NN(C2=C1)C1=CC2=NNC=C2C=C1 D62 0.332 COC1=CC=C(C=C1OC)C1=NN(C2CCN(CC2)C2=C3C=CSC3=NC(N)=N2)C(=O)[C@@H]2CC=CC[C@H]12 CK6 0.332 CNC1=NC(C)=C(S1)C1=CC=NC(NC2=CC=C(O)C=C2)=N1 826 0.332 OC1=CC=C(CN2C3=C(CCN(C3)C(=O)C3=CC=C(O)C=C3)C3=CC=CC=C23)C=C1 KTQ 0.331 COC1=CC=C(CCNC2=C(N=C3C=CC=CN23)C2=CC=C(C=C2)[N+]([O-])=O)C=C1 8HZ 0.331 CC1=CC=C(NC2=C(N=C3N2C=CC=C3C)C2=CC=C(O)C=C2)C=C1 5ES 0.33 OC1=CC=C(C=C1)C(=CC1=CC(NC2=CC=C(F)C=C2)=CC=C1)C1=CC=C(O)C=C1 5C4 0.33 CC(=C(C1=CC=C(O)C=C1)C1=CC=C(O)C=C1)C1=CC=CC(NC2=CC=CC=C2)=C1 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint 0N5 0.33 COC1=CC2=C(NC3=CC=C(NC(=O)C4=CC=CC=C4)C=C3)N=CN=C2C=C1O 70M 0.326 OC1=C(C=C2C(CCCCN3CCN(CC3)C3=CC=C(C=C3)C#N)=CNC2=C1)C#N LUO 0.325 COC1=CC=C(NC(=O)[C@H](C)NC2=NC(=O)C3=C(N2)N(N=C3)C2=CC=CC=C2C)C=C1 K0Q 0.325 OC1=CC=CN(CC(=O)NCCC2=CNC3=CC=CC=C23)C1=O 9JX 0.325 ClC1=CC=CC=C1C1=CC=CC(=C1)N1C[C@@H](CC1=O)N1CCN(CC1)C(=O)C1=NC=CS1 4G3 0.324 NC(=O)C1=CC=C(NCC2=CC(O)=CC=C2)N=C1 D4Q 0.323 NC(=O)C1=C2SC(=CC2=C(N[C@H]2CCCNC2)N=C1)C1=CC(F)=CC=C1 RY8 0.322 CC1=NC(=CS1)C1=CC=C(C=C1)C(=O)NC1=C(C)C(C)=CC(=C1)S(N)(=O)=O 1DY 0.322 COC1=CC=CC=C1NC(=O)C1=CC=C(NC(=O)CCC2=NC(=O)C3=CC=CC=C3N2)C=C1 JGZ 0.321 CC1=CC(=CC(C)=C1OC1=CC=NC(NC2CCN(CC2)C2=CC=C(C=C2)S(N)(=O)=O)=N1)C#N 9HP 0.32 OC1=CC=C(C=C1)C1=CC2=C(C=CC3=C2C=NC=C3)N=C1 O1S 0.319 CC1=NNC=C1C1=CC2=C(S1)C(=O)N=C(CN1CCCC1)N2 GUK 0.319 CC(C)NC1=CC(Cl)=NN2C(=CN=C12)C1=CC2=CNN=C2C=C1 F1Y 0.318 CCCC1=C(NC2=CC=C(C=C2)C(=O)NO)N2C=CC=CC2=N1 F45 0.317 OC1=CC=C(C=C1O)C1=NC(=CC=C1)C(=O)C1=CC=C(F)C(O)=C1 EL2 0.317 CNC(=O)C1=CC=C2N([C@@H]3CCC[C@@H](C3)NC(=O)C3=CC=C(Br)S3)C(=NC2=C1)C1=CC=CC=N1 AAI 0.317 CCCN1CCC(CC1)C1=NC2=C(C=CC=C2N1)C(N)=O CR3 0.316 NC(=N)C1=CC=C2NC(=CC2=C1)C1=C(O)C(OC2CCCC2)=CC=C1 0JA 0.316 FC1=CC=C(OC2=CC=C3N=C(NC(=O)C4CC4)SC3=N2)C=C1NC(=O)C1=C(Cl)C(=CC=C1)C1(CC1)C#N 3DX 0.315 CN1CCN(CC1)C1=CC=C(C=C1)C1=CC2=C(NC3=C2C=C(N=C3)C#N)N=C1 AI3 0.313 COC1=CC2=CN=C3C(CC4=C3C=C3OCOC3=C4)=C2C=C1OC 1NS 0.313 CS(=O)(=O)NCCC1CCN(CC1)C1=C2SC(=CC2=NC=N1)C(N)=O Q4M 0.312 COC1=CC=C2NC(=CC2=C1)C(=O)N1C[C@@H](CCl)C2=C3C=CNC3=CC=C12 ALH 0.312 CCCCC1=C(NC2=NC=CN=C12)C1=CC=C(O)C=C1 K1H 0.311 COC(=O)N1CCN(CC1)C1CCC(CC1)NC1=C2C=C(C=CC2=NC=N1)C#N E2J 0.311 CC1=C(CCN2CCC(CC2)=C(C2=CC=C(F)C=C2)C2=CC=C(F)C=C2)C(=O)N2C=CSC2=N1 O97 0.31 NC(=O)C1=CC2=C(C=CC=C2S1)C1=CC=C(S1)C(=O)NC1CC1 KTG 0.31 OC1=CC=C(CCCNC2=CC=CC3=C2C(=O)NC3=O)C=C1 4HN 0.31 CCOC1=CC=CC=C1C1=CC=C(C=C1)C1=C(C#N)C(=O)C2=CN=CC=C2N1 2WH 0.31 CCCN1C=NC(CCNC(=O)NC2=NC3=CC=C(C=C3S2)C2=CC(OC)=CN=C2)=C1 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint 9Y2 0.309 CC(C)(C)CC(=O)NC1=C(F)C=C(C=C1F)C(=O)NC1=NC=CS1 7FW 0.308 CN(CC1=CC2=NC=CN=C2C=C1)C(=O)C1=CC=C(C=C1)N(CC1=CC=CC=C1)C(=O)C1=CC=C(O)C=C1O