Greater than 170 million people worldwide have been infected with Hepatitis C virus
(HCV). Approximately 80% will progress to develop chronic liver disease and liver
cirrhosis. HCV infection is the leading cause of liver transplants and consequently a
major health burden. Current treatment of HCV uses a combination of pegylated α-
interferon and ribavirin. However treatment is only effective for about 50% of HCV
patients, has a long treatment time (48 weeks) and causes significant side effects.
New therapies are desperately needed. A combination of X-ray crystallography and
biophysical methods will be used to investigate two drugs currently in clinical trials
that are potent anti-HCV drugs. In addition several drug resistant mutations in HCV
will be studied to understand compound binding and identify modifications to these
compounds to overcome drug resistance.
The non-structural protein 5A (NS5A) is a multifunctional protein that plays key roles
in viral genome replication, particle assembly and virus-host interactions. Two
compounds BMS-790052 and AZD7295 show potent antiviral activity against HCV
replication in replicon-based assays. Both compounds are believed to target NS5A
based on characteristic NS5A resistance mutations that can be selected in replicon
assays and the results from a pull-down assay with biotinylated BMS-790052 in a
cell-based system. Recently this lab developed the first in vitro assay to measure
NS5A binding. We showed that both BMS-790052 and AZD7295 do indeed bind to
domain 1 of NS5A and interfere with RNA binding to this protein.
I will expand on this work and will include the production
and purification of various NS5A mutant proteins. I will perform
microscale thermophoresis (MST) experiments to investigate drug binding in solution
and will perform crystallisation trials to obtain crystals of various NS5A mutants in the
presence and absence of the two above drugs with and without RNA.