Discovery and Optimization of a WRN Helicase Inhibitor Series through Structure-Guided Drug Design from a Covalent Fragment Binding Insight

Momar Toure * ; Xin Cindy Yan; Yonghong Bai; Brian A. Sosa-Alvarado; Cen Gao; Theresa Baker; Erin Brophy; John R. Butler; Yuchen Yuan; Michael H. Reutershan; Anthony Tubbs; Laurie Schenkel; Giulia Bottoni; Erica Evans; Peter Hammerman; Allison Drew; Timothy Guzi; Meredeth A. McGowan

J. Med. Chem. (2026) 69 (15): 18162–18181.

https://doi.org/10.1021/acs.jmedchem.6c00443

WRN helicase activity inhibition has emerged as a promising therapeutic approach for targeting cancer cells with specific DNA repair deficiencies, especially those with microsatellite instability (MSI). Herein, we report a novel covalent WRN helicase inhibitor series discovered and optimized by leveraging insights from a covalent fragment investigation. Initial structure-based design led to potency-optimized compounds from this series that exhibited unbound cellular potency in the nanomolar range in both p21 induction and HCT116 CTG viability assays, albeit with high intrinsic warhead reactivity. Further refinement of the ADME properties by modulating the warhead reactivity yielded lead WC-2 with excellent cellular potency, low reactivity toward GSH, excellent plasma and blood stabilities, good oral bioavailability, and long in vivo half-lives in rat (T_1/2 = 7.2 h; F = 62%) and dog (T_1/2 = 18.9 h). WC-2 has the potential as a next-generation, low-dose WRN helicase clinical candidate for treating patients with MSI-classified tumors.

Discovery of a covalent FGFR2-selective inhibitor overcoming clinically-acquired resistance mutations

Huang, X., Cao, X., Zheng, L. et al. 

Nat Commun (2026). 

https://doi.org/10.1038/s41467-026-76339-0

Abnormal activation of fibroblast growth factor receptor 2 (FGFR2) drives tumorigenesis in various cancers. Clinical use of pan-FGFR inhibitors is limited due to emerging acquired resistance mutations within the FGFR2 kinase domain and adverse effects associated with FGFR1/4 off-target inhibition. Herein, we describe the structure-based discovery of LC-F2-1, an FGFR2-selective inhibitor that demonstrates irreversible covalent binding to the P-loop. Cellular assays confirm the high selectivity of LC-F2-1 for FGFR2 over FGFR1 and FGFR4, along with potent inhibition of FGFR2 signaling. LC-F2-1 maintains strong activity against clinically observed FGFR2 resistance variants, including gatekeeper, molecular brake, and activation loop variants. X-ray crystallography reveals conformational rearrangement of the kinase domain by LC-F2-1, which overcomes the recalcitrant V565F gatekeeper mutation. In vivo, LC-F2-1 induces tumor regression in xenograft models harboring FGFR2 resistance mutations without affecting serum phosphate levels. In this work, we identify LC-F2-1 as a therapeutic candidate for FGFR2-driven cancers.

A covalent PFKL activator suppresses tumor growth

Jiang, X.; Lynch, E. M.; Lyu, C.; Wilson, C. N.; Salay, L. E.; Hess, H. T.; Lyons, S. N.; Lu, M.-J.; Luo, S.; Kim, G.; Chan, H.-R.; Wolfe, W. J.; Zacharias, L. G.; Mathews, T. P.; Lin, Y.-C.; Webb, B. A.; Kollman, J. M.; Cambronne, X. A.; Hsu, K.-L. 

Nat. Chem. Biol. 2026. 

https://doi.org/10.1038/s41589-026-02289-9

Glycolysis fuels vital cellular functions, and its dysregulation has been implicated in cancer, neurodegeneration, antibiotic resistance and diabetes. The glycolytic dependency of cancer, known as the Warburg effect, represents a key vulnerability for development of targeted anticancer agents; however, the development of such agents remains challenging owing to metabolic heterogeneity and resistance. Here we developed a covalent phosphofructokinase-1 liver type (PFKL) activator that couples glycolytic activation with delivery of a cytotoxic carnitine palmitoyltransferase 2 (CPT2)-targeting payload to cancer cells in vitro and in vivo. The electrophile–drug conjugate site-specifically and proteome-wide selectively modifies K677 in the allosteric effector site to stabilize the R-state tetramer of PFKL, while concomitantly releasing a CPT2-selective inhibitor to destabilize cell metabolism. The delivery mechanism of electrophile–drug conjugates is analogous to that of antibody–drug conjugates, but differentiated by their selective covalent targeting of intracellular proteins.

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.