Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles

Abstract

Targeting noncatalytic cysteine residues with irreversible acrylamide-based inhibitors is a powerful approach for enhancing pharmacological potency and selectivity. Nevertheless, concerns about off-target modification motivate the development of reversible cysteine-targeting strategies. Here we show that electron-deficient olefins, including acrylamides, can be tuned to react with cysteine thiols in a rapidly reversible manner. Installation of a nitrile group increased the olefins’ intrinsic reactivity, yet paradoxically eliminated the formation of irreversible adducts. Incorporation of these electrophiles into a noncovalent kinase recognition scaffold produced slowly dissociating, covalent inhibitors of the p90 ribosomal protein S6 kinase, RSK. A cocrystal structure revealed specific noncovalent interactions that stabilize the complex by positioning the electrophilic carbon near the targeted cysteine. Disruption of these interactions by protein unfolding or proteolysis promoted instantaneous cleavage of the covalent bond. Our results establish a chemistry-based framework for engineering sustained covalent inhibition without accumulating permanently modified proteins and peptides.

Targeting Interleukin-2-inducible T-cell Kinase (ITK) and Resting Lymphocyte Kinase (RLK) Using a Novel Covalent Inhibitor PRN694

“…Covalent targeting of cysteine residues within the ATP binding pocket is an effective way to obtain highly potent and selective kinase inhibitors with long target residence time (24,25). Ibrutinib, for instance, irreversibly binds cysteine 481 in BTK and cysteine 442 in ITK, leading to a blockade of signaling downstream of the B-cell receptor and TCR, respectively.…”

Kinetic Template‐Guided Tethering of Fragments

“…[123] This suggested that the conjugate addition to such electrophiles was reversible, and this was conclusively demonstrated by Serafimova et al in 2012. [124] This reversibility was thought to arise from the increased acidity of the αC-H protons, and in accordance with this the Michael addition to acrylates, acrylonitriles and acrylamides was found to be irreversible, resulting in the generation of stable thioether bonds.…”

“…[123] This suggested that the conjugate addition to such electrophiles was reversible, and this was conclusively demonstrated by Serafimova et al in 2012. [124] This reversibility was thought to arise from the increased acidity of the αC-H protons, and in accordance with this the Michael addition to acrylates, acrylonitriles and acrylamides was found to be irreversible, resulting in the generation of stable thioether bonds. [124] Surprisingly, the reaction between enones and thiols was found to be freely reversible under physiological conditions and was used by Greaney et al to probe glutathione S-transferases using a dynamic combinatorial approach.…”

“…[124] This reversibility was thought to arise from the increased acidity of the αC-H protons, and in accordance with this the Michael addition to acrylates, acrylonitriles and acrylamides was found to be irreversible, resulting in the generation of stable thioether bonds. [124] Surprisingly, the reaction between enones and thiols was found to be freely reversible under physiological conditions and was used by Greaney et al to probe glutathione S-transferases using a dynamic combinatorial approach. [125,126] The irreversibility of the Michael addition of thiols to vinyl sulfonamides and acrylamides is largely assumed within the literature.…”

Synthesis and styrene copolymerization of novel trisubstituted ethylenes: 3. Alkoxy ring-substituted isopropyl 2-cyano-3-phenyl-2-propenoates

“…There are application reports exemplifying methyl 2-cyano-3-phenyl-2-propenoate, MCPP [1,2]. Thus, ring-unsubstituted MCPP was employed in studies of beta amino acid-modified and fluorescently labeled kisspeptin analogues with potent KISS1R activity [1] and in reversible targeting of noncatalytic cysteines with chemically tuned electrophiles [2]. Methoxy ring-substituted MCPP was used in photorefractive polymer composites [3] as well as in synthesis of new triazoles for potential antifungal agents applications [4].…”