Structure-based development of novel chemical scaffolds as inhibitors of the store-operated calcium entry pathway

Calcium (Ca2+) is an intracellular messenger regulating various physiological activities. The 'store-operated calcium entry' (SOCE) is a significant, almost ubiquitous Ca2+ signaling pathway and is triggered when intracellular Ca2+ stores (e.g., endoplasmic reticulum, ER) are depleted, either physiologically or pharmacologically. SOCE is mediated by Orai proteins, mainly by Orai1, the pore-forming protein of the CRAC (Calcium release-activated calcium) channel, which is activated following store-depletion by aggregated stromal interaction molecule (STIM) proteins that are localised within the ER-membrane and serve as the ER Ca2+ sensor through possession of EF hands projected towards the ER lumen. Aberrant SOCE activity has been implicated in various diseases including various forms of cancer, autoimmune and inflammatory disorders like acute pancreatitis. There have been efforts in the academia as well as the industry to develop SOCE modulators and few hits have been progressed to early phases of clinical trial. Most of the existing SOCE inhibitors originate from phenotypic screening and derivatisation of some initial hits that were not much selective against the SOCE (Orai-mediated) and non-SOCE (TRPCs-mediated) pathway. Recently, few structures of the Drosophila Orai (DmOrai) have been published and I sought out to identify novel SOCE-inhibitory chemical scaffolds utilising a more targeted approach utilising the available DmOrai structure. A homology model of human Orai1 (hOrai1) was built using DmOrai structure as the template. The model was then subjected to blind docking with some known SOCE inhibitors that are known to act via Orai1. This led to the identification of two potential inhibitor binding sites on Orai1, one of which was virtually screened against commercial lead-like libraries. Initially top 27 hits from the virtual screening with novel structures were purchased for experimental evaluation against SOCE in suitable cell-based assays. Initial screening using Fura-2 based Ca2+ imaging in RBL1-cells identified 5 potent molecules (namely compounds E1, E7, E8, E10, E11) with consistent Thapsigargin (Tg)-evoked SOCE inhibitory properties. I confirmed this by assessing the effect of the compounds on nuclear translocation of NFAT (Nuclear Factor of Activated T-cells) in HeLa cells via confocal microscopy. Out of the top 5 molecules, compound E1 possessed higher potency. Furthermore, the compound E1 significantly attenuated CRAC current (ICRAC) when tested in Jurkat-T cells using whole-cell patch-clamp electrophysiology. However, the compounds showed variations in selectivity to SOCE, with compound E1 inhibiting carbachol mediated Ca2+ release from intracellular stores as well as calcium entry from voltage-gated calcium and TRPC (Transient receptor potential canonical) channels at higher concentrations. Rest of the 4 compounds were more specific SOCE inhibitors, albeit less potent than compound E1. However, compound E1 had no effect on other Ca2+ signaling pathways at lower concentrations at which the compound retained SOCE inhibitory properties. ELISA (Enzyme-linked Immunosorbent Assay) using Jurkat-T cells revealed concentration-dependent inhibition of IL-2 production by compounds E1 and E7. In a caerulein-based cellular model of acute pancreatitis using a rat pancreatic acinar (AR42J) cell-line, E1 inhibited both Tg and caerulein-evoked SOCE at lower concentrations. Finally, few analogues of the compound E1 were purchased and screened following the methods mentioned above. Structure activity relationship (SAR) analysis revealed molecules composed of methylpyrazole ring bound to quinazoline and piperazine rings to have greater efficacy for SOCE inhibition. To conclude, a novel SOCE inhibitory scaffold with reasonable potency was identified through a structure-guided in silico screening campaign using hOrai1 homology model. This is, to my knowledge, the first example of identifying a SOCE inhibitory scaffold through a targeted approach, opposed to phenotypic screening. This compound can be subjected to chemical optimisation to improve potency and selectivity further. Besides, my methodology can be adopted for much larger scale virtual screening that could potentially reveal more novel SOCE-inhibitory scaffolds.