The Alkyne Moiety as a Latent Electrophile in Irreversible Covalent Small Molecule Inhibitors of Cathepsin K

J. Am. Chem. Soc., 2019, 141 (8), 3507–3514

DOI: 10.1021/jacs.8b11027

Irreversible covalent inhibitors can have a beneficial pharmacokinetic/pharmacodynamics profile but are still often avoided due to the risk of indiscriminate covalent reactivity and the resulting adverse effects. To overcome this potential liability, we introduced an alkyne moiety as a latent electrophile into small molecule inhibitors of cathepsin K (CatK). Alkyne-based inhibitors do not show indiscriminate thiol reactivity but potently inhibit CatK protease activity by formation of an irreversible covalent bond with the catalytic cysteine residue, confirmed by crystal structure analysis. The rate of covalent bond formation (kinact) does not correlate with electrophilicity of the alkyne moiety, indicative of a proximity-driven reactivity. Inhibition of CatK-mediated bone resorption is validated in human osteoclasts. Together, this work illustrates the potential of alkynes as latent electrophiles in small molecule inhibitors, enabling the development of irreversible covalent inhibitors with an improved safety profile.

DUckCov: a Dynamic Undocking‐based Virtual Screening Protocol for Covalent Binders

Moira Rachman Andrea Scarpino Dávid Bajusz Gyula Palfy Istvan Vida Andras Perczel Xavier Barril György M Keseru

ChemMedChem, 2019. doi:10.1002/cmdc.201900078

Thanks to recent guidelines, the design of safe and effective covalent drugs has gained significant interest. Other than targeting non‐conserved nucleophilic residues, optimizing the non‐covalent binding framework is important to improve potency and selectivity of covalent binders towards the desired target. Strong efforts have been made in extending the computational toolkits to include a covalent mechanism of protein targeting, like in the development of covalent docking methods for binding mode prediction. To highlight the value of the non‐covalent complex in the covalent binding process, here we describe a new protocol utilizing tethered and constrained docking in combination with Dynamic Undocking (DUck) as a tool to privilege strong protein binders for the identification of novel covalent inhibitors. At the end of the protocol, dedicated covalent docking methods were used to rank and select the virtual hits based on the predicted binding mode. By validating the method on JAK3 and KRas, we demonstrate how this fast iterative protocol could be applied to explore a wide chemical space and identify potent targeted covalent inhibitors.

Allosteric Inhibition of Ubiquitin-like Modifications by a Class of Inhibitor of SUMO-Activating Enzyme

Yi-Jia Li, Li Du, Jianghai Wang, Ramir Vega, Terry D. Lee, Yunan Miao, Grace Aldana-Masangkay, Eric R. Samuels, Baozong Li, S. Xiaohu Ouyang, Sharon A. Colayco, Ekaterina V. Bobkova, Daniela B. Divlianska, Eduard Sergienko, Thomas D.Y. Chung, Marwan Fakih, Yuan Chen

Cell Chemical Biology, 2019
DOI: 10.1016/j.chembiol.2018.10.026

Ubiquitin-like (Ubl) post-translational modifications are potential targets for therapeutics. However, the only known mechanism for inhibiting a Ubl-activating enzyme is through targeting its ATP-binding site. Here we identify an allosteric inhibitory site in the small ubiquitin-like modifier (SUMO)-activating enzyme (E1). This site was unexpected because both it and analogous sites are deeply buried in all previously solved structures of E1s of ubiquitin-like modifiers (Ubl). The inhibitor not only suppresses SUMO E1 activity, but also enhances its degradation in vivo, presumably due to a conformational change induced by the compound. In addition, the lead compound increased the expression of miR-34b and reduced c-Myc levels in lymphoma and colorectal cancer cell lines and a colorectal cancer xenograft mouse model. Identification of this first-in-class inhibitor of SUMO E1 is a major advance in modulating Ubl modifications for therapeutic aims.

Targeting the MKK7–JNK (Mitogen-Activated Protein Kinase Kinase 7–c-Jun N-Terminal Kinase) Pathway with Covalent Inhibitors

Targeting the MKK7–JNK (Mitogen-Activated Protein Kinase Kinase 7–c-Jun N-Terminal Kinase) Pathway with Covalent Inhibitors

Patrik Wolle, Julia Hardick, Shane J. F. Cronin, Julian Engel, Matthias Baumann, Jonas Lategahn, Josef M. Penninger, and Daniel Rauh

Journal of Medicinal Chemistry 2019

DOI: 10.1021/acs.jmedchem.9b00102

The protein kinase MKK7 is linked to neuronal development and the onset of cancer. The field, however, lacks high-quality functional probes that would allow for the dissection of its detailed functions. Against this background, we describe an effective covalent inhibitor of MKK7 based on the pyrazolopyrimidine scaffold.

A chemoproteomic portrait of the oncometabolite fumarate

Rhushikesh A. Kulkarni, Daniel W. Bak, Darmood Wei, Sarah E. Bergholtz, Chloe A. Briney, Jonathan H. Shrimp, Aktan Alpsoy, Abigail L. Thorpe, Arissa E. Bavari, Daniel R. Crooks, Michaella Levy, Laurence Florens, Michael P. Washburn, Norma Frizzell, Emily C. Dykhuizen, Eranthie Weerapana, W. Marston Linehan & Jordan L. Meier

Nature Chemical Biology, 2019
DOI: 10.1038/s41589-018-0217-y

Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth. Understanding these mechanisms thus represents a vital goal. Toward this goal, here we report a chemoproteomic map of fumarate, a covalent oncometabolite whose accumulation marks the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). We applied a fumarate-competitive chemoproteomic probe in concert with LC–MS/MS to discover new cysteines sensitive to fumarate hydratase (FH) mutation in HLRCC cell models. Analysis of this dataset revealed an unexpected influence of local environment and pH on fumarate reactivity, and enabled the characterization of a novel FH-regulated cysteine residue that lies at a key protein–protein interface in the SWI-SNF tumor-suppressor complex. Our studies provide a powerful resource for understanding the covalent imprint of fumarate on the proteome and lay the foundation for future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy.

Chemistry for Covalent Modification of Endogenous/Native Proteins: From Test Tubes to Complex Biological Systems

Tomonori Tamura^† and Itaru Hamachi^†§

*ihamachi@sbchem.kyoto-u.ac.jp

†Graduate School of Engineering, Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan

§ERATO, Japan Science and Technology Agency (JST), 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan

Journal of the American Chemical Society

Vol. 141: , Issue. 7, : Pages. 2782-2799

Publication Date (Web): December 28, 2018

https://doi.org/10.1021/jacs.8b11747

Chemical modification of proteins provides powerful tools to realize a broad range of exciting biological applications, including the development of new classes of biopharmaceuticals and functional studies of individual proteins in complex biological systems. Numerous strategies for linking desired chemical probes with target proteins have been developed in the last two decades, with most exploiting genetic protein engineering and/or bio-orthogonal chemistry that utilizes unnatural amino acids incorporated into proteins. Modification of native proteins in test tubes and biological contexts by site-specific and target-selective approaches remains challenging because appropriate organic chemistry to carry out such modifications is currently limited. Nonetheless, a variety of promising strategies have appeared recently that address this grand challenge in chemical biology. These new chemistries yield native protein-based well-defined bioconjugations, specific labeling of endogenous proteins in various biological crude milieus, and the establishment of chemical proteomics as a new research area in protein science. In this Perspective, we focus on recent remarkable progress in chemistry for native protein modification. We survey chemical characteristics of the methods and describe briefly these advanced applications to address unsolved biological issues. Current limitations and future directions of this research field are also discussed.