H49 0.307 N=C(NC1=CC=CC(CNC[C@@H]2CCCN2)=C1)C1=CC=CS1 7IP 0.306 COC1=CC=CC(=C1)C1=CC=CC(CCC2=CC=CC(N)=N2)=C1 3X7 0.306 N#CC1=CC2=C(NC3=C2C=C(C=N3)C2=CC=C(CN3CCCCC3)C=C2)C=N1 15X 0.305 COC1=CC=C(CC2=CC=C(NC(N)=O)C=C2)C(F)=C1C1=CC=CC(=C1)N(=O)=O UPX 0.304 C\\C(=N/NC1=NC=CN1)C1=CC=C(NC(=O)C2=CC3=CC=CC(=C3N2)[N+]([O-])=O)C=C1 LKG 0.304 OC1=CC=C(CC2=NN=C3C=CC(=NN23)C2=CC=CC=C2)C=C1 S79 0.303 NC1=NC(=O)C2=CC3=C(NC(NCC4=C5C=CC=CC5=CC=C4)=N3)C=C2N1 KA5 0.303 COC1=CC=C(OC)C(CNC2=C(Cl)C3=C(N)N=C(N)N=C3C=C2)=C1 5XQ 0.303 COC1=CC=CC=C1NC1=NC(=CS1)C1=C(C)N=C(NC(C)=O)S1 625 0.303 NC1=NC(CN2C(=CC=C2C2=CC=CC=C2Cl)C2=CC=C(OCCCC#N)C=C2)=CC=C1 N55 0.302 OC1=CC=C(C=C1Cl)C1=CC=C(C=C1)[C@@H](C=C)N1C=CN=C1 FZ9 0.302 C1CC(=CCN1C1=NC=NC2=NNC=C12)C1=CNC2=CC=CC=C12 2YO 0.302 O=C1N=CNC2=CC3=C(C=C12)N=C(NCCN1CCCCC1)N3 N58 0.301 CN1N=C(C=C1NC(=O)NC1=CC=C(SC2=CC=NC=C2)C=C1)C(C)(C)C EYF 0.301 COC1=CC=CC(CN2C=CC3=CC(=CN=C23)C(=O)NO)=C1 2YM 0.301 NC1=NC(=O)C2=CC3=C(NC(NCCN4CCCCC4)=N3)C=C2N1 4L7 0.3 BrC1=CC=C(S1)C(=O)[C@H]1CNC[C@@H]1C1=CC=C2C(=O)N=CNC2=C1 926 0.3 CN1C=CN=C1SC1=CC=CC(=C1)C1=CC=CC(=O)N1 Y6V 0.299 O=C(N(CC1=CC=CS1)C1CC1)C1=CC=C2NC(=O)COC2=C1 IEO 0.299 NC(=O)C1=C2NC=C(C#N)C2=C(OC2CCNCC2)C=C1 HZY 0.299 COC1=CC(C[N+]2=CC=C3C=C(OS(N)(=O)=O)C=CC3=C2)=CC(OC)=C1OC H2K 0.297 ClC1=CC(NC(=O)NC2=CN=C(C=N2)C#N)=C(O[C@@H]2CCNC2)C=C1 8E6 0.297 NC(=O)C1=C2N=C(NC2=CC(F)=C1)C1=CC2=C(CCNC2)S1 37R 0.297 NC(=O)CCCCC1=C(O)C=C2OC3=C(NC2=C1)C=CC(O)=C3 2YV 0.297 NC1=NC(=O)C2=CC3=C(NC(NCCC4=CC=CC=C4)=N3)C=C2N1 1RQ 0.296 OC1=CC=C2N=C(N(C2=C1)C1=CC=NC(N[C@@H]2CCCN(C2)C(=O)C2CC2)=N1)C1=CC2=CC=CC=C2C=C1 07Q 0.295 COC1=CC(=CC=C1)[C@@H](C)NC(=O)NC1=NC(=CS1)C1=CC=NC=C1 ZZW 0.294 ONC(=O)C1=CC2=C(C=N1)N(CC1=CC=C(F)C=C1)C1=C2C=CC=C1 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint DLN 0.294 CC(C)N1N=C(C#CC2=CC=CC(O)=C2)C2=C(N)N=CN=C12 9N2 0.294 OC1=CC(=CC=C1F)C(=O)C1=NC2=CC(=CC=C2C=C1)C#N 3KC 0.294 OC1=CC=C(C(Cl)=C1)C1=NC2=C(C=CC=C2)C2=CC=NC3=C2C1=CN3 N61 0.293 CN1N=C(C=C1NC(=O)NC1=CC=C(OC2=CC=NC=C2)C=C1)C(C)(C)C EWV 0.293 COC1=CC2=C(C=C1OC)C1=CC3=CC=C4OCOC4=C3C=[N+]1CC2 770 0.293 ClC1=CC=CC=C1C1=CC2=C(C3=C(N2)C=CC(NC=O)=C3)C2=C1C(=O)NC2=O QK0 0.292 CC(=O)NC1=NC2=CC=C(C=C2S1)C1=CC(NS(=O)(=O)C2=CC=C(F)C=C2)=C(Cl)N=C1 8TK 0.292 C[C@H](NC1=C(C#N)C(N)=NC=N1)C1=C(C(=O)N2C(C)=CSC2=C1)C1=CC=CC=C1 0WP 0.292 COC1=C2C=C(NC(=O)C3=C(F)C(NC(=O)C4=CC(=CC=C4)C(C)(C)C#N)=CC=C3F)C=NC2=NN1 P8Z 0.291 CN1CCN(CC1)C1=CC=CC=C1CNC1=C(C)C(=O)N(C)N=C1 JVT 0.291 CN1CCN(CC1)C1=CC=C(NC(=O)C2=NNC3=CC=CC=C23)C=C1 DTM 0.291 COC1=CC(CN(C)C2=CC3=C(N)N=C(N)N=C3N=C2)=CC(OC)=C1OC 9MK 0.291 CC1=CC2=CC=C(N=C2C=C1)C(=O)C1=CC=C(F)C(O)=C1 8PV 0.291 