Covalent Modifiers

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

2026-03-26T08:19:00.000-07:00

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

2026-03-24T10:43:00.000-07:00

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

2026-03-23T10:17:00.000-07:00

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

2026-03-22T12:22:00.000-07:00

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

2026-03-12T07:29:00.000-07:00

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

2026-03-11T18:04:00.000-07:00

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

2026-03-04T10:14:00.000-08:00

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

2026-02-20T09:50:00.000-08:00

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.

Discovery of a Potent, Selective, and In Vivo Efficacious Covalent Inhibitor for Lysine Methyltransferase SETD8

2026-02-19T07:54:00.000-08:00

He Chen, Rudra Prasad Dutta, Zhizhong Li, Yue Zhong, Anqi Ma, Kwang-Su Park, Jithesh Kottur, Alison Park, Nicolas Babault, Ke Wang, Dandan Wang, Yan Xiong, H. Ümit Kaniskan, Minkui Luo, Samir Parekh, and Jian Jin

Journal of Medicinal Chemistry 2026

DOI: 10.1021/acs.jmedchem.5c02958

Dysregulated signaling of SET domain-containing protein 8 (SETD8) has been implicated in tumorigenesis, yet most SETD8 inhibitors exhibited limited cellular efficacy. Herein, we developed a potent and selective SETD8 covalent inhibitor, MS2928 (3), featuring a propiolamide covalent warhead. Compound 3 potently and selectively inhibited SETD8 methyltransferase activity. The covalent inhibition mechanism of 3 was confirmed by mass spectrometry and X-ray crystallography. Moreover, 3 significantly reduced the histone H4 lysine 20 monomethylation (H4K20me1) levels in cells and robustly inhibited the proliferation of SETD8-overexpressing multiple myeloma (MM) cell lines with no significant antiproliferative effect on SETD8-low expressing MM cells and normal cells. Importantly, 3 effectively inhibited tumor growth in vivo in two xenograft mouse models of SETD8-overexpressing MM cell lines. Collectively, our results establish 3 as a valuable chemical tool for exploring the biological functions of SETD8 and pave the way for further development of novel epigenetic therapies for MM.

Exploiting human fucosyltransferase 8 allostery with a covalent inhibitor for core fucosylation suppression

2026-02-15T16:49:00.000-08:00

Jiheng Jiang, Dongyang He, Mengyu Ke, Jinhua Qin, Guang Yang, Biao Yu, Jing Wang & Pengfei Fang

Nat Commun (2026).

https://doi.org/10.1038/s41467-026-68971-7

Core fucosylation, catalyzed by fucosyltransferase 8 (FUT8), plays critical roles in cancer progression, immune evasion, and drug resistance, making it a compelling therapeutic target. However, development of selective FUT8 inhibitors has been hindered by shared substrate specificity of fucosyltransferases. Here, we report the discovery of a previously unrecognized allosteric site on FUT8 and the development of a low-toxicity covalent inhibitor, CAIF (stearic acid-N-hydroxysuccinimide ester-dimethylimidazolium bromide), through structure-based drug design. High-throughput screening and crystallographic studies reveal that small molecules such as NH125 bind to a channel-like allosteric pocket, inducing conformational changes that disrupt FUT8 activity. Leveraging these insights, we design CAIF to covalently target lysine K216 within the allosteric site. CAIF exhibits minimal cytotoxicity and significantly inhibits core fucosylation and cancer cell invasion in cellular assays. This work establishes CAIF as a lead compound for further optimization and development, offering a framework for targeting glycosyltransferases through allosteric and covalent inhibition strategies.

