Abstract
The bovine viral diarrhea virus (BVDV), a pestivirus from the family of Flaviviridae is ubiquitous and causes a range of clinical manifestations in livestock, mainly cattle. Two quinolinecarboxamide analogues were identified in a CPE-based screening effort, as selective inhibitors of the in vitro bovine viral diarrhea virus (BVDV) replication, i.e., TO505-6180/CSFCI (average EC50 = 0.07 µM, SD = 0.02 µM, CC50 > 100 µM) and TO502-2403/CSFCII (average EC50 = 0.2 µM, SD = 0.06 µM, CC50 > 100 µM). The initial antiviral activity observed for both hits against BVDV was corroborated by measuring the inhibitory effect on viral RNA synthesis and the production of infectious virus. Modification of the substituents on the quinolinecarboxamide scaffold resulted in analogues that proved about 7-fold more potent (average EC50 = 0.03 with a SD = 0.01 µM) and that were devoid of cellular toxicity, for the concentration range tested (SI = 3333). CSFCII resistant BVDV variants were selected and were found to carry the F224P mutation in the viral RNA-dependent RNA polymerase (RdRp), whereas CSFCI resistant BVDV carried two mutations in the same region of the RdRp, i.e., N264D and F224Y. Likewise, molecular modeling revealed that F224P/Y and N264D are located in a small cavity near the fingertip domain of the pestivirus polymerase. CSFC-resistant BVDV proved to be cross-resistant to earlier reported pestivirus inhibitors (BPIP, AG110, LZ37, and BBP) that are known to target the same region of the RdRp. CSFC analogues did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). CSFC analogues likely interact with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110, LZ37, and BBP. This indicates that this region is a “hot spot” for the inhibition of pestivirus replication.
Highlights
The Flaviviridae family consists of three genera—the genus Flavivirus, the genus Hepacivirus (hepatitis C virus (HCV)), and the genus Pestivirus (including veterinary pathogens such as the bovine viral diarrhea virus (BVDV), and the classical swine fever virus (CSFV))
We performed a large-scale, cytopathic effect (CPE)-based antiviral screen [21], during which we identified two quinolinecarboxamides [TO502-2403 (CSFCII)/ TO505-6180 (CSFCI), Figure 1] that resulted in selective in vitro inhibition of BVDV-1 replication. Both analogues inhibited the replication of the BVDV-1 in a Madin–Darby bovine kidney (MDBK) cell, in a dose-dependent manner
RNA-dependent RNA polymerase (RdRp), we studied the inhibitory effect of both analogues on gene, that encoded the viral RdRp, we studied the inhibitory effect of both CSFC analoguesthe on in vitro polymerase activity of the enzyme
Summary
The Flaviviridae family consists of three genera—the genus Flavivirus (including human pathogens such as dengue virus, West Nile virus, and yellow fever virus), the genus Hepacivirus (hepatitis C virus (HCV)), and the genus Pestivirus (including veterinary pathogens such as the bovine viral diarrhea virus (BVDV), and the classical swine fever virus (CSFV)). BVDV is able to establish a persistent infection in fetuses [7]. Infected animals might succumb to fatal mucosal disease if they are superinfected with a closely related BVDV strain. There are no approved antiviral drugs to control pestivirus infections. Broad-spectrum pestivirus inhibitors might be considered to control outbreaks with CSFV, in otherwise disease-free areas. This domain is, apparently a “hot spot” binding site for selective inhibitors of pestivirus replication [20,21,22,27,30]. We report on the antiviral characteristics and mode of action of a series of quinolinecarboxamide analogues as a new class of chemicals that inhibit the replication of pestiviruses
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