Re-evaluating the mechanism of action of α,β-unsaturated carbonyl DUB inhibitors b-AP15 and VLX1570: a paradigmatic example of unspecific protein crosslinking with Michael acceptor motif-containing drugs.

Jennifer A. Ward, Adan Pinto-Fernandez, Loic Cornelissen, Sarah Bonham, Laura Díaz-Sáez, Olivier Riant, Kilian V. M. Huber, Benedikt M Kessler, Olivier Feron, and Edward W. Tate

Journal of Medicinal Chemistry 2020
DOI: 10.1021/acs.jmedchem.0c00144

Deubiquitinating enzymes are a growing target class across multiple disease states, with several inhibitors now report-ed. b-AP15 and VLX1570 are two structurally related USP14/UCH-37 inhibitors. Through a proteomic approach, we demonstrate that these compounds target a diverse range of proteins, resulting in the formation of higher molecular weight complexes. Activity-based proteome profiling identified CIAPIN1 as a sub-micromolar covalent target of VLX1570, and further analysis demonstrated that high molecular weight complex formation leads to aggregation of CIAPIN1 in intact cells. Our results suggest that in addition to DUB inhibition, these compounds induce non-specific protein aggregation, providing a molecular explanation for general cellular toxicity.

Discovery of LOU064 (Remibrutinib), a Potent and Highly Selective Covalent Inhibitor of Bruton’s Tyrosine Kinase

Daniela Angst, François Gessier, Philipp Janser, Anna Vulpetti, Rudolf Wälchli, Christian Beerli, Amanda Littlewood-Evans, Janet Dawson, Barbara Nuesslein-Hildesheim, Grazyna Wieczorek, Sascha Gutmann, Clemens Scheufler, Alexandra Hinniger, Alfred Gilbert Zimmerlin, Enrico G. Funhoff, Robert Pulz, and Bruno Cenni

J. Med. Chem. 2020
https://doi.org/10.1021/acs.jmedchem.9b01916

Bruton’s tyrosine kinase (BTK), a cytoplasmic tyrosine kinase, plays a central role in immunity and is considered an attractive target for treating autoimmune diseases. The use of currently marketed covalent BTK inhibitors is limited to oncology indications based on their suboptimal kinase selectivity. We describe the discovery and preclinical profile of LOU064 (remibrutinib, 25), a potent, highly selective covalent BTK inhibitor. LOU064 exhibits an exquisite kinase selectivity due to binding to an inactive conformation of BTK and has the potential for a best-in-class covalent BTK inhibitor for the treatment of autoimmune diseases. It demonstrates potent in vivo target occupancy with an EC90 of 1.6 mg/kg and dose-dependent efficacy in rat collagen-induced arthritis. LOU064 is currently being tested in Phase 2 clinical studies for chronic spontaneous urticaria and Sjoegren’s Syndrome.

Designing Chimeric Molecules for Drug Discovery by Leveraging Chemical Biology

Chiara Borsari, Darci J. Trader, Annalisa Tait, and Maria P. Costi
Journal of Medicinal Chemistry 2020
DOI: 10.1021/acs.jmedchem.9b01456

After the first seed concept introduced in the 18th century, different disciplines have attributed different names to dual-functional molecules depending on their application, including bioconjugates, bifunctional compounds, multitargeting molecules, chimeras, hybrids, engineered compounds. However, these engineered constructs share a general structure: a first component that targets a specific cell and a second component that exerts the pharmacological activity. A stable or cleavable linker connects the two modules of a chimera. Herein, we discuss the recent advances in the rapidly expanding field of chimeric molecules leveraging chemical biology concepts. This Perspective is focused on bifunctional compounds in which one component is a lead compound or a drug. In detail, we discuss chemical features of chimeric molecules and their use for targeted delivery and for target engagement studies.

Development of a covalent inhibitor of gut bacterial bile salt hydrolases

Arijit A. Adhikari, Tom C. M. Seegar, Scott B. Ficarro, Megan D. McCurry, Deepti Ramachandran, Lina Yao, Snehal N. Chaudhari, Sula Ndousse-Fetter, Alexander S. Banks, Jarrod A. Marto, Stephen C. Blacklow & A. Sloan Devlin