O=C(NCC1=CC=CC=C1)C1=CC=CC(NCC2=NN=C(N2)C2=CC=NC=C2)=C1 VXS 0.289 OC1=CC=CC=C1CCCNC1=CC=CC2=C1C(=O)NC2=O MEY 0.289 OC1=CC(O)=C(Cl)C=C1N1C(=O)NC2=CC(CNS(=O)(=O)C3=C4C=CC=CC4=CC=C3)=CC=C12 6GE 0.289 CC1=CC=C(O)C=C1NC1=CC=NC2=CC=C(C=C12)S(C)(=O)=O 5DG 0.289 CC(=C(C1=CC=C(O)C=C1)C1=CC=C(O)C=C1)C1=CC=CC(NC2=CC=C(F)C=C2)=C1 N9W 0.288 CN1CCC2=C(C1)C1=CC=CC=C1N2CC1=CC=C(C=C1)C(=O)NO JCW 0.288 NC(=O)C1=CC(=NC=C1)C1=CC(=CN=C1F)[C@H]1C[C@@H]2CC[C@H]1N2 7DZ 0.288 CC(C)CCN1[C@@H](C)C(=O)N(C)C2=CN=C(NC3=CC(F)=C(O)C(F)=C3)N=C12 FJU 0.287 CCC1=CC(NC(=O)C2=CC3=CC=CC(O)=C3OC2=N)=CC=C1 64N 0.287 CCC(=O)NC1=CC(Cl)=C(OC2=CC=C(O)C(=C2)C2=CC=C(C=C2)C(N)=O)C(Cl)=C1 MDY 0.286 COC1=CC=CC=C1C1=CC=C(NC(=O)C(C#N)C(C)=O)C(Cl)=C1 5GD 0.286 COC1=CC(OC)=C2C(=O)N=C(NC2=C1)C1=CC(C)=C(OCCN2CCCC2)C(C)=C1 58W 0.285 OC1=CC(O)=C(Cl)C=C1NC(=O)C1(CCC1)C1=CC=CC=C1 2KL 0.285 CCNC(=O)C1=CC2=C(N=C(N)N=C2S1)C1=CC(OCCN2CCCC2)=C(Cl)C=C1Cl LXX 0.284 N1C=C2C=C(C=NC2=N1)C1=CC2=C(C=CN=C2C=C1)C1=CC=NC=C1 L26 0.284 CCC1=C(NC(C)=C1C(C)=O)C(=O)NC1=CC=CC(OCC(N)=O)=C1 5NW 0.284 COC1=CC(=CC=C1NC1=NC2=C(NCC(C)(C)C)N=CC=C2C=N1)C1=CN(C)N=C1 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint 4Q3 0.284 CC(C)N1N=C(NC(C)=O)C=C1C1=CC=C(N(C)C(=O)C2=C(F)C=CC=C2Cl)C(=C1)N1C[C@@H]2C[C@@H]2C1 17V 0.284 CC(C)N1N=CN=C1C1=CC2=C(S1)C1=CC=C(C=C1OCC2)C(N)=O 75X 0.283 CC1=C(C=CC=C1O)C1=NC2=NC(N)=NC(OCC3CCCCC3)=C2N1 GQW 0.282 ONC(=O)C1=CC=C(Cl)C(NC(=O)C2=C3SC=CC3=CC=C2)=C1 4H7 0.282 CN1CCN(CC1)C1=CC=CC=C1CNC1=CC=C(C=N1)C(N)=O GXK 0.281 ClC1=NN2C(=CN=C2C(NCC2CC2)=C1)C1=CC2=CNN=C2C=C1 G6J 0.281 CCCN1[C@H](C)C(=O)N(CCC)C2=CN=C(NC3=CC(F)=C(O)C(F)=C3)N=C12 0CV 0.281 COC1=CC2=C(N3CCNCC3)C3=C(C)NN=C3N=C2C(C)=C1 J2V 0.28 COC1=CC=C(C=N1)C1=C(NC2=CC=CN=C12)C1=CC=NC(NC(C)=O)=C1 G6T 0.28 NC1=NC(OCC2CCCCC2)=C2NC(=NC2=N1)C1=CC(=CC=C1)S(N)(=O)=O 5CC 0.279 CCC(=C(C1=CC=C(O)C=C1)C1=CC=C(O)C=C1)C1=CC=CC(NC2=CC=CC=C2)=C1 43A 0.278 COC1=CC(=CC=C1O)C1=CC=C2C(NC(=O)C2=CC2=CC=CN2)=C1 41E 0.278 COC1=CC(NC(=O)CCC2CCN(CC2)C(=O)C2=CC=C(S2)C(C)=O)=CC=C1 SV8 0.277 O=C1NCC2=C1NC=C2C1=CN=C2NC=C(CC3=CC=CC=C3)C2=C1 DUL 0.277 CCSC1=CC=CC(=C1)C1=CC=C(S1)C(N)=O V5G 0.276 NC1=NC(CCC2=CC(OCC3=CC=C4C=CC(N)=NC4=C3)=CC=C2)=CC=C1 HVU 0.276 CN1CCC(CC1)C1=CNC2=CC=C(O)C=C12 4P9 0.276 OC1=CC=C(\\C=N\\NC2=CC=C(Cl)C=C2)C(O)=C1O 43H 0.276 OB(O)C1=CN(C=C(O)C1=O)C1=CC=CC(=C1)C1=CC=CC=C1 8C1 0.275 CC(C)OC1=CC2=C(O[C@@H]3CCCNC3)C=CN=C2C=C1C(N)=O 43R 0.275 COC1=CN=CC(=C1)N1CCC2=C(C1)SC(NC(C)=O)=N2 0NV 0.275 C(N1CCC(CC1)N1C=NC2=C1C1=C(NC=C1)N=C2)C1=CC=CC=C1 99S 0.274 COC(=O)C1=CC=C(S1)C1=CC(=CC(=C1)C#N)[C@]1(C(C)C)C2=C(C)NN=C2OC(N)=C1C#N 584 0.274 CC(C)CCN1[C@H](C)C(=O)N(C)C2=CN=C(NC3=CC(F)=C(O)C(F)=C3)N=C12 KT4 0.273 OC1=CC=C(CCCNC2=CC=CC3=C2C(=O)NC3=O)C(O)=C1 