Related Experiment Video
Updated: Aug 27, 2025
Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters
Published on: September 27, 2012
CRY2 isoform selectivity of a circadian clock modulator with antiglioblastoma efficacy
Simon Miller1, Manish Kesherwani1, Priscilla Chan2
1Institute of Transformative Bio-Molecules, Nagoya University, Nagoya 464-8601, Japan.
Abstract:
The mammalian cryptochrome isoforms, CRY1 and CRY2, are core circadian clock regulators that work redundantly. Recent studies revealed distinct roles of these closely related homologs in clock output pathways. Isoform-selective control of CRY1 and CRY2 is critical for further understanding their redundant and distinct roles. KL001 was the first identified small-molecule CRY modulator that activates both CRY1 and CRY2. SHP656 is an orally available KL001 derivative and has shown efficacy in blood glucose control and inhibition of glioblastoma stem cell (GSC) growth in animal models. However, CRY isoform selectivity of SHP656 was uncharacterized, limiting understanding of the roles of CRY1 and CRY2. Here, we report the elucidation of CRY2 selectivity of SHP656. SHP656 lengthened cellular circadian period in a CRY2-dependent manner and selectively interacted with CRY2. By determining the X-ray crystal structure of CRY2 in complex with SHP656 and performing molecular dynamics simulations, we elucidated compound interaction mechanisms. SHP656 binding was compatible with the intrinsic CRY2 gatekeeper W417 "in" orientation and also a close "further in" conformation. Perturbation of W417 interaction with the lid loop resulted in a reduced effect of SHP656 on CRY2, supporting an important role of gatekeeper orientation in isoform selectivity. We also identified the R form of SHP656 (called SHP1703) as the active isomer. Treatment with SHP1703 effectively reduced GSC viability. Our results suggest a direct role of CRY2 in glioblastoma antitumorigenesis and provide a rationale for the selective modulation of CRY isoforms in the therapeutic treatment of glioblastoma and other circadian clock-related diseases.
Insights
The small molecule SHP656 selectively targets CRY2, a core circadian clock protein, influencing circadian rhythms and glioblastoma stem cell growth. This selectivity offers new therapeutic avenues for circadian clock-related diseases like glioblastoma.
Area of Science:
- Chronobiology
- Molecular Biology
- Cancer Biology
Background:
- Mammalian cryptochromes (CRY1 and CRY2) are key regulators of the circadian clock, exhibiting both redundant and distinct functions.
- Small molecules like KL001 and its derivative SHP656 modulate CRY activity, with SHP656 showing promise in preclinical models for glucose control and glioblastoma stem cell (GSC) inhibition.
- Understanding the isoform selectivity of SHP656 is crucial for elucidating the specific roles of CRY1 and CRY2 in various biological processes.
Purpose of the Study:
- To determine the isoform selectivity of the small molecule SHP656 towards CRY1 and CRY2.
- To elucidate the molecular mechanisms underlying SHP656's interaction with CRY isoforms.
- To investigate the therapeutic potential of SHP656, specifically its active isomer SHP1703, in glioblastoma treatment.
Main Methods:
- Cellular circadian period assays to assess SHP656's effect on circadian rhythm.
- X-ray crystallography to determine the structural basis of SHP656-CRY2 interaction.
- Molecular dynamics simulations to analyze compound-protein interactions.
- In vitro assays to evaluate the effect of SHP1703 on GSC viability.
Main Results:
- SHP656 was found to selectively lengthen the cellular circadian period in a CRY2-dependent manner.
- Structural analysis revealed SHP656 binds to CRY2, with its interaction influenced by the gatekeeper W417 residue orientation.
- The R-isomer of SHP656, named SHP1703, was identified as the active compound and demonstrated efficacy in reducing GSC viability.
Conclusions:
- SHP656 exhibits significant selectivity for CRY2, providing a tool to dissect CRY1 and CRY2 functions.
- The interaction mechanism highlights the importance of the CRY2 gatekeeper residue (W417) in conferring isoform selectivity.
- CRY2 plays a direct role in glioblastoma antitumorigenesis, and selective CRY2 modulators like SHP1703 represent a promising therapeutic strategy for glioblastoma and other circadian-related diseases.
More Related Videos
06:53Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures
Published on: November 11, 2016
10:33Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR
Published on: December 13, 2017
Related Concept Videos
02:19Circadian Rhythms and Gene Regulation
02:34Inhibition of Cdk Activity