Backus, K.M., Wilson, A.J.
Commun Chem 8, 266 (2025).
https://doi.org/10.1038/s42004-025-01652-6
A blog highlighting recent publications in the area of covalent modification of proteins, particularly relating to covalent-modifier drugs. @CovalentMod on Twitter, @covalentmod@mstdn.science on Mastodon, and @covalentmod.bsky.social on BlueSky
Z. Wang,
Angew. Chem. Int. Ed. 2025, e202510763
https://doi.org/10.1002/anie.202510763
Targeting surface-exposed lysines in kinases through covalent modification presents a major challenge due to their high pKa and inherently low reactivity. While current research primarily targets more reactive catalytic lysines buried in the ATP-binding pocket, no systematic rational strategy has yet been developed for selectively engaging surface-exposed lysines. Herein, we present a versatile strategy for developing cell-active covalent kinase inhibitors (CKIs) by selectively targeting unique surface-exposed lysines using squarate chemistry. By using Aurora A (AURKA) as a proof-of-concept, we systematically evaluated this approach against other well-known lysine-reactive warheads (e.g., EBA, SO2F, and OSO2F) and demonstrated, for the first time, squarates’ superior efficacy in engaging these challenging low-reactivity lysines. Amongst various AURKA CKIs, AL8 emerged as the first-in-class squarate-based, cell-active inhibitor, exhibiting excellent selectivity in both biochemical and cellular assays with long-residence time in covalently engaging endogenous AURKA. Detailed investigation of effects of leaving groups on squaric esters provided valuable insights for future development of lysine-reactive CKIs. Our finding has established squarate-containing ligands as a unique and readily tunable platform for covalent modification of surface-exposed, non-catalytic lysines in targeted kinase drug discovery.
I-Ju Yeh, Khuchtumur Bum-Erdene, Mona K. Ghozayel, Giovanni Gonzalez-Gutierrez, and Samy O. Meroueh
ACS Chemical Biology 2025
https://pubs.acs.org/doi/10.1021/acschembio.5c00283
Transcriptional enhanced associate domain transcription factors (TEAD1 to TEAD4) bind to transcriptional coactivator Yes-Associated Protein (YAP1) or its paralog transcriptional coactivator with PDZ-binding motif (TAZ) to regulate Hippo pathway target genes. The Hippo pathway is a conserved signaling pathway that regulates organ size and cell fate by controlling cell proliferation and apoptosis. Here we report small acrylamide molecules that form a covalent bond with a conserved cysteine at the TEAD palmitate pocket. Binding studies showed profound stabilization of TEADs by the small molecules, and cocrystal structures reveal that the compounds mimic the binding mode of palmitate. The small molecules achieved submicromolar binding constants and subhour reaction half-lives for all four TEADs. In mammalian cells, the compounds stabilize the TEAD•YAP1 interaction yet inhibit the TEAD transcription factor activity. Unexpectedly, several compounds degraded TEAD and YAP1 proteins and inhibited cancer cell viability. This work suggests that degradation of TEAD and YAP1 may amplify the antitumor effects of small molecules targeting the TEAD palmitate pocket, with implications for other cancer targets featuring allosteric lipid-binding sites.
Shaun O’Hare, Kateryna A. Tolmachova, and Jeffrey W. Bode
ACS Chemical Biology 2025
DOI: 10.1021/acschembio.5c00281
Covalent binders to protein targets offer a powerful approach to the generation of tool compounds and an increasingly common strategy for therapeutic development. The installation of electrophiles onto peptide binders, however, is often precluded by standard conditions for peptide synthesis, which involve strong nucleophiles, bases, and acids. The introduction of C-terminal electrophiles is further complicated by the C → N directionality of standard solid-phase peptide synthesis. Here, we employ chemoselective, site-specific functionalization of C-terminal peptide acyl hydrazides to install strong electrophiles on unprotected peptides. Using automated, high-throughput liquid handling and solid-phase extraction techniques, we have established a combinatorial workflow for the synthesis of peptide-derived covalent protease inhibitors. This methodology enables the synthesis and initial screening of inhibitor libraries in a 96-well plate format without the need for chromatographic purification prior to enzyme inhibition studies, leading to the identification of covalent Cathepsin S inhibitors active in the nanomolar range. When tested in cells, the covalent probes revealed strong off-target interactions with the protein disulfide isomerase PDIA1. These findings both underscore the role of chemoselective chemistries for covalent probe synthesis and highlight the utility of the platform for both the rapid identification of potent inhibitors and the detection of potential off-target interactions.
Holcomb, M., Llanos, M., Hansel-Harris, A. et al.