GQB 0.273 Nc1[nH]c2ccc(CNC(=O)C3=CC4=CC=CC=C4N3)cc2s1 E5M 0.273 C[C@@H]1N(C)C2=NC(NC3=CC(F)=C(O)C(F)=C3)=NC=C2N(C)C1=O COQ 0.273 COC1=CC(OC)=CC(CN(C)C2=CC3=C(N)N=C(N)N=C3N=C2)=C1 NVV 0.272 IC1=CC=CC(=C1)C(=O)[C@H]1CNC[C@@H]1C1=CC=C2C(=O)N=CNC2=C1 R8E 0.271 NC1=NC2=NNC(COC3=CC(OC4=CC(=CC(Cl)=C4)C#N)=C(Cl)C=C3)=C2C=C1 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint JZH 0.271 OC1=CC=C(C=C1)[C@H]1C2=CNC3=NC=CC(=C23)C2=CC=CC=C2NC1=O 8Q7 0.271 COC1=CC=C(CN2C3=C(C4=C(CN(CC4)C(=O)[C@@H]4CC4(F)F)S3)C3=NC=NN3C2=O)C=C1 5JN 0.271 CC1=CC(NC(=O)CC2=CC=C(OC(C)(C)C(=O)OCCO[N+]([O-])=O)C=C2)=CC(C)=C1 4FX 0.271 COC1=CC=CC=C1CNC1=CC=C(C=N1)C(N)=O NUM 0.27 OC1=CC=C(CCNC(=O)C2=CC=CC=N2)C=C1 E78 0.27 C[C@@H](C1CC1)N1CC2=C(C1=O)C(C)=CC(=C2)C1=C(C)N=C(NC(C)=O)S1 35O 0.27 COC1=CC=C(NC(=O)[C@@H](C)NC2=NC(=O)C3=C(N2)N(N=C3)C2CCCC2)C=C1 JEW 0.268 NC1=NNC(=C1)C1=CC=C2C=CN(CC3=CC=CC=C3C#N)C2=C1 5O1 0.267 COC1=CC(=CC=C1NC1=NC2=C(NC3CCCCC3)N=CC=C2C=N1)C1=CN(C)N=C1 3ND 0.267 ClC1=CC=C(C=C1)[C@@H]1CNC[C@H]1C(=O)NC1=C(Cl)C=C2C(=O)NC=CC2=C1 3DC 0.267 N(C1=CC=NC=C1)C1=NC=NN2C=C(C=C12)C1=CNN=C1 30J 0.267 C[C@@H]1CN(CCN1C1=CC=C(C=N1)C#N)C1=C2C=C(Cl)C=CC2=C(CC2=CC=CC=C2)N=N1 16L 0.267 CC1=CC(\\C=C\\C2=CC=CC(NC(=O)C=C)=C2)=CC(C)=C1O 9F4 0.266 ONC(=O)[C@@H]1[C@@H]([C@H]1C1=CC=C(C=C1)C1=NC=C(F)C=N1)C1=CC=CC=C1 3XK 0.266 CC(=O)NC1=CC=CC(=C1)C1=CC2=C(NC3=C2C=C(N=C3)C#N)N=C1 S1W 0.265 CN(C)C1=CC=CC(OC2=CN=C(NC3=CC=CC(O)=C3)N=C2)=C1 CO4 0.265 COC1=CC(=CC(OC)=C1OC)N(C)CC1=C(C)C2=C(N)N=C(N)N=C2N=C1 666 0.265 C[C@@H]1CC(=O)NN=C1C1=CC=C(NC2=C(CC3=CC=CC(I)=C3)C(=O)CCC2)C=C1 ZSO 0.264 FC1=CC=C(C=C1)[C@@H]1CCNC[C@H]1COC1=CC2=CNN=C2C=C1 LTQ 0.264 CC1=CC(=CC=C1N1CCN(CC1)S(=O)(=O)N1CSC[C@@H]1C(=O)NO)C#N 9ST 0.264 CN1C[C@@H](C[C@@H](C1)C1=CC=CC=C1)NC1=C(Br)C(=O)N(C)N=C1 5B2 0.264 NC1=NNC2=CC=C(C=C12)C1=CC=CC=C1 15F 0.264 ClC(Cl)(Cl)C(=N)OCC1=CC=[N+](CC2=CC=CC=C2)C=C1 YDJ 0.263 NC(=O)NC1=C(SC(=C1)C1=CC(F)=CC=C1)C(=O)N[C@H]1CCCNC1 RKZ 0.263 C[C@H](CN1C(C)=NC2=CC=C(N=C12)C1=CC(N)=NC(N)=C1)OC1=CC=CC=C1 6JU 0.263 ONC(=O)CN1C=C(CC2=CC=CC=C2)C2=CC(Br)=CC=C12 5H2 0.263 COC(=O)NC1=CC=C2C3=C(CCOC2=C1)C=C(S3)C1=NN=CN1C1=CC=CC=C1Cl 3FR 0.263 C[C@H](NC1=NC=C2C(C)=NN(C2=C1)C1=CC(=CC(Cl)=C1)C1=CC=CC=C1C(N)=O)C1=CC=CC=C1 304 0.263 CC[C@@H](C)NC1=NC=C(S1)C(=O)NC1=CC(=CC=C1C)C(=O)NC 92Q 0.262 CC1=CC=CC(Cl)=C1NC(=O)C1=CN=C(NC2=CC=CC(=C2)C(=O)N[C@H]2CCNC2)S1 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint ZVZ 0.261 CNC(=O)COC1=CC=C(C(CN2CCN(CC2)C2=CC=CC=N2)=C1)C1=CC=C(C=C1)C#N BER 0.261 COC1=C(OC)C2=C[N+]3=C(C=C2C=C1)C1=C(CC3)C=C2OCOC2=C1 3GN 0.261 C[C@]1(CCCN1)C1=NC2=C(C=CC=C2N1)C(N)=O S8B 0.26 CC(C)NC(=O)C1=CC=C(C=N1)C#CC1=CC=NC=C1 RT3 0.26 