Identification of Thieno[3,2‐b]Pyrrole Derivatives as Novel Small Molecule Inhibitors of Neurotropic Alphaviruses

Abstract

Neurotropic alphaviruses such as western, eastern, and Venezuelan equine encephalitis viruses cause serious and potentially fatal central nervous system infections in humans and are high-priority potential bioterrorism agents. There are currently no widely available vaccines or licensed therapies for these virulent pathogens. To identify potential novel antiviral drugs, we developed a cell-based assay with a western equine encephalitis virus replicon that expresses a luciferase reporter gene and screened a small molecule diversity library of 51,028 compounds. We identified and validated a thieno[3,2-b]pyrrole compound with a half maximal inhibitory concentration of <10 µmol/L, a selectivity index >20, and potent activity against live virus in cultured neuronal cells. Furthermore, a structure-activity relationship analysis with 20 related compounds identified several with enhanced activity profiles, including 6 with submicromolar half maximal inhibitory concentrations. In conclusion, we have identified a novel class of promising inhibitors with potent activity against virulent neurotropic alphaviruses.

The Antiviral Alkaloid Berberine Reduces Chikungunya Virus-Induced Mitogen-Activated Protein Kinase Signaling

“…4A and B) or the enzymatic activity of the viral replicase. The latter was found in a trans-replication system, an approach that had been used before to evaluate the direct effect of potential antivirals on the viral replicase (52). While berberine did not have any significant influence on replication in this system, other antiviral compounds (ABM and IVM), obtained in our earlier screen, significantly reduced the luciferase readout ( Fig.…”

Validation of a cell-based ELISA as a screening tool identifying anti-alphavirus small-molecule inhibitors

“…Select compounds inhibited virus production effectively and were not toxic to uninfected BHK cells. Cell-based assays reported in the literature include WEEV replicons and SINV expressing luciferase as a marker protein (Peng et al, 2009;Pohjala et al, 2008).…”

Novel Indole-2-Carboxamide Compounds Are Potent Broad-Spectrum Antivirals Active against Western Equine Encephalitis Virus In Vivo

“…The WEEV replicon plasmids, pWR-LUC and pWR-⌬LUC, have been previously described (19). To generate the WEEV nonstructural protein (nsP) expression plasmid, pW-nsP, we inserted the nsP1-through nsP4-coding region from pWR-LUC into pCITE-4a(ϩ) (EMD Millipore, Darmstadt, Germany).…”

“…Infectious titers for all viruses were determined by plaque assay on Vero cell monolayers as previously described (17). WEEV replicon assays used BSR-T7 cells and pWR-LUC, pW-nsP, or pWR-⌬LUC and measured fLUC accumulation as a surrogate marker for viral RNA replication as previously described (13,19). GFP-SeV replication was determined by fluorescence accumulation as previously described (24).…”

“…We previously identified a novel class of thieno [3,2-b]pyrrolebased alphavirus inhibitors via high-throughput screening using a cell-based WEEV replicon assay and a defined small-molecule diversity library (19). Through extensive structure-activity relationship (SAR) analyses and targeted medicinal-chemistry efforts, we produced second-generation indole-2-carboxamide derivatives that display increased potency and striking enantiospecific activity against WEEV and related alphaviruses in cultured cells and that also improve survival in mice infected with a neuroadapted strain of Sindbis virus (SINV) (13).…”