A screening campaign in sea urchin egg homogenate as a platform for discovering modulators of NAADP-dependent Ca2+ signaling in human cells

Abstract

The Ca2+ mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) regulates intracellular trafficking events, including translocation of certain enveloped viruses through the endolysosomal system. Targeting NAADP-evoked Ca2+ signaling may therefore be an effective strategy for discovering novel antivirals as well as therapeutics for other disorders. To aid discovery of novel scaffolds that modulate NAADP-evoked Ca2+ signaling in human cells, we have investigated the potential of using the sea urchin egg homogenate system for a screening campaign. Known pharmacological inhibitors of NAADP-evoked Ca2+ release (but not cADPR- or IP3-evoked Ca2+ release) in this invertebrate system strongly correlated with inhibition of MERS-pseudovirus infectivity in a human cell line. A primary screen of 1534 compounds yielded eighteen ‘hits’ exhibiting >80% inhibition of NAADP-evoked Ca2+ release. A validation pipeline for these candidates yielded seven drugs that inhibited NAADP-evoked Ca2+ release without depleting acidic Ca2+ stores in a human cell line. These candidates displayed a similar penetrance of inhibition in both the sea urchin system and the human cell line, and the extent of inhibition of NAADP-evoked Ca2+ signals correlated well with observed inhibition of infectivity of a Middle East Respiratory syndrome coronavirus (MERS-CoV) pseudovirus. These experiments support the potential of this simple, homogenate system for screening campaigns to discover modulators of NAADP, cADPR and IP3-dependent Ca2+ signaling with potential therapeutic value.