Comprehensive Characterization of Bruton’s Tyrosine Kinase Inhibitor Specificity, Potency, and Biological Effects: Insights into Covalent and Noncovalent Mechanistic Signatures

Antonia C. Darragh, Andrew M. Hanna, Justin H. Lipner, Alastair J. King, Nicole B. Servant, and Mirza Jahic

ACS Pharmacol. Transl. Sci. 2025
https://doi.org/10.1021/acsptsci.4c00540

Uncovering a drug’s mechanism of action and possible adverse effects are critical components in drug discovery and development. Moreover, it provides evidence for why some drugs prove more effective than others and how to design better drugs altogether. Here, we demonstrate the utility of a high-throughput in vitro screening platform along with a comprehensive panel to aid in the characterization of 15 Bruton’s tyrosine kinase (BTK) inhibitors that are either approved by the FDA or presently under clinical evaluation. To compare the potency of these drugs, we measured the binding affinity of each to wild-type BTK as well as a clinically relevant resistance mutant of BTK (BTK C481S). In doing so, we discovered a considerable difference in the selectivity and potency of these BTK inhibitors to the wild-type and mutant proteins. Some of this potentially contributes to the adverse effects experienced by patients undergoing therapy using these drugs. Overall, noncovalent BTK inhibitors showed stronger potency for both the wild-type and mutant BTK when compared with that of covalent inhibitors, with the majority demonstrating a higher specificity and less off-target modulation. Additionally, we compared biological outcomes for four of these inhibitors in human cell-based models. As expected, we found different phenotypic profiles for each inhibitor. However, the two noncovalent inhibitors had fewer off-target biological effects when compared with the two covalent inhibitors. This and similar in-depth preclinical characterization of drug candidates can provide critical insights into the efficacy and mechanism of action of a compound that may affect its safety in a clinical setting.

Rational Design of Stapled Covalent Peptide Modifiers of Oncoprotein E6 from Human Papillomavirus

ACS Chem. Biol. 2025

https://doi.org/10.1021/acschembio.4c00878

Human Papillomavirus (HPV) is linked to multiple cancers, most significantly cervical cancer, for which HPV infection is associated with nearly all cases. Essential to the oncogenesis of HPV is the function of the viral protein E6 and its role in degrading the cell cycle regulator p53. Degradation of p53, and the resultant loss of cell cycle control, is mediated by E6 recruitment of the E3 ubiquitin ligase E6AP and subsequent ubiquitination of p53. Here, we report the design of a stapled peptide that mimics the LxxLL α-helical domain of E6AP to bind and covalently label a cysteine residue specific to HPV-16 E6. Several acrylamide- and haloacetamide-based warheads were evaluated for reactivity and specificity, and a panel of hydrocarbon-stapled peptides was evaluated for enhanced binding affinity and increased proteolytic stability. Structure-based modeling was used to rationalize the observed trends in the reactivity of the warheads and the impact of the hydrocarbon staple position on the binding affinity of the stapled peptides. The development of a proteolytically stable and reactive peptide represents a new class of peptide-based inhibitors of protein–protein interactions with a potential therapeutic value toward HPV-derived cancers.

Design of Benzyl-triazolopyrimidine-Based NADPH Oxidase Inhibitors Leads to the Discovery of a Potent Dual Covalent NOX2/MAOB Inhibitor

Beatrice Noce, Sara Marchese, Marta Massari, Chiara Lambona, Joana Reis, Francesco Fiorentino, Alessia Raucci, Rossella Fioravanti, Mariana Castelôa, Alessandro Mormino, Stefano Garofalo, Cristina Limatola, Lorenzo Basile, Andrea Gottinger, Claudia Binda, Andrea Mattevi, Antonello Mai, and Sergio Valente

