Orai Channel Physiology and Pharmacology

Background Cardiovascular disease is often characterised by functional and structural changes in the blood vessel wall, usually associated with endothelial dysfunction. Therefore, interest in targeting the dysfunctional endothelial cell has heightened. Ca2+ signalling is crucial to endothelial cell physiology as it drives a number of intracellular signalling pathways. Store-operated Ca2+ entry (SOCE) mediated by Orai1 channels is of particular interest as it provides a major Ca2+ influx pathway in endothelial cells, driving cell migration and proliferation, ultimately contributing to endothelial repair and integrity, angiogenesis and wound healing events. The novel hypothesis is that modulation of Orai1 channels will have important physiological effects on endothelial cell function. Methods and Results A novel series of Orai1 inhibitors has been identified in this thesis with improved pharmacological and physicochemical properties compared to existing SOCE inhibitors. Optimisation of compounds’ structure-activity relationships (SAR) via Ca2+ measurement assays using human umbilical vein endothelial cells (HUVECs) has revealed important insights into functionally important features of SOCE blockers. In conjunction with in silico modelling, a novel binding site in the Orai1 channel located in a small extracellular pocket has been proposed. The generation and subsequent testing of a quaternary ammonium analogue of the parent compound, JPIII supports the hypothesis. The novel SOCE inhibitors suppressed endothelial cell migration and proliferation without affecting cell viability. Reassuringly, the small molecules were well tolerated in vivo, supporting further development and testing of such blockers in animal models of cardiovascular disease. Here, a novel transgenic murine model with the potential for temporal and conditional disruption of Ca2+ permeation in Orai1 channels has been generated and characterised, offering new possibilities for better understanding of the physiological and pathological roles of Orai1 and the therapeutic potential of targeting this channel. Conclusion Novel SOCE inhibitors have been characterised in vitro, their mechanism of action interrogated and their properties optimised for the next stages of in vivo testing in an animal model of cardiovascular disease.