Abstract 309: Antiviral Activity of Coxsackievirus B3 3C Protease Inhibitor
Objective: Coxsackievirus genome encodes a single polyprotein that undergoes a series of proteolytic events to produce several viral proteins. Most of this proteolytic processing is catalyzed by a cysteine protease, 3C protease (3CP). We investigated the potential for chemotherapeutic use of CVB 3CP inhibitor (3CPI).
Methods and Results: Since human rhinovirus (HRV) 3CP and CVB 3CP are similar in their catalytic structures, we have synthesized derivatives of an existing peptidomimetic inhibitor of HRV 3CP. We substituted the ethyl group at the P2′ location of the HRV 3CPI with hydrophobic aromatic rings, that can interact preferentially with the Tyr residue. The resulting derivatives showed dramatically increased inhibitory activities against CVB 3CP. Antiviral activity of CVB 3CPI derivative in vitro was evaluated by the measurement of cytotoxic effect in HeLa cells. 3CPI inhibited the proliferation of GFP-H3, a replication competent CVB3 which expresses GFP during replication. GFP expression decreased in high dose of 3CPI. We observed that 3CPI inhibited cleavage of viral capsid protein (VP2/VP4) and viral proliferation by western blotting assay. In vivo, we used an osmotic pump that releases 3CPI in 100% DMSO continuously at the rate 1 μl/hour for 72 hours (50 mM/mouse). The osmotic pump was inserted at the back of 6-week-old Balb/c male mice at day 0 and followed by intraperitoneal inoculation of 106 PFU of CVB3 (n=15 with 3CPI, n=20 without 3CPI). In CVB3- free control mice (n=10), osmostic pump were filled with 100% DMSO. The 3-week-survival rates of 3CPI-treated mice were significantly higher than those of control mice (86.7%, 30% on each group, P < 0.01 among groups). Myocardial inflammation and virus titers were all significantly reduced in the 3CPI treated groups compared to the controls.
Conclusion: 3CPI, protein structure-based drug, inhibited activity of 3C protease and had no direct cytopathic effect in high dose. Consequently, 3CPI inhibited the proliferation of CVB3. In vivo, CVB 3CPI significantly attenuated the CVB3-induced inflammation, mortality and virus replication. It could be used as a novel therapeutic agent for CVB3-induced myocarditis.