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.

 

 

First Golden Goose Award of 2016 Goes to NIH-funded Social Science Researchers

Ce0-qDSWQAIRhtTRESEARCHERS BEHIND LANDMARK ADOLESCENT HEALTH STUDY – A STUDY
THAT ALMOST DIDN’T HAPPEN – WILL RECEIVE GOLDEN GOOSE AWARD,

Five researchers whose determined pursuit of knowledge about the factors that influence
adolescent health led to one of the most influential longitudinal studies of human health—with far-reaching and often unanticipated impacts on society—will receive the first 2016 Golden Goose Award.

The researchers are Dr. Peter Bearman, Barbara Entwisle, Kathleen Mullan Harris, Ronald
Rindfuss, and Richard Udry, who worked at the University of North Carolina at Chapel Hill
(UNC) in the late 1980s and early 1990s to design and execute the National Longitudinal Study of Adolescent Health, or Add Health for short.

The social scientists’ landmark, federally funded study has not only illuminated the impact of social and environmental factors on adolescent health—often in unanticipated ways—but also continues to help shape the national conversation around human health. Their work has provided unanticipated insights into how adolescent health affects well being long into adulthood and has laid essential groundwork for research into the nation’s obesity epidemic over the past two decades.

“Four bold researchers wanted to learn more about adolescent health. Who knew that one federal study would change the way doctors approach everything from AIDS to obesity?” said Rep. Jim Cooper (D-TN), who first proposed the Golden Goose Award. “Decades later, this work is still paying off, helping Americans lead longer, healthier lives. America always comes out ahead when we invest in scientific research.”

The pathbreaking nationally representative Add Health study has answered many questions about adolescent behavior, with particular attention to sexual and other risky behaviors, but it was almost stopped in its tracks by political concerns.The study’s design grew out of the American Teenage Study, a project developed by Drs.Bearman, Entwisle, Rindfuss, and Udry. This initial adolescent sexual health study was designed to look at adolescents’ risky behaviors in a social context, rather than focusing only on individuals, in hopes of helping the nation address the growing AIDS epidemic and other public
health concerns. After two years of planning work funded by the National Institutes of Health (NIH), the American Teenage Study passed peer review and was funded by the NIH in 1991. But the grant was subsequently rescinded due to objections regarding the study’s focus on sexual behaviors.

In 1993, Congress passed legislation forbidding the NIH from funding the American Teenage Study in the future, but at the same time mandating a longitudinal study on adolescent health that would consider all behaviors related to their health – implicitly including sexual behavior.

“I congratulate Dr. Rindfuss and his colleagues on this award, which underscores the vital
importance of federal funding for research,” said Rep. David Price (D-NC), who was a key
advocate in the House of Representatives in the 1990’s for continuing to pursue this research. “Federally supported research projects not only produce new life-saving treatments and expand our understanding of the world around us, they also spur economic growth and innovation in ways we cannot always anticipate.”

In 1994, Drs. Udry and Bearman, now joined at UNC by Dr. Harris, proposed Add Health to
meet Congress’s new mandate. The new study maintained the American Teenage Study design’s strong focus on social context, but significantly expanded the scope of inquiry to include all factors influencing adolescent health. The study has followed its original cohort for over 20 years, and it is now providing valuable information about the unanticipated impacts of adolescent health on overall well-being in adulthood. For this reason, the researchers recently changed the study’s name to the National Longitudinal Study of Adolescent to Adult Health, and it is a landmark example of how longitudinal research can yield extraordinary and unexpected insights.

“Science often advances our understanding of the world in ways we could never have foreseen,” Rep. Bob Dold (R-IL) said. “Regardless of how this research began, it has served as a breakthrough for understanding the way society molds our personal health. That’s why congressional funding and support for breakthrough research is so important to push us forward as a country.”

The nationally representative sample and multifaceted longitudinal data paired with a
revolutionary open-access model have enabled more than 10,000 researchers to publish almost 3,000 research articles on human health. These scientific studies have strengthened an understanding of the importance of family connectedness to adolescent health, allowed researchers to track and scrutinize the rising tide of the obesity epidemic, and demonstrated the social, behavioral, and biological importance of adolescence to lifelong health and wellbeing.

What began as a study driven both by social science curiosity and public-health concerns has been central to shaping the national conversation around adolescent health for more than two decades.

The Golden Goose Award honors scientists whose federally funded work may have seemed odd or obscure when it was first conducted but has resulted in significant benefits to society. Drs. Bearman, Entwisle, Harris, Rindfuss and Udry are being cited for their extraordinary multidisciplinary, longitudinal study of the social and biological factors that influence adolescent health, and their work’s wide-ranging and often unexpected impacts on society. The five researchers will be honored with two other teams of researchers – yet to be named – at the fifth annual Golden Goose Award Ceremony at the Library of Congress on September 22.