Covalent inhibitor design confers activity against both GDP- and GTP-bound forms of KRAS G12C

2026-02-06T14:14:00.000-08:00

Matthew L. Condakes, Zhuo Zhang, Derek B. Danahy, Richard R. Moore, Sirish Kaushik Lakkaraju, Xiaoliang Zhuo, Yuka Amako, Robert M. Borzilleri, Srividya B. Balachander, Lisa Chourb, Rita L. Civiello, Ashok R. Dongre, Daniel P. Downes, Dieter M. Drexler, Brianne M. Dudiak, Liudmila Dzhekieva, Miriam El-Samin, Brian E. Fink, Kosea Frederick, Cherrie Huang, Javed Khan, Emma Lees, Christopher G. Levins, Courtney McCarthy, Michelle L. Stewart

Nat Commun (2026).

https://doi.org/10.1038/s41467-026-69003-0

The discovery of KRAS G12C inactive state inhibitors represented a significant advancement in the field of precision oncology. While inactive state inhibition shows promise in patients, Switch II (SWII)-binding inhibitors targeting both inactive and active states remain an underdeveloped therapeutic modality. Here, we describe the discovery of such KRAS G12C dual inhibitors that bind the SWII allosteric site using a chemically differentiated warhead to covalently modify both the KRAS G12C inactive and active states. Co-crystal structures reveal that these inhibitors perturb a key water-mediated hydrogen bonding network and trigger allosteric remodeling of the GTP-bound protein surface and SWI that prevents binding to downstream effectors. Consistent with simultaneous targeting of the active and inactive states, dual inhibitors provide rapid covalent target engagement and suppression of MAPK signaling. However, they demonstrate similar efficacy in cellular and in vivo models when compared to inactive state-selective ones despite faster target inactivation. Furthermore, both inhibitor classes show similar cellular efficacy in the presence of growth factors that drive KRAS, wt NRAS, and wt HRAS to the active state. These data provide the first detailed account of targeting both the active and inactive states of KRAS G12C and highlight the absence of a mechanistic advantage in contexts dependent on prolonged target inhibition.

Development of a Lysine-Reactive Targeted Covalent Inhibitor (TCI) for the P300/CBP-Associated Factor (PCAF) Bromodomain Through Structure-Based Design

2026-02-05T13:54:00.000-08:00

Richard Ede and Kerstin E Peterson and Richard Begyinah and Irin P Tom and Jason Ochoada and Molly Sneddon and Marcus Fischer and Anang A Shelat and William C K Pomerantz

ChemRxiv. 2026.

DOI: https://doi.org/10.26434/chemrxiv.10001717/v1

Epigenetics is defined by changes in heritable phenotypes that do not involve a change in DNA sequence. P300/CBP-associated factor (PCAF) is an important epigenetic regulatory protein that can alter chromatin through a histone acetyltransferase domain, while also serving as an epigenetic reader through a C-terminal bromodomain. PCAF promotes the transcription of the HIV-1 genome and is implicated in the development of glioblastoma. The currently reported PCAF inhibitors are non-covalent and require high concentration to maintain target occupancy. Here, we explore a new approach using covalent inhibition. Starting with a lead scaffold (BZ1), test-molecules were rationally designed for selectively targeting PCAF by installing lysine-reactive groups onto the lead scaffold to enable covalent bond formation with the non-conserved lysine residue in the PCAF bromodomain. The inhibition, selectivity, and kinetic properties (kinact/KI) of these molecules were evaluated using intact protein mass spectrometry, while biophysical, and cellular data were employed to verify covalent mechanism and in-cell target engagement. After optimization, we developed the first PCAF covalent inhibitor, 10, which labeled PCAF covalently in vitro and engages PCAF in cells. The covalent inhibitor, 10, represents a useful starting point for future inhibitor optimization and heterobifunctional molecule development.

Covalent Protein Inhibitors via Tyrosine and Tryptophan Conjugation with Cyclic Imine Mannich Electrophiles

2026-02-03T11:50:00.000-08:00

Dr. Sijie Wang, Dr. Lei Wang, Dr. Marco Hadisurya, Dr. Siavash Shahbazi Nia, Prof. Dr. W. Andy Tao, Prof. Dr. Emily C. Dykhuizen, Prof. Dr. Casey J. Krusemark

