Inhibition of the Bovine Viral Diarrhoea Virus NS3 Serine Protease by a Boron-Modified Peptidyl Mimetic of its Natural Substrate

Bovine viral diarrhoea virus (BVDV) is closely related to hepatitis C virus (HCV), and has been used as a surrogate virus in drug development for HCV infection. Similar to HCV, BVDV-encoded NS3 serine proteinase is responsible for multiple cleavages in the viral polyprotein, generating mature NS4A, NS4B, NS5A and NS5B proteins. NS3-dependent cleavage sites of BVDV contain a strictly conserved leucine at P1, and either serine or alanine at P1'. The full length BVDV NS3/4A serine protease has been cloned and expressed in bacterial cells. The enzyme has been purified from the soluble portion of Escherichia coli via a two-step purification procedure employing chromatography on heparin resin and gel filtration. The protease activity was characterized using in vitro translated BVDV NS4A/B and NS5A/B polyprotein substrates. A boronic acid analogue of the BVDV NS4A/NS4B cleavage site was synthesized and shown to be an efficient inhibitor of the NS3 serine protease in vitro. The compound, designated DPC-AB9144-00, inhibited approximately 75% of the NS3/4 activity at 10 microM with the NS4A/B substrate. However, no antiviral activity was detected with DPC-AB9144-00 in BVDV-infected Madin-Darby bovine kidney cells at concentrations as great as 90 pM, suggesting permeability or that other cellular-derived limitations were present.