Photopharmacological Manipulation of Mammalian CRY1 for Regulation of the Circadian Clock.

CRY1 and CRY2 proteins are highly conserved components of the circadian clock that controls daily physiological rhythms. Disruption of CRY functions are related to many diseases, including circadian sleep phase disorder. Development of isoform-selective and spatiotemporally controllable tools will facilitate the understanding of shared and distinct functions of CRY1 and CRY2. Here, we developed CRY1-selective compounds that enable light-dependent manipulation of the circadian clock. From phenotypic chemical screening in human cells, we identified benzophenone derivatives that lengthened the circadian period. These compounds selectively interacted with the CRY1 photolyase homology region, resulting in activation of CRY1 but not CRY2. The benzophenone moiety rearranged a CRY1 region called the "lid loop" located outside of the compound-binding pocket and formed a unique interaction with Phe409 in the lid loop. Manipulation of this key interaction was achieved by rationally designed replacement of the benzophenone with a switchable azobenzene moiety whose cis-trans isomerization can be controlled by light. The metastable cis form exhibited sufficiently high half-life in aqueous solutions and structurally mimicked the benzophenone unit, enabling reversible period regulation over days by cellular irradiation with visible light. This study revealed an unprecedented role of the lid loop in CRY-compound interaction and paves the way for spatiotemporal regulation of CRY1 activity by photopharmacology for molecular understanding of CRY1-dependent functions in health and disease.

An Isoform-Selective Modulator of Cryptochrome 1 Regulates Circadian Rhythms in Mammals.

Cryptochrome 1 (CRY1) and CRY2 are core regulators of the circadian clock, and the development of isoform-selective modulators is important for the elucidation of their redundant and distinct functions. Here, we report the identification and functional characterization of a small-molecule modulator of the mammalian circadian clock that selectively controls CRY1. Cell-based circadian chemical screening identified a thienopyrimidine derivative KL201 that lengthened the period of circadian rhythms in cells and tissues. Functional assays revealed stabilization of CRY1 but not CRY2 by KL201. A structure-activity relationship study of KL201 derivatives in combination with X-ray crystallography of the CRY1-KL201 complex uncovered critical sites and interactions required for CRY1 regulation. KL201 bound to CRY1 in overlap with FBXL3, a subunit of ubiquitin ligase complex, and the effect of KL201 was blunted by knockdown of FBXL3. KL201 will facilitate isoform-selective regulation of CRY1 to accelerate chronobiology research and therapeutics against clock-related diseases.

Isoform-selective regulation of mammalian cryptochromes.

CRY1 and CRY2 are essential components of the circadian clock controlling daily physiological rhythms. Accumulating evidences indicate distinct roles of these highly homologous proteins, in addition to redundant functions. Therefore, the development of isoform-selective compounds represents an effective approach towards understanding the similarities and differences of CRY1 and CRY2 by controlling each isoform individually. We conducted phenotypic screenings of circadian clock modulators, and identified KL101 and TH301 that selectively stabilize CRY1 and CRY2, respectively. Crystal structures of CRY-compound complexes revealed conservation of compound-binding sites between CRY1 and CRY2. We further discovered a unique mechanism underlying compound selectivity in which the disordered C-terminal region outside the pocket was required for the differential effects of KL101 and TH301 against CRY isoforms. By using these compounds, we found a new role of CRY1 and CRY2 as enhancers of brown adipocyte differentiation, providing the basis of CRY-mediated regulation of energy expenditure.

Cell-based screen identifies a new potent and highly selective CK2 inhibitor for modulation of circadian rhythms and cancer cell growth.

Compounds targeting the circadian clock have been identified as potential treatments for clock-related diseases, including cancer. Our cell-based phenotypic screen revealed uncharacterized clock-modulating compounds. Through affinity-based target deconvolution, we identified GO289, which strongly lengthened circadian period, as a potent and selective inhibitor of CK2. Phosphoproteomics identified multiple phosphorylation sites inhibited by GO289 on clock proteins, including PER2 S693. Furthermore, GO289 exhibited cell type-dependent inhibition of cancer cell growth that correlated with cellular clock function. The x-ray crystal structure of the CK2α-GO289 complex revealed critical interactions between GO289 and CK2-specific residues and no direct interaction of GO289 with the hinge region that is highly conserved among kinases. The discovery of GO289 provides a direct link between the circadian clock and cancer regulation and reveals unique design principles underlying kinase selectivity.

C-H activation generates period-shortening molecules that target cryptochrome in the mammalian circadian clock.

The synthesis and functional analysis of KL001 derivatives, which are modulators of the mammalian circadian clock, are described. By using cutting-edge C-H activation chemistry, a focused library of KL001 derivatives was rapidly constructed, which enabled the identification of the critical sites on KL001 derivatives that induce a rhythm-changing activity along with the components that trigger opposite modes of action. The first period-shortening molecules that target the cryptochrome (CRY) were thus discovered. Detailed studies on the effects of these compounds on CRY stability implicate the existence of an as yet undiscovered regulatory mechanism.

Germinated barley foodstuff ameliorates inflammation in mice with colitis through modulation of mucosal immune system.

OBJECTIVE: Germinated barley foodstuff (GBF) is a prebiotic product made from malt which contains glutamine-rich protein and hemicellulose-rich fiber. Although GBF has been observed to attenuate colonic mucosal inflammation and bowel movements in ulcerative colitis, both experimentally and clinically, the details of the immune response remain elusive. The aim of this study was to investigate the effects of GBF on the colonic epithelium immune response in a CD45RB(high) T cell chronic colitis model. MATERIAL AND METHODS: Colitis was induced by transferring CD4+ CD45RB(high) T cells to severe combined immunodeficiency (SCID) mice (control n=8, GBF n=8) and the effects of GBF on the colitis were evaluated. The evaluation included measurement of body-weight, occult blood tests, histological examination, mucosal cytokine reverse transcription-polymerase chain reaction (RT-PCR) analysis (interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta)) as well as IL-6 measurements. RESULTS: Seven weeks after transferring the above cells, body-weight loss and occult blood were significantly reduced in the mice that had been fed with GBF. In these mice, there were also significant reductions in IFN-gamma mRNA expressions and IL-6 in the colonic mucosa, as compared with the control group. GBF also significantly attenuated, mucosal damage and mucin positive goblet cell depletion. Conversely, TGF-beta expression significantly increased in the GBF group, compared with the control group. CONCLUSIONS: In this preliminary study using an experimental model in which colitis was induced by transferring CD4+ CD45RB(high) T cells to SCID mice, GBF reduced inflammation by modulating the colonic microflora.