Angewandte Chemie e16630

https://doi.org/10.1002/ange.202516630

Targeted covalent inhibitors (TCIs) are increasingly popular as drug candidates and chemical probes. Among current TCIs, the chemistry is largely limited to cysteine and lysine side chain reactivity. Here, we investigated the utility of cyclic imine Mannich electrophiles as covalent warheads to target protein tyrosine and tryptophan side chains. We characterized the intrinsic reaction rates of several cyclic imines to tyrosine and other amino acid side chains and validated reactivity using protein affinity labeling of a cyclic imine-modified trimethoprim with tyrosine and tryptophan mutants of E. coli dihydrofolate reductase. To validate the utility of the approach, we appended cyclic imine warheads to a CBX8 chromodomain inhibitor to label a non-conserved tyrosine, which improved both the potency and selectivity of the inhibitor for CBX8 in vitro and in cells. These findings indicate that Mannich electrophiles are promising and robust chemical warheads for tyrosine and tryptophan bioconjugation and development of covalent inhibitors.

Chemoproteomics discovery of a CNS-penetrant covalent inhibitor of PIKfyve

2026-02-02T08:56:00.000-08:00

Antony J. Burton, Louis S. Chupak, Alison J. Davis, Ahmed S. A. Mady, Mirco Meniconi, Barry Teobald, Bryan W. Dorsey, Lauren R. Byrne, Ryan Mulhern, Berent Lundeen, Elizabeth W. Sorensen, Bharti Patel, Sean Brennan, Dhiraj Kormocha, Ruben Tommasi, Graham L. Simpson, Jeffrey W. Keillor, Laura D’Agostino, Pearl S. Huang, Elayne Penebre

bioRxiv 2026.01.26.701341;

doi: https://doi.org/10.64898/2026.01.26.701341

PIKfyve is a lipid kinase involved in regulating protein clearance mechanisms and is a promising target for the treatment of neurodegenerative diseases. Here, we present the discovery and optimization of a CNS-penetrant covalent PIKfyve inhibitor, DUN’058, which achieves sustained target occupancy in vivo. Covalent screening hits, identified from chemoproteomics experiments performed in live cells, were rapidly optimized to deliver a brain-penetrant covalent inhibitor of PIKfyve. This covalency centered approach employed a suite of mass spectrometry, biochemical and in vivo assays to optimize compound potency, selectivity, and CNS permeability. The target nucleophile, cysteine 1970, is on a flexible loop that appears distal from the kinase active site, highlighting the power of chemoproteomics screening to identify novel nucleophilic amino acids for covalent modification. DUN’058 achieves efficient covalency at the target cysteine, as well as highly selective covalent and reversible selectivity profiles. Covalent PIKfyve inhibition results in modulation of downstream pathway activity, including activation of the transcription factor TFEB, upregulation of protein clearance pathways, and increased GPNMB transcription and secretion of exosome markers. When dosed in vivo, DUN’058 achieves sustained target occupancy in the brains of mice long after systemic compound clearance, holding promise for achieving a sustained duration of action in the CNS at low doses, without prolonged effects in the periphery. Taken together, the development of DUN’058 is a demonstration of chemoproteomics-based discovery for a high value CNS target, providing an orally bioavailable and covalent PIKfyve inhibitor.

Group Competition Strategy for Covalent Ligand Discovery

2026-01-26T14:08:00.000-08:00

Zhihao Guo, Yunzhu Meng, Boyuan Zhao, Weidi Xiao, and Chu Wang

Journal of the American Chemical Society 2026

DOI: 10.1021/jacs.5c18150

As a powerful chemoproteomic tool, activity-based protein profiling (ABPP) has been extensively used for covalent ligand discovery. However, the current ABPP-based approaches are inherently based on indirect probe labeling competed by covalent ligands, and cannot directly compare the preferences of different ligands head-to-head. Herein, we report a group competition-based ABPP strategy (GC-ABPP) to allow the direct comparison of multiple ligands’ binding ability on a proteome-wide scale. By dividing a library of fully functionalized probes (FFPs) into different subgroups and labeling the proteome simultaneously, the direct competition enables comparison of the labeling ability of different probes in drawing a global protein–ligand affinity metric. When it is applied to an expanded probe library, this strategy can be used iteratively to select the highest-affinity ligand toward a certain target protein in a multiple-round process. As a proof of concept, we synthesized 65 FFPs and employed the GC-ABPP to screen the ligand–protein reactivity for >6000 cysteine sites. After three rounds of screening, we identified high-affinity ligands targeting BCAT2 and UGDH. Our “multiple ligands versus multiple proteins” screening paradigm demonstrates great potential for applications in covalent ligand/drug discovery.

