Introduction of Reactive Thiol Handles into Tyrosine-Tagged Proteins through Enzymatic Oxidative Coupling

Paul Huang, Wendy Cao, Jennifer L. Fetzer, Nicholas S. Dolan, Matthew B. Francis

J. Am. Chem. Soc. 2025

https://doi.org/10.1021/jacs.5c06195

Site-specific protein bioconjugation methods have enabled the development of new therapeutics and materials, and further development of existing techniques has expanded the compatible library of protein substrates for bioconjugation. Among these techniques, the enzyme tyrosinase has demonstrated a promising ability to form protein–protein conjugates between exposed tyrosine and cysteine residues. In this work, we observed that the tyrosinase variant from Bacillus megaterium, termed megaTYR, has an increased tolerance for small-molecule thiol substrates, which can inhibit the activity of other tyrosinases. Among the breadth of thiol substrates that could be reliably coupled to tyrosine-tagged proteins was dithiothreitol (DTT), which effectively introduces a free thiol handle and provides a convenient method to bypass the genetic incorporation of cysteine residues for bioconjugation. Accordingly, these thiolated proteins could undergo additional coupling to commercially available maleimide probes as well as other tyrosine-tagged proteins. This was demonstrated by the conjugation of targeting proteins to drugs, fluorescent probes, and therapeutic enzymes. Of particular note and building on a previous report of a tyrosinase-sensitive tyrosine residue on the Fc region of antibodies, commercially available monoclonal antibodies (mAbs) treated with PNGase F were conjugated to DTT to produce THIOMAB equivalents. These intermediates were subsequently used to make functional antibody–drug and antibody–toxin protein conjugates. This facile method to convert accessible tyrosine residues on proteins to thiol tags extends the use of tyrosinase-mediated oxidative coupling to a broader range of protein substrates.

Diethenyl Sulfoximine (DESI) as an Irreversible Lysine-Targeting Warhead Enables the Design of Covalent Allosteric EGFR Inhibitor

Huiqi Xu, Hongjin Zhang, Suyun Jia, Yanxin Tao, Quanpeng Wei, Yingao Wang, Xuechen Liu, Yuqing Zhang, Xinpeng Ning, Yuyan Shi, Can Jin, Ke Ding, Dawei Ma, Shan Li, Mengyang Fan

Chem. Euro. J. 2025 e202501389

https://doi.org/10.1002/chem.202501389

Targeting lysine residues with covalent inhibitors is challenging due to their abundance in the proteome and the protonation of lysine's ε-amino group, which diminishes its reactivity. This study introduces diethenyl sulfoximine (DESI) as a novel bio-orthogonal aminophilic electrophile which can react with lysine via double conjugate addition to form a cyclic adduct. The second addition promotes the entire and efficient electrophilic attack by the ε-amino of lysine on the ethenyl groups. DESI exhibits superior aqueous stability, overcoming the hydrolysis issue encountered by most reported lysine-targeting covalent agents. Incorporation of DESI in the allosteric pocket binder EAI045 of oncoprotein epidermal growth factor receptor (EGFR) yields compound 4, which specifically reacts to the catalytic lysine (Lys745). Compound 4 showed potent inhibition of EGFR-driven cell proliferation with IC50 values of 0.789 µM and 1.22 µM in engineered BaF3-EGFRL858R/T790M/C797S and NCI-H1975 cells, respectively, overcoming EAI045's limitation of lack in cellular potency as a single agent. Tyrosine kinases panel profiling confirmed selectivity toward mutant EGFR while sparing the wild type with minimal off-targets. These findings highlight DESI's potential as a versatile strategy for targeting lysine residues irreversibly, offering solutions to overcome drug resistance in cancer therapy and advance next-generation precision medicines.

Diffusion Limit and the Reactivity/Affinity Conundrum: Implications for Optimization and Hit Finding for Irreversible Modulators

Bharath Srinivasan

J. Med. Chem. 2025

https://doi.org/10.1021/acs.jmedchem.4c02863

Irreversible inhibition as a therapeutic modality has come of age over the previous decade. With minimal theoretical guidance for the design of an irreversible modulator, empirical optimization efforts often involve increasing the affinity of the small molecule while reducing the reactivity of the electrophile. The latter, as per prevalent opinion, is to ensure that binding dictates engagement and the reactive electrophile does not pose a safety liability arising from off-target reactivity. Here I argue that, like the second-order kinetic rate constant kcat/Km, the parameter kinact/KI is limited by the upper physical limit imposed by the rate of diffusion. This capping ensures that any attempt to improve the affinity of the electrophile-containing small-molecule at the limit will come with an equivalent trade-off in their reactivity. This has implications for both hit finding and lead optimization within targeted irreversible inhibition, especially for intractable targets with shallow pockets where the interactions are collision-induced second-order processes.

