We propose to design and develop an assay technology platform for viral proteases possessing cis cleavage activity. As viral cis proteases are frequently responsible for the initial cleavage of the viral polyprotein, they are attractive targets for antiviral therapies. Currently, no high throughput assay format exists for cis cleavage activity of viral proteases. We will utilize our proprietary fluorescence-based assay technologies to generate a cell-based assay solution for viral cis proteases. The assay system will involve reporter fusion proteins consisting of a beta-lactamase reporter fused to the coding sequence and cleavage site for a viral cis protease. The fusion construct will also contain a specialized destabilization domain that confers short half-life upon the fusion reporter construct. Cis cleavage of the reporter within the viral sequence will cleave the destabilization domain from the beta-lactamase domain resulting in the stabilization of beta-lactamase. Stable beta-lactamase activity will then be measured using the cell-permeable fluorescent beta-lactamase substrate CCF2/AM. The use of the highly sensitive beta-lactamase-CCF2/AM system should allow us to develop robust cell-based assays that can be used in high throughput screens to identify inhibitors of cis-cleaving viral proteases with the aim of developing potent antiviral agents.
It is clear from the enormous scale and impact of hepatitis C viral infections world-wide that there is an immediate need for rapid advances in identifying and developing drugs to treat this affliction. Our intention upon successful Phase I development of an assay for HCV NS2/NS3 activity is to move rapidly to a Phase II effort involving large scale compound screening with the goal of identifying lead compounds that can be further developed into commercial antiviral drugs. In addition, completion of the research proposed here would lead to the development of a generalized assay format that can be applied to other cis proteases associated with clinically relevant viruses.
