Selective and reversible modification of kinase cysteines with chlorofluoroacetamides

Naoya Shindo, Hirokazu Fuchida, Mami Sato, Kosuke Watari, Tomohiro Shibata, Keiko Kuwata, Chizuru Miura, Kei Okamoto, Yuji Hatsuyama, Keisuke Tokunaga, Seiichi Sakamoto, Satoshi Morimoto, Yoshito Abe, Mitsunori Shiroishi, Jose M. M. Caaveiro, Tadashi Ueda, Tomonori Tamura, Naoya Matsunaga, Takaharu Nakao, Satoru Koyanagi, Shigehiro Ohdo, Yasuchika Yamaguchi, Itaru Hamachi, Mayumi Ono & Akio Ojida

Nature Chemical Biology, 2019
DOI: https://doi.org/10.1038/s41589-018-0204-3

Irreversible inhibition of disease-associated proteins with small molecules is a powerful approach for achieving increased and sustained pharmacological potency. Here, we introduce α-chlorofluoroacetamide (CFA) as a novel warhead of targeted covalent inhibitor (TCI). Despite weak intrinsic reactivity, CFA-appended quinazoline showed high reactivity toward Cys797 of epidermal growth factor receptor (EGFR). In cells, CFA-quinazoline showed higher target specificity for EGFR than the corresponding Michael acceptors in a wide concentration range (0.1–10 μM). The cysteine adduct of the CFA derivative was susceptible to hydrolysis and reversibly yielded intact thiol but was stable in solvent-sequestered ATP-binding pocket of EGFR. This environment-dependent hydrolysis can potentially reduce off-target protein modification by CFA-based drugs. Oral administration of CFA quinazoline NS-062 significantly suppressed tumor growth in a mouse xenograft model. Further, CFA-appended pyrazolopyrimidine irreversibly inhibited Bruton’s tyrosine kinase with higher target specificity. These results demonstrate the utility of CFA as a new class warheads for TCI.

Discovery of an Orally Available Janus Kinase 3 Selective Covalent Inhibitor

Liyang Shi, Zhenpeng Zhong, Xitao Li, Yiqing Zhou, and Zhengying Pan

Journal of Medicinal Chemistry 2019

DOI: 10.1021/acs.jmedchem.8b01823

JAK family kinases are important mediators of immune cell signaling and Janus Kinase 3 (JAK3) has long been indicated as a potential target for autoimmune disorders. Intensive efforts to develop highly selective JAK3 inhibitors have been underway for many years. However, because of JAK3’s strong binding preference to adenosine 5′-triphosphate (ATP), a number of inhibitors exhibit large gaps between enzymatic and cellular potency, which hampers efforts to dissect the roles of JAK3 in cellular settings. Using a targeted covalent inhibitor approach, we discovered compound 32, which overcame ATP competition (1 mM) in the enzymatic assay, and demonstrated significantly improved inhibitory activity for JAK3-dependent signaling in mouse CTLL-2 and human peripheral blood mononuclear cells. Compound 32 also exhibited high selectivity within the JAK family and good pharmacokinetic properties. Thus, it may serve as a highly valuable tool molecule to study the overlapping roles of JAK family kinases in complex biological settings. Our study also suggested that for covalent kinase inhibitors, especially those targeting kinases with low Km ATP values, the reversible interactions between molecules and proteins should be carefully optimized to improve the overall potency.

Photoredox Alkenylation of Carboxylic Acids and Peptides: Synthesis of Covalent Enzyme Inhibitors

Lisa Marie Kammer, Benjamin Lipp, and Till Opatz
The Journal of Organic Chemistry, 2019
DOI: 10.1021/acs.joc.8b02759

The synthesis of vinyl sulfones and (α,β-unsaturated) nitriles from carboxylic acids was realized through oxidative decarboxylation with 1,4-dicyanoanthracene as an organic photoredox catalyst. Various types of C-radicals are generated and used to construct three different classes of potential covalent protease inhibitors. The procedure is functional group tolerant and applicable to natural products and druglike scaffolds. It may serve for the rapid construction of screening candidates as demonstrated by a three-step synthesis of the known protease inhibitor K11777.

TAS‐120 cancer target binding; defining reactivity and revealing the first FGFR1 irreversible structure

Kalyukina, M. , Yosaatmadja, Y. , Middleditch, M. ., Patterson, A. , Smaill, J. . and Squire, C.
ChemMedChem, 2019
doi:10.1002/cmdc.201800719

TAS‐120 is an irreversible inhibitor of the fibroblast growth factor receptor (FGFR) family that is currently under phase I/II clinical trials in patients with confirmed advanced metastatic solid tumours harbouring FGFR aberrations. This inhibitor specifically targets the P‐loop of the FGFR tyrosine kinase domain, forming a covalent adduct with a cysteine side chain of the protein. Our mass spectrometry experiments characterise an exceptionally fast chemical reaction in forming the covalent complex. The structural basis of this reactivity is revealed by a sequence of three X‐ray crystal structures, a free ligand structure, a reversible FGFR1 structure, and the first reported irreversible FGFR1‐adduct structure. We hypothesise that the most significant reactivity feature of TAS‐120 is its inherent ability to undertake conformational sampling of the FGFR P‐loop. In designing novel covalent FGFR inhibitors, such a phenomenon presents an attractive strategy requiring appropriate positioning of an acrylamide group similarly to that of TAS‐120.

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

Tomonori Tamura and Itaru Hamachi

Journal of the American Chemical Society Just Accepted Manuscript

DOI: 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.

Live-Cell Imaging and Profiling of c-Jun N-Terminal Kinases with Covalent Inhibitor-Derived Probes

Linghui Qian,  Sijun Pan,  Jun-Seok Lee,  Jingyan Ge,  Lin Li  and  Shao Yao

Chem. Commun., 2018
DOI: 10.1039/C8CC09558B

c-Jun N-terminal kinases (JNKs) are involved in critical cellular functions. Herein, small-molecule JNK-targeting probes are reported based on a covalent inhibitor. Together with newly developed two-photon fluorescence Turn-ON reporters and chemoproteomic studies, we showed some probes may be suitable for live-cell imaging and profiling of JNKs.