Biophysics on World Hepatitis Day 2016

July 28 is World Hepatitis Day. Viral hepatitis is inflammation of the liver caused by a virus. There are five different hepatitis viruses, hepatitis A, B, C, D and E. Hepatitis C affects approximately 250 million people worldwide. We spoke with Jiawen Li, University of Texas at Austin, Institute of Cellular and Molecular Biology, about her research related to hepatitis C, for which there is currently no vaccination.  

What is the connection between your research and hepatitis C?

Here in the Johnson lab we use transient-state kinetic approaches to characterize viral polymerases, specifically to measure nucleotide specificity, polymerase fidelity and dynamics. More importantly, we apply these methods to understand the mechanisms of action of nucleoside analogs and non-nucleoside inhibitors that are developed to target viral polymerases. For example, to combat HIV, reverse transcriptase is primarily targeted for anti-AIDS therapy. As the RNA-dependent RNA polymerase for Hepatitis C virus, NS5B is considered an important target for effective antivirals as well. Thus the focus of our research is to develop assays to determine kinetic parameters governing RNA dependent RNA replication by NS5B and establish the mechanisms of action and efficiency of various clinically relevant anti-HCV drugs.

Why is your research important to those concerned about hepatitis C?

Hepatitis C affects approximately 250 million people worldwide and chronic infection can lead to hepatitis, liver cirrhosis, and cancer. There is no vaccine available, but combination therapies with direct-acting antivirals including nucleoside analogs and non-nucleoside inhibitors targeting NS5B have been recently advanced and have dramatically improved the potency of HCV treatment. Surprisingly, besides the identification of binding site on NS5B, very little is known about the inhibition mechanisms of drugs that are currently on the market. Two pharmaceutical companies, Gilead Science and Alios Biopharma, have generously provided us with some of their inhibitors to study. Our primary goal is to analyze a handful of these inhibitors in depth to establish their mechanisms of inhibition and to set evaluation guidelines for the effectiveness of each class of inhibitor. Ultimately, we want to apply our methods to each FDA-approved inhibitor for HCV treatment to aid information for the development of even better therapeutics.

How did you get into this area of research?

With a bachelor’s degree in Biochemistry, I was accepted into the Biochemistry graduate program at UT Austin in 2011. During my rotation in the Kenneth Johnson lab, I was fascinated by transient-state kinetic methods such as combining rapid quench-flow and stopped-flow techniques to accurately measure and analyze nucleotide incorporation by HIV RT. Of course I immediately joined the lab and I was very enthusiastic to work on other viral polymerases. The hepatitis C viral RNA-dependent RNA polymerase, NS5B, is known to catalyze de novo RNA synthesis, which means RNA replication is divided into two distinct mechanistic phases: initiation and elongation. Previous studies in our lab along with other groups in the field have made tremendous efforts to develop assays for efficient NS5B replication, but were always hindered by the slow and inefficient initiation phase. Therefore, although the crystal structure of NS5B was solved a decade ago, kinetic characterization of enzyme mechanism, specificity and fidelity are limited, and little is known about the mechanistic basis for inhibition. Finally in 2012, Zhinan Jin, who graduated from our lab and worked for Roche at the time, succeeded in developing conditions for formation of highly active HCV elongation complex. I then continued the work he has accomplished and further optimized the kinetic assays for NS5B inhibition analysis.

How long have you been working on it?

It has been four years since I started working on HCV NS5B in 2012 as a second year graduate student here at UT Austin. I know several lab members had tried to establish conditions for efficient NS5B replication over a decade ago. I am glad this project is brought back to life again!

Do you receive public funding for this work? If so, from what agency?

Yes, we received funding from the Welch Foundation and the National Institutes of Health.

Have you had any surprise findings thus far?

Yes, we have had several surprise findings along the way. Firstly, we now have successfully developed robust kinetic assays to monitor RNA replication by NS5B from initiation to elongation. To our surprise, once the elongation complex is formed, it is extremely stable with half-life of more than a week, which makes the crystal structure of NS5B ternary complex highly promising to obtain in the near future.

Secondly, we have been able to establish modes of action for four classes of non-nucleoside inhibitors. One class of NNIs, the thumb site II inhibitors (NNI2) were shown to be most interesting. NNI2 do not significantly block HCV initiation or elongation; rather they act as allosteric inhibitors to block NS5B transition from initiation to elongation, which is thought to occur with a significant change in enzyme structure. To further examine this allosteric inhibition, we collaborated with Dr. Patrick Wintrode from the University of Maryland and his postdoc Daniel Degrede who mapped the effect of NNI2 inhibitors on the conformational dynamics of NS5B using hydrogen-deuterium exchange kinetics. HDX shows that NNI2 rigidifies an allosteric network extending up to 40 Å from the inhibitor binding site to enzyme active site, providing the rational for blocking NS5B transition at the molecular dynamics level.

NNI2-NS5B HDX (Jiawen Li)

Peptic fragments resulted in significant decrease in HDX upon NNI2 (magenta sticks) binding are shown in dark blue. Rigidification of a large network of enzyme dynamics was observed starting from inhibitor binding site throughout the protein, especially surrounding the enzyme active site, suggesting a long range allosteric effect from inhibitor binding on NS5B conformational change.

Meanwhile, we also explored the mechanisms of NS5B inhibition by nucleotide analogs. We found that both pyrophosphate and NTP mediated excision of incorporated nucleoside analogs were relatively fast reactions, suggesting the important role of pyrophosphorolysis in evaluating the effectiveness of chain-terminating inhibitors. In fact, wild-type NS5B polymerase catalyzes the nucleotide-dependent excision reaction faster than mutants of HIV reverse transcriptase that have evolved to overcome inhibition of nucleoside analogs. This is a significant problem for design of nucleoside analogs to treat HCV infections. We are in the process of publishing this work soon.

What is particularly interesting about the work from the perspective of other researchers?

Our detailed mechanistic studies have provided a fundamental understanding of RNA-dependent RNA replication by HCV NS5B and established the mechanisms of action of different anti-HCV drugs. We hope our experimental and analytical methods will benefit other researchers for studying HCV polymerase or similar viral polymerases and eventually assist screening and design of more effective inhibitors to combat HCV and other viral diseases.

What is particularly interesting about the work from the perspective of the public?

It is great news knowing that more and more anti-HCV drugs are being developed and approved by FDA. With the platform we built for inhibitor analysis, we would like to incorporate more inhibitors into our study and determine their biochemical role of inhibition. We think our work will help providing insights for the development of drugs that are safer and effective against broader range of HCV genotypes.

 

 

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