State-of-the-art covalent virtual screening with AlphaFold3

Yoav Shamir, Nir London

bioRxiv 2025.03.19.642201;

doi: https://doi.org/10.1101/2025.03.19.642201

Recent years have seen an explosion in the prominence of covalent inhibitors as research and therapeutic tools. However, a lag in application of computational methods for covalent docking slows progress in this field. AI models such as AlphaFold3 have shown accuracy in ligand pose prediction but were never assessed for virtual screening. We show that AlphaFold3 reaches near-perfect classification (average AUC=98.3%) of covalent active binders over property-matched decoys, dramatically outperforming classical covalent docking tools. We identify a predicted metric that allows to reliably assign a probability of binding and demonstrate it also improves non-covalent virtual screening.

Methods for Kinetic Evaluation of Reversible Covalent Inhibitors from Time-Dependent IC50 Data

L. Mader and J. W. Keillor, 

RSC Med. Chem., 2025

DOI: 10.1039/D5MD00050E

Potent reversible covalent inhibitors are often slow in establishing their covalent modification equilibrium, resulting in time-dependent inhibition. While these inhibitors are commonly assessed using IC50 values, there are no methods available to analyze their time-dependent IC50 data to provide their inhibition (Kiand Ki*) and covalent modification rate (k5and k6) constants, leading to difficulty in accurately ranking drug candidates. Herein, we present an implicit equation that can estimate these constants from incubation time-dependent IC50 values and a numerical modelling method, EPIC-CoRe, that can fit these kinetic parameters from pre-incubation time-dependent IC50 data. The application of these new methods is demonstrated by the evaluation of a known inhibitor, saxagliptin, providing results consistent with those obtained by other known methods. This work introduces two new practical methods of evaluation for time-dependent reversible covalent inhibitors, allowing for rigorous characterization to enable the fine-tuning of their binding and reactivity.

Discovery of YJZ5118: A Potent and Highly Selective Irreversible CDK12/13 Inhibitor with Synergistic Effects in Combination with Akt Inhibition

Jianzhang Yang, Yu Chang, Kaijie Zhou, Weixue Huang, Jean Ching-Yi Tien, Pujuan Zhang, Wenyan Liu, Licheng Zhou, Yang Zhou, Xiaomei Ren, Rahul Mannan, Somnath Mahapatra, Yuping Zhang, Rudana Hamadeh, Grafton Ervine, Zhen Wang, George Xiaoju Wang, Arul M. Chinnaiyan, and Ke Ding

J. Med. Chem. 2025
https://doi.org/10.1021/acs.jmedchem.5c00127

Cyclin-dependent kinases 12 and 13 (CDK12/13) have emerged as promising therapeutic targets for castration-resistant prostate cancer (CRPC) and other human cancers. Despite the development of several CDK12/13 inhibitors, challenges remain in achieving an optimal balance of potency, selectivity and pharmacokinetic properties. Here, we report the discovery of YJZ5118, a novel, potent and highly selective covalent inhibitor of CDK12/13 with reasonable pharmacokinetic profiles. YJZ5118 effectively inhibited CDK12 and CDK13 with IC50 values of 39.5 and 26.4 nM, respectively, while demonstrating high selectivity over other CDKs. Mass spectrometry analysis, cocrystal structure determination, and pulldown-proteomic experiments confirmed the compound’s covalent binding mode with CDK12/13. Functionally, YJZ5118 efficiently suppressed the transcription of DNA damage response genes, induced DNA damage, and triggered apoptosis. Moreover, the compound significantly inhibited the proliferation of multiple tumor cell lines, particularly prostate cancer cells. Notably, YJZ5118 exhibited synergistic effects with Akt inhibitors both in vitro and in vivo.

Structure-based development of a covalent inhibitor targeting Streptococcus pyogenes over Staphylococcus aureus sortase A

Hailing Zhou, Ziqi Yuan, Xiang-Na Guan, Chuan Yue, Wei Wu, Lefu Lan, Jianhua Gan, Tao Zhang, Cai-Guang Yang

Chemistry. 2025

https://doi.org/10.1002/chem.202500464

Sortase A (SrtA), a cysteine transpeptidase critical for surface protein anchoring in Gram-positive pathogens, represents an attractive antivirulence target. While covalent SrtA inhibitors show therapeutic potential, existing compounds lack species selectivity. Through structure-guided design, we developed T10, a covalent inhibitor selectively targeting Streptococcus pyogenes SrtA (SpSrtA) over Staphylococcus aureus SrtA (SaSrtA). Molecular docking revealed that shortening a "C=C" bond in lead compound ML346 eliminated SaSrtA inhibition due to steric hindrance from W194, while maintaining SpSrtA binding. X-ray crystallography confirmed T10's covalent modification of Cys208 in SpSrtA. T10 demonstrated two fold enhanced inhibitory potency and species-specific disruption of M-protein anchoring and biofilm formation in Streptococcus pyogenes, without affecting Staphylococcus aureus viability. In a Galleria mellonella infection model, T10 conferred potent protection against lethal infection. This work demonstrates the development of narrow-spectrum antivirulence agents through a structure-based rational strategy.