CC1=CC=C2N(CCCC2=C1)C(=O)SCC(=O)NC1=CC=C(C=C1Cl)S(N)(=O)=O GQ5 0.26 C[C@H]1CCCN1CC1=NC2=CC(NC(=O)C3=CC=C4N(C)N=CC4=C3)=CC=C2N1 3L0 0.26 C[C@H]1N2C(COC3=CC(C4=CC=CC=C4)=C(C=C23)N(C)C2(C)CN(C)C2)=NNC1=O 3E1 0.26 CC(=O)NC1=CC=C(O)C(=C1)C1=CC(NC(C)=O)=CC=C1O 18A 0.26 CCC(=O)NC1=CC=C(\\C=C\\C2=CC(C)=C(O)C(C)=C2)C=C1 M94 0.258 CNCCC1=CC=CC(OCC2=CC3=NC(N)=CC=C3C=C2)=C1 5B4 0.258 CN1C=C(C=N1)C1=CC(OC2=CC(F)=C(NC(=O)C3(CC3)C(=O)NC3=CC=C(F)C=C3)C=C2F)=CC=N1 1NR 0.258 CC(C)(C)C(=O)NCCC1CCN(CC1)C1=C2SC(=CC2=NC=N1)C(N)=O A27 0.257 CCCC(=O)NC1=NNC2=CC(C3=CC=C(O)C=C3)=C(C=C12)C1=CC=CC=C1 BAI 0.256 NC(=N)C1=CC=C2NC(CC3=NC4=CC=CC=C4N3)=NC2=C1 5H5 0.256 CC(C)N1N=CN=C1C1=CN2CCOC3=CC(O[C@@H](C)C(N)=O)=CC=C3C2=N1 LKQ 0.255 COC1=NN(C)C=C1NC1=NC=C(C)C(=N1)C1=CNC2=C(NC(=O)C3=CC=CN=C3C)C=CC=C12 7DQ 0.255 CC(C)C1=CC=C(CC(=O)N2CCN(CC2)C2=CC=C(N=N2)C#N)C=C1 R69 0.254 CN(C)C1=CC=C(O)C(=C1)C(=O)C1=CC=C(C=C1)C(=O)N[C@@H]1CCCNC[C@H]1NC(=O)C1=CC=NC=C1 JA1 0.254 CCCCCCC1=CC=C(OC2=CC=C(C=C2)[N+]([O-])=O)C(O)=C1 EMW 0.254 COC1=CC=C(NC(=O)N2CCN([C@@H](C)C2)C2=C3C=NN(C)C3=NC(N)=N2)C(C)=C1 ABZ 0.254 NC1=NC(NC2=CC=C(C=C2)C#N)=NC(CC2=C3C=CNC3=CC=C2Cl)=N1 9LP 0.254 OS(=O)(=O)OC1CCC(CC1)NC(=O)[C@H](C1CCCCC1)N1C2=CC(F)=C(F)C=C2N=C1C1=CC=C(Cl)C=C1 KT1 0.253 COC1=CC(O)=CC=C1CCCNC1=CC=CC2=C1C(=O)NC2=O 68M 0.253 C[C@H](CC1=CC2=CC(=CC=C2N=C1N)C1=NC=CC=C1C)C(=O)NCCC(C)(C)C 58G 0.253 NC(=O)C1=CC=C(N[C@@H]2CC[C@@H](C2)C2=CC=CC=C2)N=C1 50B 0.253 COC1=C(OC)C2=C[N+]3=C(C(CCCC(C4=CC=CC=C4)C4=CC=CC=C4)=C2C=C1)C1=CC2=C(OCO2)C=C1CC3 SK6 0.252 CC1=CC(CCC2=CC(=CC(NCCC3=CC=CC=N3)=C2)C#N)=NC(N)=C1 4G4 0.252 CN(C)C1=CC=CC=C1CNC1=CC=C(C=N1)C(N)=O Z7H 0.251 COC1=CC=C(OC2=C(Cl)C=C(NC(=O)CC3=CC=CN=C3)C=C2Cl)C=C1C(C)C U1T 0.251 CC1=C2C=C(NC(=O)C2=CC=C1)C1=CC=C(CN2CCCC2)C=C1 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint MXI 0.251 COC1=CC2=CC=C(C=C2C=C1)C1=C(NC(=N1)C(C)(C)C)C1=CC=NC(N)=C1 CTI 0.251 COC1=C(OC)C2=C(C=C1)C1=C(C3=C(C=C1)C=C1OCOC1=C3)[N+](C)=C2 78P 0.251 C[C@@]1(CCCN1)C1=NC2=C(C=CC=C2N1)C(N)=O 3FV 0.251 ClC1=CN(N=C1C(=O)NC1=CNN=C1C1=CC=CC=N1)C1CCNCC1 WXV 0.25 CNC(=O)C1=NN(C)C2=C1C=CC1=C2N=C(NC2CCN(CC2)S(C)(=O)=O)N=C1 NVS 0.25 COC1=CC=C(C=C1)N1CC2=CC=C(O)C=C2OC1=O EX7 0.25 OC1=C2N=C(CCC3=CC(Cl)=CC=C3)NC2=CC=C1 43G 0.25 OC1=CN(C=CC1=O)C1=CC(=CC=C1)C1=CC=CC=C1 JC8 0.249 NC(=O)C1=CN=C(C=C1)C1=CC(=CN=C1F)[C@H]1C[C@@H]2CC[C@H]1N2 E89 0.249 COC1=CC=C(CCC2=C3NC=NC3=CC3=C2NC(N)=NC3=O)C=C1 C51 0.249 C[C@H](C1CCC(CC1)C1=CC=NC2=CC=CC=C12)C(=O)NC1=CC=C(C=C1)C#N RKK 0.248 CCCCN1C(CCCC)=NC2=CC=C(N=C12)C1=CC(N)=NC(N)=C1 9ME 0.248 OC1=CC(=CC=C1F)C(=O)C1=NC2=CC=CC=C2C=C1 6GH 0.248 NC1=NC(=CC(=N1)C1=CSC=C1)N(CC1=CC=CC=N1)CC1=CC=CC=N1 924 0.248 NC1=C2C(SC=C2C2=CC3=C(C=C2)N(CC3)C(=O)CC2=CC=CC=C2)=CC=N1 