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.