Covalent Modifiers

Overcoming EGFR Resistance by Monovalent and Bident Inhibitors Targeting Cys775

2026-10-29T06:22:06.269-07:00

Zhengnian Li, Jie Jiang, Yaning Wang, Scott B. Ficarro, Stephen J. Collins, Tyler S. Beyett, Ilse K. Schaeffner, Isidoro Tavares, Felix H. Gottlieb, Dhiraj Suda, Prafulla C. Gokhale, Leah M. Black-Holmes, Dimitris Gazgalis, Michael J. Eck, Pasi A. Jänne, Jarrod A. Marto, Jianwei Che, Nathanael S. Gray, Tinghu Zhang;

ACS Cent. Sci. 28 October 2026; 12 (10): 1647–1658. https://doi.org/10.1021/acscentsci.6c01219

Covalent targeting of EGFR cysteine 797 by osimertinib is one of the most successful breakthroughs in targeted therapy, fundamentally transforming the treatment landscape for non-small-cell lung cancer (NSCLC) patients. However, resistance driven by the mutation of C797 remains a major clinical challenge. Developing novel covalent strategies beyond C797 targeting presents a compelling opportunity for next-generation EGFR inhibitors. We first demonstrated that cysteine 775, located deep within the ATP-binding pocket, is accessible by a rationally designed covalent molecule ZNL-3, which as the first-in-class covalent cysteine 775 inhibitor exhibited strong efficacy in osimertinib-resistant mouse models. To further enhance resilience to resistance-causing mutations, we developed a dual-warhead, bident compound YNW-1 which covalently targets both cysteines 775 and 797 simultaneously. YNW-1 is the first intramolecular lock to exhibit balanced reactive efficiency on both cysteines, rendering single-site mutations ineffective at conferring resistance. This study establishes the therapeutic potential of an EGFR covalent inhibitor through unprecedented targeting of cysteine 775. It also provides the first evidence that dual-cysteine engagement offers superior efficacy to conventional covalent inhibitors by delaying the emergence of resistance. Further optimization for clinical translation is currently ongoing in our laboratory.

Ninhydrin as a Covalent Warhead for Chemical Proteomic-Enabled Discovery and Selective Engagement of Reactive Arginines

2026-10-29T06:14:26.833-07:00

Andrew K. Ecker, Andreas Langen, Chloe S. Fields, José L. Montaño, Minh Tran, Ian B. Seiple, Balyn W. Zaro;

ACS Cent. Sci. 2026; 12 (10): 1610–1624.

https://doi.org/10.1021/acscentsci.6c00922

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 from one cell line. These studies uncovered potential modification sites on disease-relevant proteins, including reactive arginines within catalytic sites that are essential for function and reactive arginines on proteins lacking reactive cysteines. By endowing a reversible small-molecule binder of cyclophilin A with a ninhydrin warhead, we achieved selective, covalent engagement in cell lysates and attenuation of enzymatic activity in vitro. These findings establish ninhydrin as a warhead for studying arginine reactivity and identifying at scale arginines amenable to covalent modification.

Characterization of F-30 as an FGFR2/4-selective covalent inhibitor with activity against clinically relevant mutations

2026-10-22T22:08:16.875-07:00

Lin Zhang, Xueru Liu, Wuqing Deng, Tanyuxuan Yang, Ke Gui, Yuepiao Cai, Yongheng Chen, Xiaojuan Chen,

Biochemical Pharmacology, 2026, 118560,

https://doi.org/10.1016/j.bcp.2026.118560.

