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

Abstract

The Ca 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 Ca 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 Ca 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 Ca release (but not cADPR- or IP-evoked Ca 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 Ca release. A validation pipeline for these candidates yielded seven drugs that inhibited NAADP-evoked Ca release without depleting acidic Ca 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 Ca 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 IP-dependent Ca signaling with potential therapeutic value.

Mining of Ebola virus entry inhibitors identifies approved drugs as two-pore channel pore blockers

“…2). Our data is consistent with a recent screening campaign published while this work was in progress, which also identified phenothiazines as inhibitors of NAADP-induced Ca 2+ release from sea urchin egg homogenates [63]. Indeed, the IC 50 values for fluphenazine, 42 μM (Fig.…”

“…Indeed, the IC 50 values for fluphenazine, 42 μM (Fig. 3B) and~11 μM [63], are comparable. This congruence highlights the power of our in silico strategy for drug identification which, together with our electrophysiological analyses, points mechanistically to drug action on the TPC pore.…”

Two-pore channels as master regulators of membrane trafficking and endocytic well-being

“…Notably, the relative potency of the drugs in electrophysiological studies directly correlated with their potencies for inhibition of EBOV VLP entry [ 42 • ]. Gunaratne et al undertook an independent screening campaign using sea urchin egg homogenates and identified several novel inhibitors of NAADP-dependent Ca 2+ signalling that were active in mammalian cells [ 45 ]. Importantly, the compounds blocked NAADP-evoked Ca 2+ signals and MERS-CoV pseudovirus translocation with similar potencies [ 24 ].…”

The protein interaction networks of mucolipins and two-pore channels

“…TPC inhibition prevents infectivity of recombinant vesicular stomatitis virus strains (VSV) bearing filovirus glycoproteins (Ebola and Marburgvirus), but not VSV, Lassa virus, Venezuelan equine encephalitis virus, nor Rabies virus [29]. Recently, the middle east respiratory syndrome (MERS) coronavirus was also shown to depend on TPC1, TPC2, and NAADP signalling for endosomal trafficking and infectivity [75,76]. It remains unclear which other viruses may require TPC for uptake and transport, and which are TPC-independent (for instance, candidates from virus families Arenaviridae, Rhabdoviridae, and Togaviridae were shown not to) [29].…”