Data-driven design of chiral covalent fragments using highthroughput chemoproteomics and machine learning

McCarthy, William J.; Nightingale, Luke; Biggs, George S.; Cawood, Emma E.; Dudley-Fraser, Jane; Werner, Thilo; Riziotis, Ioannis G.; Pillay, Timesh D.; Lambert, Hugues; Pogány, Peter; van der Zouwen, Antonie J.; Pettinger, Jonathan; Boulton, Simon J.; House, David; Skehel, J. Mark; Bush, Jacob T.; Rittinger, Katrin

ChemRxiv.  2026.
DOI: https://doi.org/10.26434/chemrxiv.15002030/v1

A significant barrier in translating biological insights into therapeutic targets is the limited availability of high-quality chemical probes for target validation. Chemoproteomic profiling of covalent small molecules has dramatically accelerated the discovery of ligandable binding sites across the human proteome. However, the limited specificity and selectivity of initial hits often hinders their effectiveness in evaluating the functional consequences of ligand binding. To address this challenge, we developed a data-driven strategy that integrates chemoproteomic profiling of enantiomerically pure pairs of cysteine-targeting electrophilic fragments (enantiopairs) with machine learning (ML) to design fragment libraries optimised for proteome-wide selectivity. ML-guided library evolution produced a second generation enantiopair library markedly enriched in selective and stereospecific interactions relative to the first generation library. This approach identified high-quality enantioselective binding events with 205 cysteines, the majority not previously liganded. These findings establish a general framework for designing covalent fragment libraries to deliver higherquality initial hits.

Deciphering covalent kinase inhibitor binding landscape through structural kinome profiling

Zheng Zhao, Philip E. Bourne

European Journal of Medicinal Chemistry, 312, 2026, 118872

https://doi.org/10.1016/j.ejmech.2026.118872

Significant progress in kinase-targeted drug discovery has been made over the past two decades, with 100 FDA-approved kinase-targeted drugs and a substantial number of bioactive kinase inhibitors under preclinical study. However, given that more than 180 kinases have been implicated in disease, there remains a considerable need for continued kinase-targeted drug discovery. Covalent kinase inhibitors (CKIs) are a class of kinase inhibitors that form covalent interactions with kinase targets, valued for the potential for enhanced selectivity through anchoring nucleophiles. Here, we collate all the kinase structures from the PDB into dedicated structural kinome resources, containing: (i) the kinase domain structure database (6969 PDB structures); (ii) the kinase ligand-binding structure database (6122 PDB structures); and (iii) the kinase-CKI complex structure database (325 PDB structures). With these data, we systematically investigate the binding modes of CKIs, the fingerprint characteristics of kinase-CKI interactions, 21 types of electrophilic warheads, and 64 nucleophilic amino acids distributed in 15 corresponding spatial positions in kinase domains. We also mentioned covalent degraders and multi-warhead CKIs. Together, our results offer a comprehensive structural kinase resource and in-depth insights into CKI binding properties, supporting future kinase-targeted drug design. The databases are freely accessible at https://zhengzhster.github.io/KinaseStructureDatabase/.

A Fragment Screen Identifies Acrylamide Covalent Inhibitors of the TEAD•YAP Protein-Protein Interaction

Khuchtumur Bum-Erdene, Mona K. Ghozayel, Mark J. Zhang, Giovanni Gonzalez-Gutierrez, Samy O. Meroueh

bioRxiv 2026.03.18.712694; 

doi: https://doi.org/10.64898/2026.03.18.712694

TEA domain (TEAD) proteins bind co-activator Yes-associated protein (YAP) to regulate the expression of target genes of the Hippo pathway. The TEAD•YAP protein-protein interaction is not druggable, but TEADs possess a unique and deep palmitate pocket with a highly conserved cysteine located outside the TEAD•YAP protein-protein interaction interface. Here, we screen a fragment library of acrylamide electrophiles and identify a fragment that forms an adduct with the conserved palmitate pocket cysteine and inhibits TEAD4 binding to YAP. Synthesis of a focused set of derivatives and time- and concentration-dependent studies with four TEADs provide reaction rates and binding constants. Co-crystal structures of fragments bound to TEAD2 and TEAD3 reveal reaction at the conserved palmitate pocket cysteine but also at another less conserved cysteine located in the palmitate pocket of TEAD2 closer to the TEAD•YAP interface. These fragments provide a starting point for the development of allosteric acrylamide small-molecule covalent TEAD•YAP inhibitors.

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.