Covalent Recruitment of NEDD4 for Targeted Protein Degradation: Rational Design of Small Molecular Degraders

Xiaoqiang He, Shihan Zeng, Yalei Wen, Tao Yang, Chaoming Huang, Yifang Li, Zhang Zhang, Ke Ding, Tongzheng Liu, Yi Tan, and Zhengqiu Li

J. Am. Chem. Soc. 2025, 147, 25, 21512–21525

https://doi.org/10.1021/jacs.4c18083

Targeted protein degradation (TPD) has emerged as a promising therapeutic strategy for treating various diseases. However, current small molecule degraders predominantly rely on a limited set of E3 ubiquitin ligases, such as CRBN and VHL, which restricts their applications. Here, we report that incorporation of the 2H-azirine chemical handle into the EGFRL858R/T790M/C797S inhibitor induced remarkable degradation of the targeted protein. Proteomic profiling and functional validation confirmed that the NEDD4 E3 ligase was covalently recruited by 2H-azirine through engagement of C1286 residue, facilitating target degradation. Furthermore, the 2H-azirine moiety demonstrated versatility by acting as a small molecular degrader when conjugated to various ligands, effectively mediating the degradation of CDK4, PDE5, BTK and Brd4. More importantly, using the identical protein ligand scaffold, we demonstrated that the 2H-azirine based probe can degrade proteins resistant to degradation by CRBN or VHL recruitment. This approach provides a rational strategy for developing novel small molecular degraders that target alternative E3 ubiquitin ligases. Notably, these degraders significantly outperformed their parent kinase inhibitor in suppressing cancer cell growth.

Discovery of IHMT-15130 as a Highly Potent Irreversible BMX Inhibitor for the Treatment of Myocardial Hypertrophy and Remodeling

Shuang Qi, Jiangyan Cao, Ting Wu, Chenliang Shi, Junjie Wang, Beilei Wang, Ziping Qi, Hong Wu, Yun Wu, Aoli Wang, Jing Liu, Wenchao Wang, and Qingsong Liu

ACS Chem. Biol. 2025, 20, 6, 1181–1194

https://doi.org/10.1021/acschembio.4c00875

Cardiac hypertrophy is usually accompanied by many forms of heart disease, including hypertension, vascular disease, ischemic disease, and heart failure, and thus effectively predicts the increased cardiovascular morbidity and mortality. Bone marrow kinase in chromosome X (BMX) has been reported to be the major signaling transduction protein in cardiac arterial endothelial cells and is thought to be involved in the pathology of cardiac hypertrophy. We report here the discovery of a potent irreversible BMX kinase inhibitor, IHMT-15130, which covalently targets cysteine 496 of BMX and exhibits potent inhibitory activity against BMX kinase (IC50: 1.47 ± 0.07 nM). Compared to recently approved BTK/BMX dual inhibitor Ibrutinib, IHMT-15130 displayed selectivity over CSK kinase (IC50 > 25,000 nM), targeting of which may cause severe atrial fibrillation and bleeding. IHMT-15130 effectively reduced the secretion of inflammatory cytokines, inhibited the inflammatory signaling pathway in vitro and in vivo, and alleviated angiotensin II (Ang II)-induced myocardial hypertrophy in a murine model. This study provides further experimental evidence for the application of BMX kinase inhibitors in the treatment of cardiac hypertrophy.

Synthesis and functionalization of vinyl sulfonimidamides and their potential as electrophilic warheads

Yu Tung Wong,  Charles Bell, and  Michael C. Willis

Chem. Sci., 2025

DOI

https://doi.org/10.1039/D5SC02420J

Covalent inhibitor design is dominated by the use of electrophilic acrylamide warheads. One limitation of acrylamides is that there are limited opportunities to modify their electrophilicity, and hence reactivity, by simple structural changes. Here we show that vinyl sulfonimidamides are effective electophilic groups for reaction with both sulfur- and nitrogen-based biologically relevant nucleophiles. The parent N–H vinyl sulfonimidamides are prepared in a single step from an aryl-ONSO reagent, a vinyl organometallic, and an appropriate amine. Imidic N-functionalisation is straightforward, providing a collection of electrophilic fragments of varied reactivity. We demonstrate that the electrophilicity of these new reagents can be modulated by choice of the imidic N-substituent, and when this is used in combination with alkene substituents, allows for a reactivity range both above and below that of the parent acrylamide.

