ABHD17 enzymes regulate dynamic plasma membrane palmitoylation and N-Ras-dependent cancer growth [@MJNiphakis]

Jarrett R Remsberg, Radu M Suciu, Noemi A Zambetti, Thomas W Hanigan, Ari J Firestone, Anagha Inguva, Amy Long, Nhi Ngo, Kenneth M Lum, Cassandra L Henry, Stewart K Richardson, Marina Predovic, Ben Huang, Amy R Howell, Micah J Niphakis, Kevin Shannon, Benjamin F Cravatt
bioRxiv, 2020
 doi: https://doi.org/10.1101/2020.05.21.108316

A subset of Ras proteins, including N-Ras, depend on a palmitoylation/depalmitoylation cycle to regulate their subcellular trafficking and oncogenicity. General lipase inhibitors such as Palmostatin M block N-Ras depalmitoylation, but lack specificity and target several enzymes displaying depalmitoylase activity. Here, we describe ABD957, a potent and selective covalent inhibitor of the ABHD17 family of depalmitoylases, and show that this compound impairs N-Ras depalmitoylation in human acute myeloid leukemia (AML) cells. ABD957 produced partial effects on N-Ras palmitoylation compared to Palmostatin M, but was much more selective across the proteome, reflecting a plasma membrane-delineated action on dynamically palmitoylated proteins. Finally, ABD957 impaired N-Ras signaling and the growth of NRAS-mutant AML cells in a manner that synergizes with MEK inhibition. Our findings uncover a surprisingly restricted role for ABHD17 enzymes in modulating the N-Ras palmitoylation cycle and suggest that ABHD17 inhibitors may have value as targeted therapies for NRAS-mutant cancers

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Structure and Characterization of a Covalent Inhibitor of Src Kinase [@kenwestover]

Gurbani Deepak, Du Guangyan, Henning Nathaniel J., Rao Suman, Bera Asim K., Zhang Tinghu, Gray Nathanael S., Westover Kenneth D.

Front. Mol. Biosci.,  2020 
DOI: https://doi.org/10.3389/fmolb.2020.00081

Unregulated Src activity promotes malignant processes in cancer, but no Src-directed targeted therapies are used clinically, possibly because early Src inhibitors produce off-target effects leading to toxicity. Improved selective Src inhibitors may enable Src-directed therapies. Previously, we reported an irreversible Src inhibitor, DGY-06-116, based on the hybridization of dasatinib and a promiscuous covalent kinase probe SM1-71. Here, we report biochemical and biophysical characterization of this compound. An x-ray co-crystal structure of DGY-06-116: Src shows a covalent interaction with the kinase p-loop and occupancy of the back hydrophobic kinase pocket, explaining its high potency, and selectivity. However, a reversible analog also shows similar potency. Kinetic analysis shows a slow inactivation rate compared to other clinically approved covalent kinase inhibitors, consistent with a need for p-loop movement prior to covalent bond formation. Overall, these results suggest that a strong reversible interaction is required to allow sufficient time for the covalent reaction to occur. Further optimization of the covalent linker may improve the kinetics of covalent bond formation.

Identification of a Covalent Molecular Inhibitor of Anti-apoptotic BFL-1 by Disulfide Tethering

Edward P.Harvey, Zachary J. Hauseman, Daniel T. Cohen, T. Justin Rettenmaier, Susan Lee, Annissa J.Huhn, Thomas E. Wales, Hyuk-Soo Seo, James Luccarelli1, Catherine E.Newman, Rachel M.Guerra, Gregory H.Bird, Sirano Dhe-Paganon, John R.Engen, James A.Wells, Loren D. Walensky

Cell Chemical Biology, 2020

https://doi.org/10.1016/j.chembiol.2020.04.004

The BCL-2 family is composed of anti- and pro-apoptotic members that respectively protect or disrupt mitochondrial integrity. Anti-apoptotic overexpression can promote oncogenesis by trapping the BCL-2 homology 3 (BH3) “killer domains” of pro-apoptotic proteins in a surface groove, blocking apoptosis. Groove inhibitors, such as the relatively large BCL-2 drug venetoclax (868 Da), have emerged as cancer therapies. BFL-1 remains an undrugged oncogenic protein and can cause venetoclax resistance. Having identified a unique C55 residue in the BFL-1 groove, we performed a disulfide tethering screen to determine if C55 reactivity could enable smaller molecules to block BFL-1's BH3-binding functionality. We found that a disulfide-bearing N-acetyltryptophan analog (304 Da adduct) effectively targeted BFL-1 C55 and reversed BFL-1-mediated suppression of mitochondrial apoptosis. Structural analyses implicated the conserved leucine-binding pocket of BFL-1 as the interaction site, resulting in conformational remodeling. Thus, therapeutic targeting of BFL-1 may be achievable through the design of small, cysteine-reactive drugs.

Alkynyl Benzoxazines and Dihydroquinazolines as Cysteine Targeting Covalent Warheads and Their Application in Identification of Selective Irreversible Kinase Inhibitors

Kirsten McAulay, Emily A Hoyt, Morgan Thomas, Marianne Schimpl, Michael Steven Bodnarchuk, Hilary Jane Lewis, Derek Barratt, Deepa Bhavsar, David M. Robinson, Michael J. Deery, Derek Ogg, Gonçalo J.L. Bernardes, Richard A Ward, Michael J. Waring, and Jason Grant Kettle
Journal of the American Chemical Society 2020
DOI: 10.1021/jacs.9b13391

With a resurgence in interest in covalent drugs, there is need to identify new moieties capable of cysteine bond formation that are differentiated from commonly employed systems such as acrylamide. Herein, we report on the discovery of new alkynyl benzoxazine and dihydroquinazoline moieties capable of covalent reaction with cysteine. Their utility as alternative electrophilic warheads for chemical biological probes and drug molecules is demonstrated through site-selective protein modification and incorporation into kinase drug scaffolds. A potent covalent inhibitor of JAK3 kinase was identified with superior selectivity across the kinome and improvements in in vitro pharmacokinetic profile relative to the related acrylamide-based inhibitor. In addition, the use of a novel heterocycle as cysteine reactive warhead is employed to target Cys788 in c-KIT where acrylamide has previously failed to form covalent interactions. These new reactive and selective heterocyclic warheads supplement the current repertoire for cysteine covalent modification whilst avoiding some of the limitations generally associated with established moieties.

In vivo imaging of the tumor‐associated enzyme NCEH1 with a covalent PET probe

Chang, J., Bhuiyan, M., Tsai, H., Zhang, H., Li, G., Fathi, S., McCutcheon, D., Leoni, L., Freifelder, R., Chen, C. and Moellering, R.
Angew. Chem. Int. Ed. 2020
doi:10.1002/anie.202004762

Here we report the development of an 18F‐labeled, activity‐based small molecule probe targeting the cancer‐associated serine hydrolase NCEH1. We undertook a focused medicinal chemistry campaign to simultaneously preserve potent and specific NCEH1 labeling in live cells and animals, while permitting facile 18F radionuclide incorporation required for PET imaging. The resulting molecule, [18F]JW199, labels active NCEH1 in live cells at nM concentrations and greater than 1,000‐fold selectivity relative to other serine hydrolases. [18F]JW199 displays rapid, NCEH1‐dependent accumulation in mouse tissues. Finally, we demonstrate that [18F]JW199 labels aggressive cancer tumor cells in vivo, which uncovered localized NCEH1 activity at the leading edge of triple‐negative breast cancer tumors, suggesting roles for NCEH1 in tumor aggressiveness and metastasis. More generally, these data support the broader development of potent and specific covalent PET probes to visualize localized, active enzymes in live animals.

Covalent Immune Recruiters: Tools to Gain Chemical Control Over Immune Recognition

Lake B, Serniuck N, Kapcan E, Wang A, Rullo AF.
ACS Chemical Biology. 2020.
https://doi.org/10.1021/acschembio.0c00112

Unprecedented progress made in the treatment of cancer using the body’s own immune system has encouraged the development of synthetic molecule based immunotherapeutics. An emerging class of these compounds, called Antibody Recruiting Molecules (ARMs) or Antibody Engagers (AEs), functions by reversibly binding antibodies naturally present in human serum and recruiting these to cancer cells. The recruited antibodies then engage immune cells to form quaternary complexes that drive cancer eradication. Despite their promise, the requirement to form quaternary complexes governed by multiple equilibria complicates an understanding of their in vivo efficacy. Particularly problematic are low endogenous serum antibody concentrations and rapid clearance of AEs from circulation. Here we describe a new class of trifunctional chemical tools we call covalent immune recruiters (CIRs). CIRs covalently label specific serum antibodies in a selective manner with a target protein binding ligand. CIRs thereby exert well-defined control over antibody recruitment and simplify quaternary complex equilibrium, enabling probing of the resultant effects on immune recognition. We demonstrate CIRs can selectively covalently label anti-DNP IgG, a natural human antibody, directly in human serum to drive efficient immune cell recognition of targets. We expect CIRs will be useful tools to probe how quaternary complex stability impacts the immune recognition of cancer in vivo, revealing new design principles to guide the development of future AEs.

A Fast and Clean BTK Inhibitor [@london_lab]

Ronen Gabizon, Nir London
J. Med. Chem. 2020
https://doi.org/10.1021/acs.jmedchem.0c00597

Bruton’s tyrosine kinase (BTK) is a major drug target for B-cell related malignancies; however, existing BTK inhibitors approved for cancer treatment have significant off-targets that limit their use for autoimmune and inflammatory diseases. Remibrutinib (LOU064) is a novel covalent BTK inhibitor that binds an inactive BTK conformation, which affords it unprecedented selectivity. Its optimization led to rapid BTK engagement in vivo and fast clearance, further limiting systemic exposure. Remibrutinib is currently in phase 2 clinical trials for treatment of chronic urticaria and Sjoegren’s syndrome.