Modulation of the Circadian Period: Searching for Isoform-Selective Cyclophilin Inhibitors

Abstract

A dynamic, transcription-based oscillator coordinates physiological and behavioral processes with specific times in the 24-hour day, ultimately acting as a cellular clock. The transcription factor complex known as CLOCK:BMAL1 is at the core of this oscillator that generates circadian (about a day) rhythms. BMAL1 possesses a transactivation domain (TAD) that acts as a molecular switch to control the activation or repression state of CLOCK:BMAL1 and modulate circadian period. Recent findings from our lab have demonstrated that cis/trans isomerization about a Trp-Pro imide bond in the TAD is necessary for proper 24-hour circadian timekeeping. Members of the cyclophilin family, which possess peptidyl-prolyl isomerase (PPIase) activity, typically catalyze this intrinsically slow process in vivo. Use of the broad specificity cyclophilin inhibitor Cyclosporin A lengthens circadian rhythms and its persistent use as an immunosuppressant in transplant recipients is associated with deregulation of circadian rhythms. We found that a number of nuclear-localized cyclophilins exhibit robust catalytic activity on the BMAL1 TAD in vitro, yet the absence of isoform-selective inhibitors has made it difficult to tease out their respective contributions to circadian timekeeping. Here we present our initial studies on three nuclear-localized PPIases: PPIA, PPIE, and PPIH. Fluorescence polarization binding assays have allowed us to explore substrate binding in conjunction with 15N/1H ZZ NMR exchange assays that probe catalytic activity. Utilizing in silico ligand docking against high-resolution x-ray structures and NMR-based fragment-based drug discovery (FBDD), we aim to find isoform-selective inhibitors to better understand their role in circadian rhythms.