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.