Commun Chem 8, 242 (2025).
https://doi.org/10.1038/s42004-025-01606-y
uan F. Tamez-Fernández, Craig F. Steven, Jade Nguyen, and Pablo Rivera-Fuentes
Journal of the American Chemical Society 2025
DOI: 10.1021/jacs.5c07109Zanon, P. R. A.; Yu, F.; Musacchio, P.; Lewald, L.; Zollo, M.; Krauskopf, K.; Mrdović, D.; Raunft, P.; Maher, T. E.; Cigler, M.; Chang, C.; Lang, K.; Toste, F. D.; Nesvizhskii, A. I.; Hacker, S. M.
ChemRxiv 2021.
https://doi.org/10.26434/chemrxiv-2021-w7rss-v2
Qian Wen Tan, Subramanyam Vankadara, Jia Yi Fong, Yi Yang See, Nithya Baburajendran, Pearly Shuyi Ng, Weijun Xu, Yee Khoon Yeo, Weiling Wang, Choon Heng Low, Li Hong Tan, Eileen Gui Ju Tay, Yun Xuan Wong, Chuhui Huang, Sandra Sim, Shi Hua Ang, Hannah Hui Min Toh, Juliana Mohammad, Gang Wang, Boping Liu, Shu Ting Tan, Perlyn Zekui Kwek, Monique Danielle Dawson, Qin Yao Oh, Xiaoying Koh, Joma Joy, May Ann Lee, Walter Stunkel, Vishal Pendharkar, Hannes Hentze, Siew Pheng Lim, Kantharaj Ethirajulu, C. S. Brian Chia, and Joseph Cherian
J. Med. Chem. 2025
Resulting in several million deaths globally, the COVID-19 pandemic has highlighted the criticality of antiviral drugs during a viral pandemic. Herein, we describe our efforts toward targeting SARS-CoV-2 Mpro, a key viral protease, which led to the discovery of compound 18, a reversible covalent inhibitor with potent antiviral activity against several clinical variants of SARS-CoV-2. Compound 18 demonstrated dose-dependent efficacy in a mouse-adapted SARS-CoV-2 infection model, with favorable pharmacokinetic profiles in mice, rats, dogs, and monkeys.
Ng, K.-Y.; Koo, T.-Y.; Huang, I. B.; Lee, T. K.-W.; Fong, T.-L.; Gao, Y.; Wong, T.-L.; Gao, Y.; Yun, J.-P.; Guan, X.-Y.; Liu, M.; Chung, C. Y.-S.; Ma, S.
Sci. Transl. Med.17,eadn9472(2025).
DOI:10.1126/scitranslmed.adn9472
The development of cancerous cells leads to considerable changes in metabolic processes to meet the demands of tumor growth. Tumor lineage plasticity has been identified as a key factor in therapy resistance and tumor recurrence. Herein, we showed one aspect of this plasticity to be abnormal glycerophospholipid metabolism, specifically the presence of a metabolic protein called 1-acylglycerol-3-phosphate o-acyltransferase 4 (AGPAT4). We identified AGPAT4 as an oncofetal protein that is abundant in embryonic stem cells and hepatocellular carcinoma (HCC) tumor cells but is low or absent in most normal tissues. We demonstrated that AGPAT4 is a functional regulator of tumor lineage plasticity, which correlates with enhanced metastasis and resistance to sorafenib. Heightened plasticity was induced as a result of increased AGPAT4-mediated conversion of LPA (lysophosphatidic acid) to phosphatidic acid (PA), which then acts on its downstream mTOR/S6K/S6 signaling pathway. Inhibition of Agpat4 by the AAV8-mediated liver-directed strategy in an immunocompetent HCC mouse model reduced tumorigenicity and stemness and sensitized tumors to sorafenib. Through a chemical biology approach, a cysteine-reacting compound that specifically targets AGPAT4 at the Cys228 residue and therefore hinders its acyltransferase activity was identified and found to work synergistically with sorafenib in suppressing HCC in tumor xenograft models derived from patients with preclinical HCC and sorafenib-resistant HCC. Toxicological analysis revealed minimal side effects associated with the covalent inhibitor. In conclusion, the plasticity of tumor lineages induced by AGPAT4 represents a potential target for HCC treatment and could expand the effectiveness of sorafenib treatment, offering new possibilities for HCC therapy.
