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).

Discovery and Optimization of a Covalent AKR1C3 Inhibitor

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.

DCAF16-Based Covalent Degradative Handles for the Modular Design of Degraders

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

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.

Proteomic Ligandability Maps of Phosphorus(V) Stereoprobes Identify Covalent TLCD1 Inhibitors

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.

Discovery and Optimization of a Covalent AKR1C3 Inhibitor

R. Justin Grams, Wesley J. Wolfe, Robert J. Seal, James Veccia, and Ku-Lung Hsu

Journal of Medicinal Chemistry 2025

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.

Discovery of RNA-Reactive Small Molecules Guides Design of Electrophilic Modules for RNA-Specific Covalent Binders

Noah A. Springer, Patrick R. A. Zanon, Amirhossein Taghavi, Kisu Sung, Matthew D. Disney

bioRxiv 2025.04.22.649986; 

doi: https://doi.org/10.1101/2025.04.22.649986

RNA is a key drug target that can be modulated by small molecules, however covalent binders of RNA remain largely unexplored. Using a high-throughput mass spectrometry screen of 2,000 electrophilic compounds, we identified ligands that react with RNA in a binding-dependent manner. RNA reactivity was influenced by both the reactive group and the RNA-binding scaffold. Electrophilic modules such as 3-chloropivalamide, bis(2-chloroethyl)amine, chloroacetamide, and N-acylimidazole that react with proteins also cross-linked to RNA, especially when paired with aromatic heterocycles, particularly those with a thieno[3,2-c]pyridinium core. These results suggest that electrophiles commonly used for protein targeting can also covalently modify RNA, potentially contributing to both on- and off-target effects. This insight enabled the design of an RNA-specific covalent compound by modifying a Hoechst scaffold, originally identified to bind DNA, to react selectively with the expanded triplet repeat RNA, r(CUG)exp, that causes myotonic dystrophy type 1 (DM1). Selectivity appears to arise from binding to the RNA major groove near the reactive site. Overall, this study highlights the potential of rationally designing covalent RNA-targeting small molecules.

Identification of a phenyl ester covalent inhibitor of caseinolytic protease and analysis of the ClpP1P2 inhibition in mycobacteria

Genhui Xiao, Yumeng Cui, Liangliang Zhou, Chuya Niu, Bing Wang, Jinglan Wang, Shaoyang Zhou, Miaomiao Pan, Chi Kin Chan, Yan Xia, Lan Xu, Yu Lu, Shawn Chen

mLife, 2025

DOI: https://doi.org/10.1002/mlf2.12169

The caseinolytic protease complex ClpP1P2 is crucial for protein homeostasis in mycobacteria and stress response and virulence of the pathogens. Its role as a potential drug target for combating tuberculosis (TB) has just begun to be substantiated in drug discovery research. We conducted a biochemical screening targeting the ClpP1P2 using a library of compounds phenotypically active against Mycobacterium tuberculosis (Mtb). The screening identified a phenyl ester compound GDI-5755, inhibiting the growth of Mtb and M. bovis BCG, the model organism of mycobacteria. GDI-5755 covalently modified the active-site serine residue of ClpP1, rendering the peptidase inactive, which was delineated through protein mass spectrometry and kinetic analyses. GDI-5755 exerted antibacterial activity by inhibiting ClpP1P2 in the bacteria, which could be demonstrated through a minimum inhibitory concentration (MIC) shift assay with a clpP1 CRISPRi knockdown (clpP1-KD) mutant GH189. The knockdown also remarkably heightened the mutant's sensitivity to ethionamide and meropenem, but not to many other TB drugs. On the other hand, a comparative proteomic analysis of wild-type cells exposed to GDI-5755 revealed the dysregulated proteome, specifically showing changes in the expression levels of multiple TB drug targets, including EthA, LdtMt2, and PanD. Subsequent evaluation confirmed the synergistic activity of GDI-5755 when combined with the TB drugs to inhibit mycobacterial growth. Our findings indicate that small-molecule inhibitors targeting ClpP1P2, when used alongside existing TB medications, could represent novel therapeutic strategies.