Table 1. Summary of compounds from primary screen with >80% inhibition of NAADP-evoked Ca2+ release.
Compounds were ranked (#1 through #18) in order of maximum average inhibition of NAADP-evoked Ca2+ release in the primary screen (Figure 3). Hits were then further assessed in concentration-response and 32P-NAADP binding experiments in sea urchin egg homogenate (Figure 4). Counter-screening activities were performed in U2OS cells, with the cut-off threshold for non-pursuit of the candidates (shaded boxes, red text) being >30% decrease in fluorescence ratios in either Ca2+ release or LTR assays. Validation assays represent extent of inhibition of NAADP-evoked Ca2+ release signals, and MERS pseudovirus translocation by the remaining seven candidates (bold in compound list). IPFME (rank #8): 3-(1H-Imidazol-4-yl)propyl di(p-fluorophenyl)methyl ether hydrochloride.
| Compound | 1° Screen | 2° Screen | Counterscreen | Validation | |||
|---|---|---|---|---|---|---|---|
| (sea urchin) | (sea urchin) | (human cells) | (human cells) | ||||
| Ca2+ release | Ca2+ release | NAADP binding | Ca2+ release | LTR signal | Ca2+ release | MERS | |
| (% NAADP) | log(IC50) | (% inhibition) | (% GPN) | (% control) | (% NAADP) | (% control) | |
| PF-543 | 6.0 ± 0.4 | −5.44 ± 0.18 | 5.9 ± 2.2 | 94.0 ± 4 | 95.3 ± 3 | 14.1 ± 3.0 | 18.8 ± 2.1 |
| SKF 96365 | 6.3 ± 4.5 | −5.29 ± 0.06 | −5.3 ± 5.7 | 98.1 ± 6 | 98.5 ± 5 | 12.4 ± 8.9 | 22.1 ± 7.8 |
| Fluphenazine dihydrochloride | 7.7 ± 8.3 | −4.97 ± 0.03 | 3.2 ± 1.5 | 67.6 ± 14 | 69.8 ± 10 | ||
| GBR-12935 dihydrochloride | 10.7 ± 7.0 | −4.90 ± 0.13 | 3.4 ± 3.3 | 61.1 ± 15 | 67.5 ± 11 | ||
| Racecadotril | 11.5 ± 6.0 | −5.80 ± 0.03 | −8.3 ± 0.2 | 110.0 ± 4 | 92.8 ± 3 | 16.6 ± 10 | 20.3 ± 4.1 |
| A-315456 | 13.0 ± 5.0 | −4.44 ± 0.01 | 8.4 ± 1.2 | 97.1 ± 3 | 95.2 ± 4 | 41.1 ± 14 | 56.7 ± 9.3 |
| Clemastine fumarate | 13.7 ± 15 | −4.78 ± 0.09 | 8.2 ± 3.6 | 69.5 ± 13 | 70.9 ± 15 | 40.8 ± 3.6 | 44.2 ± 4.2 |
| IPFME | 13.7 ± 11 | −5.22 ± 0.08 | 2.9 ± 2.1 | 51.3 ± 18 | 56.9 ± 8 | ||
| Prochlorperazine dimaleate | 15.0 ± 12 | −4.62 ± 0.27 | 0.0 ± 3.4 | 20.2 ± 17 | 24.3 ± 14 | ||
| Thioridazine hydrochloride | 15.3 ± 17 | −4.77 ± 0.02 | −3.3 ± 1.5 | 20.5 ± 12 | 26.2 ± 18 | ||
| Dilazep hydrochloride | 16.0 ± 3.0 | −5.03 ± 0.11 | −10.9 ± 8.1 | 65.1 ± 5 | 69.6 ± 9 | ||
| LY-310,762 hydrochloride | 16.3 ± 8.7 | −4.75 ± 0.05 | −2.8 ± 3.8 | 108.1 ± 11 | 90.2 ± 8 | 42.9 ± 5.1 | 82.2 ± 5.1 |
| ST-148 | 16.7 ± 3.5 | −4.80 ± 0.08 | 0.2 ± 4.3 | 66.6 ± 5 | 69.8 ± 13 | ||
| TMB-8 hydrochloride | 18.3 ± 3.2 | −5.12 ± 0.06 | −16.6 ± 10.3 | 68.8 ± 5 | 71.8 ± 8 | ||
| PDMP | 18.3 ± 17 | −4.80 ± 0.05 | 6.0 ± 0.5 | 91.4 ± 2 | 91.6 ± 13 | 43.7 ± 15 | 65.4 ± 7.3 |
| Salmeterol xinafolate | 18.7 ± 12 | −4.94 ± 0.04 | −2.3 ± 13.6 | 97.4 ± 5 | 97.3 ± 7 | 26.7 ± 7.2 | 32.9 ± 4.7 |
| Oxybutynin Chloride | 19.0 ± 10 | −5.07 ± 0.05 | −6.0 ± 5.2 | 62.1 ± 4 | 59.2 ± 9 | ||
| Trifluoperazine dihydrochloride | 19.7 ± 15 | −5.13 ± 0.10 | −13.7 ± 13.7 | 5.8 ± 11 | 15.2 ± 9 | ||
| PPADS (positive control) | 40.0 ± 13 | −5.27 ± 0.01 | 84.8 ± 1.6 | 91.3 ± 16 | 92.1 ± 7 | 24.2 ± 2.1 | 26.0 ± 3.8 |
| DMSO (negative control) | 94.5 ± 2 | n/a | 2.1 ± 4.8 | 93.0 ± 3 | 97.6 ± 2 | 94.5 ± 13 | 97.3 ± 2.1 |