Lysine-Targeted Covalent Inhibition of eIF4E with an Anionic Electrophile Warhead

Paramita Pal, Erika M. James, Caitlyn E. Given, Hong Yue, Rebecca J. Metivier, Jianwei Che, Eric S. Fischer, Lyn H. Jones; 

ACS Chem. Biol. 2026

https://doi.org/10.1021/acschembio.6c00714

Covalent strategies offer a powerful route to expand the druggable proteome, yet selective lysine targeting remains difficult due to the high pKa and protonation of the amine side chain. The translation initiation factor eIF4E, an mRNA cap-binding protein that controls nuclear export and translation of oncogenic transcripts, has long been considered an undruggable RNA-binding protein. We apply an anionic electrophile strategy that converts a carboxylic acid within a reversible eIF4E ligand into a vinyl acylsulfonamide warhead, preserving salt-bridge and hydrogen bonding interactions in the cap-binding pocket and facilitating Lys162 deprotonation to enable site-specific engagement. This single-step transformation yields JL-03, a potent covalent eIF4E inhibitor with excellent stability toward biological nucleophiles, consistent with context-dependent activation of the warhead in the binding site. In cells, JL-03 achieves robust target engagement and suppresses translation of the oncogenic driver cyclin D. JL-03 reduces the viability of HOXA9-high acute myeloid leukemia cells with greater potency than reversible eIF4E inhibitors and synergizes with the menin inhibitor revumenib, demonstrating that coordinated transcriptional and translational blockade exhausts leukemic dependencies. These findings establish eIF4E as a tractable covalent target and highlight anionic electrophile installation as a generalizable strategy for lysine-directed covalent inhibitor design.

Chemoproteomics Discovery of a Central Nervous System-Penetrant Covalent Inhibitor of PIKfyve

Antony J. Burton, Louis S. Chupak, Alison J. Davis, Ahmed S. A. Mady, Mirco Meniconi, Barry Teobald, Bryan W. Dorsey, Lauren R. Byrne, Ryan Mulhern, Berent Lundeen, Elizabeth W. Sorensen, Bharti Patel, Sean Brennan, Dhiraj Kormocha, Ruben Tommasi, Graham L. Simpson, Jeffrey W. Keillor, Laura D’Agostino, Elayne Penebre, Pearl S. Huang;

ACS Chem. Biol. 2026; https://doi.org/10.1021/acschembio.6c00795

PIKfyve is a lipid kinase involved in regulating protein clearance mechanisms and is a promising target for the treatment of neurodegenerative diseases. We present the discovery and optimization of a central nervous system (CNS)-penetrant covalent PIKfyve inhibitor, DUN’058, which achieves sustained target occupancy in vivo. Covalent screening hits, identified from chemoproteomics experiments performed in live cells, were rapidly optimized to deliver a brain-penetrant covalent inhibitor of PIKfyve. This covalency centred approach employed a suite of mass spectrometry, biochemical and in vivo assays to optimize compound potency, selectivity, and CNS permeability. The target nucleophile, cysteine 1970, is on a flexible loop that appears distal from the kinase active site, highlighting the power of chemoproteomics screening to identify novel nucleophilic amino acids for covalent modification. DUN’058 achieves efficient covalency at the target cysteine, as well as highly selective covalent and reversible selectivity profiles. Covalent PIKfyve inhibition results in modulation of downstream pathway activity, including activation of the transcription factor TFEB, upregulation of protein clearance pathways, and increased GPNMB transcription and secretion of exosome markers. When dosed in vivo, DUN’058 achieves sustained target occupancy in the brains of mice long after systemic compound clearance. The development of DUN’058 is a demonstration of chemoproteomics-based discovery for a high value CNS target, providing an orally bioavailable and covalent PIKfyve inhibitor.

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.