H5G 0.247 OC1=CC=CC(CC(=O)NC2=CC=CC(Cl)=C2)=C1 52D 0.247 O=C(C1=CC=NC=C1)C1=CC=C(OCCN2CCCC2)C=C1 19Z 0.247 NC(=O)C1=CC(=CC=C1NCC1=CC=C(O)C=C1)[N+]([O-])=O QV5 0.246 CC(C)OC1=CC(=CC=C1C1=C(NC2=CC(F)=C(Cl)C=C12)C(N)=O)N1CCNCC1 H0M 0.246 OC1=CC(CC(=O)NC2=CC=CC=N2)=CC=C1 D90 0.246 CN1CCC2=C(C1)SC(=N2)C(=O)NC1=CC=CC=C1NC(=O)C1=CC2=CC(Cl)=CC=C2N1 6WW 0.246 CN([C@H]1CC[C@H]2[C@@H]3CCC4=CC(O)=CC=C4[C@H]3CC[C@]12C)C1=CC=CC=C1 6BU 0.246 O=C(NC1=NNC2=CC(=CC=C12)C1=CC(NS(=O)(=O)C2CC2)=CC=C1)C1CC1 2HV 0.246 CC(=O)NC1=NC(=CS1)C(=O)NC1=CC=CC=C1N1CCCCC1 9HJ 0.244 CCCN(C)C1=NC2=CC(OC)=C(OC)C=C2C(NC2CCN(C)CC2)=N1 8ZZ 0.244 CC1=CC=CC(Cl)=C1NC(=O)C1=CN=C(NC2=CC=CC(=C2)C(=O)N[C@@H]2CCNC[C@H]2F)S1 8ZH 0.244 CC1=CC=CC(Cl)=C1NC(=O)C1=CN=C(NC2=CC=CC(=C2)C(=O)NC2CCNCC2)S1 65O 0.244 CC1=C(SC2=CC=CC3=CC=CC=C23)SC2=NC(N)=NC(N)=C12 2UQ 0.244 N=CC1=NC=C(C=N1)C(=O)NCC1=CC=CC=C1 VXV 0.243 OC1=CC(Cl)=CC(CCCNC2=CC=CC3=C2C(=O)NC3=O)=C1 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint FAZ 0.243 CN(C(=O)C1=CC2=C(S1)C1=CC=C(NC(C)=O)C=C1OCC2)C1=CC=CC=C1Cl C4Q 0.243 C(N1CCNCCNCCNCC1)C1=C2C=CC=CC2=NC=C1 45R 0.243 COC1=CC(=CC=C1C(=O)NC1=CC=C2CCNCCC2=C1)C1=CNN=C1 Z68 0.242 NC1=C(SC(NC2CCCCC2)=N1)C(=O)C1=CC(=CC=C1)[N+]([O-])=O QPC 0.242 CC(C)(C)CNC(=O)CC1=CC=C(NC2=C(C=NC(=N2)C2=CC=CC=C2)C(N)=O)C=C1 JHT 0.242 CN(C)C1=CC=CC(CNCC2=CC3=NC(N)=CC=C3C=C2)=C1 9P1 0.242 CN(C)C1=CC=C(CNCC2=CC3=NC(N)=CC=C3C=C2)C=C1 76B 0.242 CC1=CC(=CC=N1)C1=NN=C(CCNC(=O)C2=CC(=CC(O)=C2O)C2=CC=C(F)C=C2)N1 7U6 0.241 NC(=O)C1=C2N=C(NC2=CC=C1)C(=O)N1CCNCC1 5UO 0.241 ClC1=CC=C(C=C1)C1CCN(CCC2=CN(N=C2)C2=C3NC=NC(=O)C3=CC=N2)CC1 516 0.241 CN[C@@H](C)C(=O)N[C@@H](C1CCCCC1)C(=O)N1CCC[C@H]1C1=NC(=CS1)C1=CC=CC2=CC=CC=C12 W4G 0.24 O=C1N(CC2=CC=CC=C2)C=NC2=CC(=CC=C12)C1=CNN=C1 T61 0.24 ONC(=O)C1=CC=C(Cl)C(NC(=O)C2=CC=CC=C2)=C1 IDK 0.24 O=C(NC1=NNC2=CC(=CC=C12)C1=CC(NS(=O)(=O)CC2CC2)=CC=C1)C1CC1 8CG 0.24 CC[C@H]1[C@@H](COC2=C3C=C(OC)C(=CC3=CC=N2)C(N)=O)NC(=O)[C@H]1F RFM 0.239 O=C(CN1N=NC2=CC=CC=C12)N(CC1=CSC=C1)C1=CC=C(NC(=O)C2=CC=CC=C2)C=C1 O1H 0.239 ClC1=C2N=NN(C2=CC(=C1)C1=CC=NN1CC1=CC=C(C=C1)C#N)C1=CC2=NNC=C2C=C1 KZQ 0.239 CC1=C([C@@H](NC(=O)N1)C1=CC=C(F)C=C1)C(=O)NC1=CC2=CNN=C2C=C1 426 0.239 NC(=N)C1=CC2=CC=C(C=C2C=C1)C(=O)NC1=CC=CC(OC2CCCC2)=C1 VUG 0.238 CC1=CC=CC(=C1)C1CCC(CC1)N1CCN(CC1)C1=CC=CN=C1 4G0 0.238 CN(C)C1=CC=CC(CNC2=CC=C(C=N2)C(N)=O)=C1 VXM 0.237 OC1=CC=C(F)C=C1CNC1=CC=CC2=C1C(=O)NC2=O QWI 0.237 C1C[C@H](CC2(CCN(CC2)C2=C3NC=NC3=NC=N2)N1)C1=CC=CC=C1 IXF 0.237 CC(C)C1=CC=C(C=C1)N(C)C1=CC2=C(N)N=C(N)N=C2N=C1 7O3 0.237 OC1=CC=C(Cl)C(NC2=C3C=CC(NC(=O)CCl)=CC3=NC=N2)=C1 TWP 0.236 ONC(=O)C1=CC=C(CN2C=CC3=CC=CC=C23)C=C1 PRD 0.236 COC1=CC=C(OC)C(CN(C)C2=CC3=C(N)N=C(N)N=C3N=C2)=C1 