The cryptochrome inhibitor KS15 enhances E-box-mediated transcription by disrupting the feedback action of a circadian transcription-repressor complex

Mar 15, 2018Life sciences

The cryptochrome blocker KS15 increases E-box gene activity by interfering with a circadian clock repressor

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Abstract

KS15 significantly reduces the amplitude and delays the phase of molecular circadian rhythms in fibroblast cultures.

KS15 inhibits the interaction between cryptochromes (CRYs) and Brain-Muscle-Arnt-Like protein 1 (BMAL1).
This inhibition impairs the feedback actions of CRYs on E-box-dependent transcription regulated by the CLOCK:BMAL1 heterodimer.
Structure-activity relationship analyses identified important structural features of KS15 for its effects on CRY-evoked regulation.
Several derivatives of KS15 also show similar effects on circadian rhythms.

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AIMS: We have previously identified a chemical scaffold possessing 2-ethoxypropanoic acid (designated as KS15) that directly binds to the C-terminal region of cryptochromes (CRYs: CRY1 and CRY2) and enhances E-box-mediated transcription. However, it is still unclear how KS15 impairs the feedback actions of the CRYs and which chemical moieties are functionally important for its actions.

MAIN METHODS: The E-box-mediated transcriptional activities were mainly used to examine the effects of KS15 and its derivatives. Co-immunoprecipitation assays accompanied by immunoblotting were employed to monitor protein-protein associations. We also examined the effects of KS15 and selected derivatives on circadian molecular rhythms in cultured cells.

KEY FINDINGS: The present study shows that KS15 inhibits the interaction between CRYs and Brain-Muscle-Arnt-Like protein 1 (BMAL1), thereby impairing the feedback actions of CRYs on E-box-dependent transcription by CLOCK:BMAL1 heterodimer, an indispensable transcriptional regulator of the mammalian circadian clock. Subsequent structure-activity relationship analyses using a well-designed panel of derivatives identified the structural requirements for the effects of KS15 on CRY-evoked regulation of E-box-mediated transcription. We found that KS15 and several derivatives significantly reduce the amplitude and delayed the phase of molecular circadian rhythms in fibroblast cultures.

SIGNIFICANCE: Taken together, our results provide valuable information on the molecular mode-of-action as well as the chemical components of the CRYs inhibitor that pharmacologically impact on the transcriptional activity of the CLOCK:BMAL1 heterodimer.

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The cryptochrome inhibitor KS15 enhances E-box-mediated transcription by disrupting the feedback action of a circadian transcription-repressor complex

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