Size-Dependent Target Engagement of Covalent Probes

László Petri, Ronen Gabizon, György G. Ferenczy, Nikolett Péczka, Attila Egyed, Péter Ábrányi-Balogh, Tamás Takács, and György M. Keserű

Journal of Medicinal Chemistry 2025 68 (6), 6616-6632

DOI: 10.1021/acs.jmedchem.5c00017

Labeling proteins with covalent ligands is finding increasing use in proteomics applications, including identifying nucleophilic residues amenable for labeling and in the development of targeted covalent inhibitors (TCIs). Labeling efficiency is measured by the covalent occupancy of the target or by biochemical activity. Here, we investigate how these observed quantities relate to the intrinsic parameters of complex formation, namely, noncovalent affinity and covalent reactivity, and to experimental conditions, including incubation time and ligand concentration. It is shown that target engagement is beneficially driven by noncovalent recognition for lead-like compounds, which are appropriate starting points for targeted covalent inhibitors owing to their easily detectable occupancy and fixed binding mode, facilitating optimization. In contrast, labeling by fragment-sized compounds is inevitably reactivity-driven as their small size limits noncovalent affinity. They are well-suited for exploring ligandable nucleophilic residues, while small fragments are less appropriate starting points for TCI development.

O-Cyanobenzaldehydes Irreversibly Modify Both Buried and Exposed Lysine Residues in Live Cells

Huan Ling, Lin Li, Liping Duan, Weixue Huang, Jiangnan Zheng, Shijie Zhang, Xinling Li, Xiaorong Qiu, Yang Zhou, Nan Ma, Xiaomei Ren, Jinwei Zhang, Zhen Wang, Yujun Zhao, Ruijun Tian, Zhi-Min Zhang, and Ke Ding

Journal of the American Chemical Society 2025

DOI: 10.1021/jacs.4c18006

Lysine residue represents an attractive site for covalent drug development due to its high abundance (5.6%) and critical functions. However, very few lysines have been characterized to be accessible to covalent ligands and perturb the protein functions, owing to their protonation state and adjacent steric hindrance. Herein, we report a new lysine bioconjugation chemistry, O-cyanobenzaldehyde (CNBA), that enables selective modification of the lysine ε-amine to form iso-indolinones under physiological conditions. Activity-based proteome profiling enabled the mapping of 3451 lysine residues and 85 endogenous kinases in live cells, highlighting its potential for modifying hyper-reactive lysines within the proteome or buried catalytic lysines within the kinome. Further protein crystallography and mass spectrometry confirmed that K271_ABL1 and K162_AURKA are covalently targetable sites in kinases. Leveraging a structure-based drug design, we incorporated CNBA into the core structure of Nutlin-3 to irreversibly inhibit the MDM2-p53 interaction by targeting an exposed lysine K94 on the surface of murine double minute 2. Importantly, we have demonstrated the potential application of CNBA as a lysine-recognized bioconjugation agent for developing new antibody-drug conjugates. The results collectively validate CNBA as a new selective and efficient modifying agent with broad applications for both buried and exposed lysine residues in live cells.

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.