Dual-Site Covalent Targeting Enables BD2-Selective BET Inhibition With Potent Antitumor Activity in Mice

Jibo Kang, Xuan Wang, Hong Zhang, Jieying Lin, Peng Chen, Zuqin Wang, Zengjun Hao, Fengfei Miao, Fengcai Zhang, Tao Li, Yusheng Xie, Junjian Wang, Zhi-Min Zhang, Shao Q. Yao, Xiaoyun Lu

Angewandte Chemie International Edition 2026 e1224416

https://doi.org/10.1002/anie.1224416

Bromodomain and extra-terminal (BET) proteins are validated therapeutic targets for cancer, but clinical translation of pan-BET inhibitors is limited by dose-limiting toxicities from non-selective inhibition of BD1/BD2 domains. Herein, we report the first-ever domain-selective covalent inhibitor, named ipAE1, of BET BD2 domains by installing an epoxide warhead onto the scaffold of ABBV-744. ipAE1 was shown to irreversibly modify Glu438 and His437 (to a lesser extent) located within the BRD4(2) binding pocket via a dual-covalent mechanism, as evidenced by its chemical probe pAE1. Furthermore, ipAE1 exhibited exceptional potency against BRD4(2) (Kd = 0.096 nM) with > 1900-fold selectivity over BRD4(1), leading to potent and sustained antiproliferative activity in MV4-11 cells (GI50 = 1.5 nM). Subsequent live-cell proteome-wide profiling validated BRD4 as the primary cellular target of ipAE1. Consistent with cellular activities, ipAE1 possessed a significantly enhanced antitumor efficacy in an MV4-11 xenografted mouse model compared to ABBV-744, presumably due to its on-target covalent engagement in vivo. Our study thus establishes for the first time a novel targeted covalent inhibition (TCI) strategy that engages two weakly nucleophilic residues within a single bromodomain, an approach generalizable to other compounds targeting E/H, providing a highly selective platform for future development of next-generation BET inhibitors.

Isoform-Selective Targeting of Akt Through Covalent Allosteric Inhibition

'Angelo, Giovanni Danilo; Pervanidis, Kosmas Alexandros; Athanasiadis, Ioannis; Lukianchikov, Vladimir; Scrima, Andrea; Depta, Laura; Stier, Sara; Müller, Matthias P.; Farin, Henner F.; Quambusch, Lena; Rauh, Daniel. 

Angewandte Chemie International Edition (2026), e3567206. 

https://doi.org/10.1002/anie.3567206

The Akt family of serine/threonine kinases plays a crucial role in various cellular processes, including proliferation, survival, and metabolism. Three Akt isoforms (Akt1, Akt2, and Akt3) have distinct physiological roles, and while individual isoform dysregulation is disease-linked, unselective Akt inhibition leads to side effects. Here, we report the development of selective covalent-allosteric Akt inhibitors (CAAIs) targeting Akt2 and Akt3 while sparing Akt1. Guided by protein x-ray crystallography and molecular modeling, key structural differences within the allosteric pockets of the isoforms were identified and exploited in a structure-based design strategy. By stabilizing the inactive kinase conformation, CAAIs overcome the intrinsic selectivity limitations of ATP-competitive inhibitors. After biological characterization, the pyrazole-containing inhibitors emerged as the most potent and selective Akt2 inhibitors, while inhibitors with pyridines as an isoform-selective element were predominantly targeting Akt3 selectively. Importantly, these new inhibitors were also evaluated in patient-derived colorectal cancer organoids. Co-crystal structures of inhibitors bound to an engineered construct mimicking the Akt2 allosteric pocket elucidated the molecular basis of isoform selectivity, guiding further optimization. This work not only establishes a framework for the development of isoform-selective therapeutics but also highlights the potential for unraveling isoform-specific functions in signaling pathways relevant to cancer biology.

An enantioselective covalent inhibitor of BAX confers cytoprotection in vivo

Peiwen Shi, Bruno Melillo, Matthew W. McHenry, Christina M. Camara, Ka Yang, Evert Njomen, Marina Godes, Maria F. Pazyra-Murphy, Mary Rose Branch, Bethany Tesar, Rosalind A. Segal, Lee L. Rubin, Michael D. Cameron, Gregory H. Bird, Thomas E. Wales, Steven P. Gygi, Benjamin F. Cravatt, and Loren D. Walensky.

