Benzothiazole and Pyrrolone Flavivirus Inhibitors Targeting the Viral Helicase

Abstract

The flavivirus nonstructural protein 3 (NS3) is a protease and helicase, and on the basis of its similarity to its homologue encoded by the hepatitis C virus (HCV), the flavivirus NS3 might be a promising drug target. Few flavivirus helicase inhibitors have been reported, in part, because few specific inhibitors have been identified when nucleic acid unwinding assays have been used to screen for helicase inhibitors. To explore the possibility that compounds inhibiting NS3-catalyzed ATP hydrolysis might function as antivirals even if they do not inhibit RNA unwinding in vitro, we designed a robust dengue virus (DENV) NS3 ATPase assay suitable for high-throughput screening. Members of two classes of inhibitory compounds were further tested in DENV helicase-catalyzed RNA unwinding assays, assays monitoring HCV helicase action, subgenomic DENV replicon assays, and cell viability assays and for their ability to inhibit West Nile virus (Kunjin subtype) replication in cells. The first class contained analogues of NIH molecular probe ML283, a benzothiazole oligomer derived from the dye primuline, and they also inhibited HCV helicase and DENV NS3-catalyzed RNA unwinding. The most intriguing ML283 analogue inhibited DENV NS3 with an IC50 value of 500 nM and was active against the DENV replicon. The second class contained specific DENV ATPase inhibitors that did not inhibit DENV RNA unwinding or reactions catalyzed by HCV helicase. Members of this class contained a 4-hydroxy-3-(5-methylfuran-2-carbonyl)-2H-pyrrol-5-one scaffold, and about 20 μM of the most potent pyrrolone inhibited both DENV replicons and West Nile virus replication in cells by 50%.

Broad-spectrum agents for flaviviral infections: dengue, Zika and beyond

“…The compound had low cytotoxicity, inhibited viral replication of DENV-1-4 and YFV (but not WNV or JEV) in various cell types, and reduced viral load in a mouse model of DENV infection 156 . A pyrrolone derivative (compound 25) inhibited WNV and DENV replication in cell culture, albeit with low selectivity index values, by targeting helicasecatalysed ATP hydrolysis, but had no effect on HCV helicase 157 . Ivermectin has also demonstrated inhibitory activity against YFV, DENV-2 and WNV helicases in the upper nanomolar range 158 , and is a weak inhibitor of DENV protease 159 , as discussed in detail below.…”

Inhibitors compounds of the flavivirus replication process

“…In addition, pyrrolone specifically inhibits NS3 capacity to cleave ATP, as well as 50% of DENV and WNV (subtype Kunjin) replication in cells [57]. …”

“…Cytotoxicity in cell culture is unknown.Peptide WCW-NH2DENVPrusis et al, 2013 [50]Peptide WCW-NH2 inhibited protease in the four serotypes.ARDP0006 and ARDP0009DENVTonlimson and Watowich, 2011 [51]ARDP0006 and ARDP0009 inhibited DENV-2 virus replication in cell culture. Promising compounds for future research.AprotininDENVNoble et al, 2010 [53]Aprotinin envelops the enzyme and prevents the substrate from accessing the protease active site.NS3- HelicasEHalogenated benztriolesWNVSampath and Padmanabhan 2009 [3]Halogenated benztrioles were good, and selective inhibitor of the West Nile virus.IvermectinDENV, JEV, YFVMastrangelo et al, 2012 [55]; Sweeney et al, 2015 [57]Promising compound as the first specific therapy against flaviviruses (patent application EP2010/065880).ST-610DENVLim et al, 2013a [38]; Sweeney et al, 2015 [57]ST610 potently and selectively inhibited all four DENV serotypes in cell culture. For future trials, the pharmacokinetic properties of this compound should be improved.SuraminDENVBasavannacharya, Vasudeban, 2014 [58]; Sweeney et al, 2015 [57]Suramin acted as a potent NS3 helicase inhibitor of dengue virus by a non-competitive mode of inhibition.Analogues ML283DENVSweeney et al, 2015 [57]Analogues ML28331 were potent inhibitors of DENV NS3- catalyzed ATP hydrolysis.…”

“…For future trials, the pharmacokinetic properties of this compound should be improved.SuraminDENVBasavannacharya, Vasudeban, 2014 [58]; Sweeney et al, 2015 [57]Suramin acted as a potent NS3 helicase inhibitor of dengue virus by a non-competitive mode of inhibition.Analogues ML283DENVSweeney et al, 2015 [57]Analogues ML28331 were potent inhibitors of DENV NS3- catalyzed ATP hydrolysis. These trials serve as a tool to find more inhibitory compounds.PyrroloneDENV, WNVSweeney et al, 2015 [57]Pyrrolone inhibited DENV replicon and WNV replication in cell culture. These trials serve as a tool to find more inhibitory compounds.NS4 BSDM25NDENVVan Cleef et al, 2013 [59]SDM25N showed antiviral activity against wild-type DENV2 in both Hela and BHK-21 cells, but not in the C6/36 cell line.…”

Structure of the NS3 helicase from Zika virus

“…Also, the close proximity of the two pockets may lend to the design of inhibitors that can span both sites. Several inhibitors have been reported for DENV helicase 19 . It will be interesting to see if they bind the ZIKV helicase and mitigate the ability of the virus to replicate.…”