Protein tyrosine phosphatase inactivation by electrophilic tyrosine modification

2026-01-20T07:25:00.000-08:00

Madeleine L. Ware, David M. Leace, Zihan Qu, Quentin Schaefer, Sagar D. Vaidya, Mikayla L. Horvath, Zhihong Li, Yunpeng Bai, Zhong-Yin Zhang, and Ku-Lung Hsu

Chem. Sci., 2026

https://doi.org/10.1039/D5SC07398G

Covalent protein tyrosine phosphatase (PTP) inhibitors principally target the catalytic cysteine, which is highly conserved and presents challenges for achieving selectivity across the PTP family. Here, we identified a tyrosine-reactive covalent inhibitor for SHP2 (DML189) with secondary molecular glue activity via a ligand induced protein tethering (LIPT) mechanism. We detected ligand binding at Y279, which is in proximity to the catalytic cysteine on SHP2 and has known functional and pathogenic properties. Covalent SHP2 modification by DML189 induced reversible disulfide tethering and monomer loss that was not observed to the same extent on PTP1B, LYP, or SHP1. Crosslinking mass spectrometry detected unique tethering events involving regulatory cysteines after DML189 modification on SHP2. Together, we discovered a tyrosine reactive inhibitor that targets functional sites on SHP2 and exhibits molecular glue activity through LIPT.

Covalent Peptide-Encoded Libraries Enable Discovery of Inhibitors of Epidermal Growth Factor Receptor (EGFR)

2026-01-18T18:18:00.000-08:00

Ching-Pei Hsu, Michael Desgagné, Simon L. Rössler, Nathalie M. Grob, Charlotte E. Farquhar, Andrei Loas, Zena D. Jensvold, Hannah T. Baddock, Matthew Bratkowski, Aaron H. Nile, and Bradley Pentelute

ChemRxiv, 2026

doi:10.26434/chemrxiv-2026-z6vkt

The use of encoding tags in combinatorial libraries accelerates hit generation by enabling high-throughput identification of small-molecule ligands. Peptide-encoded libraries (PELs) support the selection of structurally diverse small-molecule binders to proteins of interest. Here, we introduce a covalent PEL (coPEL) platform that incorporates cysteine-reactive scaffolds to identify irreversible protein binders. We leverage the chemical stability of PELs and the selective reactivity of palladium catalysts derived from dialkylbiaryl phosphine ligands to enable solid-phase Heck coupling reactions to rapidly diversify covalent acrylamide warheads. The optimized reaction conditions are high-yielding across a broad range of (hetero)aryl halides, ensuring robust performance and versatility within the coPEL platform. Screening a coPEL against the epidermal growth factor receptor (EGFR) tyrosine kinase, a key oncology target, yielded covalent small-molecule inhibitors with low-micromolar potency in vitro. This approach provides a complementary strategy for targeting diverse proteins and developing new classes of covalent inhibitors.

Structure-Guided Optimization of 4-Chloro-Pyrazolopyridine Analogs for Covalent PREP Inhibition

2026-01-12T05:46:00.000-08:00

Kalyani Thakur, Ian Fucci, Joshua Pandian, Kiall F. Suazo, Diana C. F. Monteiro, and Euna Yoo