Fibroblast growth factor receptors FGFR2 and FGFR4 are oncogenic drivers in multiple cancers, yet most approved FGFR inhibitors lack subtype selectivity and are prone to acquired resistance. Here we identify F-30 as, to our knowledge, the first covalent inhibitor with preferential activity toward both FGFR2 and FGFR4 through covalent targeting of a conserved P-loop cysteine. Structural analyses reveal that differential engagement of the P-loop and surrounding binding environment contributes to paralog discrimination. Consistent with these structural observations, F-30 showed an approximately 12-fold lower KI and 13-fold higher kinact/KI for FGFR2 than for FGFR1, whereas their kinact values were comparable. These results indicate that the preference for FGFR2 over FGFR1 is primarily established during pre-covalent recognition. F-30 retained strong activity against multiple clinically relevant FGFR2 resistance-associated mutations but showed reduced potency against gatekeeper mutations. In cellular models, F-30 preferentially inhibited the proliferation of FGFR2/4–driven cancer cells, while substantially weaker pathway inhibition was observed in FGFR1- and FGFR3-driven models than in FGFR2-dependent cells. Taken together, these findings identify structural and kinetic features that contribute to the subtype-selective profile of F-30 and may guide the design of covalent FGFR inhibitors targeting the conserved P-loop cysteine.

Covalent-Allosteric Akt Inhibitors: Targeting Akt3 through Molecular Hybridization and Expanding the Isoform-Selective Toolbox

2026-10-16T06:15:09.843-07:00

Kosmas Alexandros Pervanidis § ; Giovanni Danilo D’Angelo § ; Ioannis Athanasiadis § ; Hannah Jantzen; Andrea Scrima; Katharina Schäfer; Lena Quambusch # ; Matthias P. Müller; Daniel Rauh *

ACS Chem. Biol. (2026) 21 (10): 2572–2596.

https://doi.org/10.1021/acschembio.6c00415

Akt is a central serine/threonine kinase in the PI3K/Akt/mTOR pathway that regulates cell survival, growth, and metabolic homeostasis. Although Akt1, Akt2, and Akt3 share high sequence homology, they perform distinct physiological and pathological functions, motivating the development of small-molecule isoform-selective chemical probes. Here, we first applied a molecular hybridization strategy to combine structural features that lead to Akt3-selectivity with the covalent pharmacophore of established covalent-allosteric Akt inhibitors, enabling selective and covalent targeting of Akt3. We then expanded the chemical space of covalent-allosteric Akt inhibitors to investigate how subtle structural differences influence selectivity across all three isoforms. Guided by structure–activity relationships and high-resolution co-crystal structures, systematic modifications of substituent patterns, linker geometry, and scaffold architecture revealed distinct isoform-selectivity profiles. Biochemical target inhibition, cellular target engagement, and covalent-binding studies further characterized these compounds and their selectivity. Collectively, these findings expand the covalent-allosteric Akt inhibitor toolbox, provide insight into the structural determinants governing Akt isoform selectivity, and establish molecular hybridization as a strategy for selective covalent targeting of Akt3.

Sub-stoichiometric degradation is dispensable for potent PROTACs as demonstrated by irreversible covalent BTK degraders

2026-10-11T09:45:54.635-07:00

Ran Cheng, Hanfeng Lin, Xin Yu, Shrilekha Misra, Andrew D. Mitchell, Jennifer A. Woyach, Xiaoli Qi & Jin Wang

Nat Commun (2026). https://doi.org/10.1038/s41467-026-78239-9

Proteolysis-targeting chimeras (PROTACs) represent a transformative therapeutic modality, yet the viability of covalent PROTACs remains debated, as irreversible binding seemingly contradicts the catalytic mechanism of action. Here, we develop and characterize PSIRC3, a potent covalent PROTAC for Bruton’s tyrosine kinase (BTK). PSIRC3 induces selective BTK degradation with a sub-nanomolar DC50 of 0.75 nM and a Dmax of 85%, while its non-covalent counterpart is inactive. Such activity requires covalent bond formation with Cys481, as efficacy is abolished against the C481S BTK mutant. PSIRC3 achieves maximum BTK degradation within 30 min, a kinetic profile linked to rapid cell permeation and efficient ternary complex formation. In vivo, a single administration of PSIRC3 leads to substantial BTK degradation in both PBMCs ( > 80%) and splenocytes ( > 50%). Computational modeling, parameterized with our experimental data, reveals that degradation efficacy is governed by a balance between E3 ligase and target affinities: excessively high E3 affinity is detrimental by inducing a hook effect, while higher target affinity is generally beneficial. Our findings provide strong evidence that covalent engagement can drive potent and selective protein degradation, challenging the notion that catalytic turnover is indispensable for PROTAC efficacy, and open avenues for targeting intractable proteins.

