Development of novel systems for monitoring Hepatitis C Virus genotype 4a Non-Structural 3/4A protease activity

Abstract

Hepatitis C virus (HCV) represents a serious worldwide health care problem. No protective vaccines against HCV have been developed yet due to the fact that HCV is rapidly mutable, allowing the virus to escape from the neutralizing antibodies. Understanding of HCV was initially hampered by the inability to achieve viral replication in cell culture. Over the past 10 years, the growth in scientific understanding of the HCV life cycle and new technologies to measure HCV replication played a critical role in the development of a number of models to study the HCV lifecycle and assess the potency of drugs that disrupt it. Given its essential roles in viral polyprotein processing and immune evasion, HCV NS3/4A protease is a prime target for antiviral chemotherapy. We aimed to establish in vivo cell-based assay system for monitoring the activity of NS3/4A protease from HCV genotype 4a, the predominant genotype in Egypt and the Middle East. Furthermore, the developed system was used to evaluate the inhibitory potency of a series of computer-designed chemically-synthesized compounds (7a [BE113], 7b [BE114], 8a [BE115] and 8b [BE116]) against NS3/4A protease from HCV genotype 4a. Native as well as mutant cleavage sites to NS3/4A protease were cloned in frame into β-galactosidase gene of TA cloning vector. The target specificity of HCV NS3/4A was evaluated by co-expression of β-galactosidase containing the protease cleavage site with NS3/4A protease construct in bacterial cells. The activity of β-galactosidase was colorimetrically estimated in the cell lysate using ortho-nitro phenyl β-D-galactopyanoside (ONPG) as a substrate. We successfully developed an efficient cell-based system based on the blue/white selection of bacterial cells that are able to express functional/non-functional β-galactosidase enzyme. Our system showed a specific activity of NS3/4A protease towards its native cleavage site with no significant specificity towards the mutant one. Two out of the four tested compounds (7b [BE114] and 8a [BE115]) showed inhibitory potency against NS3/4A protease.