Wei Zhang, Lizhen Yuan, Rui Liu, Yanbo Jing, Shijun Lin, Hao Fang, Yuxuan Li, Xiaohui Zhang, Jun Dai, Tao Liu, Fan Xia, and Xiaoding Lou
Journal of the American Chemical Society 2025
DOI: 10.1021/jacs.5c07041
Peptides have demonstrated great potential in drug development. However, their broader application in modalities such as proteolysis-targeting chimeras (PROTACs) remains limited by the lack of real-time efficacy feedback and poor pharmacokinetic stability. Herein, we develop a covalent self-reporting peptide degrader (Co-SPeD) by integrating a fluorine-substituted aryl fluorosulfate warhead and a rotor fluorophore derived from stilbene derivatives, which allows for covalent binding to target proteins via sulfur(VI) fluoride exchange chemistry and emitting activatable fluorescence. Co-SPeD is found to covalently bind to the K51 residue of the MDM2 protein, enabling real-time monitoring of targeted MDM2 degradation. By swapping the targeting peptide and screening rotor fluorophores, the Co-SPeD platform is successfully extended to other oncogenic proteins, including BCL-xL, GRP78, and KRAS (G12D). Additionally, Co-SPeD demonstrates significant antitumor efficacy in preclinical tumor models. More importantly, real-time in vivo monitoring of MDM2 degradation using Co-SPeD plays a crucial role in guiding cisplatin combination administration, leading to a 50% increase in tumor growth inhibition compared to nonguided treatment groups. This approach provides a targeted endogenous protein degradation strategy with real-time monitoring, offering a powerful and generalizable platform for next-generation PROTAC design, the advancement of peptide-based therapeutics, and the rational optimization of cancer therapy.
Bing Zhao, Sha Xu, Shiqing Zhou, Xiangru Jiang, Ailin Jiang, Hongrui Lei, Xin Zhai
Bioorganic Chemistry, 163, 2025, 108800,
https://doi.org/10.1016/j.bioorg.2025.108800
https://doi.org/10.1016/j.bioorg.2025.108800.Over the past two decades, covalent inhibitors have undergone a remarkable resurgence in drug discovery. Currently, targeting non-catalytic cysteine residues with acrylamide and other α,β-unsaturated carbonyl compounds is a predominate strategy, especially in the protein kinase field. Several cysteine-targeting covalent inhibitors (e.g. Ibrutinib, Afatinib) have demonstrated significant clinical efficacy. Covalent inhibitors have also enabled targeting of traditionally undruggable targets, highlighting the unique advantages of covalent strategies over non-covalent ligands. The rapid recent development of covalent strategies has prompted researchers to make significant efforts to develop novel reversible and irreversible covalent binding warheads targeting non-cysteine residues, thereby opening up new chemical space for covalent strategies. This article reviews the research advancements in specific and promiscuous warheads, as well as their covalent ligands, targeting three alkaline amino acids (lysine, arginine, and histidine), which will provide more opportunities for covalent fragment approaches targeting residues beyond cysteine.
Xue Li, Ru Li, Xiaoxia Zhu et al.
Research Square Preprint, 23 July 2025,
https://doi.org/10.21203/rs.3.rs-7058001/v1
Orelabrutinib is a highly selective, irreversible inhibitor of Bruton’s tyrosine kinase (BTK). It has shown promising results in animal models, indicating potential for treating systemic lupus erythematosus (SLE). A multicentre, double-blind, randomised, placebo-controlled, phase Ib/IIa trial (NCT04305197) was conducted. Sixty SLE patients were randomised 1:1:1:1 to receive oral orelabrutinib (50, 80, 100 mg) or placebo once daily for 12 weeks. A total of 55 patients completed the trial. In all evaluable patients, the SRI-4 rates at week 12 were 50%, 62%, and 64% for orelabrutinib at 50 mg, 80 mg, and 100 mg, respectively, compared with 36% for placebo. Among patients with baseline SLEDAI-2K > 8, significantly higher SRI-4 responses were noted with orelabrutinib at 50 mg (80%, p = 0·048), 80 mg (83%, p = 0·048), and 100 mg (100%, p = 0·029) compared to placebo (0%). Adverse events were mostly mild or moderate in the study. In summary, orelabrutinib was effective and well-tolerated in SLE patients.
Yoav Shamir, Nir London
bioRxiv 2025.03.19.642201
doi: https://doi.org/10.1101/2025.03.19.642201
Recent years have seen an explosion in the prominence of covalent inhibitors as research and therapeutic tools. However, a lag in application of computational methods for covalent docking slows progress in this field. AI models such as AlphaFold3 have shown accuracy in ligand pose prediction but were never assessed for virtual screening. We show that AlphaFold3 reaches near-perfect classification (average AUC=98.3%) of covalent active binders over property-matched decoys, dramatically outperforming classical covalent docking tools. We identify a predicted metric that allows to reliably assign a probability of binding and demonstrate it also improves non-covalent virtual screening.
Fernando, M. C., Craven, G. B., & Shokat, K. M.