Sulfonyl-Imidazopyridines Decouple Reactivity from Stability for Ligandability Mapping and Covalent Inhibitor Discovery

2026-10-08T06:00:09.139-07:00

Zhihong Li, Madeleine L. Ware, Phillip W. Gingrich, Bissan Al-Lazikani, Ku-Lung Hsu;

Journal American Chemical Society (2026) 148 (39): 41908–41920.

https://doi.org/10.1021/jacs.6c07487

Covalent probes and therapeutics must balance electrophilicity and stability for function in biological systems, yet gains in stability can sacrifice proteome coverage and limit ligand discovery. Here, we show that single-atom N-to-C substitutions of sulfonyl purines at the nucleofuge position decouple reactivity from stability. Systematic chemical proteomic profiling identifies sulfonyl-imidazopyridines as a class of electrophiles with enhanced cellular and in vivo stability while retaining tunable reactivity toward functional tyrosine and lysine sites. These electrophiles expand access to proteomic sites not engaged by parent sulfonyl-purines or related -triazoles. Importantly, the differential binding of N7- vs N9-sulfonyl-imidazopyridine regioisomer pairs expedites the discovery of proteome-wide-selective inhibitors of metabolic targets, including lanosterol synthase (LSS), phosphoglycerate mutase 1 (PGAM1), and DCTP pyrophosphatase 1 (DCTPP1). Collectively, this work establishes a general strategy for stabilizing electrophiles and introduces a platform for global ligandability mapping guided by regioselective recognition.

β-d-Arabinofuranose-configured Cyclitol Aziridines as Selective, Brain-penetrant Covalent GBA2 Inhibitors

2026-10-04T04:22:22.263-07:00

Rob F. Lammers, Qin Su, Laura Mazo, Wendy A. Offen, Roelof Ottenhoff, Mats J. Bulterman, Martijn van der Lienden, Maria J. Ferraz, Florian Küllmer, Zahid Ilhan, Hans van den Elst, Ingeborg van der Made, Cindy P. A. A. van Roomen, Anne de Jongh, Adrianus M. C. H. van den Nieuwendijk, Rolf G. Boot, Noam Zelcer, Gideon J. Davies, Carme Rovira, Herman S. Overkleeft, Johannes M. F. G. Aerts, Marta Artola;

Journal American Chemical Society, 2026

https://doi.org/10.1021/jacs.6c09754

Neutral glucosylceramidase GBA2 is a retaining β-d-glucosidase involved in sphingolipid metabolism and implicated in neurological lysosomal storage disorders, yet the lack of highly selective chemical inhibitors has hindered efforts to disentangle its function from that of glucosylceramide synthase (GCS) and lysosomal glucosylceramidase (GBA1). Here, we report the development of the first brain-penetrant, covalent inhibitor that selectively inactivates GBA2 in vivo. Building on β-d-arabinofuranose-configured (β-d-Araf) cyclitol aziridine activity-based probes previously shown to covalently and selectively label GBA2, we designed and synthesized a focused library of electrophile-bearing cyclitols, including epoxides, cyclic sulfates, and a diverse set of N-alkyl aziridines. Biochemical evaluation of their inhibitory activity against GBA1, GBA2, GBA3 and GCS revealed that several N-alkyl aziridines are highly selective, nanomolar GBA2 inhibitors. Lead compounds also demonstrated metabolic stability in murine plasma, robust cellular activity and high selectivity in living cells, with no detectable GCS inhibition. Notably, octyl-alkyne aziridine 5 selectively inhibited GBA2 in mouse brain, representing the first covalent GBA2 inhibitor with demonstrated in vivo activity and brain permeability. Together, these results establish β-d-Araf cyclitol aziridine 5 as a versatile inhibitor for selective, covalent GBA2 inhibition, well suited to dissect GBA2 biology in vivo, particularly in neurological lysosomal storage disorders.

