Structure Activity Relationships in Cadpr

Graham, Steven

Abstract

Release of calcium from intracellular stores (calcium signaling) plays a fundamental role in regulating cellular function. Cyclic adenosine diphosphate ribose (cADPR) is one of a family of small molecule phosphates known to cause calcium release, and recent evidence points to a role for cADPR in the regulation of insulin secretion, diabetes, immune suppression, cell cycle arrest, and fertilization. Studies using cADPR and analogs have elucidated parts of this signaling pathway, but the details of the interactions between cADPR, cADPR analogs, and their cognate proteins remain unclear. While some of the solution structural features of cADPR itself have been reported, very little is known about the analog solution structures and even less about the structure of the relevant cADPR-binding protein complexes. This application proposes to establish the structure activity relationships for cADPR. To achieve this, cADPR analogs will be rationally designed and synthesized so that their conformation behavior in solution will vary in a defined, predictable, and quantifiable fashion. The structural modifications include altering the ribose hydroxyl group configurations, systematically replacing the hydroxyl groups with fluorine, alterations to the periphery of the adenine moiety, and the preparation of carbocyclic (cyclopentane) and adenine ring-extended analogs. The fluorosubstituted analogs are especially important, as fluorine is known to have pronounced yet predictable effects on sugar conformations. Analysis of high quality NMR data will allow determination of the sugar conformations, their relative populations, and the sugar-phosphate backbone torsion angles (pseudorotational analysis). Once the solution structures have been determined, the analogs will be tested for Ca2+-releasing activity in order to establish the structure-activity relationships. Finally, isotopically labeled cADPR and analogs will be prepared so that previously inaccessible structural features (e.g. glycosidic torsion angles) and the extent of transmission of stereoelectronic effects between sugars, backbone and adenine base can be revealed.