Comprehensive Characterization of Bruton’s Tyrosine Kinase Inhibitor Specificity, Potency, and Biological Effects: Insights into Covalent and Noncovalent Mechanistic Signatures

Antonia C. Darragh, Andrew M. Hanna, Justin H. Lipner, Alastair J. King, Nicole B. Servant, and Mirza Jahic

ACS Pharmacol. Transl. Sci. 2025
https://doi.org/10.1021/acsptsci.4c00540

Uncovering a drug’s mechanism of action and possible adverse effects are critical components in drug discovery and development. Moreover, it provides evidence for why some drugs prove more effective than others and how to design better drugs altogether. Here, we demonstrate the utility of a high-throughput in vitro screening platform along with a comprehensive panel to aid in the characterization of 15 Bruton’s tyrosine kinase (BTK) inhibitors that are either approved by the FDA or presently under clinical evaluation. To compare the potency of these drugs, we measured the binding affinity of each to wild-type BTK as well as a clinically relevant resistance mutant of BTK (BTK C481S). In doing so, we discovered a considerable difference in the selectivity and potency of these BTK inhibitors to the wild-type and mutant proteins. Some of this potentially contributes to the adverse effects experienced by patients undergoing therapy using these drugs. Overall, noncovalent BTK inhibitors showed stronger potency for both the wild-type and mutant BTK when compared with that of covalent inhibitors, with the majority demonstrating a higher specificity and less off-target modulation. Additionally, we compared biological outcomes for four of these inhibitors in human cell-based models. As expected, we found different phenotypic profiles for each inhibitor. However, the two noncovalent inhibitors had fewer off-target biological effects when compared with the two covalent inhibitors. This and similar in-depth preclinical characterization of drug candidates can provide critical insights into the efficacy and mechanism of action of a compound that may affect its safety in a clinical setting.

Rational Design of Stapled Covalent Peptide Modifiers of Oncoprotein E6 from Human Papillomavirus

ACS Chem. Biol. 2025

https://doi.org/10.1021/acschembio.4c00878

Human Papillomavirus (HPV) is linked to multiple cancers, most significantly cervical cancer, for which HPV infection is associated with nearly all cases. Essential to the oncogenesis of HPV is the function of the viral protein E6 and its role in degrading the cell cycle regulator p53. Degradation of p53, and the resultant loss of cell cycle control, is mediated by E6 recruitment of the E3 ubiquitin ligase E6AP and subsequent ubiquitination of p53. Here, we report the design of a stapled peptide that mimics the LxxLL α-helical domain of E6AP to bind and covalently label a cysteine residue specific to HPV-16 E6. Several acrylamide- and haloacetamide-based warheads were evaluated for reactivity and specificity, and a panel of hydrocarbon-stapled peptides was evaluated for enhanced binding affinity and increased proteolytic stability. Structure-based modeling was used to rationalize the observed trends in the reactivity of the warheads and the impact of the hydrocarbon staple position on the binding affinity of the stapled peptides. The development of a proteolytically stable and reactive peptide represents a new class of peptide-based inhibitors of protein–protein interactions with a potential therapeutic value toward HPV-derived cancers.

Design of Benzyl-triazolopyrimidine-Based NADPH Oxidase Inhibitors Leads to the Discovery of a Potent Dual Covalent NOX2/MAOB Inhibitor

Beatrice Noce, Sara Marchese, Marta Massari, Chiara Lambona, Joana Reis, Francesco Fiorentino, Alessia Raucci, Rossella Fioravanti, Mariana Castelôa, Alessandro Mormino, Stefano Garofalo, Cristina Limatola, Lorenzo Basile, Andrea Gottinger, Claudia Binda, Andrea Mattevi, Antonello Mai, and Sergio Valente

Journal of Medicinal Chemistry 2025

DOI: 10.1021/acs.jmedchem.4c02644

NADPH oxidases (NOXs) are enzymes dedicated to reactive oxygen species (ROS) production and are implicated in cancer, neuroinflammation, and neurodegenerative diseases. VAS2870 is a covalent inhibitor of mainly NOX2 and NOX5. It alkylates a conserved active-site cysteine, blocking productive substrate binding. To enhance potency and selectivity toward NOXs, we conducted some chemical modifications, leading to the discovery of compound 9a that preferentially inhibits NOX2 with an IC50 of 0.155 μM, and only upon its preactivation. We found that 9a, bearing a pargyline moiety, is also able to selectively inhibit MAOB over MAOA (465-fold) with an IC50 of 0.182 μM, being the first-in-class dual NOX2/MAOB covalent inhibitor. Tested in the BV2 microglia neuroinflammation model, 9a decreased ROS production and downregulated proinflammatory cytokines as iNOS, IL-1β, and IL-6 expression more efficiently than the single target inhibitors (rasagiline for MAOB and VAS2870 for NOXs) but also, more importantly, than their combination.

