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

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.

RdRp (RNA-dependent RNA polymerase): A key target providing anti-virals for the management of various viral diseases

“…It was observed that although the derivatives inhibited the wild-type replication complexes, they could not inhibit mutant replication complexes. Interestingly, the derivative ( 2) did inhibit the activity of replication complexes but they could not inhibit the purified RdRp [46] .

Figure 4 SAR profile of compound 2 as anti-BVDV agent.

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Non-structural proteins of bovine viral diarrhea virus

“…These results suggest that NS5B is a promising target for antiviral research. As reported, Quinoline carboxamide analogs can be used as selective inhibitors of BVDV replication in vitro by inhibiting the activity of BVDV RdRp [ 2 , 80 ], whereas guanosine triphosphate (GTP) can stimulate the RdRp activity of NS5B. However, the synergistic effect of NS3 and NS5B in the host is required to synthesize viral RNA (Table 1 ).…”

Design and Optimization of Quinazoline Derivatives: New Non-nucleoside Inhibitors of Bovine Viral Diarrhea Virus

“…Molecular modeling studies performed with most previously reported BVDV NNI inhibitors, such as TSC (Castro et al, 2011), BPIP (Paeshuyse et al, 2007), VP32947 (Baginski et al, 2000), LZ37 (Paeshuyse et al, 2009), benzimidazole (Asthana et al, 2013), and CSFCII (Musiu et al, 2020), have shown that these compounds bind to a hydrophobic cavity limited by amino acids F224, I390, A392, and L225. This allosteric site was defined as a "hot spot" for the inhibition of virus replication.…”

“…The use of antiviral agents could be an attractive strategy as a stopgap measure to prevent infection in naïve calves (Newcomer et al, 2014). Moreover, antivirals could also be used to treat valuable animals infected with pestiviruses in zoological collections and in in-breeding programs and in vitro embryo production, to cure established cell lines contaminated with pestiviruses, which are used, for example, to produce interferons and vaccines for medical use (Stringfellow et al, 2005;Newcomer and Givens, 2013;Newcomer et al, 2014;Musiu et al, 2020). However, no antivirals are currently available for controlling BVDV infections in either laboratories or farms.…”