Small GTPases, 2024 15(1), 1–7.
https://doi.org/10.1080/21541248.2025.2505441
KRAS is the most frequently mutated oncogene in human cancer. In multiple types of cancer, a missense mutation at codon 12 substitutes a glycine for a cysteine, causing hyperactivation of the GTPase and enhanced MAPK signalling. Recent drug discovery efforts culminating from work during the past decade have resulted in two FDA-approved inhibitors, sotorasib and adagrasib, which target the KRASG12C mutant allele. Ongoing medicinal chemistry efforts across academia and industry have continued developing more potent and efficacious KRASG12C inhibitors. One agent in late-stage clinical trials, divarasib, has demonstrated robust overall response rates, in some cases greater than currently approved agents. Divarasib also exhibits enhanced covalent target engagement in vitro and significant specificity for KRASG12C, yet the structural details of its binding have not been published. Here we report a high-resolution crystal structure of cysteine-light KRAS-4BG12C in complex with divarasib. Though it binds in the same allosteric pocket as sotorasib and adagrasib, the switch-II loop in each crystal structure takes on a distinct conformation differing as much as 5.6 Å between the Cα atom of residue 65 with sotorasib. Additionally, we highlight structural features of the drug complex that may guide future medicinal chemistry efforts targeting various KRAS alleles.
Nathanael Schiander Gray,
Angew. Chem. Int. Ed. 2025, e202508427.
https://doi.org/10.1002/anie.202508427
Targeted protein degradation (TPD) is an emerging therapeutic approach for the selective elimination of disease-related proteins. While molecular glue degraders exhibit drug-like properties, their discovery has traditionally been serendipitous and often requires post-hoc rationalization. In this study, we demonstrate the rational, mechanism-guided design of molecular glue degraders using gluing moieties. Building on established principles, by appending a chemical gluing moiety to several small molecule inhibitors, we successfully transformed them into degraders, obviating the need for a specific E3 ubiquitin ligase recruiter. Specifically, we found that incorporating a hydrophobic aromatic ring or a double bond into a cyclin-dependent kinase 12 and 13 (CDK12/13) dual inhibitor enabled the recruitment of DNA damage-binding protein 1 (DDB1), thereby transforming a high-molecular-weight bivalent CDK12 degrader into a potent monovalent CDK12/13 molecular glue degrader. We also showcase that attaching a cysteine-reactive warhead to a bromodomain-containing protein 4 (BRD4) inhibitor converts it into a degrader by recruiting the DDB1 and CUL4 associated factor 16 (DCAF16) E3 ligase.
Yuto Unoh, Keiichiro Hirai, Shota Uehara, Sho Kawashima, Haruaki Nobori, Jun Satom, Hiromitsu Shibayama, Akihiro Hori, Kenji Nakahara, Kana Kurahashi, Masayuki Takamatsu, Shiho Yamamoto, Qianhui Zhang, Miki Tanimura, Reiko Dodo, Yuki Maruyama, Hirofumi Sawa, Ryosuke Watari, Tetsuya Miyano, Teruhisa Kato, Takafumi Sato,Yuki Tachibana
J. Med. Chem. 2025
https://doi.org/10.1021/acs.jmedchem.5c00754
The coronavirus disease 2019 (COVID-19) pandemic crisis has been mitigated by worldwide efforts to develop vaccines and therapeutic drugs. However, there remains concern regarding public health and an unmet need for therapeutic options. Herein, we report the discovery of S-892216, a second-generation SARS-CoV-2 3C-like protease (3CLpro) inhibitor, to treat COVID-19. S-892216 is a reversible covalent 3CLpro inhibitor with highly potent antiviral activity and an EC50 value of 2.48 nM against SARS-CoV-2 infected cells. Structure-based design of a covalent modifier for compound 1 revealed that introducing a nitrile warhead increased 3CLpro inhibition activity by 180-fold. Subsequent optimization efforts yielded S-892216, which combined a favorable pharmacokinetic profile and high off-target selectivity. S-892216 exhibited antiviral activity against diverse SARS-CoV-2 variants, including major mutations reducing antiviral activities of nirmatrelvir and ensitrelvir. In SARS-CoV-2-infected mice, S-892216 inhibited viral replication in the lungs similar to ensitrelvir, although at a 30-fold lower dose.
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.
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.
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.
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.
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.
Yu Tung Wong, Charles Bell, and Michael C. Willis
Chem. Sci., 2025
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
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.
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
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.
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.
Kyle Ghaby, Benoît Roux
bioRxiv 2025.05.28.656658;
doi: https://doi.org/10.1101/2025.05.28.656658
Sara Amberntsson, Alison J Foster, Bhavik Chouhan, Stephen Wilkinson, Stephanie Harlfinger, Graham Smith, Jason G Kettle, Michael Niedbala, Stefan Kavanagh, Dominic P Williams
Toxicology Research, 2025, 14, 3, tfaf054,
https://doi.org/10.1093/toxres/tfaf054
Interest in inhibiting target proteins through covalent binding mechanisms has increased in the last decade due to the potential for beneficial pharmacological properties. However, the inherent targeted covalent inhibitor (TCI) adverse off-target reactivity risk requires a mitigation strategy early during drug discovery. The aim of this research was to design a pre-clinical hepatic safety assessment strategy for TCIs considering risk associated with electrophilic warhead reactivity and reactive metabolites formation at clinically-relevant plasma concentrations.