Journal of Medicinal Chemistry 2025

DOI: 10.1021/acs.jmedchem.4c02644

NADPH oxidases (NOXs) are enzymes dedicated to reactive oxygen species (ROS) production and are implicated in cancer, neuroinflammation, and neurodegenerative diseases. VAS2870 is a covalent inhibitor of mainly NOX2 and NOX5. It alkylates a conserved active-site cysteine, blocking productive substrate binding. To enhance potency and selectivity toward NOXs, we conducted some chemical modifications, leading to the discovery of compound 9a that preferentially inhibits NOX2 with an IC50 of 0.155 μM, and only upon its preactivation. We found that 9a, bearing a pargyline moiety, is also able to selectively inhibit MAOB over MAOA (465-fold) with an IC50 of 0.182 μM, being the first-in-class dual NOX2/MAOB covalent inhibitor. Tested in the BV2 microglia neuroinflammation model, 9a decreased ROS production and downregulated proinflammatory cytokines as iNOS, IL-1β, and IL-6 expression more efficiently than the single target inhibitors (rasagiline for MAOB and VAS2870 for NOXs) but also, more importantly, than their combination.

‘Direct-to-biology’ drives optimisation of a cell-active covalent 1 inhibitor of WRN helicase

1S. M. Rowe, A. Price, D. J. Murphy, J. Lin, E. N. Nartey, A. Chaikuad, K. Wong, J. E. Cottom, N. O. Concha, R. A. Reid, E. R. Dickinson, M. Jundt, K. Kammerer, M. Steidel, T. Mathieson, T. Werner, E. K. Grant, C. K. Stanborough, M. Rouah, J. Wojno-Picon, P. Pogány, J. Pettinger, D. J. Norman, H. Wilders, F. Rianjongdee, G. Valdes-Garcia, N. Nevins, R. Shenje, R. K. Thalji, C. Chung, H. C. Eberl, G. Neubauer, D. House, Y. Rao, M. P. Martino and J. T. Bush, 

ChemRxiv, 2025

https://doi.org/10.26434/chemrxiv-2025-tvdzn

We report a ‘direct-to-biology’ (D2B) approach for optimising covalent acrylamide binders of protein targets and apply this to the identification of a selective and cell-active inhibitor of Werner (WRN) helicase. Inhibition of WRN helicase activity exhibits a synthetic lethal relationship with cancers displaying high microsatellite instability (MSI-H) and is being pursued as a therapeutic strategy in the clinic. Using intact-protein liquid chromatography-mass spectrometry (LC-MS) screening, we identified acrylamide fragment binders of the WRN helicase domain and then used covalent D2B chemistry to optimise these initial hits. Our efforts ultimately afforded a potent covalent inhibitor of WRN-mediated DNA unwinding, which displays selective, concentration-dependent cellular engagement of WRN, and demonstrates synthetic lethality in an MSI-H setting. Furthermore, our inhibitor targets a distinct conformation of WRN helicase compared to the current clinical covalent inhibitor, presenting a complementary approach for covalent inhibition of WRN helicase. This work demonstrates how D2B chemistry platforms can be used to explore structure-activity relationships in a modular fashion, while reducing investment of human and material resources.

Morita–Baylis–Hillman Adduct Chemistry as a Tool for the Design of Lysine-Targeted Covalent Ligands

Marco Paolino, Giusy Tassone, Paolo Governa, Mario Saletti, Matteo Lami, Riccardo Carletti, Filippo Sacchetta, Cecilia Pozzi, Maurizio Orlandini, Fabrizio Manetti, Massimo Olivucci, and Andrea Cappelli

ACS Medicinal Chemistry Letters 2025

DOI: 10.1021/acsmedchemlett.4c00479 

The use of Targeted Covalent Inhibitors (TCIs) is an expanding strategy for the development of innovative drugs. It is driven by two fundamental steps: (1) recognition of the target site by the molecule and (2) establishment of the covalent interaction by its reactive group. The development of new TCIs depends on the development of new warheads. Here, we propose the use of Morita–Baylis–Hillman adducts (MBHAs) to covalently bind Lys strategically placed inside a lipophilic pocket. A human cellular retinoic acid binding protein II mutant (M2) was selected as a test bench for a library of 19 MBHAs. The noncovalent interaction step was investigated by molecular docking studies, while experimentally the entire library was incubated with M2 and crystallized to confirm covalent binding with the target lysine. The results, rationalized through covalent docking analysis, support our hypothesis of MBHAs as reactive scaffolds for the design of lysine-TCIs.

