Discovery of Covalent Ligands with AlphaFold3

Yoav Shamir, Ronen Gabizon, Adi Rogel, David Yin-wei Lin, Amy H. Andreotti, and Nir London

Journal of the American Chemical Society 2026

DOI: 10.1021/jacs.5c22222

Covalent inhibitors are a prominent modality for research and therapeutic tools. However, a scarcity of computational methods for their discovery slows progress in this field. AI models such as AlphaFold3 (AF3) have shown accuracy in ligand pose prediction, but their applicability for virtual screening campaigns was not assessed. We show that AF3 cofolding predictions and an associated predicted confidence metric ranks true covalent binders with near-optimal classification over property-matched decoys, significantly outperforming state-of-the-art covalent docking tools for a set of protein kinases. In a prospective virtual screening campaign against the model kinase BTK, we discovered a chemically distinct, novel, covalent small molecule that displays potent inhibition in vitro and in cells while maintaining marked kinome and proteomic selectivity. Co-crystallography validated the subangstrom accuracy of the predicted AF3 binding mode. These results demonstrate that AF3 can be practically used to discover novel chemical matter for kinases, one of the most prolific families of drug targets.

Acrylamide Bioisosterism: Alkenyl Aromatic Heterocycles as Reactivity-Tunable Warheads for Covalent BTK Inhibitors

Zeyue Huang, Xiuqi Hu, Zheng Liu, Hongxuan Cao, Yunjie Xiang, Jian Wan, Ivailo Slavchev, Li Rao, Ivanka Nikolova, Petar Grozdanov, Nadya Nikolova, Georgi M. Dobrikov, and Yanliang Ren

Journal of Medicinal Chemistry 2026

DOI: 10.1021/acs.jmedchem.5c03394

Targeted covalent inhibitors (TCIs) are powerful tools in drug discovery, but the high intrinsic reactivity of conventional warheads often compromises selectivity and increases the off-target liability. Here, we reported nitrodiphenyl-ether compounds as a novel irreversible and released-type covalent warhead with exceptionally low reactivity that potently inhibits coronavirus HCoV-OC43 infection. To identify their molecular targets, we designed a panel of active and inactive alkyne-tagged probes and performed chemical proteomic profiling in human host cells. An integrated approach combining activity- and inactivity-based proteome profiling (AIBPP), competitive ABPP, LC–MS/MS, and fluorescence polarization (FP) assays identified low-density lipoprotein receptor adapter protein 1 (LDLRAP1) as the primary target, modified selectively at C119, thereby disrupting the LDLR–LDLRAP1 interaction. Inhibition of this interaction strongly correlated with antiviral efficacy, confirming LDLRAP1 as the functional target. Collectively, this study establishes LDLRAP1 as an unexploited host antiviral target and expands the repertoire of cysteine-targeted covalent warheads for host-directed therapy.

Covalent JAK3 inhibitors based on 2-arylamino and 7H-pyrrolo[2,3-d]pyrimidine scaffold: design, synthesis, and biological evaluation for the potential treatment of Bortezomib-resistant multiple myeloma

Tian, L.; Li, J.; Yu, J.; Han, Q.; Bolghanabadi, N.; Wang, K.; Chen, Z.; Zheng, X.; Chu, P.; Chen, L.

Euro J Med Chem, 2026

DOI: https://doi.org/10.1016/j.ejmech.2026.118764

Bortezomib, as a first-generation proteasome inhibitor, is one of the cornerstone drugs in the treatment of multiple myeloma. However, its long-term clinical efficacy is severely limited by both primary and acquired resistance. Studies have shown that the Janus kinase 3/Signal transducer and activator of transcription (JAK/STAT) signaling pathway may be persistently activated in certain bortezomib-resistant myeloma cells. Herein, we designed, synthesized, and evaluated a series of acrylamide group-bearing 2-arylaminopyrimidine derivatives as potent Janus kinase 3 (JAK3) inhibitors. Among them, 7n, a promising compound, exhibited a strong combining capability with JAK3 (half-maximal inhibitory concentration [IC50] = 0.7473 nM) and effective antiproliferative activities against Bortezomib-resistant KM3 cells (IC50 = 0.2452 μM). The results of the pharmacokinetics analysis showed that 7n presented good oral bioavailability with an F value of 39.11%. Furthermore, 7n showed notable inhibition of tumor growth in a murine Bortezomib-resistant KM3 cell xenograft model. Additionally, the analysis of the mechanism of action validated that compound 7n inhibited cell migration, promoted cell apoptosis and arrested the JAK–signal transducers and activators of the transcription pathway. Notably, 7n displayed the strongest inhibitory activities against JAK3 in 76 kinase profiles with the inhibitory rate of 96.87% at the concentration of 5 nM. Altogether, these findings suggest that JAK3 is a potential target to develop the inhibitor for treating Bortezomib-resistant multiple myeloma and 7n can be considered a promising candidate for further research.

