Quinolinecarboxamides Inhibit the Replication of the Bovine Viral Diarrhea Virus by Targeting a Hot Spot for the Inhibition of Pestivirus Replication in the RNA-Dependent RNA Polymerase.

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.

Towards the design of combination therapy for the treatment of enterovirus infections.

We report here on a comparative study of the activity of 10 enterovirus inhibitors against poliovirus 1, enterovirus 71 and human rhinovirus 14. Three of the selected molecules (Pleconaril, BTA-798 and V-073) are in clinical development. The in vitro antiviral activity of pairwise combinations of inhibitors indicated that most combinations resulted in an additive to slightly synergistic antiviral activity. However, the combination of ribavirin with a nucleoside polymerase inhibitor resulted in a pronounced antagonistic effect.