‘Direct-to-biology’ drives optimisation of a cell-active covalent 1 inhibitor of WRN helicase

1S. M. Rowe, A. Price, D. J. Murphy, J. Lin, E. N. Nartey, A. Chaikuad, K. Wong, J. E. Cottom, N. O. Concha, R. A. Reid, E. R. Dickinson, M. Jundt, K. Kammerer, M. Steidel, T. Mathieson, T. Werner, E. K. Grant, C. K. Stanborough, M. Rouah, J. Wojno-Picon, P. Pogány, J. Pettinger, D. J. Norman, H. Wilders, F. Rianjongdee, G. Valdes-Garcia, N. Nevins, R. Shenje, R. K. Thalji, C. Chung, H. C. Eberl, G. Neubauer, D. House, Y. Rao, M. P. Martino and J. T. Bush, 

ChemRxiv, 2025

https://doi.org/10.26434/chemrxiv-2025-tvdzn

We report a ‘direct-to-biology’ (D2B) approach for optimising covalent acrylamide binders of protein targets and apply this to the identification of a selective and cell-active inhibitor of Werner (WRN) helicase. Inhibition of WRN helicase activity exhibits a synthetic lethal relationship with cancers displaying high microsatellite instability (MSI-H) and is being pursued as a therapeutic strategy in the clinic. Using intact-protein liquid chromatography-mass spectrometry (LC-MS) screening, we identified acrylamide fragment binders of the WRN helicase domain and then used covalent D2B chemistry to optimise these initial hits. Our efforts ultimately afforded a potent covalent inhibitor of WRN-mediated DNA unwinding, which displays selective, concentration-dependent cellular engagement of WRN, and demonstrates synthetic lethality in an MSI-H setting. Furthermore, our inhibitor targets a distinct conformation of WRN helicase compared to the current clinical covalent inhibitor, presenting a complementary approach for covalent inhibition of WRN helicase. This work demonstrates how D2B chemistry platforms can be used to explore structure-activity relationships in a modular fashion, while reducing investment of human and material resources.

Morita–Baylis–Hillman Adduct Chemistry as a Tool for the Design of Lysine-Targeted Covalent Ligands

Marco Paolino, Giusy Tassone, Paolo Governa, Mario Saletti, Matteo Lami, Riccardo Carletti, Filippo Sacchetta, Cecilia Pozzi, Maurizio Orlandini, Fabrizio Manetti, Massimo Olivucci, and Andrea Cappelli

ACS Medicinal Chemistry Letters 2025

DOI: 10.1021/acsmedchemlett.4c00479 

The use of Targeted Covalent Inhibitors (TCIs) is an expanding strategy for the development of innovative drugs. It is driven by two fundamental steps: (1) recognition of the target site by the molecule and (2) establishment of the covalent interaction by its reactive group. The development of new TCIs depends on the development of new warheads. Here, we propose the use of Morita–Baylis–Hillman adducts (MBHAs) to covalently bind Lys strategically placed inside a lipophilic pocket. A human cellular retinoic acid binding protein II mutant (M2) was selected as a test bench for a library of 19 MBHAs. The noncovalent interaction step was investigated by molecular docking studies, while experimentally the entire library was incubated with M2 and crystallized to confirm covalent binding with the target lysine. The results, rationalized through covalent docking analysis, support our hypothesis of MBHAs as reactive scaffolds for the design of lysine-TCIs.

Orally Bioavailable and Site-Selective Covalent STING Inhibitor Derived from a Macrocyclic Marine Diterpenoid

Guang-Hao Niu, Wan-Chi Hsiao, Po-Hsun Lee, Li-Guo Zheng, Yu-Shao Yang, Wei-Cheng Huang, Chih-Chien Hsieh, Tai-Yu Chiu, Jing-Ya Wang, Ching-Ping Chen, Chen-Lung Huang, May-Su You, Yi-Ping Kuo, Chien-Ming Wang, Zhi-Hong Wen, Guann-Yi Yu, Chiung-Tong Chen, Ya-Hui Chi, Chun-Wei Tung, Shu-Ching Hsu, Teng-Kuang Yeh, Ping-Jyun Sung, Mingzi M. Zhang, and Lun Kelvin Tsou

Journal of Medicinal Chemistry 2025

DOI: 10.1021/acs.jmedchem.4c02665

Pharmacological inhibition of the cGAS-STING-controlled innate immune pathway is an emerging therapeutic strategy for a myriad of inflammatory diseases. Here, we report GHN105 as an orally bioavailable covalent STING inhibitor. Late-stage diversification of the briarane-type diterpenoid excavatolide B allowed the installation of solubility-enhancing functional groups while enhancing its activity as a covalent STING inhibitor against multiple human STING variants, including the S154 variant responsible for a genetic autoimmune disease. Selectively engaging the membrane-proximal Cys91 residue of STING, GHN105 dose-dependently inhibited cGAS-STING signaling and type I interferon responses in cells and in vivo. Moreover, orally administered GHN105 exhibited on-target engagement in vivo and markedly reversed key pathological features in a delayed treatment of the acute colitis mouse model. Our study provided proof of concept that the synthetic briarane analog GHN105 serves as a safe, site-selective, and orally active covalent STING inhibitor and devises a regimen that allows long-term systemic administration.