The mitigation strategy was applied to compound 35, a potent irreversible inhibitor to KRASG12C. Drug induced liver injury was assessed in primary human hepatocyte spheroids. GSH and ATP depletion were investigated for compound 35 and 6 other marketed TCIs containing an acrylamide warhead which binds irreversibly to cysteine-containing target proteins. None of the TCIs showed GSH depletion prior to ATP depletion after 7-days exposure, suggesting that GSH depletion was not driving cytotoxicity in the spheroids. The calculated hepatotoxicity margin towards plasma exposure of 2.5 for compound 35 was found to be in the same range as for the two KRASG12Cinhibitors adagrasib and sotorasib, with clinically reported treatment-related adverse aminotransferase elevations leading to dose modifications. The safety evaluation reported here suggests no negative discrepancy in liver toxicity for compound 35 versus similar approved TCI’s. Finally, the risk associated with detected oxidative metabolites was further mitigated as the pan-CYP450 inhibitor 1-aminobenzotriazole (ABT) had no effect on the cytotoxicity response following incubation of compound 35 in the presence and absence of ABT.
Birgit Wilding, Lydia Woelflingseder, Anke Baum, Krzysztof Chylinski, Gintautas Vainorius, Neil Gibson, Irene C. Waizenegger, Daniel Gerlach, Martin Augsten, Fiona Spreitzer, Yukina Shirai, Masachika Ikegami, Sylvia Tilandyová, Dirk Scharn, Mark A. Pearson, Johannes Popow, Anna C. Obenauf, Noboru Yamamoto, Shunsuke Kondo, Frans L. Opdam, Annemarie Bruining, Shinji Kohsaka, Norbert Kraut, John V. Heymach, Flavio Solca, Ralph A. Neumüller
Cancer Discov (2025) 15 (1): 119–138.
https://doi.org/10.1158/2159-8290.CD-24-0306
Mutations in ERBB2 (encoding HER2) occur in 2% to 4% of non–small cell lung cancer (NSCLC) and confer poor prognosis. ERBB-targeting tyrosine kinase inhibitors, approved for treating other HER2-dependent cancers, are ineffective in HER2-mutant NSCLC due to dose-limiting toxicities or suboptimal potency. We report the discovery of zongertinib (BI 1810631), a covalent HER2 inhibitor. Zongertinib potently and selectively blocks HER2, while sparing EGFR, and inhibits the growth of cells dependent on HER2 oncogenic driver events, including HER2-dependent human cancer cells resistant to trastuzumab deruxtecan. Zongertinib displays potent antitumor activity in HER2-dependent human NSCLC xenograft models and enhances the activities of antibody–drug conjugates and KRASG12C inhibitors without causing obvious toxicities. The preclinical efficacy of zongertinib translates in objective responses in patients with HER2-dependent tumors, including cholangiocarcinoma (SDC4–NRG1 fusion) and breast cancer (V777L HER2 mutation), thus supporting the ongoing clinical development of zongertinib.
Significance: HER2-mutant NSCLC poses a challenge in the clinic due to limited options for targeted therapies. Pan-ERBB blockers are limited by wild-type EGFR–mediated toxicity. Zongertinib is a highly potent and wild-type EGFR–sparing HER2 inhibitor that is active in HER2-driven tumors in the preclinical and clinical settings.
Dandan Zhu, Zijian Zheng, Huixin Huang, Xiaojuan Chen, Shuhong Zhang, Zhuchu Chen, Ting Liu, Guangyu Xu, Ying Fu, Yongheng Chen,
European Journal of Medicinal Chemistry, 2025
https://doi.org/10.1016/j.ejmech.2025.117795
Fibroblast growth factor receptors (FGFRs) represent promising therapeutic targets in various malignancies, yet the clinical application of FGFR covalent inhibitors has been impeded by several significant challenges, including unquantifiable target engagement, undefined off-target effects, and the emergence of drug resistance. In this study, we designed and synthesized a series of FGFR activity-based probes (ABPs) derived from FIIN-2, a pioneering selective, next-generation irreversible covalent FGFR inhibitor with demonstrated efficacy against gatekeeper mutations. Among them, FP1 exhibited comparable inhibitory potency to FIIN-2. FP1 could facilitate precise in vitro and in situ labeling and visualization of both FGFR1-4 and their mutants. Utilizing FP1, we successfully mapped the target spectrum of FIIN-2 in MDA-MB-453 cells through activity-based protein profiling (ABPP), and established a robust framework for employing our probe as a generalizable tool to systematically evaluate the on- and off-target activities of prospective FGFR covalent inhibitors. Overall, the FGFR ABP offers a promising strategy for elucidating the engagement of FGFR, profiling the target specificity and mechanisms of covalent FGFR inhibitors, and offering potential avenues for overcoming drug resistance.
