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closeFig 3 and its conclusion
Posted by liplos on 27 Aug 2018 at 14:37 GMT
Niclosamide is a known potent inhibitor of Firefly luciferase (Thorne et al. (2012), Chem & Biol, 8, 1060-72), with a potency of IC50 of 0.2 uM. It is surprised to use a Firefly luciferase (FLuc)-based replicon to investigate the inhibition of replication by a Fluc inhibitor. It is even more surprised that niclosamide at 1 uM did not inhibit the Fluc reporter. Also most virus inhibition data for niclosamide were done at >= 2 uM, whereas in the Fluc reporter experiment, 1 uM was used and the conclusion made based on this one concentration may be problematic.
RE: Fig 3 and its conclusion
cflin replied to liplos on 28 Aug 2018 at 14:28 GMT
Thanks for your concerns and the authors do our best to answer your questions. As you mentioned, Thorne and colleagues (Chemistry & Biology 19:1060-1072) reported a panel of inhibitors against firefly luciferase (FLuc). This study provides important suggestion for searching the potential compounds targeting the proposed factors by using FLuc-based assays. According to their works, they screened approximately 360,864 compounds and categorized the concentration response curves (CRCs) of selected compounds into classes such that class 1a CRCs (5,306 compounds) exhibit full inhibition of enzyme activity, class 1b (2,191 compounds) are partially inhibitory at the highest concentration tested, and classes 2a (5,772 compounds), 2b (19,938 compounds), and 3 (10,062 compounds) have incomplete CRCs. In summary, this work found that a total of 43,885 compounds (~12% of the library) potentially inhibited FLuc, with a significant fraction of this activity (~30%) associated with potent and efficacious CRCs (e.g., class 1a, 1b, and 2a CRCs). Especially in this paper, they had characterized the selected 151 inhibitors with different inhibitory modes against FLuc. As you stated, PubChem CID 4477 (known as Niclosamide) is also listed in Supporting Table S2 (a total of 12,628 compounds) as one of potential compound against FLuc. However, only assayed once has been done for the listed compounds in this study. The blockade effects of selected compounds on cell-based FLuc assay may be executed by multiple modes directly and indirectly through the actions on FLuc and any factors related to cellular activity. Further validations for niclosamide-mediated inhibition on FLuc needs to prove accordingly. In our study, we showed no inhibitory effects of niclosamide on BHK21-based FLuc assay by using non-cytotoxic dose. These results indicate that blocking DENV infection with niclosamide had no direct inhibitory effects on FLuc activity in BHK-D2-Fluc-SGR-Neo-1 cells (please see corrected version). Cell types and treatment time points may cause different cellular response to niclosamide. Importantly, we showed that niclosamide effectively inhibited dsRNA replication and virus release by reducing endosomal acidification in this study. The potential mechanisms for niclosamide-mediated antiviral activity have been discussed in our paper. Thanks again for your questions.