BBO-10203 inhibits tumor growth without inducing hyperglycemia by blocking RAS-PI3Kα interaction

Dhirendra K. Simanshu, Rui Xu, James P. Stice, Daniel J. Czyzyk, Siyu Feng, John-Paul Denson, Erin Riegler, Yue Yang, Cathy Zhang, Sofia Donovan, Brian P. Smith, Maria Abreu-Blanco, Ming Chen, Cindy Feng, Lijuan Fu, Dana Rabara, Lucy C Young, Marcin Dyba, Wupeng Yan, Ken Lin, Samar Ghorbanpoorvalukolaie, Erik K. Larsen, Wafa Malik, Allison Champagne, Katie Parker, Jin Hyun Ju, Stevan Jeknic, Dominic Esposito, David M. Turner, Felice C. Lightstone, Bin Wang, Paul M. Wehn, Keshi Wang, Andrew G. Stephen, Anna E. Maciag, Aaron N. Hata, Kerstin W. Sinkevicius, Dwight V. Nissley, Eli M. Wallace, Frank McCormick, Pedro J. Beltran

Science, eadq2004

DOI:10.1126/science.adq2004

BBO-10203 is an orally available drug that covalently and specifically binds to the RAS-binding domain of phosphoinositide 3-kinase α (PI3Kα), preventing its activation by HRAS, NRAS, and KRAS. It inhibited PI3Kα activation in tumors with oncogenic mutations in KRAS or PIK3CA, and in tumors with human epidermal growth factor receptor 2 (HER2) amplification or overexpression. In preclinical models, BBO-10203 caused significant tumor growth inhibition across multiple tumor types and showed enhanced efficacy in combination with inhibitors of cyclin-dependent kinase 4/6 (CDK4/6), estrogen receptor (ER), HER2 and KRAS-G12C mutant, including in tumors harboring mutations in Kelch-like ECH-associated protein 1 (KEAP1) and Serine/Threonine Kinase 11 (STK11). Notably, these antitumor effects occurred without inducing hyperglycemia, as insulin signaling does not depend on RAS-mediated PI3Kα activation to promote glucose uptake.

Allosteric Covalent Inhibitors of the STAT3 Transcription Factor from Virtual Screening

Tibor Viktor Szalai, Vincenzo di Lorenzo, Nikolett Péczka, Levente M. Mihalovits, László Petri, Qirat F. Ashraf, Elvin D. de Araujo, Viktor Honti, Dávid Bajusz, and György M. Keserű

ACS Medicinal Chemistry Letters 2025 16 (6), 991-997

DOI: 10.1021/acsmedchemlett.4c00622

The STAT family of transcription factors are important signaling hubs, with several of them, particularly STAT3, being emerging oncotargets already investigated in clinical trials. The modular structure of STAT3 nominates several of its protein domains as possible drug targets, but their exploitation with potential small-molecule inhibitors has been unevenly distributed so far, with past efforts highly favoring the conserved SH2 domain. Here, we have targeted a sparsely studied binding site at the junction of the coiled-coil and DNA-binding domains and discovered several new lead-like covalent inhibitors by virtual screening. The most favorable hit compound has been explored via structure-guided hit expansion and optimized into a low micromolar inhibitor. This compound can serve as a chemical biology tool against this site in future exploratory studies or form the basis of a more advanced stage of lead optimization.