Danyang Hu, Hairong Zeng, Wenxuan Li, Ya Zhang, Xiaoqian Chi, Xiaoyu Liu, Haijing Zhang, Guangbo Ge, Xiaozhen Jiao, and Ping Xie
Journal of Medicinal Chemistry 2025
DOI: 10.1021/acs.jmedchem.5c00563
Mammalian carboxylesterases play an important role in the hydrolysis of both endogenous substrates and xenobiotics bearing ester or amide bond(s). We previously reported that bysspectin A and its derivative LC-20W were potent reversible hCES2A inhibitors. Here, a series of bysspectin A derivatives were designed and synthesized using LC-20W as the leading compound. Compound 9d was identified as a potent serine-targeting covalent inhibitor of hCES2A (IC50 = 0.12 nM), which was much more potent than that of LC-20W. Further chemoproteomics and docking simulations showed that 9d could selectively modify hCES2A at the catalytic serine (Ser228), thereby blocking its catalytic activity. Notably, 9d showed good cell-membrane permeability and was capable of inhibiting intracellular hCES2A in living cells. In vivo tests showed that 9d significantly alleviates irinotecan-induced diarrhea and dextran sulfate sodium-induced colitis. Collectively, a novel serine-targeting covalent inhibitor against hCES2A was developed, offering a promising candidate for treating drug-induced diarrhea and ulcerative colitis.
Nafizul Haque Kazi, Nikolas Klink, Kai Gallant, Gian-Marvin Kipka & Malte Gersch
Nat Struct Mol Biol, 2025
https://doi.org/10.1038/s41594-025-01534-4
The mitochondrial deubiquitinase ubiquitin-specific protease (USP) 30 negatively regulates PINK1–parkin-driven mitophagy. Whether enhanced mitochondrial quality control through inhibition of USP30 can protect dopaminergic neurons is currently being explored in a clinical trial for Parkinson’s disease. However, the molecular basis for specific inhibition of USP30 by small molecules has remained elusive. Here we report the crystal structure of human USP30 in complex with a specific inhibitor, enabled by chimeric protein engineering. Our study uncovers how the inhibitor extends into a cryptic pocket facilitated by a compound-induced conformation of the USP30 switching loop. Our work underscores the potential of exploring induced pockets and conformational dynamics to obtain deubiquitinase inhibitors and identifies residues facilitating specific inhibition of USP30. More broadly, we delineate a conceptual framework for specific USP deubiquitinase inhibition based on a common ligandability hotspot in the Leu73 ubiquitin binding site and on diverse compound extensions. Collectively, our work establishes a generalizable chimeric protein-engineering strategy to aid deubiquitinase crystallization and enables structure-based drug design with relevance to neurodegeneration.
A.E. Maciag,Y. Yang,A.K. Sharma,D.M. Turner,C.J. DeHart,H. Abdelkarim,L. Fan,B.P. Smith,V. Kumari,M. Dyba,M. Rigby,J.A. Castillo Badillo,L. Adams,L. Fornelli,S. Fox,A. Brafman,T. Turbyville,W. Gillette,S. Messing,[...]& F. McCormick,
Proc. Natl. Acad. Sci. 2025 122 (19) e2410766122,
https://doi.org/10.1073/pnas.2410766122
RAS is the most frequently mutated oncogene in cancer. RAS proteins show high sequence similarities in their G-domains but are significantly different in their C-terminal hypervariable regions (HVR). These regions interact with the cell membrane via lipid anchors that result from posttranslational modifications (PTM) of cysteine residues. KRAS4b is unique as it has only one cysteine that undergoes PTM, C185. Small molecule covalent modification of C185 would block any form of prenylation and subsequently inhibit attachment of KRAS4b to the cell membrane, blocking its biological activity. We translated this concept to the discovery and development of disulfide tethering screen hits into irreversible covalent modifiers of C185. These compounds inhibited proliferation of KRAS4b-driven mouse embryonic fibroblasts, but not cells driven by N-myristoylated KRAS4b that harbor a C185S mutation and are not dependent on C185 prenylation. Top–down proteomics was used to confirm target engagement in cells. These compounds bind in a pocket formed when the HVR folds back between helix 3 and 4 in the G-domain (HVR-α3-α4). This interaction can happen in the absence of small molecules as predicted by molecular dynamics simulations and is stabilized in the presence of C185 binders as confirmed by small-angle X-ray scattering and solution NMR. NOESY-HSQC, an NMR approach that measures internuclear distances of 6 Å or less, and structure analysis identified the critical residues and interactions that define the HVR-α3-α4 pocket. Further development of compounds that bind to this pocket could be the basis of a new approach to targeting KRAS cancers.
