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.

Identification of Covalent Cyclic Peptide Inhibitors Targeting Protein–Protein Interactions Using Phage Display

 Sijie Wang, Franco F. Faucher, Matilde Bertolini, Heeyoung Kim, Bingchen Yu, Li Cao, Katharina Roeltgen, Scott Lovell, Varun Shanker, Scott D. Boyd, Lei Wang, Ralf Bartenschlager, and Matthew Bogyo

Journal of the American Chemical Society 2025

DOI: 10.1021/jacs.4c15843

Peptide macrocycles are promising therapeutics for a variety of disease indications due to their overall metabolic stability and potential to make highly selective binding interactions with targets. Recent advances in covalent macrocycle peptide discovery, driven by phage and mRNA display methods, have enabled the rapid identification of highly potent and selective molecules from large libraires of diverse macrocycles. However, there are currently limited examples of macrocycles that can be used to disrupt protein–protein interactions and even fewer examples that function by formation of a covalent bond to a target protein. In this work, we describe a directed counter-selection method that enables identification of covalent macrocyclic ligands targeting a protein–protein interaction using a phage display screening platform. This method utilizes binary and ternary screenings of a chemically modified phage display library, employing the stable and weakly reactive aryl fluorosulfate electrophile. We demonstrate the utility of this approach using the SARS-CoV-2 spike-ACE2 protein–protein interaction and identify multiple covalent macrocyclic inhibitors that disrupt this interaction. The resulting compounds displayed antiviral activity against live virus that was irreversible after washout due to the covalent binding mechanism. These results highlight the potential of this screening platform for developing covalent macrocyclic drugs that disrupt protein–protein interactions with long lasting effects.

Site-specific activation of the proton pumpinhibitor rabeprazole by tetrathiolate zinccentres

Teresa Marker, Raphael R. Steimbach, Cecilia Perez-Borrajero, Marcin Luzarowski, Eric Hartmann, Sibylle Schleich, Daniel Pastor-Flores, Elisa Espinet, Andreas Trumpp, Aurelio A. Teleman, Frauke Gräter, Bernd Simon, Aubry K. Miller & Tobias P. Dick 

Nat. Chem. (2025). https://doi.org/10.1038/s41557-025-01745-8

Proton pump inhibitors have become top-selling drugs worldwide. Serendipitously discovered as prodrugs that are activated by protonation in acidic environments, proton pump inhibitors inhibit stomach acid secretion by covalently modifying the gastric proton pump. Despite their widespread use, alternative activation mechanisms and potential target proteins in non-acidic environments remain poorly understood. Employing a chemoproteomic approach, we found that the proton pump inhibitor rabeprazole selectively forms covalent conjugates with zinc-binding proteins. Focusing on DENR, a protein with a C4 zinc cluster (that is, zinc coordinated by four cysteines), we show that rabeprazole is activated by the zinc ion and subsequently conjugated to zinc-coordinating cysteines. Our results suggest that drug binding, activation and conjugation take place rapidly within the zinc coordination sphere. Finally, we provide evidence that other proton pump inhibitors can be activated in the same way. We conclude that zinc acts as a Lewis acid, obviating the need for low pH, to promote the activation and conjugation of proton pump inhibitors in non-acidic environments.

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, Molham Nassir, Tamara El-Hayek Ewing, Bruno Melillo, J Fernando Bazan, Phil S. Baran, Benjamin F. Cravatt

bioRxiv 2025.01.31.635883; 

doi: https://doi.org/10.1101/2025.01.31.635883

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.

Covalent Destabilizing Degrader of AR and AR-V7 in Androgen-Independent Prostate Cancer Cells

Charlotte M Zammit, Cory Nadel, Ying Lin, Sajjan Koirala, Patrick Ryan Potts, Daniel K Nomura

bioRxiv 2025.02.12.637117; 

doi: https://doi.org/10.1101/2025.02.12.637117

Androgen-independent prostate cancers, correlated with heightened aggressiveness and poor prognosis, are caused by mutations or deletions in the androgen receptor (AR) or 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, that only possess the 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 leveraged a cysteine-reactive covalent ligand library in a cellular screen to identify 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 an intrinsically disordered cysteine C125 in AR and AR-V7. EN1441 causes significant and selective destabilization of AR and AR-V7, leading to 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 to 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.