 Nat Chem Biol (2026). 

https://doi.org/10.1038/s41589-026-02297-9

No therapies directly block apoptosis in tissue injury or the many diseases driven by cell loss. The BCL-2 family protein BAX is a central mediator of this pathway and C126 resides within a key regulatory region where physiologic or pharmacologic ligands can activate or inhibit its function. Here, we report enantioselective covalent BAX inhibitors that site-specifically react with C126 and confer cytoprotection across multiple cell types. These ligands constrain BAX conformation and suppress apoptosis in a strictly BAX-dependent manner. Medicinal chemistry optimization yielded covalent BAX inhibitor 3 (CBI-3), an analog with pharmacokinetics suitable for in vivo studies. In a murine model of Fas-induced fulminant hepatic failure, CBI-3 reduced hepatocyte apoptosis and preserved liver histology and survival. CBI-3 also conferred cytoprotection of motor neurons derived from human induced pluripotent stem cells of healthy and amyotrophic lateral sclerosis donors. These findings establish covalent BAX inhibition as a therapeutic strategy to directly block pathologic cell death.

Enhancing De Novo Designed Peptides and Proteins via Irreversible Covalent Isoquinolinium Capture

Paul M. Levine, Patrick W. Erickson, Timothy W. Craven, Aaron T. Balana, Derrick R. Hicks, Green Ahn, Chan J. Kim, Lisa S. Brandenburg, Wei Yang, Danielle P. Johnson Erickson,

ACS Chem. Biol. (2026)

https://doi.org/10.1021/acschembio.6c00584

Irreversible covalent inhibitors have garnered significant attention in recent years. Despite encouraging progress, the vast majority contain electrophiles that target the least abundant amino acid, cysteine, substantially limiting target inhibitor design for therapeutic intervention. Here, we generalize 2-ethynylbenzaldehyde as a proximity-induced electrophile for generating irreversible covalent peptide and protein inhibitors that specifically target native lysine residues. Leveraging this warhead, we designed a covalent de novo peptide that potently engages MCL1 to block its interaction with Bak. We show it is faster, more site-selective, and increases potency by 61-fold for MCL1 relative to a sulfonyl fluoride warhead. Additionally, with the guide of a computational script to predict "reactive hotspots" at the protein level, we developed a minibinder that labels PD-L1 in vitro and in live cells, displays a slower off-rate, and potently blocks the native PD-1 and PD-L1. These results establish isoquinolinium capture as a promising strategy to inhibit protein–protein interactions and for the development of novel covalent peptide and protein therapeutics.

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)

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.

Development of Covalent Inhibitors of Chikungunya Virus nsP2 Cysteine Protease Enabled by Direct-to-Biology Synthesis and Screening D

Zhengjun Cai § ; Kan Li § ; Sainetra Sridhar; Haozhou Tan; Hiwot Demssie; Gaungjin Fan; Wenyi Zhang; Bobby Brooke Herrera; Jun Wang

J. Med. Chem. (2026)

https://doi.org/10.1021/acs.jmedchem.5c03672

Chikungunya virus (CHIKV), an arthropod-borne alphavirus, has emerged as a global health threat due to its rapid transmission and the lack of effective antiviral therapies. The cysteine protease activity of the virus-encoded nonstructural protein 2 (nsP2) is critical for CHIKV replication, as it processes viral polyproteins and counteracts host antiviral defenses, establishing it as a highly attractive target for therapeutic intervention. In this study, we present a rapid drug development platform that integrates covalent docking with direct-to-biology (D2B) synthesis and screening to identify nsP2 inhibitors. Candidates prioritized by in silico docking were synthesized and directly tested in FRET enzymatic assays without purification. This approach led to the identification of several nsP2 inhibitors with diverse chemical scaffolds, potent enzymatic inhibition, and antiviral activity. Together, these findings establish a streamlined strategy for covalent inhibitor development and provide promising leads for CHIKV antiviral development.

Covalent allosteric inhibition of AARS1 lactyltransferase

Yunyuan Huang, Siying Liu, Luyang Tian, Yang Tang, Yilin Dou, Huiling Wang, Tao Zheng, Mingyang Wang, Runhao Li, Zhi Wang, Zhaocai Zhou, Xin Chen & Jinrong Min 

Nature Communications (2026) 

https://doi.org/10.1038/s41467-026-76732-9

Alanyl-tRNA synthetase 1 (AARS1) was recently identified as a lactyltransferase responsible for protein lactylation, a modification associated with epigenetic regulation and metabolic adaptation. Here, we report compound XY353, which covalently binds C184 of AARS1, inducing steric clashes with F175 and triggering structural rearrangements in the region that displace W176, a key residue for lactate binding. By means of a combination of a variety of biophysical and enzymatic assays with MD simulations and structural analysis, we show that XY353 and its derivative XY353-1 inhibit AARS1 by competing with lactate via the C184–F175–W176 relay, supporting a covalent allosteric mechanism of inhibition, which is further confirmed by our cellular data that XY353-1 reduces lactylation of the AARS1 substrate YAP and suppresses the proliferation of HGC-27 cells. Collectively, these findings identify a covalent allosteric mechanism for AARS1 inhibition and provide chemical tools to explore its biological functions.