Ludovica S. Sirocchi, Maximilian Scharnweber, Sarah Oberndorfer, Gabriella Siszler, Krzysztof M. Zak, Klaus Rumpel, Ralph A. Neumüller, and Birgit Wilding
Journal of the American Chemical Society 2025 147 (18), 15787-15795
DOI: 10.1021/jacs.5c03562
Xinyuan Wu, Jiayi Pan, Rufeng Fan, Yiwei Zhang, Chao Wang, Guoliang Wang, Jiaxiang Liu, Mengqing Cui, Jinfeng Yue, Rui Jin, Zhiqiang Duan, Mingyue Zheng, Lianghe Mei, Lu Zhou, Minjia Tan, Jing Ai, and Xiaojie Lu
Journal of the American Chemical Society 2025 147 (18), 15469-15481
DOI: 10.1021/jacs.5c01712
Wang, P., Sun, X., He, X., Kang, D., Liu, X., Liu, D., Li, A., Yang, G., Lin, Y., Li, S., Wang, Y., & Wang, Y.
Cancer research communications, 2025
https://doi.org/10.1158/2767-9764.CRC-25-0001
Clinical studies have demonstrated the antitumor efficacy of covalent KRAS G12C inhibitors in treating advanced/metastatic cancers. In the current study, we report the preclinical characteristics of a specific KRAS p.G12C covalent inhibitor, glecirasib. Glecirasib exhibited high potency against KRAS G12C, along with a high level of selectivity over the wild-type KRAS, HRAS, and NRAS in biochemical assays. On the cellular level, it substantially reduced downstream ERK phosphorylation, AKT phosphorylation and inhibited the viability of cancer cells harboring the KRAS p.G12C mutation, and demonstrated high selectivity over non-KRAS p.G12C cancer cells. Glecirasib could effectively inhibit HRAS G12C, NRAS G12C, and several G12C-inclusive KRAS double mutants that showed resistance to adagrasib. In vivo research suggested that once-daily dosing of glecirasib can robustly inhibit ERK phosphorylation for at least 24 h and induced tumor regression in several xenograft models, including the NCI-H1373-luciferase intracranial model. Glecirasib in combination with cetuximab or JAB-3312 (sitneprotafib, a clinical-stage SHP2 inhibitor developed by Jacobio) greatly enhanced antitumor activity both in vitro and in vivo. Collectively, these results suggest that glecirasib is a potent and selective covalent inhibitor of KRAS G12C, shows potent antitumor activity as monotherapy and synergizes with either EGFR blockade or SHP2 inhibition. A new drug application for glecirasib has been submitted in China, seeking approval for the treatment of non-small cell lung cancer, supported by a pivotal phase 2 single-arm study (NCT05009329). Additionally, glecirasib is being explored in clinical trials in combination with cetuximab (phase 2, NCT05194995) and JAB-3312 (phase 3, NCT06416410).
R. Justin Grams, Wesley J. Wolfe, Robert J. Seal, James Veccia, and Ku-Lung Hsu
Journal of Medicinal Chemistry 2025
DOI: 10.1021/acs.jmedchem.5c00050
Aldo-keto reductase family 1 member C3 (AKR1C3) is a member of the AKR superfamily of enzymes that metabolize androgen, estrogen, and prostaglandin substrates that drive proliferation in hormone-dependent cancers. Interest in developing selective inhibitors has produced tool compounds for the inactivation or degradation of AKR1C3 with varying degrees of selectivity among the 14 known AKR proteins. Selectivity of AKR1C3 inhibitors across the AKR family is critical since a clinical candidate failed due to hepatotoxicity from off-target inhibition of AKR1D1. Here, we report development of a sulfonyl-triazole (SuTEx) covalent AKR1C3 inhibitor (RJG-2051) that selectively engages a noncatalytic tyrosine residue (Y24) on AKR1C3. Importantly, RJG-2051 exhibited negligible cross-reactivity with AKRs or other proteins across 1800+ tyrosine and lysine sites quantified by chemical proteomics. Our disclosure of a covalent inhibitor for potent AKR1C3 inactivation with proteome-wide selectivity in cells will expedite cell biological studies for testing the therapeutic potential of this metabolic target.