Structure-Guided Design of a Brain-Penetrant Covalent Inhibitor of Human Butyrylcholinesterase with In Vivo Procognitive Efficacy

2026-10-02T07:02:26.911-07:00

Peter Mastnak-Sokolov, Urban Košak, Anže Meden, Damijan Knez, Simon Žakelj, Selena Horvat, Anja Pišlar, Jakob Kljun, Alexandre Igert, José Dias, Florian Nachon, Xavier Brazzolotto, Martin Novak, Vendula Rydlova, Jana Zdarova Karasova, Ondřej Soukup, Aleksandra Manik, Jan Detka, Kinga Sałat, Stanislav Gobec

J. Med. Chem. 2026

https://doi.org/10.1021/acs.jmedchem.6c01737

Butyrylcholinesterase (BChE) has emerged as a validated therapeutic target for the management of Alzheimer’s disease symptoms, particularly in advanced stages when acetylcholinesterase activity declines. Despite the numerous BChE inhibitors reported, only the cymserine derivative bisnorcymserine has progressed to clinical evaluation. Here, we report the design and characterization of a novel selective human BChE (hBChE) inhibitor, N-benzylpyrrolidine carbamate (S)-(+)-14. Kinetic analyses and X-ray crystallography of the hBChE−inhibitor complex confirm a covalent mechanism of inhibition. Compound (S)-(+)-14 demonstrates favorable metabolic stability and achieves brain exposure following intraperitoneal administration in mice. In a scopolamine-induced cognitive impairment model, (S)-(+)-14 improves cognitive performance without causing cholinergic adverse effects or motor deficits. Collectively, these findings identify (S)-(+)-14 as a structurally validated, brain-penetrant covalent hBChE inhibitor and highlight its promise as a lead compound for the treatment of Alzheimer’s disease.

Fragment-Based Covalent Targeting of Lysines at the Allosteric Latch Site of SHP2.

2026-09-22T08:16:51.325-07:00

Vincenzo Di Lorenzo, Noémi Csorba, Renáta Szabó, Levente Kollár, Yvette Roske, Ivan Rand̵elović, Krisztina Balázs, Tibor Viktor Szalai, Nina-Louisa Efrém, Levente Mihalovits, Tímea Imre, József Simon, József Tóvári, Marc Nazaré, Oliver Daumke, Péter Ábrányi-Balogh, György M. Keserű;

J. Med. Chem. 2026; https://doi.org/10.1021/acs.jmedchem.6c01030

Covalent mechanism of action is a powerful way to modulate challenging drug targets. Here, we present a streamlined workflow that combines covalent fragment screening (electrophile first approach) and ligand-first strategy to identify covalent inhibitors targeting lysine residues in the allosteric latch site of SHP2 phosphatase. Supported by complementary computational and experimental analyses, this strategy enabled us to identify the first potent cell active allosteric covalent inhibitors acting at this site. Demonstrating the covalent tractability of the latch site opens further avenues for future optimization and therapeutic exploration of high-value allosteric SHP2 inhibitors.

Dual-Site Covalent Targeting Enables BD2-Selective BET Inhibition With Potent Antitumor Activity in Mice

2026-09-13T22:32:34.365-07:00

Jibo Kang, Xuan Wang, Hong Zhang, Jieying Lin, Peng Chen, Zuqin Wang, Zengjun Hao, Fengfei Miao, Fengcai Zhang, Tao Li, Yusheng Xie, Junjian Wang, Zhi-Min Zhang, Shao Q. Yao, Xiaoyun Lu