Nature Chemical Biology (2020)
https://doi.org/10.1038/s41589-020-0467-3

Bile salt hydrolase (BSH) enzymes are widely expressed by human gut bacteria and catalyze the gateway reaction leading to secondary bile acid formation. Bile acids regulate key metabolic and immune processes by binding to host receptors. There is an unmet need for a potent tool to inhibit BSHs across all gut bacteria to study the effects of bile acids on host physiology. Here, we report the development of a covalent pan-inhibitor of gut bacterial BSHs. From a rationally designed candidate library, we identified a lead compound bearing an alpha-fluoromethyl ketone warhead that modifies BSH at the catalytic cysteine residue. This inhibitor abolished BSH activity in conventional mouse feces. Mice gavaged with a single dose of this compound displayed decreased BSH activity and decreased deconjugated bile acid levels in feces. Our studies demonstrate the potential of a covalent BSH inhibitor to modulate bile acid composition in vivo.

Cyclization Reaction-Based Turn-on Probe for Covalent Labeling of Target Proteins

Simon A. Hawley, Fiona A. Ross, Fiona M. Russell, Abdelmadjid Atrih, Douglas J. Lamont, D. Grahame Hardie
Cell Chem. Biol. 2020
DOI: https://doi.org/10.1016/j.chembiol.2020.01.006

Cordycepin (3-deoxyadenosine) is a major bioactive agent in Cordyceps militaris, a fungus used in traditional Chinese medicine. It has been proposed to have many beneficial metabolic effects by activating AMP-activated protein kinase (AMPK), but the mechanism of activation remained uncertain. We report that cordycepin enters cells via adenosine transporters and is converted by cellular metabolism into mono-, di-, and triphosphates, which at high cordycepin concentrations can almost replace cellular adenine nucleotides. AMPK activation by cordycepin in intact cells correlates with the content of cordycepin monophosphate and not other cordycepin or adenine nucleotides. Genetic knockout of AMPK sensitizes cells to the cytotoxic effects of cordycepin. In cell-free assays, cordycepin monophosphate mimics all three effects of AMP on AMPK, while activation in cells is blocked by a γ-subunit mutation that prevents activation by AMP. Thus, cordycepin is a pro-drug that activates AMPK by being converted by cellular metabolism into the AMP analog cordycepin
monophosphate.

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The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity

Jude Canon, Karen Rex, Anne Y. Saiki, Christopher Mohr, Keegan Cooke, Dhanashri Bagal,
Kevin Gaida, Tyler Holt, Charles G. Knutson, Neelima Koppada, Brian A. Lanman, Jonathan
Werner, Aaron S. Rapaport, Tisha San Miguel, Roberto Ortiz, Tao Osgood, Ji-Rong Sun,
Xiaochun Zhu, John D. McCarter, Laurie P. Volak, Brett E. Houk, Marwan G. Fakih, Bert H.
O’Neil, Timothy J. Price, Gerald S. Falchook, Jayesh Desai, James Kuo, Ramaswamy Govindan, David S. Hong, Wenjun Ouyang, Haby Henary, Tara Arvedson, Victor J. Cee & J. Russell Lipford

Nature 575, 217–223 (2019).
DOI: https://doi.org/10.1038/s41586-019-1694-1

KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours. The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity. Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRASG12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRASG12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking.

Structure-based design of a potent and selective covalent inhibitor for SRC kinase that targets a p-Loop cysteine

Guangyan Du, Suman Rao, Deepak Gurbani, Nathaniel J. Henning, Jie Jiang, Jianwei Che, Annan Yang, Scott B Ficarro, Jarrod A. Marto, Andrew J. Aguirre, Peter K. Sorger, Kenneth Dale Westover, Tinghu Zhang, and Nathanael S Gray.

J. Med. Chem. 2020.
DOI: https://doi.org/10.1021/acs.jmedchem.9b01502 

SRC is a major regulator of many signaling pathways and contributes to cancer development. However, development of a selective SRC inhibitor has been challenging, and FDA-approved SRC inhibitors, dasatinib and bosutinib, are multitargeted kinase inhibitors. Here, we describe our efforts to develop a selective SRC covalent inhibitor by targeting cysteine 277 on the P loop of SRC. Using a promiscuous covalent kinase inhibitor (CKI) SM1-71 as a starting point we developed covalent inhibitor 15a, which discriminates SRC from other covalent targets of SM1-71 including TAK1 and FGFR1. As an irreversible covalent inhibitor, compound 15a exhibited sustained inhibition of SRC signaling both in vitro and in vivo. Moreover, 15a exhibited potent anti-proliferative effects in non-small cell lung cancer cell lines harboring SRC activation, thus providing evidence that this approach may be promising for further drug development efforts.