Journal of Medicinal Chemistry 2025

DOI: 10.1021/acs.jmedchem.5c02680

Prolyl endopeptidase (PREP) is a dynamic serine protease that cleaves proline-containing peptides. PREP is also involved in numerous pathophysiological processes through modulation of protein–protein interactions and has been extensively studied in neurodegenerative diseases. In this study, we report the structure-based design and synthesis of covalent PREP inhibitors built on a 4-chloro-pyrazolopyridine (CPzP) scaffold, previously identified through chemoproteomic screening to target a noncatalytic cysteine residue within the active site. Guided by crystallographic data and molecular docking studies, we optimized initial hits to develop a potent inhibitor exhibiting nanomolar potency in both biochemical and cellular assays, with high selectivity over related serine proteases FAP and DPP4. Molecular dynamics simulations indicated that modulation of the conformational flexibility of a dynamic A-loop within PREP by CPzP analogs may contribute to inhibitory potency. Collectively, this work introduces a new class of structurally distinct inhibitors and provides tools to explore the diverse biological roles of PREP.

Ninhydrin as a covalent warhead for chemical proteomic-enabled discovery and selective engagement of reactive arginines

2026-01-06T10:11:00.000-08:00

Andrew Ecker, Andreas Langen, Chloe Fields, José Luis Montaňo, Minh Tran, Ian Bass Seiple, Balyn W Zaro

bioRxiv 2026.01.05.697388;

doi: https://doi.org/10.64898/2026.01.05.697388

Covalent molecules have emerged as next-generation therapeutics and as powerful tools for perturbing fundamental biological processes. Chemical proteomic methods to screen for reactive proteinaceous amino acids have transformed small-molecule discovery pipelines, but their application remains mostly limited to sites where reactive cysteines and lysines are present. Here we report a ninhydrin-based warhead that selectively modifies arginine residues, thus expanding the repertoire of amino acids targetable by covalent molecules. Specifically, we developed alkyne-functionalized variants of ninhydrin to establish an arginine-specific chemical proteomics platform, enabling the classification of more than 6,800 unique reactive arginines. These studies uncovered potential modification sites on disease-relevant proteins, including reactive arginines within catalytic sites that are essential for function. By endowing a reversible small molecule inhibitor of cyclophilin A with a ninhydrin warhead, we achieved selective, covalent engagement, and attenuation of enzymatic activity, highlighting the potential for targeting arginines in future therapeutic development campaigns. These findings establish ninhydrin as a warhead for studying arginine reactivity and modulating protein function.

Peptide-based covalent inhibitor of tubulin detyrosination promotes mesenchymal-to-epithelial transition in lung cancer cells

2026-01-03T21:28:00.000-08:00

Hathaichanok Impheng, Ghislain Gillard, Nuttanid Numnoi, and Krzysztof Rogowski

PNAS 123 (1) e2514990123

https://doi.org/10.1073/pnas.2514990123

Detyrosination is a reversible posttranslational modification specific to α-tubulin, which has been implicated in cancer progression and invasiveness by promoting epithelial-to-mesenchymal transition. The members of the vasohibin family, VASH1 and VASH2, were previously identified as the first class of enzymes involved in catalyzing this modification. Here, we report the development of a covalent VASH inhibitor, which is characterized by high specificity and low toxicity. By combining the use of a new compound with molecular approaches in lung cancer cell lines, we find that tubulin detyrosination plays an important role in the maintenance of mesenchymal properties. We show that in the absence of VASH activity, collective cell migration and 3D spheroid formation are severely compromised. Moreover, we demonstrate that the observed phenotypes are caused by the accumulation of the important epithelial marker E-cadherin with simultaneous reduction in mesenchymal markers N-cadherin and vimentin. Taken together, our study establishes tubulin detyrosination as a promising target for the future development of anticancer treatment.

Discovery of a First-in-Class Covalent Allosteric SHP1 Inhibitor with Immunotherapeutic Activity

2025-12-29T10:57:00.000-08:00

Zihan Qu, Frederick Nguele Meke, Zheng Zhang, Aaron D. Krabill, Christine S. Muli, Brenson A. Jassim, Jiajun Dong, Quyen D. Nguyen, Yunpeng Bai, Jinyue Li, Yiming Miao, Bardia Asadi, Levi M. Johnson, Jinmin Miao, Darci J. Trader, W. Andy Tao, Zhong-Yin Zhang

Angew. Chem. Int. Ed. 2025, e25126

https://doi.org/10.1002/anie.202525126

Src homology 2 domain-containing phosphatase 1 (SHP1), encoded by PTPN6, is a key intracellular mediator of inhibitory immune signals. SHP1 is garnering attention as a potential immunotherapeutic target since SHP1 deletion elicits strong antitumor activity by boosting both innate and adaptive immunity. Unfortunately, no quality SHP1 inhibitor exists to demonstrate its translatability owing to the challenges posed by the chemistry of the phosphatase active site. Herein, we describe the discovery of a first-in-class, phenyl chloroacetamide-based covalent allosteric SHP1 inhibitor M029 through covalent fragment screening and multiparameter optimization. M029 inactivates SHP1 by covalently binding to a non-conserved and cryptic Cys480 far away from the active site, thus uncovering a novel allosteric mechanism for SHP1 inhibition. In addition, M029 is highly selective for SHP1 and exhibits robust cellular target engagement. Importantly, M029 is orally active and blocks tumor progression in a syngeneic cancer model by activating natural killer cells and cytotoxic CD8+ T cells, along with reduced T cell exhaustion. Together, this study reveals a ligandable Cys that can be exploited for allosteric inhibition of SHP1, which has been refractory to targeted pharmacologic manipulation. The work also demonstrates small-molecule SHP1 inhibition as a compelling approach for new cancer immunotherapy.

Covalent Drug Binding in Live Cells Monitored by Mid-Infrared Quantum Cascade Laser Spectroscopy: Photoactive Yellow Protein as a Model System

2025-12-22T23:45:00.000-08:00

Srijit Mukherjee, Steven D. E. Fried, Nathalie Y. Hong, Nahal Bagheri, Jacek Kozuch, Irimpan I. Mathews, Jacob M. Kirsh, and Steven G. Boxer

Journal of the American Chemical Society 2026

DOI: 10.1021/jacs.5c14498

The detection of drug-target interactions in live cells enables analysis of therapeutic compounds in a native cellular environment. Recent advances in spectroscopy and molecular biology have facilitated the development of genetically encoded vibrational probes like nitriles that can sensitively report on molecular interactions. Nitriles are powerful tools for measuring electrostatic environments within condensed media like proteins, but such measurements in live cells have been hindered by low signal-to-noise ratios. In this study, we design a spectrometer based on a double-beam quantum cascade laser (QCL)-based transmission infrared (IR) source with balanced detection that can significantly enhance sensitivity to nitrile vibrational probes embedded in proteins within cells compared to a conventional FTIR spectrometer. Using this approach, we detect small-molecule binding in Escherichia coli, with particular focus on the interaction between para-Coumaric acid (pCA) and nitrile-incorporated photoactive yellow protein (PYP). This system effectively serves as a model for investigating covalent drug binding in a cellular environment. Notably, we observe large spectral shifts of up to 15 cm–1 for nitriles embedded in PYP between the unbound and drug-bound states directly within bacteria, in agreement with observations for purified proteins. Such large spectral shifts are ascribed to the changes in the hydrogen-bonding environment around the local environment of nitriles, accurately modeled through high-level molecular dynamics simulations using the AMOEBA force field. Our findings underscore the QCL spectrometer’s ability to enhance sensitivity for monitoring drug–protein interactions, offering new opportunities for advanced methodologies in drug development and biochemical research.

A practical method for determining the rate of covalent modification of fragments and leads

2025-12-19T18:28:00.000-08:00

Janice Jeon, Svetlana A. Kholodar, Brian H. Tran, Kimberly E. Mallinger, Daniel A. Erlanson & Robert A. Everley

Nat Commun 16, 11234 (2025).

https://doi.org/10.1038/s41467-025-66924-0

The clinical success of covalent drugs such as sotorasib has renewed interest in covalency for rational drug design. The most rigorous potency metric for covalent modifiers is the second-order rate constant kinact/KI. However, existing methods for measuring kinact/KI are resource-intensive and involve complex data interpretation. We describe the diagonal dose-response time-course (dDRTC), an efficient mass spectrometry-based method for determining kinact/KI, enabling routine kinact/KI quantification earlier in programs and accelerating SAR interpretation for lead discovery. We apply dDRTC to a dozen covalent fragment and lead-like modifiers for three targets, KRASG12C and two E3 ligase complexes. Kinetic simulations comparing a range of kinact and KI values establish recommended parameters for dDRTC and reveal that the approach is particularly suited for covalent fragments and leads. Our results demonstrate accurate determination of kinact/KI values across three orders of magnitude with eight-fold increased throughput, reduced protein consumption, and simplified data analysis.

Stereoselective Degradation of Diacylglycerol Kinases Potentiate T cell Activation and Tumor Cell Cytotoxicity

2025-12-12T21:40:00.000-08:00

Minhaj Shaikh, Surya P Mookherjee, Claire Weckerly, Adam H Libby, Aizhen Xiao, Yunge Zhao, Sagar D Vaidya, AeRyon Kim, Zhihong Li, Madeleine L Ware, Michelle Marants, Olivia Murtagh, Wesley J Wolfe, Timothy N Bullock, Benjamin W Purow, Gerald R Hammond, Ken Hsu

bioRxiv 2025.12.09.692983;

doi: https://doi.org/10.64898/2025.12.09.692983

Stereoselective recognition is a powerful means to differentiate selective versus non-specific activity of small molecules in complex biological systems. Here, we disclose stereochemically defined, sulfonyl-triazole inhibitors of the lipid enzyme diacylglycerol kinase-alpha (DGKA), a key metabolic checkpoint for T cell effector function. Acute treatment with the covalent DGKA inhibitor AHL-7160 recruited endogenous DGKA to the plasma membrane in a stereoselective and isozyme-specific manner. The membrane translocation activity of AHL-7160 correlated with blockade of cellular phosphatidic acid production and potentiation of primary T cell-mediated killing of a glioblastoma cell line. Quantitative chemoproteomics revealed Y669 and K411 as sites of AHL-7160 modification on endogenous DGKA in cells. Extended treatments resulted in proteasome-dependent and proteome-wide selective degradation of DGKA in T cells. Collectively, these findings establish covalent DGKA ligands as potent molecular glues with translational potential in immunotherapy.

Identification of VVD-214/RO7589831, a Clinical-Stage, Covalent Allosteric Inhibitor of WRN Helicase for the Treatment of MSI-High Cancers

2025-12-10T18:11:00.000-08:00

Shota Kikuchi, Jason C. Green, Don C. Rogness, Betty Lam, Zachary A. Owyang, Robert D. Malmstrom, Ali Tabatabaei, Aaron N. Snead, Melissa A. Hoffman, Steffen M. Bernard, Paige Ashby, Kelsey N. Lamb, Benjamin D. Horning, Kristen A. Baltgalvis, Kent T. Symons, Thomas A. Glaza, Chu-Chiao Wu, Xiaodan Song, Martha K. Pastuszka, John J. Sigler, Jonathan Pollock, Laurence Burgess, Gabriel M. Simon, Matthew P. Patricelli, and David S. Weinstein

Journal of Medicinal Chemistry 2025

DOI: 10.1021/acs.jmedchem.5c01805

Werner syndrome helicase (WRN) has emerged as a compelling therapeutic target for microsatellite instability-high (MSI-H) cancers, owing to its selective dependency on the helicase activity of WRN. Despite the inherent challenges in targeting helicases, our chemoproteomics approach enabled the identification of compounds that covalently engage C727 within an allosteric pocket of WRN, thereby inhibiting its ability to unwind DNA. Through optimization of each molecular component, particularly focusing on the vinyl sulfone warhead and C2 substitution at the pyrimidine core, an optimal balance of intrinsic reactivity, inhibitory potency, and metabolic stability was achieved, culminating in the identification of VVD-214/RO7589831. This process underscored the tunability of the vinyl sulfone warhead and its effectiveness in covalent drug discovery. VVD-214 induced tumor regression in MSI-H colorectal cancer models and is being evaluated as a promising therapeutic candidate for MSI-H cancers.