Discovery of Potent Broad Spectrum Antivirals Derived from Marine Actinobacteria

Abstract

Natural products provide a vast array of chemical structures to explore in the discovery of new medicines. Although secondary metabolites produced by microbes have been developed to treat a variety of diseases, including bacterial and fungal infections, to date there has been limited investigation of natural products with antiviral activity. In this report, we used a phenotypic cell-based replicon assay coupled with an iterative biochemical fractionation process to identify, purify, and characterize antiviral compounds produced by marine microbes. We isolated a compound from Streptomyces kaviengensis, a novel actinomycetes isolated from marine sediments obtained off the coast of New Ireland, Papua New Guinea, which we identified as antimycin A1a. This compound displays potent activity against western equine encephalitis virus in cultured cells with half-maximal inhibitory concentrations of less than 4 nM and a selectivity index of greater than 550. Our efforts also revealed that several antimycin A analogues display antiviral activity, and mechanism of action studies confirmed that these Streptomyces-derived secondary metabolites function by inhibiting the cellular mitochondrial electron transport chain, thereby suppressing de novo pyrimidine synthesis. Furthermore, we found that antimycin A functions as a broad spectrum agent with activity against a wide range of RNA viruses in cultured cells, including members of the Togaviridae, Flaviviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Finally, we demonstrate that antimycin A reduces central nervous system viral titers, improves clinical disease severity, and enhances survival in mice given a lethal challenge with western equine encephalitis virus. Our results provide conclusive validation for using natural product resources derived from marine microbes as source material for antiviral drug discovery, and they indicate that host mitochondrial electron transport is a viable target for the continued development of broadly active antiviral compounds.

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

“…3A). In initial time course experiments, all three viruses showed peak production by 24 hpi after a low-inoculum (MOI ϭ 0.1) infection, although the titer magnitude varied by almost 100,000-fold, from 10 5 PFU/ml for EMCV to 10 10 PFU/ml for VEEV (17). Both third-generation compounds suppressed the production of infectious VEEV, CEV, and EMCV, although CCG205432 was more potent than CCG206381 (Fig.…”

“…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).…”

“…2F and Table 1). We infected cells at a low inoculum (MOI ϭ 0.1) and used predetermined optimal time points to harvest supernatants and analyze WEEV titers (17). Both compounds suppressed infectious WEEV production in all cell lines tested, although CCG205432 was generally more active than CCG206381 50 , and selectivity indices (SI) for each compound in the WEEV replicon assay are shown for reference (13,19,20).…”

“…(F) Virion production in various mammalian cells. Individual cell lines were infected with WEEV at an MOI of 0.1 and simultaneously treated with DMSO or the indicated compound at a concentration of 25 M. Virus titers in tissue culture supernatants were determined by plaque assay at 24 hpi for BE(2)-C, BE(2)-C/m, BHK-21, and Vero cells, 48 hpi for CHO, HEK293, and SH-SY5Y cells, and 72 hpi for Huh-7 and U87 cells, which represented predetermined optimal time periods for WEEV virion production in individual cell lines (17). *, P Ͻ 0.05; **, P Ͻ 0.005 compared to results for DMSO-treated controls.…”

“…Furthermore, the combination of active vaccination plus antiviral therapy may be a more effective response in the setting of an outbreak caused either by natural transmission or by intentional exposure to one of these viral pathogens (12). Although numerous compounds have been reported to inhibit alphavirus replication in cultured cells, only a select few have shown any activity in animal models (13)(14)(15)(16)(17). Thus, there is a pressing need to identify new antiviral compounds and drug targets as part of a comprehensive medical-countermeasure strategy to prevent or mitigate illness, suffering, and death resulting from infections caused by these virulent pathogens (18).…”