About the Golden Goose Award
The Golden Goose Award is the brainchild of Rep. Jim Cooper, who first had the idea for the award when the late Senator William Proxmire (D-WI) was issuing the Golden Fleece Award to target wasteful federal spending and often targeted peer-reviewed science because it sounded odd. Rep. Cooper believed such an award was needed to counter the false impression that odd sounding research was not useful. In 2012, a coalition of business, university, and scientific organizations created the Golden Goose Award. Like the bipartisan group of Members of Congress who support the Golden Goose Award, the founding organizations believe that federally funded basic scientific research is the cornerstone of American innovation and essential to our economic growth, health, global competitiveness, and national security. Award recipients are selected by a panel of respected scientists and university research leaders.

The Biophysical Society has been a sponsor of the award for the past three years.

Rheumatoid Arthritis and Biophysics

July has been designated Juvenile Arthritis Awareness Month by the Arthritis Foundation. NIAMS estimates that about 294,000 American children under age 18 have arthritis or other rheumatic conditions. The Biophysical Society is taking this opportunity to highlight how advances in basic research contribute to our understanding of this disease. We spoke with BPS member Christine Beeton, Baylor College of Medicine, about her research related to rheumatoid arthritis, the most common form of arthritis in children. Her work focuses on targeting potassium channels for the treatment of chronic diseases including multiple sclerosis, rheumatoid arthritis, and type 1 myotonic dystrophy, and using antioxidant nanomaterials for the treatment of T lymphocyte-mediated autoimmune diseases (multiple sclerosis and rheumatoid arthritis).

What is the connRA-FLS bright fieldection between your research and arthritis?

The term arthritis encompasses a number of diseases that affect joints; my focus is on rheumatoid arthritis (RA) that is characterized as a systemic inflammatory and chronic disease. In the last decades the role of resident joint cells, the fibroblast-like synoviocytes (FLS), has come to light in this disease. However, no therapeutic currently specifically targets these cells. We have identified KCa1.1 (BK, KCNMA1) as the major potassium channels at the plasma membrane of FLS isolated from patients with RA and have shown that blocking this channel inhibits pathogenic functions of FLS ex vivo and reduces the severity of two animal models of RA (Hu et al. J. Biol. Chem. 2012; Tanner et al. Arthritis Rheumatol. 2015). We are currently investigating the roles of these channels in the pathogenic functions of FLS and also as a potential target for therapy.

Why is your research important to those concerned about arthritis?

Current therapeutics for RA have significantly improve the wellbeing of the patients. However, most induce immunosuppression and therefore place the patients at risk for infections and tumor development. Our ability to target FLS has the potential of development effective treatments for RA that do not immunocompromised the patients. In addition, targeting both immune cells and FLS by combination therapy may offer the added benefit of using reduced amounts of drugs if the effect is synergistic.

How did you get into this area of research?

During my PhD in Immunology, I had the opportunity to study Kv1.3 channels in T lymphocytes, cells involved in autoimmune diseases. As a student my work focused on multiple sclerosis but as a postdoctoral fellow I extended this work to RA. When testing Kv1.3 blockers in animal models of RA and analyzing potassium channel expression in synovium biopsies from patients with RA I became interested in the potassium channel phenotype of other cells involved in RA. When I started my own laboratory I started a collaboration with Dr. Gulko, a rheumatologist now at Mount Sinai in New York, and focused my attention to FLS. This lead to the identification of KCa1.1 as the major potassium channel at the plasma membrane of these cells.

How long have you been working on it?

I started working on potassium channels in FLS from patients with RA in 2008, soon after starting my laboratory at Baylor College of Medicine.

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

This work is not funded by the NIH but we have received funding fromX-rays the Alkek Foundation for Medical Research and from Baylor College of Medicine.

Have you had any surprise findings thus far?

Many. The happiest was to find out that FLS from patients with RA express one major potassium channel at their plasma membrane and not a combination of many channels, which would have made the work of identifying them and defining their roles a lot more complicated. The most puzzling was the finding, repeated many times, that blocking KCa1.1 induces a calcium transient in these cells. This really intrigued us and started us into studying the cell signaling downstream of the channel.

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

Over the last few years, FLS have emerged as important players in the pathogenesis of RA. While many signaling molecules have been identified as regulators of various pathogenic functions of these cells, nothing was known about the expression and function of potassium channels. Our work therefore brings a new cell signaling regulator to light.

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

Getting a better understanding of the different cell types that play a role in rheumatoid arthritis will help design better drugs to combat this disease. In addition, understanding the roles of KCa1.1 channels in the function of FLS during RA may help understand the roles of these and other potassium channels in other tissues and diseases.

Do you have a cool image you want to share with the blog post related to this research?

I am showing two cool images; one of an FLS, isolated from the joint of a patient with RA who had to undergo therapeutic joint surgery due to disease severity. The other shows X-ray images of the hind paws of rats with a model of RA induced by the injection of pristane. In the top X-ray, joint damage (yellow arrows) is visible; In contrast, the joints are much healthier in the paw of the rat that was treated with a KCa1.1 blocker for 21 days, starting at onset of clinical signs.