Angewandte Chemie International Edition 2026 e1224416

https://doi.org/10.1002/anie.1224416

Bromodomain and extra-terminal (BET) proteins are validated therapeutic targets for cancer, but clinical translation of pan-BET inhibitors is limited by dose-limiting toxicities from non-selective inhibition of BD1/BD2 domains. Herein, we report the first-ever domain-selective covalent inhibitor, named ipAE1, of BET BD2 domains by installing an epoxide warhead onto the scaffold of ABBV-744. ipAE1 was shown to irreversibly modify Glu438 and His437 (to a lesser extent) located within the BRD4(2) binding pocket via a dual-covalent mechanism, as evidenced by its chemical probe pAE1. Furthermore, ipAE1 exhibited exceptional potency against BRD4(2) (Kd = 0.096 nM) with > 1900-fold selectivity over BRD4(1), leading to potent and sustained antiproliferative activity in MV4-11 cells (GI50 = 1.5 nM). Subsequent live-cell proteome-wide profiling validated BRD4 as the primary cellular target of ipAE1. Consistent with cellular activities, ipAE1 possessed a significantly enhanced antitumor efficacy in an MV4-11 xenografted mouse model compared to ABBV-744, presumably due to its on-target covalent engagement in vivo. Our study thus establishes for the first time a novel targeted covalent inhibition (TCI) strategy that engages two weakly nucleophilic residues within a single bromodomain, an approach generalizable to other compounds targeting E/H, providing a highly selective platform for future development of next-generation BET inhibitors.

Isoform-Selective Targeting of Akt Through Covalent Allosteric Inhibition

2026-09-13T21:48:17.662-07:00

'Angelo, Giovanni Danilo; Pervanidis, Kosmas Alexandros; Athanasiadis, Ioannis; Lukianchikov, Vladimir; Scrima, Andrea; Depta, Laura; Stier, Sara; Müller, Matthias P.; Farin, Henner F.; Quambusch, Lena; Rauh, Daniel.

Angewandte Chemie International Edition (2026), e3567206.

https://doi.org/10.1002/anie.3567206

The Akt family of serine/threonine kinases plays a crucial role in various cellular processes, including proliferation, survival, and metabolism. Three Akt isoforms (Akt1, Akt2, and Akt3) have distinct physiological roles, and while individual isoform dysregulation is disease-linked, unselective Akt inhibition leads to side effects. Here, we report the development of selective covalent-allosteric Akt inhibitors (CAAIs) targeting Akt2 and Akt3 while sparing Akt1. Guided by protein x-ray crystallography and molecular modeling, key structural differences within the allosteric pockets of the isoforms were identified and exploited in a structure-based design strategy. By stabilizing the inactive kinase conformation, CAAIs overcome the intrinsic selectivity limitations of ATP-competitive inhibitors. After biological characterization, the pyrazole-containing inhibitors emerged as the most potent and selective Akt2 inhibitors, while inhibitors with pyridines as an isoform-selective element were predominantly targeting Akt3 selectively. Importantly, these new inhibitors were also evaluated in patient-derived colorectal cancer organoids. Co-crystal structures of inhibitors bound to an engineered construct mimicking the Akt2 allosteric pocket elucidated the molecular basis of isoform selectivity, guiding further optimization. This work not only establishes a framework for the development of isoform-selective therapeutics but also highlights the potential for unraveling isoform-specific functions in signaling pathways relevant to cancer biology.

An enantioselective covalent inhibitor of BAX confers cytoprotection in vivo

2026-08-31T06:48:19.135-07:00

Peiwen Shi, Bruno Melillo, Matthew W. McHenry, Christina M. Camara, Ka Yang, Evert Njomen, Marina Godes, Maria F. Pazyra-Murphy, Mary Rose Branch, Bethany Tesar, Rosalind A. Segal, Lee L. Rubin, Michael D. Cameron, Gregory H. Bird, Thomas E. Wales, Steven P. Gygi, Benjamin F. Cravatt, and Loren D. Walensky.

Nat Chem Biol (2026).

https://doi.org/10.1038/s41589-026-02297-9

No therapies directly block apoptosis in tissue injury or the many diseases driven by cell loss. The BCL-2 family protein BAX is a central mediator of this pathway and C126 resides within a key regulatory region where physiologic or pharmacologic ligands can activate or inhibit its function. Here, we report enantioselective covalent BAX inhibitors that site-specifically react with C126 and confer cytoprotection across multiple cell types. These ligands constrain BAX conformation and suppress apoptosis in a strictly BAX-dependent manner. Medicinal chemistry optimization yielded covalent BAX inhibitor 3 (CBI-3), an analog with pharmacokinetics suitable for in vivo studies. In a murine model of Fas-induced fulminant hepatic failure, CBI-3 reduced hepatocyte apoptosis and preserved liver histology and survival. CBI-3 also conferred cytoprotection of motor neurons derived from human induced pluripotent stem cells of healthy and amyotrophic lateral sclerosis donors. These findings establish covalent BAX inhibition as a therapeutic strategy to directly block pathologic cell death.

