Small molecule inhibitors of the Orai1 channel as potential therapeutics for abdominal aortic aneurysm

Ca2+ is involved in a wide range of cellular processes including signalling, mitochondrial regulation, motility and apoptosis. Orai1 is a highly Ca2+-selective plasma membrane channel which facilitates store-operated calcium entry (SOCE) and is the main source of Ca2+ influx into non-excitable eukaryotic cells. It plays a vital role in the mammalian immune system and has been proposed as a drug target in the areas of immunology, inflammatory disease and cancer, with other indications still emerging. Unpublished research from the University of Leeds links the upregulation of Orai1 with abdominal aortic aneurysm (AAA) development. AAA is a progressive disease of aging which is characterised by expansion and inflammation of the aorta, extracellular matrix destruction and vascular smooth muscle cell death, and has no current drug treatment.
This work consists of a review of current small molecule Orai1 channel inhibitors, many of which were profiled in a standardised Ca2+ addback assay. Analogues of the novel Orai1 inhibitor JPIII have been synthesised and assessed for their ability to inhibit SOCE, with some analogues having improved potency, aqueous solubility and half-life compared to the lead compound. Select compounds were also screened for their selectivity, plasma protein binding and membrane permeability. Computational and synthetic methods were used in attempts to discover novel and structurally distinct inhibitors to improve pharmacokinetic properties and to predict their binding.
Due to the limited structural data currently available, work was undertaken to overexpress and purify tagged Orai1 protein in mammalian cells towards the structural elucidation of human Orai1 by cryogenic electron microscopy. This could then allow for binding site determination of inhibitors and the discovery of novel inhibitors by virtual screening or other computational methods. Transient expression in HEK293T and HEK293F cells was ultimately unsuccessful, so a stable tetracycline-inducible expression system was established in HEK293S GnTI- cells.