Covalent Protein Inhibitors via Tyrosine and Tryptophan Conjugation with Cyclic Imine Mannich Electrophiles

Dr. Sijie Wang, Dr. Lei Wang, Dr. Marco Hadisurya, Dr. Siavash Shahbazi Nia, Prof. Dr. W. Andy Tao, Prof. Dr. Emily C. Dykhuizen, Prof. Dr. Casey J. Krusemark

Angewandte Chemie e16630

https://doi.org/10.1002/ange.202516630

Digital Object Identifier (DOI)

Targeted covalent inhibitors (TCIs) are increasingly popular as drug candidates and chemical probes. Among current TCIs, the chemistry is largely limited to cysteine and lysine side chain reactivity. Here, we investigated the utility of cyclic imine Mannich electrophiles as covalent warheads to target protein tyrosine and tryptophan side chains. We characterized the intrinsic reaction rates of several cyclic imines to tyrosine and other amino acid side chains and validated reactivity using protein affinity labeling of a cyclic imine-modified trimethoprim with tyrosine and tryptophan mutants of E. coli dihydrofolate reductase. To validate the utility of the approach, we appended cyclic imine warheads to a CBX8 chromodomain inhibitor to label a non-conserved tyrosine, which improved both the potency and selectivity of the inhibitor for CBX8 in vitro and in cells. These findings indicate that Mannich electrophiles are promising and robust chemical warheads for tyrosine and tryptophan bioconjugation and development of covalent inhibitors.

Chemoproteomics discovery of a CNS-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, Pearl S. Huang, Elayne Penebre

bioRxiv 2026.01.26.701341; 

doi: https://doi.org/10.64898/2026.01.26.701341

PIKfyve is a lipid kinase involved in regulating protein clearance mechanisms and is a promising target for the treatment of neurodegenerative diseases. Here, we present the discovery and optimization of a 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 centered 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, holding promise for achieving a sustained duration of action in the CNS at low doses, without prolonged effects in the periphery. Taken together, 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.

Group Competition Strategy for Covalent Ligand Discovery

Zhihao Guo, Yunzhu Meng, Boyuan Zhao, Weidi Xiao, and Chu Wang

Journal of the American Chemical Society 2026

DOI: 10.1021/jacs.5c18150

As a powerful chemoproteomic tool, activity-based protein profiling (ABPP) has been extensively used for covalent ligand discovery. However, the current ABPP-based approaches are inherently based on indirect probe labeling competed by covalent ligands, and cannot directly compare the preferences of different ligands head-to-head. Herein, we report a group competition-based ABPP strategy (GC-ABPP) to allow the direct comparison of multiple ligands’ binding ability on a proteome-wide scale. By dividing a library of fully functionalized probes (FFPs) into different subgroups and labeling the proteome simultaneously, the direct competition enables comparison of the labeling ability of different probes in drawing a global protein–ligand affinity metric. When it is applied to an expanded probe library, this strategy can be used iteratively to select the highest-affinity ligand toward a certain target protein in a multiple-round process. As a proof of concept, we synthesized 65 FFPs and employed the GC-ABPP to screen the ligand–protein reactivity for >6000 cysteine sites. After three rounds of screening, we identified high-affinity ligands targeting BCAT2 and UGDH. Our “multiple ligands versus multiple proteins” screening paradigm demonstrates great potential for applications in covalent ligand/drug discovery.

Protein tyrosine phosphatase inactivation by electrophilic tyrosine modification

Madeleine L. Ware, David M. Leace, Zihan Qu, Quentin Schaefer, Sagar D. Vaidya, Mikayla L. Horvath, Zhihong Li, Yunpeng Bai, Zhong-Yin Zhang, and Ku-Lung Hsu 

Chem. Sci., 2026

https://doi.org/10.1039/D5SC07398G

Covalent protein tyrosine phosphatase (PTP) inhibitors principally target the catalytic cysteine, which is highly conserved and presents challenges for achieving selectivity across the PTP family. Here, we identified a tyrosine-reactive covalent inhibitor for SHP2 (DML189) with secondary molecular glue activity via a ligand induced protein tethering (LIPT) mechanism. We detected ligand binding at Y279, which is in proximity to the catalytic cysteine on SHP2 and has known functional and pathogenic properties. Covalent SHP2 modification by DML189 induced reversible disulfide tethering and monomer loss that was not observed to the same extent on PTP1B, LYP, or SHP1. Crosslinking mass spectrometry detected unique tethering events involving regulatory cysteines after DML189 modification on SHP2. Together, we discovered a tyrosine reactive inhibitor that targets functional sites on SHP2 and exhibits molecular glue activity through LIPT.