Enhancing De Novo Designed Peptides and Proteins via Irreversible Covalent Isoquinolinium Capture

2026-08-30T20:45:49.711-07:00

Paul M. Levine, Patrick W. Erickson, Timothy W. Craven, Aaron T. Balana, Derrick R. Hicks, Green Ahn, Chan J. Kim, Lisa S. Brandenburg, Wei Yang, Danielle P. Johnson Erickson,

ACS Chem. Biol. (2026)

https://doi.org/10.1021/acschembio.6c00584

Irreversible covalent inhibitors have garnered significant attention in recent years. Despite encouraging progress, the vast majority contain electrophiles that target the least abundant amino acid, cysteine, substantially limiting target inhibitor design for therapeutic intervention. Here, we generalize 2-ethynylbenzaldehyde as a proximity-induced electrophile for generating irreversible covalent peptide and protein inhibitors that specifically target native lysine residues. Leveraging this warhead, we designed a covalent de novo peptide that potently engages MCL1 to block its interaction with Bak. We show it is faster, more site-selective, and increases potency by 61-fold for MCL1 relative to a sulfonyl fluoride warhead. Additionally, with the guide of a computational script to predict "reactive hotspots" at the protein level, we developed a minibinder that labels PD-L1 in vitro and in live cells, displays a slower off-rate, and potently blocks the native PD-1 and PD-L1. These results establish isoquinolinium capture as a promising strategy to inhibit protein–protein interactions and for the development of novel covalent peptide and protein therapeutics.

Sulfonyl-Imidazopyridines Decouple Reactivity from Stability for Ligandability Mapping and Covalent Inhibitor Discovery

2026-08-30T09:38:03.614-07:00

Zhihong Li; Madeleine L. Ware; Phillip W. Gingrich; Bissan Al-Lazikani; Ku-Lung Hsu *

Journal American Chemical Society (2026)

https://doi.org/10.1021/jacs.6c07487

Covalent probes and therapeutics must balance electrophilicity and stability for function in biological systems, yet gains in stability can sacrifice proteome coverage and limit ligand discovery. Here, we show that single-atom N-to-C substitutions of sulfonyl purines at the nucleofuge position decouple reactivity from stability. Systematic chemical proteomic profiling identifies sulfonyl-imidazopyridines as a class of electrophiles with enhanced cellular and in vivo stability while retaining tunable reactivity toward functional tyrosine and lysine sites. These electrophiles expand access to proteomic sites not engaged by parent sulfonyl-purines or related -triazoles. Importantly, the differential binding of N7- vs N9-sulfonyl-imidazopyridine regioisomer pairs expedites the discovery of proteome-wide-selective inhibitors of metabolic targets, including lanosterol synthase (LSS), phosphoglycerate mutase 1 (PGAM1), and DCTP pyrophosphatase 1 (DCTPP1). Collectively, this work establishes a general strategy for stabilizing electrophiles and introduces a platform for global ligandability mapping guided by regioselective recognition.

Development of Covalent Inhibitors of Chikungunya Virus nsP2 Cysteine Protease Enabled by Direct-to-Biology Synthesis and Screening D

2026-08-25T17:20:18.875-07:00

Zhengjun Cai § ; Kan Li § ; Sainetra Sridhar; Haozhou Tan; Hiwot Demssie; Gaungjin Fan; Wenyi Zhang; Bobby Brooke Herrera; Jun Wang

