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Potent and selective caspase-2 inhibitor prevents MDM-2 cleavage in reversine-treated colon cancer cells.

ABSTRACT: Most caspases can be positioned unambiguously within the regulated cell death networks of apoptosis and pyroptosis, but the role of caspase-2, a highly conserved protease within the family, remains enigmatic. This is mainly due to lack of selective chemical and biochemical tools for the investigation of this protease. In this study, we used our hybrid combinatorial substrate library (HyCoSuL) approach to broadly profile caspase-2 substrate specificity using peptide scanning libraries. This screen uncovered previously unknown caspase-2 peptidyl substrate preferences, which were further used to develop caspase-2 selective fluorogenic substrates and covalent, irreversible AOMK inhibitors. Finally, we used the champion inhibitor (NH-23-C2) in reversine-treated HCT-116 colon cancer cells to selectively block caspase-2 activity and caspase-2-mediated MDM-2 cleavage. In addition, we showed that NH-23-C2 does not block caspase-3 or caspase-8, which makes it a powerful chemical tool to dissect the true role of caspase-2 in various biological setups.

SUBMITTER: Poreba M

PROVIDER: S-EPMC7224280 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Potent and selective caspase-2 inhibitor prevents MDM-2 cleavage in reversine-treated colon cancer cells.

Poreba Marcin M Rut Wioletta W Groborz Katarzyna K Snipas Scott J SJ Salvesen Guy S GS Drag Marcin M

Cell death and differentiation 20190411 12

Most caspases can be positioned unambiguously within the regulated cell death networks of apoptosis and pyroptosis, but the role of caspase-2, a highly conserved protease within the family, remains enigmatic. This is mainly due to lack of selective chemical and biochemical tools for the investigation of this protease. In this study, we used our hybrid combinatorial substrate library (HyCoSuL) approach to broadly profile caspase-2 substrate specificity using peptide scanning libraries. This scree ...[more]

PMID: 30976094

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Project description:The dedifferentiation agent "reversine" [2-(4-morpholinoanilino)-N(6)-cyclohexyladenine 2] was found to be a moderately potent antagonist for the human A(3) adenosine receptor (AR) with a K(i) value of 0.66 microM. This result prompted an exploration of the structure-activity relationship of related derivatives, synthesized via sequential substitution of 6-chloro-2-fluoropurine with selected nucleophiles. Optimization of substituents at these two positions identified 2-(phenylamino)-N(6)-cyclohexyladenine (12), 2-(phenylamino)-N(6)-cycloheptyladenine (19), and 2-phenylamino-N(6)-endo-norbornyladenine (21) as potent A(3) AR ligands with K(i) values of 51, 42, and 37 nM, respectively, with 30-200-fold selectivity in comparison to A(1) and A(2A) ARs. The most selective A(3) AR antagonist (>200-fold) was 2-(phenyloxy)-N(6)-cyclohexyladenine (22). 9-Methylation of 12, but not 19, was well-tolerated in A(3) AR binding. Extension of the 2-phenylamino group to 2-benzyl- and 2-(2-phenylethylamino) reduced affinity. In the series of 2-(phenylamino), 2-(phenyloxy), and 2-(phenylthio) substitutions, the order of affinity at the A(3) AR was oxy > or = amino > thio. Selected derivatives, including reversine (K(B) value of 466 nM via Schild analysis), competitively antagonized the functional effects of a selective A(3) AR agonist, i.e., inhibition of forskolin-stimulated cAMP production in stably transfected Chinese hamster ovary (CHO) cells. These results are in agreement with other studies suggesting the presence of a lipophilic pocket in the AR binding site that is filled by moderately sized cycloalkyl rings at the N(6) position of both adenine and adenosine derivatives. Thus, the compound series reported herein comprise an important new series of selective A(3) AR antagonists. We were unable to reproduce the dedifferentiation effect of reversine, previously reported, or to demonstrate any connection between A(3) AR antagonist effects and dedifferentiation.

Dataset Information

Potent and selective caspase-2 inhibitor prevents MDM-2 cleavage in reversine-treated colon cancer cells.

Publications

Potent and selective caspase-2 inhibitor prevents MDM-2 cleavage in reversine-treated colon cancer cells.

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