Covalent Destabilizing Degrader of AR and AR-V7 in Androgen-Independent Prostate Cancer Cells

Charlotte M. Zammit, Cory M. Nadel, Ying Lin, Sajjan Koirala, Elnaz Ahani, Patrick Ryan Potts, and Daniel K. Nomura

Journal of the American Chemical Society 2025

DOI: 10.1021/jacs.5c02801

Androgen-independent prostate cancers, correlated with heightened aggressiveness and poor prognosis, are caused by mutations or deletions in the androgen receptor (AR) or the expression of truncated variants of AR that are constitutively activated. Currently, drugs and drug candidates against AR target the steroid-binding domain to antagonize or degrade AR. However, these compounds cannot therapeutically access largely intrinsically disordered truncated splice variants of AR, such as AR-V7, which only possess the N-terminal transactivation domain and DNA-binding domain and are missing the ligand-binding domain. Targeting intrinsically disordered regions within transcription factors has remained challenging and is considered “undruggable”. Herein, we leverage a cysteine-reactive covalent ligand library in a cellular screen to identify the degraders of AR and AR-V7 in androgen-independent prostate cancer cells. We identified a covalent compound, EN1441, that selectively degrades AR and AR-V7 in a proteasome-dependent manner through direct covalent targeting of intrinsically disordered cysteine C125 in the N-terminal transactivation domain of AR and AR-V7. EN1441 causes significant and selective destabilization of AR and AR-V7, leading to the aggregation of AR/AR-V7 and subsequent proteasome-mediated degradation. Consistent with targeting both AR and AR-V7, we find that EN1441 completely inhibits total AR transcriptional activity in androgen-independent prostate cancer cells expressing both AR and AR-V7 compared with AR antagonists or degraders that only target the ligand-binding domain of full-length AR, such as enzalutamide and ARV-110. Our results put forth a pathfinder molecule EN1441 that targets an intrinsically disordered cysteine within AR to destabilize, degrade, and inhibit both AR and AR-V7 in androgen-independent prostate cancer cells and highlights the utility of covalent ligand discovery approaches in directly targeting, destabilizing, inhibiting, and degrading classically undruggable transcription factor targets.

Advancing Covalent Ligand and Drug Discovery beyond Cysteine

Gibae Kim, R. Justin Grams, and Ku-Lung Hsu

Chemical Reviews 2025

DOI: 10.1021/acs.chemrev.5c00001

Targeting intractable proteins remains a key challenge in drug discovery, as these proteins often lack well-defined binding pockets or possess shallow surfaces not readily addressed by traditional drug design. Covalent chemistry has emerged as a powerful solution for accessing protein sites in difficult to ligand regions. By leveraging activity-based protein profiling (ABPP) and LC-MS/MS technologies, academic groups and industry have identified cysteine-reactive ligands that enable selective targeting of challenging protein sites to modulate previously inaccessible biological pathways. Cysteines within a protein are rare, however, and developing covalent ligands that target additional residues hold great promise for further expanding the ligandable proteome. This review highlights recent advancements in targeting amino acids beyond cysteine binding with an emphasis on tyrosine- and lysine-directed covalent ligands and their applications in chemical biology and therapeutic development. We outline the process of developing covalent ligands using chemical proteomic methodology, highlighting recent successful examples and discuss considerations for future expansion to additional amino acid sites on proteins.

Kinetic Modeling of Covalent Inhibition: Effects of Rapidly Fluctuating Intermediate States

Kyle Ghaby, Benoît Roux

bioRxiv 2025.05.28.656658; 

doi: https://doi.org/10.1101/2025.05.28.656658

There is increasing interest in the discovery of small-molecule inhibitors that form covalent bonds with their targets for therapeutic applications. Nevertheless, identifying clear rational design principles remains challenging because the action of these molecules cannot be understood as common noncovalent inhibitors. Conventional kinetic models often reduce the binding of covalent inhibitors to a two-step irreversible process, overlooking rapid complex dynamics of the associated unlinked inhibitor before the formation of the covalent bond with its target. In the present analysis, we expand the intermediate state into two conformations—reactive (E·I) and nonreactive (E··I). To illustrate the consequences of such simplification, the expanded kinetic model can be reduced to an effective two-step scheme expressed in terms of the equilibrium probability of the unlinked inhibitor to form either conformation. A mass-action-based numerical workflow is implemented to simulate time-dependent kinetics, overcoming the common limitations of empirical models. The numerical workflow helps relate microscopic states observed in molecular dynamics simulations to macroscopic observables like EC50 and the apparent rate of covalent inhibition, showing the impact of transient intermediates on dissociation rates and potency. The proposed framework refines the interpretation of dose-response data, aiding medicinal chemists in optimizing covalent inhibitors and provides a quantitative platform for relating molecular conformational distributions to empirical parameters.