Gunaratne, Gihan2019-02-122019-02-122018-09https://hdl.handle.net/11299/201699University of Minnesota Ph.D. dissertation. 2018. Major: Pharmacology. Advisors: Jonathan Marchant, Timothy Walseth. 1 computer file (PDF); 148 pages.Middle East Respiratory Syndrome coronavirus (MERS-CoV) infections are associated with a significant mortality rate, and existing drugs show poor efficacy. Identifying novel targets/pathways required for MERS-CoV infectivity is therefore important for developing novel therapeutics. As an enveloped virus, translocation through the endolysosomal system provides one pathway for cellular entry of MERS-CoV. In this context, Ca2+-permeable channels within the endolysosomal system regulate both the luminal environment and trafficking events, meriting investigation of their role in regulating processing and trafficking of MERS-CoV. Knockdown of endogenous two-pore channels (TPCs), targets for the Ca2+ mobilizing second messenger NAADP, impaired infectivity in a MERS-CoV spike pseudovirus translocation assay. This effect was selective as knockdown of the lysosomal cation channel mucolipin-1 (TRPML1) was without effect. Pharmacological inhibition of NAADP-evoked Ca2+ release using several bisbenzylisoquinoline alkaloids also blocked MERS pseudovirus translocation. Knockdown of TPC1 (biased endosomally) or TPC2 (biased lysosomally) decreased the activity of furin, a protease which facilitates MERS-CoV fusion with cellular membranes. Pharmacological or genetic inhibition of TPC1 activity also inhibited endosomal motility impairing pseudovirus progression through the endolysosomal system. Following this initial validation of NAADP-gated TPCs supporting pseudotyped MERS-CoV infectivity, a high throughput drug screen of 1534 compounds was conducted in a sea urchin egg homogenate system, yielding 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 strongly correlated with observed inhibition of infectivity of a MERS-CoV pseudovirus. This work highlights the utility of targeting regulators of intracellular trafficking as a novel therapeutic strategy, and provide strong support for the execution of a higher throughput screening campaign using the sea urchin egg homogenate system to discover new ligands for manipulation of NAADP-gated TPC signaling.encalcium signalingmembrane traffickingNAADPTwo-pore channelsviral entryThesis or Dissertation