J. Med. Chem. (2026)

https://doi.org/10.1021/acs.jmedchem.5c03672

Chikungunya virus (CHIKV), an arthropod-borne alphavirus, has emerged as a global health threat due to its rapid transmission and the lack of effective antiviral therapies. The cysteine protease activity of the virus-encoded nonstructural protein 2 (nsP2) is critical for CHIKV replication, as it processes viral polyproteins and counteracts host antiviral defenses, establishing it as a highly attractive target for therapeutic intervention. In this study, we present a rapid drug development platform that integrates covalent docking with direct-to-biology (D2B) synthesis and screening to identify nsP2 inhibitors. Candidates prioritized by in silico docking were synthesized and directly tested in FRET enzymatic assays without purification. This approach led to the identification of several nsP2 inhibitors with diverse chemical scaffolds, potent enzymatic inhibition, and antiviral activity. Together, these findings establish a streamlined strategy for covalent inhibitor development and provide promising leads for CHIKV antiviral development.

Covalent allosteric inhibition of AARS1 lactyltransferase

2026-08-23T18:54:50.497-07:00

Yunyuan Huang, Siying Liu, Luyang Tian, Yang Tang, Yilin Dou, Huiling Wang, Tao Zheng, Mingyang Wang, Runhao Li, Zhi Wang, Zhaocai Zhou, Xin Chen & Jinrong Min

Nature Communications (2026)

https://doi.org/10.1038/s41467-026-76732-9

Alanyl-tRNA synthetase 1 (AARS1) was recently identified as a lactyltransferase responsible for protein lactylation, a modification associated with epigenetic regulation and metabolic adaptation. Here, we report compound XY353, which covalently binds C184 of AARS1, inducing steric clashes with F175 and triggering structural rearrangements in the region that displace W176, a key residue for lactate binding. By means of a combination of a variety of biophysical and enzymatic assays with MD simulations and structural analysis, we show that XY353 and its derivative XY353-1 inhibit AARS1 by competing with lactate via the C184–F175–W176 relay, supporting a covalent allosteric mechanism of inhibition, which is further confirmed by our cellular data that XY353-1 reduces lactylation of the AARS1 substrate YAP and suppresses the proliferation of HGC-27 cells. Collectively, these findings identify a covalent allosteric mechanism for AARS1 inhibition and provide chemical tools to explore its biological functions.

Sulfonyl-Imidazopyridines Decouple Reactivity from Stability for Ligandability Mapping and Covalent Inhibitor Discovery

2026-08-22T12:51:53.693-07:00

Zhihong Li; Madeleine L. Ware; Phillip W. Gingrich; Bissan Al-Lazikani; Ku-Lung Hsu *

Journal American Chemical Society (2026)

https://doi.org/10.1021/jacs.6c07487

Covalent probes and therapeutics must balance electrophilicity and stability for function in biological systems, yet gains in stability can sacrifice proteome coverage and limit ligand discovery. Here, we show that single-atom N-to-C substitutions of sulfonyl purines at the nucleofuge position decouple reactivity from stability. Systematic chemical proteomic profiling identifies sulfonyl-imidazopyridines as a class of electrophiles with enhanced cellular and in vivo stability while retaining tunable reactivity toward functional tyrosine and lysine sites. These electrophiles expand access to proteomic sites not engaged by parent sulfonyl-purines or related -triazoles. Importantly, the differential binding of N7- vs N9-sulfonyl-imidazopyridine regioisomer pairs expedites the discovery of proteome-wide-selective inhibitors of metabolic targets, including lanosterol synthase (LSS), phosphoglycerate mutase 1 (PGAM1), and DCTP pyrophosphatase 1 (DCTPP1). Collectively, this work establishes a general strategy for stabilizing electrophiles and introduces a platform for global ligandability mapping guided by regioselective recognition.

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

2026-08-15T20:06:26.845-07:00

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.

Science 393,408-416(2026).

DOI:10.1126/science.adx7219

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.

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

2026-08-13T13:43:55.556-07:00

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

2026-08-09T15:44:57.798-07:00

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

2026-08-06T05:28:11.786-07:00

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

2026-07-27T21:41:06.091-07:00

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

Structured Abstract

INTRODUCTION

RATIONALE

RESULTS

CONCLUSION

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

2026-07-24T11:54:57.432-07:00

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

2026-07-23T07:28:28.551-07:00

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

2026-07-22T05:53:53.958-07:00

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.