Covalent Peptide-Encoded Libraries Enable Discovery of Inhibitors of Epidermal Growth Factor Receptor (EGFR)

Ching-Pei Hsu, Michael Desgagné, Simon L. Rössler, Nathalie M. Grob, Charlotte E. Farquhar, Andrei Loas, Zena D. Jensvold, Hannah T. Baddock, Matthew Bratkowski, Aaron H. Nile, and Bradley Pentelute

ChemRxiv, 2026

doi:10.26434/chemrxiv-2026-z6vkt 

The use of encoding tags in combinatorial libraries accelerates hit generation by enabling high-throughput identification of small-molecule ligands. Peptide-encoded libraries (PELs) support the selection of structurally diverse small-molecule binders to proteins of interest. Here, we introduce a covalent PEL (coPEL) platform that incorporates cysteine-reactive scaffolds to identify irreversible protein binders. We leverage the chemical stability of PELs and the selective reactivity of palladium catalysts derived from dialkylbiaryl phosphine ligands to enable solid-phase Heck coupling reactions to rapidly diversify covalent acrylamide warheads. The optimized reaction conditions are high-yielding across a broad range of (hetero)aryl halides, ensuring robust performance and versatility within the coPEL platform. Screening a coPEL against the epidermal growth factor receptor (EGFR) tyrosine kinase, a key oncology target, yielded covalent small-molecule inhibitors with low-micromolar potency in vitro. This approach provides a complementary strategy for targeting diverse proteins and developing new classes of covalent inhibitors.

Structure-Guided Optimization of 4-Chloro-Pyrazolopyridine Analogs for Covalent PREP Inhibition

Kalyani Thakur, Ian Fucci, Joshua Pandian, Kiall F. Suazo, Diana C. F. Monteiro, and Euna Yoo

Journal of Medicinal Chemistry 2025

DOI: 10.1021/acs.jmedchem.5c02680

Prolyl endopeptidase (PREP) is a dynamic serine protease that cleaves proline-containing peptides. PREP is also involved in numerous pathophysiological processes through modulation of protein–protein interactions and has been extensively studied in neurodegenerative diseases. In this study, we report the structure-based design and synthesis of covalent PREP inhibitors built on a 4-chloro-pyrazolopyridine (CPzP) scaffold, previously identified through chemoproteomic screening to target a noncatalytic cysteine residue within the active site. Guided by crystallographic data and molecular docking studies, we optimized initial hits to develop a potent inhibitor exhibiting nanomolar potency in both biochemical and cellular assays, with high selectivity over related serine proteases FAP and DPP4. Molecular dynamics simulations indicated that modulation of the conformational flexibility of a dynamic A-loop within PREP by CPzP analogs may contribute to inhibitory potency. Collectively, this work introduces a new class of structurally distinct inhibitors and provides tools to explore the diverse biological roles of PREP.

Ninhydrin as a covalent warhead for chemical proteomic-enabled discovery and selective engagement of reactive arginines

Andrew Ecker, Andreas Langen, Chloe Fields, José Luis Montaňo, Minh Tran, Ian Bass Seiple, Balyn W Zaro

bioRxiv 2026.01.05.697388; 

doi: https://doi.org/10.64898/2026.01.05.697388

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. These studies uncovered potential modification sites on disease-relevant proteins, including reactive arginines within catalytic sites that are essential for function. By endowing a reversible small molecule inhibitor of cyclophilin A with a ninhydrin warhead, we achieved selective, covalent engagement, and attenuation of enzymatic activity, highlighting the potential for targeting arginines in future therapeutic development campaigns. These findings establish ninhydrin as a warhead for studying arginine reactivity and modulating protein function.