Lauren M Orr, Sydney J Tomlinson, Hannah R Grupe, Melissa Lim, Emily Ho, Halime Yilmaz, Grace Zhou, Barbara Leon, James A Olzmann, Daniel K Nomura
ACS Cent. Sci. 2025
https://doi.org/10.1021/acscentsci.5c00959
bioRxiv 2025.04.25.650514;
doi: https://doi.org/10.1101/2025.04.25.650514
Targeted protein degradation (TPD) is a powerful strategy for targeting and eliminating disease-causing proteins. While heterobifunctional Proteolysis-Targeting Chimeras (PROTACs) are more modular, the rational design of monovalent or molecular glue degraders remains challenging. In this study, we generated a small library of BET-domain inhibitor JQ1 analogs bearing elaborated electrophilic handles to identify permissive covalent degradative handles and E3 ligase pairs. We identified an elaborated fumaramide handle that, when appended onto JQ1, led to the proteasome-dependent degradation of BRD4. Further characterization revealed that the E3 ubiquitin ligase CUL4(DCAF16), a common E3 ligase target of electrophilic degraders, was responsible for BRD4 loss by covalently targeting C173 on DCAF16. While this original fumaramide handle, when appended onto other protein-targeting ligands, did not accommodate the degradation of other neo-substrates, a truncated version of this handle attached to JQ1 was still capable of degrading BRD4, now through targeting both C173 and C178. This truncated fumaramide handle, when appended on various protein targeting ligands, and was also more permissive in degrading other neo-substrates, including CDK4/6, SMARCA2 and SMARCA4, and the androgen receptor (AR). We further demonstrated that this optimized truncated fumaramide handle, when transplanted onto an AR DNA binding domain-targeting ligand, could degrade both AR and the undruggable truncation variant of AR, AR-V7, in androgen-independent prostate cancer cells in a DCAF16-dependent manner. Overall, we have identified a unique DCAF16-targeting covalent degradative handle that can be transplanted across several protein-targeting ligands to induce the degradation of their respective targets for the modular design of monovalent or bifunctional degraders.
Hayden A. Sharma, Michael Bielecki, Meredith A. Holm, Ty M. Thompson, Yue Yin, Jacob B. Cravatt, Timothy B. Ware, Alex Reed, Molhm Nassir, Tamara El-Hayek Ewing, Bruno Melillo, J. Fernando Bazan, Phil S. Baran, and Benjamin F. Cravatt
Journal of the American Chemical Society 2025
DOI: 10.1021/jacs.5c01944
Activity-based protein profiling (ABPP) of stereoisomerically defined sets of electrophilic compounds (‘stereoprobes’) offers a versatile way to discover covalent ligands for proteins in native biological systems. Here we report the synthesis and chemical proteomic characterization of stereoprobes bearing a P(V)-oxathiaphospholane (OTP) reactive group. ABPP experiments identified numerous proteins in human cancer cells that showed stereoselective reactivity with OTP stereoprobes, and we confirmed several of these liganding events with recombinant proteins. OTP stereoprobes engaging the poorly characterized transmembrane protein TLCD1 impaired the incorporation of monounsaturated fatty acids into phosphatidylethanolamine lipids in cells, a lipidomic phenotype that mirrored genetic disruption of this protein. Using AlphaFold2, we found that TLCD1 structurally resembles the ceramide synthase and fatty acid elongase families of coenzyme A-dependent lipid processing enzymes. This structural similarity included conservation of catalytic histidine residues, the mutation of which blocked the OTP stereoprobe reactivity and lipid remodeling activity of recombinant TLCD1. Taken together, these data indicate that TLCD1 acts as a lipid acyltransferase in cells, and that OTP stereoprobes function as inhibitors of this enzymatic activity. Our findings thus illuminate how the chemical proteomic analysis of electrophilic compounds can facilitate the functional annotation and chemical inhibition of a key lipid metabolic enzyme in human cells.
R. Justin Grams, Wesley J. Wolfe, Robert J. Seal, James Veccia, and Ku-Lung Hsu
https://doi.org/10.1021/acs.jmedchem.5c00050
Aldo-keto reductase family 1 member C3 (AKR1C3) is a member of the AKR superfamily of enzymes that metabolize androgen, estrogen, and prostaglandin substrates that drive proliferation in hormone-dependent cancers. Interest in developing selective inhibitors has produced tool compounds for the inactivation or degradation of AKR1C3 with varying degrees of selectivity among the 14 known AKR proteins. Selectivity of AKR1C3 inhibitors across the AKR family is critical since a clinical candidate failed due to hepatotoxicity from off-target inhibition of AKR1D1. Here, we report development of a sulfonyl-triazole (SuTEx) covalent AKR1C3 inhibitor (RJG-2051) that selectively engages a noncatalytic tyrosine residue (Y24) on AKR1C3. Importantly, RJG-2051 exhibited negligible cross-reactivity with AKRs or other proteins across 1800+ tyrosine and lysine sites quantified by chemical proteomics. Our disclosure of a covalent inhibitor for potent AKR1C3 inactivation with proteome-wide selectivity in cells will expedite cell biological studies for testing the therapeutic potential of this metabolic target.
Backus, K.M., Wilson, A.J. Commun Chem 8 , 266 (2025). https://doi.org/10.1038/s42004-025-01652-6