Overcoming EGFR Resistance by Monovalent and Bident Inhibitors Targeting Cys775

 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

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

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

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

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

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

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.