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American Liver Foundation
39 Broadway, Suite 2700
New York, New York 10006
Herman Lopata Memorial Hepatitis Postdoctoral Research Fellowship
Stanford University, Stanford, California
Development of novel host-factor-based antivirals for hepatitis C
The hepatitis C virus (HCV) forms a structure called the “membranous web” in order to replicate. One component of the cellular membranes that are hijacked to form the membranous web is
phosphatidylinositol 4,5-bisphosphate (PIP2), a particular membrane lipid molecule. The enzyme that synthesizes PIP2, PI4K IIIα, is required for HCV to replicate. The nonstructural 5A protein (NS5A) of HCV binds specifically to PIP2. This binding appears to take place at the N-terminal amphipathic helix (AH, an alpha-helix with one charged face and one hydrophobic face) of NS5A. Mutating the AH or sequestering the PIP2 with neomycin disrupts the binding and prevents HCV replication.
Dr. Gelman’s hypothesis is that a novel class of anti-HCV therapeutics that disrupt the PIP2-NS5A interaction can be discovered. One way to disrupt the interaction is to bind either NS5A AH or PIP2. Another approach is to inhibit PI4K IIIα. Dr. Gelman will pursue both of these approaches. His specific aims are (1) to develop a fluorescence polarization (FP) assay for inhibition of binding of fluorescence-tagged PIP2 to NS5A (AH peptide, full length protein, or biotin-tethered AH peptide), (2) to use this assay to screen thousands of compounds in the Stanford High Throughput Biosciences Center, (3) to validate compounds identified in the screen for activity against HCV replication using luciferase-reporter and focus-reduction assays in cell culture based on the Huh7 or Huh7.5 cell lines, and (4) to screen known and candidate inhibitors of PI4K IIIα to identify compounds that inhibit HCV replication without unacceptable cytotoxicity.
Dr. Gelman expects to identify lead compounds both for disruption of the PIP2-NS5A interaction and for inhibition of PI4K IIIα. Further work will focus on application of organic chemistry expertise to these compounds to synthesize and evaluate promising structural analogues. Since PIP2 is a host factor and PI4K IIIα is a host enzyme, the barrier to development of resistance may be high.