Use of new approach methodology for hepatic safety assessment of covalent inhibitor drug candidates

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.

Zongertinib (BI 1810631), an Irreversible HER2 TKI, Spares EGFR Signaling and Improves Therapeutic Response in Preclinical Models and Patients with HER2-Driven Cancers

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.

Design, synthesis and biological evaluation of the activity-based probes for FGFR covalent inhibitor

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.

Discovery of a Novel Serine-Targeting Covalent Inhibitor against HCES2A for Treating Drug-induced Diarrhea and Ulcerative Colitis

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.

Chimeric deubiquitinase engineering reveals structural basis for specific inhibition of the mitophagy regulator USP30

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.

Blocking C-terminal processing of KRAS4b via a direct covalent attack on the CaaX-box cysteine

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.

Discovery of Carbodiimide Warheads to Selectively and Covalently Target Aspartic Acid in KRASG12D

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

Targeted covalent inhibitors are known to be successful therapeutics used in various indications. Covalent drugs typically target cysteine, as cysteine is well suited due to its high nucleophilicity. However, its low abundance in protein binding sites represents a major limitation. As a result, there is a need to covalently target additional nucleophilic amino acids. Recent literature has reported covalent inhibitors labeling aspartic acid in KRASG12D. However, these compounds also covalently bind to KRASG12C, indicating their cross-reactivity to cysteine along with aspartic acid. We report here carbodiimides as a novel reactive group to selectively target aspartic acid. Covalent inhibitors bearing a carbodiimide moiety are shown to covalently label KRASG12D in biochemical and cellular assays. A high-resolution X-ray crystal structure was obtained, which illustrates the mechanism of the covalent bond formation with KRASG12D. Carbodiimide warheads show selectivity toward KRASG12D over other KRAS alleles and represent a new covalent warhead suitable for covalently binding to aspartic acid in a biochemical and cellular context.

Residue-Selective Inhibitors Discovery via Covalent DNA-Encoded Chemical Libraries with Diverse Warheads

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

Covalent small molecule drugs have emerged as a crucial support in precision therapy due to their high selectivity and robust potency. Covalent DNA-encoded chemical library (CoDEL) technology is an advanced platform for covalent drug discovery. However, the application of CoDELs is constrained by a single-residue focus and limited warhead diversity. Here we report a method to identify residue-selective inhibitors using CoDELs with diverse warheads targeting multiple distinct residues. We systematically evaluated the reactivity of 17 warheads with 9 nucleophilic amino acids of FGFR2 and then constructed CoDELs comprising 24.8 million compounds. These CoDELs enabled the identification of active covalent inhibitors targeting cysteine, lysine, arginine, or glutamic acid. The lysine-targeting inhibitor engaged a novel reactive site. The arginine-targeting inhibitor demonstrated subtype selectivity and overcame drug resistance. The glutamic acid-targeting inhibitor validated the druggability of this unconventional covalent residue site. These findings suggest that our work could potentially expand the target space of covalent drugs and promote precision therapy by harnessing the power of the CoDELs.

Glecirasib, a potent and selective covalent KRAS G12C inhibitor exhibiting synergism 2 with cetuximab or SHP2 inhibitor JAB-3312

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).