DEH 0.236 COC1=CC=C2C=C3N(CCC4=C3C=C(O)C(OC)=C4)C=C2C1=[OH+] 8QE 0.236 CC(C)(C)NC1=C(C#N)C(N)=CC(NC2=CC=C(C=C2)C(N)=O)=N1 TMU 0.235 COC1=CC=C(CNC(=O)NC2=NC=C(S2)[N+]([O-])=O)C=C1 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint M8G 0.235 ONC(=O)\\C=C\\C1=CC=CC=C1NC(=O)C1=CC=CC=C1OC1=CC=CC=C1 K8M 0.235 FC1=CC(=CC(=C1)C1=CC=NC=C1)C(=O)N1C[C@@H](CC(F)(F)C1)C(=O)NC1=CC=C(Cl)C=C1 5UL 0.235 ClC1=CC=C(CC2CCN(CCC3=CN(N=C3)C3=C4NC=NC(=O)C4=CC=N3)CC2)C=C1 XU2 0.234 O=C(NC1=CC=C(NC2=CC=NC3=C2C2=C(C=CC=C2)C(=O)N3)C=C1)C1=CC=CC=C1 X5M 0.234 CCN(CC)C1=CC=C(C=C1[N](O)=O)C1=NNC(=O)C[C@H]1C WDY 0.234 O=C(NC1=CC=CC(=C1)C1=NNC=C1)C1=CC=CC2=CNN=C12 TJH 0.234 OC1=CC=CC2=C1C(=O)C=C(NCCNC1=C3C=CC(Cl)=CC3=NC3=C1CCCC3)C2=O HJM 0.234 CC[C@H]1C[C@@H]2C[C@@H]3[C@H]1N(C2)CCC1=C3NC2=CC=C(OC)C=C12 5Q2 0.234 CN1CCN(CC1)C1=CC=C(NC(=O)C2=C(NCCC3=CC=C(Cl)C=C3)C=CNC2=O)C=C1 16X 0.234 CC1=C(SC(N)=N1)C1=CC=NC(NC2=CC=CC(=C2)N2OO2)=N1 0P8 0.234 COC1=C(CNCCCNC2=CC(=O)C3=CC=CC=C3N2)C=C(Br)C=C1Br S9H 0.233 COC1=CC=C(NC2=NC=CC(=N2)C2=CC=C3NC(=O)CCCC3=C2)C=C1 279 0.233 CCNC(=O)C1=CN2N=CN=C(NC3=CC(=CC=C3C)C(=O)NOC)C2=C1C Q26 0.232 CCOC1=CC=C(C=C1C1=CC2=NC(N)=NC(N)=C2C=C1)C1=CC=CC(NS(C)(=O)=O)=C1 KSY 0.232 OC1=CC=CC(CCCNC2=CC=CC3=C2C(=O)NC3=O)=C1 E1D 0.232 C[C@H]1N(C)C2=NC(NC3=CC(F)=C(O)C(F)=C3)=NC=C2N(C)C1=O 9S1 0.232 NC1=CC=C2NC(=CC2=N1)C1C=NN(C1=O)C1=CC(CNC(=O)NC2=CC=CC=C2)=CC=C1 QRX 0.231 CCCN1C(CNC2=CC=CC(=C2)C(=O)NCC2=C(F)C=CC=C2F)=NN=C1C1=CC=NC=N1 LKT 0.231 ClC1=CC=CC(N2CC[C@@H](C2)[C@H](CC#N)N2C=C(C=N2)C2=C3C=CNC3=NC=N2)=C1C#N LD2 0.231 O=C1COC2=CC=C(C=C2N1)C(\\CC1=CC=CC=C1)=N\\N1CN=NC1=S K4A 0.231 CCN1N=CC(=C1C)C1=NC2=C(N=CN=C2N1C)N1CCC(CC1)N1C(=O)NC2=NC=CC=C12 KTM 0.23 OC1=CC=CC=C1CNC1=CC=CC2=C1C(=O)NC2=O GVK 0.23 ClC1=CC=C(C=C1)C1(CCNCC1)C1=CC=C(C=C1)C1=C2N=CNC2=NC=N1 FH3 0.23 CN1C=C(C(C)=N1)C1=NC2=NC=C(Cl)C(N3CC[C@@H](CNC(C)=O)C3)=C2N1 7KZ 0.23 CCCCCCNC1=NC2=CC(OC)=C(OC)C=C2C(NC2CCN(C)CC2)=N1 65C 0.23 CC1=CNC(=N1)C1=CN=C(NCCNC2=CC=C(C=N2)C#N)N=C1C1=CC=C(Cl)C=C1Cl 4YU 0.23 COC(=O)C=CCNC(=O)C1=C(C)N(C)C2=CC=C(OC)C=C12 W45 0.229 SCC(=O)NCCCCCN1C=CC2=CC(Cl)=C(Cl)C=C12 328 0.228 COC1=CC=NC(NC2CCN(CC2)C(=O)C2=CC=C(C=C2)C#N)=C1 SM6 0.227 CCOC(=O)C1=C(NC2=CC3=CCC(NO)=C3C=C2)C2=CC=NC=C2S1 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint JCE 0.227 NC1=NNC(=C1)C1=CC=C2C=CN(CC3=CC=CC=N3)C2=C1 4TV 0.227 CN1C=C(C=N1)C1=CC=C(C=C1)C1=CN=CC(Cl)=C1N1CCC2(CCNC2=O)CC1 T27 0.226 CC1=CC(\\C=C\\C#N)=CC(C)=C1NC1=CC=NC(NC2=CC=C(C=C2)C#N)=N1 JDQ 0.226 NC1=NNC(=C1)C1=CC=C2C=CN(CC3=CC=CC=C3)C2=C1 81F 0.226 CC1=CC=CC(=C1)C1CCC(CC1)N1CCN(CC1)C1=CC(Br)=CN=C1 5TD 0.226 O=C(CCC1CCN(CC1)C1=CC=C(C=C1)C#N)N1CCCC1 56Y 0.226 