A Global Ligandability Map of Tryptoline Butynamide Stereoprobes Identifies Covalent Inhibitors of the Actin Maturation Protease ACTMAP

Yijun Xiong, Christopher J. Reinhardt, Tracey Nguyen, Melissa A. Hoffman, Gabriel M. Simon, Bruno Melillo, Benjamin F. Cravatt

bioRxiv, 2026

doi: https://doi.org/10.64898/2026.02.21.707170

Covalent chemistry coupled with activity-based protein profiling (ABPP) offers a versatile approach for small-molecule ligand discovery in native biological contexts. The covalent ligandability maps generated by ABPP that target cysteine have frequently leveraged the acrylamide as a reactive group due to its tempered electrophilicity and presence in many advanced tool compounds and therapeutics. More recently, alternative cysteine-directed reactive groups such as the butynamide have emerged as an additional source of covalent probes and drugs, but their global reactivity with the proteome remains largely unexplored. Here, we compare the ligandability maps of stereochemically defined acrylamide and butynamide compounds (stereoprobes) built from a common tryptoline core and find that the butynamides, despite exhibiting attenuated intrinsic and proteome-wide reactivity, preferentially engage a diverse set of proteins in human cancer cells. Among the butynamide-preferring proteins was C19orf54/ACTMAP, a cysteine protease required for the post-translational maturation of actin. We show that (1S, 3R)-tryptoline butynamides stereoselectively react with the catalytic nucleophile of ACTMAP, leading to accumulation of N-terminally unprocessed actin in cancer cells. Our findings support reactive group diversification as a strategy for expanding the ligandability of the human proteome and the butynamide, more specifically, as a differentiated cysteine-directed electrophile for chemical probe discovery.

Development and Structural Characterization of UTE-156, a Covalent Inhibitor of the VCP/p97 AAA+ ATPase

 Daniela Tamayo-Jaramillo, Subramanya Hegde, Xuan Jia, Kimberly Coffman, Hariprasad Vankayalapati, David Bearss, Kevin B. Jones, Alex W. Stark, Peter S. Shen 

Advanced Science (2026): e20545. 

https://doi.org/10.1002/advs.202520545

The AAA+ ATPase valosin-containing protein (VCP/p97) is a central regulator of protein homeostasis that is well characterized for its role in extracting and remodeling ubiquitinated substrates. Dysregulation of VCP activity contributes to the pathogenesis of neurodegenerative diseases and cancer, making it an important therapeutic target. Here, we report the development and characterization of UTE-156, a novel covalent small-molecule inhibitor that modifies Cys522 within the D2 ATPase domain of VCP. UTE-156 potently inhibits VCP ATPase activity, while losing activity against a C522A mutant, supporting a covalent mechanism of action. High-resolution cryo-electron microscopy (cryo-EM) structures reveal that UTE-156 occupies the D2 nucleotide-binding site, sterically blocking ATP binding and inducing conformational remodeling of the pocket. Biochemical and cell-based assays demonstrate strong inhibitory potency but limited solubility and rapid metabolic turnover. These pharmacochemical limitations preclude immediate therapeutic use but underscore its value as a chemical probe. Together, these findings establish UTE-156 as a powerful tool for dissecting VCP function and provide a framework for future optimization of covalent modulators of protein homeostasis.

Discovery and Characterization of Divarasib (GDC-6036), a Potent Covalent Inhibitor of KRAS G12C

Nicholas F. Endres, Steven Do, Rana Mroue, Jack A. Terrett, Matt Saabye, Angela Oh, Thomas Hunsaker, Emily Chan, John C. Tran, Lan K. Nguyen, Qihui Lian, Taylur P. Ma, Thomas Garner, Luca Gerosa, Maureen Beresini, Aaron Boudreau, Sarah M. Bronner, Patrick Cyr, Noriko Ishisoko, Yevgeniy Izrayelit, Fan Jiang, Terry Kellar, Hank La, Sharada Labadie, Matthew Lardy, Liling Liu, Wendy Liu, Sarah Miller, Joachim Rudolph, Emile Plise, Benjamin D. Sellers, Cheng Shao, Weiru Wang, Yanguang Wang, Wentao Wei, Susan Wong, Christine Yu, Kebing Yu, Po-Wai Yuen, Richard Zang, Chenghong Zhang, Yuhui Zhou, Xiaoyu Zhu, John G. Quinn, Xin Ye, James R. Kiefer, Jialin Mao, Marie Evangelista, Mark Merchant, Matthew L. Landry, Sushant Malhotra, and Hans E. Purkey

Journal of Medicinal Chemistry 2026

DOI: 10.1021/acs.jmedchem.5c02272

KRAS G12C is one of the most prevalent oncogenic mutations in nonsmall cell lung cancer. Herein we describe the discovery and optimization of divarasib (GDC-6036), an orally available, highly potent, and selective covalent KRAS G12C inhibitor. We demonstrate a significant noncovalent binding component of divarasib that contributes to its potency and rapid kinetics. Divarasib has greater potency and kinetics of alkylation compared with other KRAS G12C inhibitors in vitro and shows robust tumor growth inhibition in multiple KRAS G12C-positive cell lines.

A Tandem Bioorthogonal Retro-Cope and Cope Elimination for the Activation of Covalent Inhibitors with an Acrylamide or Vinylsulfonamide Warhead in Live Cells

Yan Huang, Miao Liu, Dongguang Fan, Fan Xu, Fushuang Xiang, Qingqiang Min, and Xingyue Ji

Journal of the American Chemical Society 2026

DOI: 10.1021/jacs.6c00226

Precisely controlling the activation of covalent inhibitors through the caging and decaging of their reactive warheads is pivotal, yet this strategy is rarely pursued due to its formidable technical challenges. In this contribution, we report a novel tandem bioorthogonal retro-Cope and Cope elimination designed for efficient and selective activation of the covalent inhibitors bearing an acrylamide or vinylsulfonamide warhead in live cells. Notably, this strategy can be simultaneously tailored to coactivate both the covalent inhibitor and a fluorescent reporter, enabling real-time monitoring of prodrug activation. We successfully demonstrate the proof of concept through on-demand activation of two distinct EGFR covalent inhibitors and a BRD4-targeting molecular glue in live cells. This approach allows precise control over antiproliferative activity or induced protein degradation exclusively upon triggering via the tandem bioorthogonal reaction. We anticipate that this methodology will open new avenues for the selective delivery and controlled activation of covalent inhibitors, with broad potential in chemical biology and targeted therapy.