In vitro characterization of CLOCK-interacting small molecules that changes the phase of the circadian rhythm

Circadian rhythms, the intrinsic 24-hour biological cycles, govern a myriad of physiological processes, including sleep/wake sequences, hormonal oscillations, thermoregulation, and blood pressure fluctuations. These rhythms are predominantly conserved across a broad spectrum of organisms, from prokaryotic cyanobacteria to complex eukaryotes such as mammals and plants, and they enhance an organism's adaptive capabilities in relation to their environmental context. Within mammals, the regulation of these rhythmic patterns occurs on two primary levels: the organismal level, where the master clock situated in the suprachiasmatic nuclei (SCN) synchronizes the circadian clock through a delicate interplay of neuronal and hormonal cues, and the molecular level, where intricate transcriptional-translational feedback loops (TTFLs) modulate the clock mechanism. Key proteins such as CLOCK, BMAL1, CRYs, and PERs are integral to the functionality of the clock mechanism. The protein interaction begins with transcription factors CLOCK and BMAL1 forming a heterodimer that binds to the E-BOX of clock-controlled genes, such as Cry and Per, thereby initiating their transcription. Following accumulation of PERs and CRYs in the cytosol, a heterodimer forms and, upon binding with Casein Kinase Iε, translocates into the nucleus to inhibit CLOCK:BMAL1 driven transcription. Subsequent degradation of CRYs and PERs through ubiquitin-dependent proteasomal degradation lifts this transcriptional repression, triggering the commencement of a new cycle. Disruptions to this carefully orchestrated rhythm can have significant health implications, potentially precipitating severe pathologies like metabolic and cardiovascular diseases, cancer, sleep disorders, depression, and Alzheimer's disease. Consequently, the search for small molecules capable of regulating the circadian clock holds promising potential. The objective of this study is to identify novel small molecules that can engage, and subsequently modulate the activity of the CLOCK protein, utilizing structure-based drug design. Following an in silico analysis of millions of molecules, candidate drug molecules were chosen through a comprehensive evaluation process. These selected molecules were synthesized and subjected to in vitro screening to assess their circadian rhythm attributes. The resulting phase-altering small molecules were then further characterized to elucidate their potential in treating circadian rhythm-related pathologies.