Discovery and characterization of small molecules stabilizing the cryptochrome in the mammalian circadian clock

Circadian clock mechanism is found in most of the living organisms and helps to maintain a daily rhythm of sleep/wake cycle, metabolism, body temperature and many other physiological processes. The mammalian circadian clock mechanism is intrinsic in organs and tissues and it is synchronized by the suprachiasmatic nuclei (SCN) located in anterior hypothalamus. Since many physiological processes are related to the circadian clock, in numerous studies it has been shown that the disruption of the clock can lead to certain diseases. At the molecular level, circadian clock mechanism consists of a transcriptional-translational autoregulatory feedback loop. In the core loop, BMAL1 (Brain and muscle Arntl-1 like) and CLOCK (the circadian locomotor output cycles kaput), which are transcription factors, initiate the transcription of clock controlled genes including the Cry1, Cry2, Per1, and Per2 genes. Afterwards, CRY (cryptochrome) and PER (period) proteins interact and then translocate back to nucleus where they repress the activity of BMAL1:CLOCK, hence repress their own expression. Ubiquitin ligases SCF-FBXL3 and SCF-FBXL21 (SKP1–CUL1–F-box protein complexes) regulate the degradation and stabilization of CRY1 and CRY2 proteins. The stability of CRY proteins is important because they can alter the period and amplitude of the circadian rhythm, in turn, affecting metabolism. The aim of this thesis is to identify small molecules that enhance CRY stability considering they can be effective in type 2 diabetes treatment. Glucose metabolism is under the regulation of circadian clock mechanism along with other mechanisms. The crosstalk between gluconeogenesis pathway and CRY proteins can be exploited to discover small molecules for treatment of type 2 diabetes or other glucose metabolism related diseases. In previous studies, it was discovered that increase in CRY1 protein levels inhibit glucagon-induced gluconeogenesis. This inhibition is caused by CRY1 binding to the α subunit of the G protein (Gsα) which blocks GPCR-mediated increases in cAMP. In order to identify CRY stabilizers, around 2 million small molecules were previously screened in our laboratory for their interaction with the FBXL3/21 binding pocket in silico. In this thesis, 32 candidate small molecules were tested for their effects on CRY stability, circadian clock, and gluconeogenesis. Among these candidates, small molecules TW63 and TW68 have shown promising results by increasing the stability of CRY1/2 and lengthening the period of circadian rhythm in Bmal1-dLuc U2OS cells. Moreover, TW68 caused the repression of gluconeogenic genes Pck1 and G6pc, and lowered the glucose production during glucagon-induced gluconeogenesis in hepatic HepG2 cells. These results signify the therapeutic potential of TW68, a CRY stabilizer, for the treatment of type 2 diabetes mellitus.