Orally Bioavailable and Site-Selective Covalent STING Inhibitor Derived from a Macrocyclic Marine Diterpenoid

Guang-Hao Niu, Wan-Chi Hsiao, Po-Hsun Lee, Li-Guo Zheng, Yu-Shao Yang, Wei-Cheng Huang, Chih-Chien Hsieh, Tai-Yu Chiu, Jing-Ya Wang, Ching-Ping Chen, Chen-Lung Huang, May-Su You, Yi-Ping Kuo, Chien-Ming Wang, Zhi-Hong Wen, Guann-Yi Yu, Chiung-Tong Chen, Ya-Hui Chi, Chun-Wei Tung, Shu-Ching Hsu, Teng-Kuang Yeh, Ping-Jyun Sung, Mingzi M. Zhang, and Lun Kelvin Tsou

Journal of Medicinal Chemistry 2025

DOI: 10.1021/acs.jmedchem.4c02665

Pharmacological inhibition of the cGAS-STING-controlled innate immune pathway is an emerging therapeutic strategy for a myriad of inflammatory diseases. Here, we report GHN105 as an orally bioavailable covalent STING inhibitor. Late-stage diversification of the briarane-type diterpenoid excavatolide B allowed the installation of solubility-enhancing functional groups while enhancing its activity as a covalent STING inhibitor against multiple human STING variants, including the S154 variant responsible for a genetic autoimmune disease. Selectively engaging the membrane-proximal Cys91 residue of STING, GHN105 dose-dependently inhibited cGAS-STING signaling and type I interferon responses in cells and in vivo. Moreover, orally administered GHN105 exhibited on-target engagement in vivo and markedly reversed key pathological features in a delayed treatment of the acute colitis mouse model. Our study provided proof of concept that the synthetic briarane analog GHN105 serves as a safe, site-selective, and orally active covalent STING inhibitor and devises a regimen that allows long-term systemic administration.

Discovery of Elironrasib (RMC-6291), a Potent and Orally Bioavailable, RAS(ON) G12C-Selective, Covalent Tricomplex Inhibitor for the Treatment of Patients with RAS G12C-Addicted Cancers

James Cregg, Kristof Pota, Aidan C. A. Tomlinson, Jason Yano, Abby Marquez, Yang Liu, Christopher J. Schulze, Kyle J. Seamon, Matthew Holderfield, Xing Wei, Yongxian Zhuang, Yu Chi Yang, Jingjing Jiang, Yue Huang, Ruiping Zhao, Yun Ling, Zhican Wang, Michael Flagella, Zhengping Wang, Mallika Singh, John E. Knox, Robert Nichols, David Wildes, Jacqueline A. M. Smith, Elena S. Koltun, and Adrian L. Gill

Journal of Medicinal Chemistry 2025

DOI: 10.1021/acs.jmedchem.4c02313

The discovery of elironrasib (RMC-6291) represents a significant breakthrough in targeting the previously deemed undruggable GTP-bound, active KRASG12C. To target the active state of RAS (RAS(ON)) directly, we have employed an innovative tri-complex inhibitor (TCI) modality involving formation of a complex with an inhibitor, the intracellular chaperone protein CypA, and the target protein KRASG12C in its GTP-bound form. The resulting tri-complex inhibits oncogenic signaling, inducing tumor regressions across various preclinical models of KRASG12C mutant human cancers. Here we report structure-guided medicinal chemistry efforts that led to the discovery of elironrasib, a potent, orally bioavailable, RAS(ON) G12C-selective, covalent, tri-complex inhibitor. The investigational agent elironrasib is currently undergoing phase 1 clinical trials (NCT05462717, NCT06128551, NCT06162221), with preliminary data indicating clinical activity in patients who had progressed on first-generation inactive state-selective KRASG12C inhibitors.