CCC1=C(NC(C)=C1C(C)=O)C(=O)N(C)CC1=CC=CC(=C1)C(N)=O C9J 0.225 CN1C=CC2=CC=C(N=C2C1=O)C1=CN(N=N1)C1=CC=C(O)C(F)=C1 6WV 0.225 C[C@]12CC[C@H]3[C@@H](CCC4=CC(O)=CC=C34)[C@@H]1CC[C@@H]2NC1=CC=CC=C1 YKC 0.224 O=C(N[C@@H]1C2=CC=CC=C2C2=C(C=CC=C12)C1=NC2=CC=NC=C2N1)C1=CC=NC=C1 VM4 0.224 C[C@@H](SC1=NC2=C(C=NN2C)C(=O)N1C1=CC=CC=C1)C1=CC=CC(O)=C1 L74 0.224 ClC1=CC=C(C=C1)N1CCN(CC2=CNC3=NC=CC=C23)CC1 H6K 0.224 O=C(NC1=NNC(=C1)C1=CC=CC=C1)NC1=CN=C(C=N1)C#N EKT 0.224 CC(C)CCN1C2=C(C=NC(NC3=CC(F)=C(O)C(F)=C3)=N2)N(C)C(=O)C1(C)C EDH 0.224 CCCN1C(NC(=O)C2=CC=CC(=C2)C(N)=O)=NC2=CC=CC=C12 C73 0.224 NC(=O)NC1=C(C=C(S1)C1=CC=C(Cl)C=C1)C(=O)N[C@H]1CCCNC1 WR2 0.223 CNC(=O)[C@@H](NC(=O)[C@H](CC(C)C)[C@H](CNC(=O)C1=NC=CS1)C(=O)NO)C(C)(C)C RK8 0.223 C[C@@H](CN1C(C)=NC2=CC=C(N=C12)C1=CC(N)=NC(N)=C1)OC1=CC=CC(Br)=N1 QKQ 0.223 COC1=CC=C(C=C1)C1=CN=CC2=CC=C(N=C12)C(=O)NCCCN1C=CN=C1 7Z0 0.223 COC1=NNC2=NC=C(C=C12)C#CC1=C(F)C=CC(NS(=O)(=O)C2=CC=CC(Br)=C2)=C1F 64O 0.223 CCC(=O)NC1=CC(Cl)=C(OC2=CC=C(O)C(=C2)C2=CC=C(NC(N)=O)C=C2)C(Cl)=C1 172 0.223 NC1=NC2=CC(O)=CC=C2N1 7AH 0.222 FC1=CC=C(CC(=O)NC2=CC=C3NC=C(C4CCNCC4)C3=C2)C=C1 6GM 0.222 NC1=NC(=CC(=N1)C1=CC=C(O)C=C1)N(CC1=CC=CC=N1)CC1=CC=CC=N1 1WQ 0.222 C[C@@H]1OC2=CC=C(CN3CCC(=CC3)C3=CC=C(Cl)C=C3)C=C2NC1=O 0MH 0.222 COC1=CC=CC=C1C1=NOC(C)=C1C(=O)N1CCN(CC1)C1=CC(NC(=O)C2=CC=CS2)=C(C=C1Cl)[N+]([O-])=O A42 0.221 COC1=CC(OC)=C(Cl)C=C1NC(=O)NC1=CN=C(C=N1)C#N 5EZ 0.221 ClC1=C(OC[C@@H]2CCNC2)C=C2C=CNC(=O)C2=C1 NT5 0.22 C[C@@H]1CC[C@@H](COC2=CC=C(F)C=N2)CN1C(=O)C1=CC(C)=CC=C1C1=NC=CC=N1 K9Y 0.22 CC1=CC=C(C=C1)N1N=C(C=C1NC(=O)NC1=CC=C(C=C1)N1C=NC2=C(N)N=CN=C12)C(C)(C)C .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint JDS 0.22 O\\N=C/C1=NC(CCCCNC2=C3C=C(Cl)C=CC3=NC3=C2CCCC3)=CC=C1O HWQ 0.22 CN(CCC#N)C1=C2SC=CC2=NC(=N1)N(C)C1CCNCC1 D5Z 0.22 COC1=CC=C(C=C1OC)C1=NN(C2CCN(CC2)C2=C3C=CC=CC3=NC(N)=N2)C(=O)[C@@H]2CC=CC[C@H]12 3MW 0.22 NC1=CC=C(C=C1)C(=O)N[C@@H](C(=O)NO)C1=CC=C(C=C1)N1C=CC=N1 WYE 0.219 CN1C=C(\\C=C2/OC3=CC(O)=CC(O)=C3C2=O)C2=C(C=CN=C12)N1CCN(C)CC1 SCQ 0.219 BrC1=C2C=C(C=C(NCC3=CN=CN=C3)N2N=C1)C1=CC=CC=C1 QXQ 0.219 CCC(CC)(C=O)C(=O)NC1=CC=CC(CN2CCC3=CC4=C(OCO4)C=C3C2)=C1 NTI 0.219 CC(=O)OC1=CC=CC=C1C(=O)NC1=NC=C(S1)[N+]([O-])=O NJD 0.219 COC1=NC(NCCC2=CNC3=CC=CC=C23)=NC(NC2=CC3=CNN=C3C=C2)=N1 LTJ 0.219 C\\C=C\\C(=O)N[C@H]1CCCN(C1)C1=C2C(C)=C(C)NC2=C(C=C1F)C(N)=O C9G 0.219 CN1C=CC2=CC=C(N=C2C1=O)C1=CN(N=N1)C1=CC=C(O)C=C1 5G3 0.219 OC1=CC=C(C=C1)C(=N/C1=CC=CC=C1Cl)\\C1=CC=C(O)C=C1O 5G4 0.218 CC1=CC=CC=C1\\N=C(/C1=CC=C(O)C=C1)C1=CC=C(O)C=C1O 30M 0.218 CC(=O)NCCCCN1C2=C(C3=CC=CC=C13)C(=O)NCC2 G9G 0.217 CN1CCN(CC1)C1=NC2=C(NC(=O)C3=C2C=CC=C3)C=C1 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint