Late-stage functionalization with strain-release warheads enables tunable covalent inhibition

Zachary P. Shultz, Ansar Lee-Sam, Yun-Pu Chang, Luxin Sun, Dylan Grassie, Alessio Gabellini, Kyle Pedretty, Thomas Scattolin, Victoria Izumi, Bin Fang, Samer Sansil, Ramu Kakumanu, Lukasz Wojtas, John Koomen, Ernst Schönbrunn, Andrii Monastyrskyi, Derek Duckett, and Justin M. Lopchuk P. Shultz et al.

Science393,408-416(2026).DOI:10.1126/science.adx7219

Structured Abstract

INTRODUCTION

Covalent drugs are transforming targeted therapy by forming durable bonds with disease-driving proteins, yet most rely on a narrow set of reactive groups, particularly acrylamides. These conventional approaches, although effective, can lead to off-target interactions and restrict broader application by their limited structural design. Expanding covalent drug design beyond these established chemotypes is essential to improve both selectivity and therapeutic performance.

RATIONALE

We sought to establish a general platform for replacing acrylamide-based covalent reactive groups in complex drug molecules with alternative chemotypes that offer improved control over reactivity. Our approach centers on bicyclobutanes that are integrated with sulfur-based functional groups commonly used in medicinal chemistry. To enable broad application, we developed a reagent-based strategy that allows these strain-release elements to be installed at the final stage of a synthesis from widely accessible amine precursors. This modular S(IV)-based platform provides a unified entry to multiple sulfur oxidation states and connectivity patterns, enabling systematic tuning of covalent reactivity and target engagement while preserving the parent-drug architecture. By design, this approach allows direct, head-to-head comparison with established covalent inhibitors.

RESULTS

We developed stable, scalable reagents that enable efficient late-stage installation of strain-release bicyclobutane groups across a wide range of clinically relevant scaffolds, including multiple approved kinase inhibitors. This strategy enables direct bioisosteric replacement of acrylamide warheads without modifying the underlying pharmacophore. These S(VI) strain-release groups are highly chemoselective for thiols, and their intrinsic reactivity can be tuned over a broad range through structural modification.

Notably, compounds with similar intrinsic reactivity displayed markedly different levels of target inhibition, demonstrating that productive covalent engagement depends not only on electrophile reactivity but also on molecular orientation within the protein binding site. In cellular systems, the modified inhibitors retained potent activity and effectively suppressed target signaling. Structural analysis confirmed covalent bond formation at the intended site. Across kinase panels and proteome-wide profiling experiments, these strain-release analogs displayed improved selectivity and reduced off-target interactions relative to their acrylamide counterparts. Importantly, these advances translated beyond in vitro systems, with strain-release analogs demonstrating favorable pharmacokinetic properties and efficacy in preclinical in vivo mice models.

CONCLUSION

This work establishes a reagent-enabled, late-stage functionalization platform for the bioisosteric replacement of acrylamides using strain-release bicyclobutanes, bridging chemical innovation to preclinical validation. More broadly, it further supports that effective covalent inhibition is governed not only by intrinsic electrophile reactivity but also by its integration with molecular recognition, providing a framework for designing more selective and clinically effective covalent therapies.

Peptidic Phosphonates as Irreversible Covalent Inhibitors of Plasmodium falciparum Serine Protease PfSUB1.

Armands Kazia, Elina Lidumniece, Chrislaine Withers-Martinez, Liva Eglite, Owain Donnelly, David A. Fidock, Michael J. Blackman, Aigars Jirgensons; 

ACS Med. Chem. Lett. 2026

https://doi.org/10.1021/acsmedchemlett.6c00268

Malaria, caused by Plasmodium parasites, remains a major global health challenge, exacerbated by the widespread emergence of drug-resistant plasmodium strains. Subtilisin-like serine protease SUB1 triggers escape of the parasite from the red cell via a process called egress, rendering the enzyme a prospective antimalarial drug target. While several SUB1 inhibitors have been developed, irreversible covalent inhibition has not been explored so far. In this work, we report our studies of peptidic inhibitors bearing covalent serine traps such as β-lactam, β-lactone, epoxide, and diaryl phosphonate. Out of these, peptidic diaryl phosphonates were found to be irreversible PfSUB1 inhibitors, with the best inhibitor 3b showing a PfSUB1 inhibitory potency (IC50) of 167 nM.

CHARMM-GUI Covalent Ligand Docker as a Web-based Molecular Docking Platform for Covalent Ligands

Lingyang Kong, Donghyuk Suh, Wonpil Im

bioRxiv 2026.07.13.738313; doi: https://doi.org/10.64898/2026.07.13.738313

Covalent inhibitor research is an emerging topic in drug discovery due to its superior performance in specificity and inhibition effects. While molecular docking is a popular strategy in prediction and assessment of ligand conformations or poses in receptor proteins, covalent ligand docking requires nontrivial preparation efforts, as the ligand structure changes during the covalent complex formation. In order to facilitate molecular docking for covalent ligands, we have developed CHARMM-GUI Covalent Ligand Docker (CGUI-CLD), a new module for covalent ligand docking supported by AutoDock4. CGUI-CLD automates ligand preparation, supports ligand modification, implements docking simulation, and presents results through an intuitive user interface. A knowledge-based library built in CGUI-CLD currently supports 66 warheads and 8 amino acids, which can be used to automate the covalent ligand transformation from a pre-reaction to a post-reaction adduct form seamlessly. Moreover, CHARMM-GUI High-Throughput Simulator is integrated for rapid generation of multiple molecular dynamics simulation systems. CGUI-CLD is expected to significantly reduce a massive workload of covalent ligand docking and advance covalent ligand research.

Covalent remodeling of CRBN creates a non-canonical neosubstrate interface with NTAQ1

Andres H. de la Peña, Justin T. Cruite, Jianwei Che, Mary E. Matyskiela, Philip P. Chamberlain, Eric S. Fischer, Lyn H. Jones

bioRxiv 2026.07.14.738385; 

doi: https://doi.org/10.64898/2026.07.14.738385

Molecular glue degrader EM12-FS covalently modifies cereblon (CRBN) His353, enabling selective recruitment of the neosubstrate NTAQ1 to the CRL4CRBN ubiquitin ligase. We determined the cryo-EM structure of the NTAQ1–EM12-FS–CRBN–DDB1 complex, revealing a non-canonical neosubstrate interface created by covalent remodeling of the CRBN sensor loop. Imidazylation repositions His353 to eliminate the steric clash that prevents NTAQ1 engagement by reversible IMiDs, and the engineered interface is stabilized by a distinctive T-shaped C-H/π interaction between sulfated His353 and NTAQ1 Phe126. Biochemical and mutational analyses define the determinants of ternary complex formation and ubiquitination. These findings show that site-specific synthetic modification of CRBN can reprogram induced-proximity pharmacology, expanding specificity beyond the G-loop degron and establishing a framework for covalent engineering of new degrader modalities.

A covalent irreversible inhibitor binds in two mutually exclusive conformations to the active-site cysteine residue of human aldehyde dehydrogenase 1A3

Daniela Covaleda, David Vizarraga, Tulsi Upadhyay, Jiyun Zhu, Daniel Abegg, Raquel Pequerul, Martín Hugo, Alexander Adibekian, Ignacio Fita, Xavier Parés, Francesc Xavier Avilés, Matthew Bogyo, Jaume Farrés

doi: https://doi.org/10.64898/2026.07.14.738401

Aldehyde dehydrogenases (ALDH) are enzymes that catalyze the NAD(P)+-dependent oxidation of aldehydes into carboxylic acids, playing roles in detoxification, biosynthesis, and regulatory functions. Dysfunction of ALDH is associated with serious conditions such as alcohol intolerance, cancer, cardiovascular problems, and neurological disorders. In humans, ALDH1A1 and ALDH1A3 isoforms act as retinaldehyde dehydrogenases and are overexpressed in various cancers, where high levels are associated with increased tumor malignancy, cancer stem cell traits, and therapeutic resistance. ALDH1A3 is recognized as a promising target for anticancer therapies, with several inhibitors, mainly reversible, developed to specifically target it or the enzyme family.

Since ALDH enzymes can also display esterase activity, we used this property to develop an in vitro assay specifically targeting the esterase function of ALDH1A3. A highly conserved active-site cysteine in ALDH1A3 is located at the bottom of two converging channels, which define the substrate- and cofactor-binding pockets. To target this catalytic cysteine, we screened a library of 3,200 cysteine-focused covalent fragments. This led to the identification of Z3405279217 (Z34), an acrylamide-based covalent compound that inhibits both ALDH1A1 and ALDH1A3 at sub-micromolar levels. Biochemical and biophysical tests confirmed that Z34 acts as a time-dependent, covalent, and irreversible binder to the active-site cysteine. In this work, we determined the Cryo-EM structure of the ALDH1A3-Z34 complex at 2.26 Å resolution, confirming the covalent attachment to the catalytic cysteine of Z34. Notably, two mutually exclusive covalent binding modes were observed: one occupying the substrate-binding pocket and the other the cofactor-binding region. Z34 displayed unexpected binding modes within the active site and holds promise as a lead compound for future drug development.

A covalent inhibitor targeting Cys-349 of LIMK1 confers selectivity over LIMK2

Jon B. Patteson,  Ioannis Manolaridis, Mee Ra Hong,  Jennifer M. Johnston, Samaneh Mesbahi-Vasey,  Michael C. Gregory ,Daniel V. Iwamoto John C. Reid John M. Sanders, Ditte Lovatt, Terrence P. McDonald, Valerie W. Shurtleff, Sandra B. Gabelli,  Marina Bukhtiyarova 

Journal of Biological Chemistry, 2026, 113322,

https://doi.org/10.1016/j.jbc.2026.113322

LIM domain kinase 1 (LIMK1) has been identified as a promising therapeutic target for a variety of conditions, such as chronic pain, open-angle glaucoma, various cancers, schizophrenia, and Fragile X syndrome. However, identifying inhibitors that selectively inhibit LIMK1 over LIM domain kinase 2 (LIMK2) has proven to be challenging. A viable strategy to overcome this difficulty is the development of covalent inhibitors, which can offer both potency and selectivity for LIMK1 due to a reactive cysteine, C349, near the active site absent in its paralog LIMK2. Here we identify an irreversible covalent inhibitor of LIMK1 (cLIMK1i), which is highly selective for LIMK1 over both LIMK2 and a panel of over 100 kinases. A crystal structure of LIMK1 soaked with cLIMK1i reveals it is a type I inhibitor occupying the ATP-binding site with its acrylamide moiety oriented toward the P-loop where C349 resides. Computational modeling supports that the P-loop of LIMK1 can adopt a conformation compatible with covalent bond formation. Biochemical and biophysical characterization of the interaction of cLIMK1i with LIMK1 demonstrates that the covalent bond with LIMK1-C349 is essential for its potent inhibition. These results support covalent inhibition of LIMK1 as a viable strategy for selectively inhibiting LIMK1 over LIMK2 and other kinases.

Machine learning-guided discovery of covalent sortase A inhibitors targeting MRSA virulence

Xu-liang Xu, Ti-ti Ying, Xiao-wen Wu, Yun-jun Chen, Gang-ao Hu, Yu-tian Guan, Shi-yi Liu, He Wang, Mohamed Seif, Mahmoud Emam, Hong Wang, Wei Hou, Bin Wei,

European Journal of Medicinal Chemistry, 2026, 119139,

https://doi.org/10.1016/j.ejmech.2026.119139

The global rise of methicillin-resistant Staphylococcus aureus (MRSA) has highlighted the urgent need for alternative therapeutic strategies beyond conventional bactericidal antibiotics. Targeting bacterial virulence rather than viability represents a promising approach to mitigate selective pressure and delay resistance development. Sortase A (SrtA), a membrane-associated transpeptidase responsible for anchoring virulence-associated surface proteins, is an attractive anti-virulence target due to its non-essential role in bacterial survival. Here, we report a machine learning-guided strategy for the discovery of novel covalent SrtA inhibitors based on a 1,2-benzoselenazol-3-one (BSEA) scaffold featuring a tunable electrophilic Se–N bond. A scaffold-aware classification model with a Tanimoto similarity constraint trained on 529 SrtA inhibitors enabled prospective virtual screening of over 35,000 BSEA and BTA derivatives, leading to a high hit rate of 89% upon experimental validation. Representative compounds exhibited submicromolar SrtA inhibition (IC50 = 0.84–1.04 μM) while showing minimal effects on bacterial growth (MIC = 8–32 μM), indicating effective functional decoupling of virulence and viability. Mechanistic studies demonstrated time-dependent irreversible inhibition kinetics, supported by jump dilution assays and Nano-LC-MS/MS identification of covalent modification at the catalytic residue Cys184. These inhibitors effectively disrupted MRSA biofilm formation at sub-inhibitory concentrations and significantly improved host survival in a Galleria mellonella infection model. Collectively, this study establishes a data-driven framework integrating machine learning and covalent chemistry for anti-virulence drug discovery and provides promising lead compounds targeting SrtA to combat MRSA infections.

A chemoproteomic atlas of the human purine interactome for regioselective ligand discovery

Zhihong Li, Hsiao-Kuei Tsai, Adam H. Libby, Michael W. Founds, Olivia L. Murtagh, Madeleine L. Ware, David M. Leace, Wesley J. Wolfe, Phillip W. Gingrich, Bissan Al-Lazikani, Chin-Yuan Chang & Ku-Lung Hsu

Nat Commun (2026). 

https://doi.org/10.1038/s41467-026-73407-3

Purines are essential bioactive molecules that interact with a large fraction of the human proteome. Despite their importance, the scope of actionable purine-binding pockets for ligand discovery remains limited. Here, we develop a quantitative chemoproteomics platform using sulfonyl-purine (SuPUR) chemistry to produce a massive and functional map of the human purine interactome. The SuPUR platform captures 31,000+ targetable tyrosine and lysine sites, representing the most comprehensive beyond cysteine chemoproteomics database for enabling protein ligand discovery. SuPUR ligands that bind through a regioselective fashion serve as enabling starting points for developing potent (nanomolar) and proteome-wide-selective modulators of enzymatic and protein-protein interaction function. Phenotypic screening identifies a site-specific (Y237) and regioselective SuPUR ligand of ACAT2 to reveal an unexpected metabolic dependency in cancer cells. A crystal structure of SuPUR ligand-bound ACAT2 reveals the purine group binds deep in the CoA pocket forming key interactions with catalytic residues via a water bridge to guide future structure-based ligand design.

Linking of fragments in neighboring binding sites is one of the optimization strategies in fragment-based drug discovery, where additive or even more substantial bioactivity improvements can be realized. However, such efforts present a considerable challenge when one fragment binds covalently to the target protein, as small modifications can influence the correct positioning of the covalent warhead toward the targeted nucleophilic residue. Here, we present a case study of fragment linking that yielded single-digit micromolar, covalent inhibitors of the SARS-CoV-2 main protease, starting from fragments that were inactive in the biochemical assay. Using structural information from a recent, high-throughput crystallographic fragment screen, we show that the success of fragment linking in the design of targeted covalent inhibitors is heavily impacted by several factors, including the warhead type, the labeling chemistry, and even subtle changes in the designed linker. Notably, we observe that induced fit effects might override the original fragment orientations in the linked molecule, highlighting the need for reliable structure verification, especially in consecutive rounds of fragment elaboration.

Levente Kollár, Levente M. Mihalovits, Dávid Bajusz, DamijanKnez, József Simon, Blake H. Balcomb, Daren Fearon, Stanislav Gobec, György M. Keserű,

ChemMedChem 2026, 21, e202501108. 

https://doi.org/10.1002/cmdc.202501108

Linking of fragments in neighboring binding sites is one of the optimization strategies in fragment-based drug discovery, where additive or even more substantial bioactivity improvements can be realized. However, such efforts present a considerable challenge when one fragment binds covalently to the target protein, as small modifications can influence the correct positioning of the covalent warhead toward the targeted nucleophilic residue. Here, we present a case study of fragment linking that yielded single-digit micromolar, covalent inhibitors of the SARS-CoV-2 main protease, starting from fragments that were inactive in the biochemical assay. Using structural information from a recent, high-throughput crystallographic fragment screen, we show that the success of fragment linking in the design of targeted covalent inhibitors is heavily impacted by several factors, including the warhead type, the labeling chemistry, and even subtle changes in the designed linker. Notably, we observe that induced fit effects might override the original fragment orientations in the linked molecule, highlighting the need for reliable structure verification, especially in consecutive rounds of fragment elaboration.

Characterization of the Second-Generation Covalent Fragment Library (CovLib Gen2): Thiol Reactivity Profiling and p53-Y220C Rescue

Schwer, M., Aldea, S. R., Engelhardt, M. U., Stahlecker, J., Rheinganz, J., Langkamp, A., & Boeckler, F. M. 

Drug Design, Development and Therapy, (2026). 20. 

https://doi.org/10.2147/DDDT.S598622

Purpose

Covalent Fragment-Based Drug Discovery (FBDD) has emerged as a powerful strategy for unlocking challenging pharmacological targets and engaging shallow or “cryptic” binding pockets. In this study, we present the design and characterization of the Second Generation Covalent Fragment Library (CovLib Gen2), an expanded collection of 81 structurally diverse electrophiles tailored for Covalent Fragment-Based Drug Discovery (FBDD) using an electrophile-first approach. The library spans five distinct warhead classes, including epoxides, vinyl sulfones, acrylamides, α-cyanoacrylamides, and a core set of SɴAr-reactive heteroarenes.

Methods

We comprehensively profiled the library for physicochemical properties and intrinsic thiol reactivity using high-throughput 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) and high-performance liquid chromatography (HPLC)-based glutathione (GSH) reactivity assays. To demonstrate the library’s utility, we performed differential scanning fluorimetry (DSF) screening against the oncogenic, thermally unstable p53-Y220C mutant and subsequent specificity testing with two control mutants.

Results

The library exhibited a broad dynamic range of reactivities with a clear correlation between the assay methods. Additionally, we identified 12 fragments with desirable mild reactivity profiles (t1/2GSH = 1–10 h). The DSF screen yielded 15 hits, primarily SɴAr-reactive heteroarenes and vinyl sulfones. Notably, the fragment SN054 emerged as the most potent stabilizer, inducing a maximal thermal shift of 4.5 °C. Specificity was confirmed using a cysteine-light variant (T-p53C-Y220C-CL), where SN054 retained significant stabilizing activity.

Conclusion

Our findings validate CovLib Gen2 as a versatile tool for ligand discovery, including electrophilic fragments covering a broad range of reactivity, and provide tractable starting points for the pharmacological rescue of p53-Y220C.

Accelerating SuFEx Reactions via Aryl Fluorosulfate Structural Engineering for Enhanced Covalent Targeted Cancer Therapy

Gao, W.; Zhang, C.; Li, D.; Liu, Y.; Zhao, M.; Xia, X.-X.; Huang, W.; Xia, X.; Yan, D. 

Angew. Chem. Int. Ed. 2026

https://doi.org/10.1002/anie.4497440

 https://onlinelibrary.wiley.com/doi/full/10.1002/anie.4497440 Aryl fluorosulfate warheads, possessing sulfur(VI) fluoride exchange (SuFEx) reaction, hold significant promise for the development of covalent protein drugs. However, their SuFEx reactivity remains limited within the complex microenvironment of protein interactions. To address this challenge, we sought to enhance their reactivity by adjusting the electronic and steric properties of warheads. Herein, we synthesized various maleimide-functionalized aryl fluorosulfate (MFS) bearing different substituents (e.g., o-F, o-CF3, o-NO2, o-CH3, o-OCH3, o-Cl, o-Br, o-I, 2,6-diF, and m-F), which were then chemically conjugated to Adnectin (an EGFR-targeting protein). The SuFEx reactivity of the resulting xMFS-modified Adnectin was systematically investigated by comparing their covalent cross-linking efficiency to EGFR. Notably, the meta-Fluoro-substituted MFS warhead, featuring moderate electrophilicity and minimal steric hindrance, exhibited the highest reactivity, achieving a 3.5-fold increase in cross-linking efficiency compared to unsubstituted control. The m-F MFS-modified Adnectin was further attached to the surface of albumin-bound DXd. Leveraging its enhanced SuFEx reactivity, the resulting covalent albumin-bound drug exhibited 6.4-fold higher intracellular accumulation, 3.0-fold greater tumor retention, and 4.0-fold higher antitumor efficiency compared to unsubstituted control. Overall, fine-adjusting the electronic and steric properties of warheads significantly enhances their SuFEx reactivity, enabling the rational design of SuFEx-based warheads and facilitating the application in covalent protein drugs.

Covalent Inhibitors of Monoacylglycerol Lipase Induce Conformational Changes and Proteasomal Degradation

Jordan A. Pham, Thanawat Thaingtamtanha, William McLeish, David Lefebvre, Spencer M. Uguccioni, Roxana Filip, Francesco Gentile, and John Paul Pezacki

J. Am. Chem. Soc. 2026

https://doi.org/10.1021/jacs.5c22859

Monoacylglycerol lipase (MGLL) is a key serine hydrolase that regulates 2-arachidonoylglycerol (2-AG) and eicosanoid signaling. Inhibition of MGLL blocks the conversion of 2-AG into arachidonic acid (AA) with broad therapeutic implications in inflammation, cancer, and viral infection. Carbamate/urea inhibitors such as MJN110, JZL184, and SAR629 are widely used to irreversibly inhibit MGLL through covalent modification of the catalytic serine residue. Here, we demonstrate that this inhibitor class also induces proteasome-dependent degradation of MGLL, functioning as monovalent degraders. We show that loss of MGLL following covalent inhibitor treatment is dependent on the 26S proteasome, while detailed simulations of MGLL dynamics following inhibitor binding suggest that these inhibitors do not destabilize their protein target but instead induce conformational changes that likely facilitate polyubiquitination by exposing two lysine residues. Taken together, these findings establish carbamate/urea inhibitors as dual functional molecules with the propensity to both covalently inhibit their target and act as structural degraders, potentially functioning as monovalent molecular glues, highlighting the need to evaluate the degradation potential alongside inhibitory potency in future screening and drug discovery.

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

Tamayo-Jaramillo D, Hegde S, Jia X, Coffman K, Vankayalapati H, Bearss D, Jones KB, Stark AW, Shen PS. 

Adv Sci. 2026; 13(25):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.

Nitro-Diphenyl Ethers as Emerging Cysteine-Targeting Covalent Warheads Enable Identification of Novel Target LDLRAP1 for Anticoronaviral Activity

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.

Shifting the PPARγ conformational ensemble toward a transcriptionally repressive state improves covalent inhibitor efficacy

Liudmyla Arifova, Brian S MacTavish, Zane Laughlin, Mithun Nag, Karadi Giridhar, Jinsai Shang, Min-Hsuan Li, Xiaoyu Yu, Di Zhu, Theodore M Kamenecka, Douglas J Kojetin,

eLife 2025 14:RP106697

https://doi.org/10.7554/eLife.106697.2

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) regulates transcription in response to ligand binding at an orthosteric pocket within the ligand-binding domain (LBD). We previously showed that two covalent ligands, T0070907 and GW9662—extensively used as PPARγ inhibitors to assess off-target activity—weaken but do not completely block ligand binding via an allosteric mechanism associated with pharmacological inverse agonism (Shang et al., 2024). These covalent inhibitors shift the LBD towards a repressive conformation, where the activation function-2 (AF-2) helix 12 occupies the orthosteric pocket, competing with orthosteric ligand binding. Here, we provide additional support for this allosteric mechanism using two covalent inverse agonists, SR33065 and SR36708, which better stabilize the repressive LBD conformation and are more effective inhibitors of—but also do not completely inhibit—ligand cobinding. Furthermore, we show that ligand cobinding can occur with a previously reported PPARγ dual-site covalent inhibitor, SR16832, which appears to weaken ligand binding through a direct mechanism independent of the allosteric mechanism. These findings underscore the complex nature of the PPARγ LBD conformational ensemble and highlight the need to develop alternative methods for designing more effective covalent inhibitors.

Don’t Lose Your (War)head: Structure–Activity Relationships of Covalent Warheads as Substrates for GST-Catalyzed Glutathione Conjugation

Lavleen K. Mader, Jessica E. Borean, and Jeffrey W. Keillor

Journal of Medicinal Chemistry 2026

DOI: https://doi.org/10.1021/acs.jmedchem.6c00826

The resurgence of targeted covalent inhibitors (TCIs) has expanded the diversity of electrophilic warheads used in drug discovery. TCIs must balance efficient target engagement with resistance to rapid metabolic clearance. In drug development campaigns, intrinsic reactivity toward glutathione (GSH) is commonly used to estimate metabolic liability; however, in vivo GSH conjugation is primarily catalyzed by glutathione S-transferases (GSTs), a phase II metabolic pathway that is not captured by intrinsic reactivity measurements. Here, we establish a quantitative assay to determine GST kcat and KM values across a panel of structurally diverse warheads. We show that their intrinsic reactivities correlate poorly with GST-catalyzed conjugation rates, which are instead governed by warhead- and scaffold-dependent enzyme–substrate interactions. In contrast, GST kcat/KM values correlate closely with compound half-lives in human liver cytosol. Together, these findings establish GST susceptibility as a structurally tunable determinant of metabolic GSH conjugation and provide new principles for the optimization of TCIs.

Efficacy and safety of branebrutinib (BMS-986195), an irreversible Bruton's tyrosine kinase inhibitor, for the treatment of rheumatoid arthritis: a phase 2a, randomised, double-blind, placebo-controlled study

 Østergaard M, Haavardsholm E, Nowak M et al.

The Lancet Rheumatology, 2026

https://doi.org/10.1016/S2665-9913(25)00374-1

1. Background

   Branebrutinib, an oral, highly selective, and irreversible Bruton's tyrosine kinase inhibitor, is a potential candidate for rheumatoid arthritis treatment as Bruton's tyrosine kinase has a role in B-cell activation, autoantibody production, and proinflammatory cytokine release, all of which are implicated in rheumatoid arthritis disease activity and progression. This study assessed the efficacy and safety of branebrutinib in patients with rheumatoid arthritis and an inadequate response to methotrexate.

   Methods

   This phase 2a, randomised, double-blind, placebo-controlled study was designed to assess the efficacy and safety of branebrutinib in patients with rheumatoid arthritis, systemic lupus erythematosus, or primary Sjögren's disease. Here, we report the results of the rheumatoid arthritis substudy, done in the USA, Poland, and Spain across 24 sites. The study included a 12-week double-blind treatment period followed by an additional 12-week open-label period with abatacept treatment. Only data for the double-blind treatment period are reported here. Eligible patients were aged 18–75 years, met the 2010 American College of Rheumatology (ACR)–European Alliance of Associations for Rheumatology criteria for rheumatoid arthritis, had disease duration less than 4 years, and had inadequate response to methotrexate. Patients were randomly assigned (3:1) to receive branebrutinib 9 mg once daily or placebo for 12 weeks. Randomisation was carried out centrally according to a computer-generated block randomisation scheme using interactive response technology. All parties were masked to treatment allocation. The primary endpoint was the proportion of patients who had 50% improvement in the ACR response criteria (ACR50) at week 12, assessed in all participants randomly assigned to treatment (full analysis set). Safety was assessed in patients who received at least one dose of branebrutinib or placebo. This trial was registered with ClinicalTrials.gov, NCT04186871. Patients with lived experience of rheumatoid arthritis were not involved in the study design.

   Findings

   Between Jan 7, 2020, to Dec 5, 2022, 85 patients were randomly assigned to receive branebrutinib (n=64) or placebo (n=21). 63 (74%) of 85 patients were female, 22 (26%) were male, 80 (94%) were White, and the mean age was 49·1 years (SD 12·0). The primary endpoint of ACR50 response at week 12 was not met; 12 (19%) of 64 patients had an ACR50 response in the branebrutinib group compared with seven (33%) of 21 patients in the placebo group (p=0·16). Adverse events were similar between the two groups (30 [47%] of 64 in the branebrutinib group and 8 [38%] of 21 in the placebo group), with no reported serious adverse events or deaths.

   Interpretation

   There was no significant difference between branebrutinib and placebo for any clinical efficacy measures. The 12-week safety profiles were similar between treatment groups, and branebrutinib was well tolerated with a favourable safety profile.

   Funding

   Bristol Myers Squibb.

A Novel Covalent Inhibitor Fragment for the SARS-CoV-2 Main Protease Identified by Target-Specific Deep Learning

Weijun Zhou, Angel D′Oliviera, Xuhang Dai, Jeffrey S. Mugridge, and Yingkai Zhang

ACS Chemical Biology 2026

DOI: 10.1021/acschembio.6c00120

The SARS-CoV-2 main protease (Mpro, also known as 3CLpro) is an attractive antiviral drug target due to its essential role in viral replication and absence of human homologues. Development of new coronavirus-specific Mpro inhibitors will be important as SARS-CoV-2 continues to evolve. Leveraging the rapidly expanding pool of diverse, experimental Mpro-inhibitor data, we developed a target-specific deep learning workflow to accelerate the discovery of new Mpro inhibitor compounds and fragment-like starting points. This workflow combined a fine-tuned inhibitor prediction model with solubility (logS) and lipophilicity (logP) models, molecular similarity analysis, and literature mining to prioritize novel, drug-like candidates. Applied to a purchasable library of over 500,000 compounds, the approach rapidly identified 24 candidates for experimental testing. Biochemical assays revealed a novel, small covalent inhibitor fragment (A02) with an apparent IC50 of 1.5 μM, prior to any synthetic optimization or derivatization. A 1.76 Å crystal structure of Mpro bound to A02 confirmed covalent modification of the catalytic Mpro cysteine (C145), unique engagement of the underutilized Mpro S3′ pocket, and the potential for derivatives of this scaffold to interact with additional Mpro pockets in future optimization efforts. Together, these results demonstrate the potential for target-specific deep learning approaches to guide the rapid screening and discovery of new inhibitor leads or drug scaffolds.

Electrophilic compound screening identifies GPX4-dependent ferroptosis as a senescence vulnerability

Mariantonietta D’Ambrosio, Matthew E. H. White, Efthymios S. Gavriil, Laura Bousset, Jodie Birch, Aleksandra Gruevska, Emiliano Pasquini, Manuel Colucci, Winnie Fong, Simone Mosole, Aurora Valdata, Dimitris Veroutis, Katie Tyson, Vikas Ranvir, Sandra Prokosch, Joaquim Pombo, Aoki Ardisson, Sanjay Khadayate, George Young, Alex Montoya, Georgia Roumelioti, Jack Houghton, Jianan Lu, Pavel V. Shliaha, Elena De Vita, Santiago Vernia, Vassilis G. Gorgoulis, Suchira Gallage, Mathias Heikenwälder, Zoe Hall, Andrea Alimonti, Iain A. McNeish, Edward W. Tate, Jesús Gil

Nature Chemical Biology, 2026

Journal: Nature Cell BiologySenescent cells drive ageing and age-related pathologies, including cancer. Consequently, senolytics, drugs that selectively kill senescent cells, have broad therapeutic appeal. Here we report a senolytic screen of a library of 10,480 electrophilic compounds. Among 38 identified hits, we found a subset of chloroacetamides with broad senolytic activity. Activity-based protein profiling, coupled with functional assays, identified the glutathione peroxidase GPX4 as a target. We show that senescent cells are primed for ferroptosis, displaying high levels of oxidative stress and intracellular Fe2+, but also upregulate GPX4, which prevents the accumulation of oxidized lipids. Treatment with senolytic chloroacetamides or GPX4 inhibitors selectively kills senescent cells by ferroptosis. The combination of anticancer therapies with GPX4 inhibitors eliminated senescent tumour cells in models of melanoma, prostate and ovarian cancer. Our results show that senescent cells rely on GPX4 to prevent ferroptosis and that GPX4 inhibitors kill senescent cells.

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.