Infectious Curiosity

The Hepatitis C virus NS3 serine protease (in gray)

James Griffith, Vertex Pharmaceuticals

The Scientist
Volume 24 Issue 12 Page 24
Date: 2010-12-01
By Jessica Wapner

The course of virologist Charlie Rice's career changed with one phone call in 1989. Then at Washington University in St. Louis, Rice was the country's leading yellow fever expert. The voice on the other end of the line belonged to Stephen Feinstone, an FDA scientist asking about a vaccine for the disease that had just won agency approval. Yellow fever virus is a flavivirus. Feinstone wanted to know if Rice could help develop a vaccine to protect against another flavivirus: hepatitis C. “I can get interested in pretty much anything, I guess,” says Rice.

Today, more than 20 years after Rice took that call, hepatitis C virus (HCV) infects about 170 million people worldwide, but those statistics may soon take a downward turn. Two protease inhibitors that recently completed late-stage clinical trials—telaprevir and boceprevir—are curing significant numbers of patients who may otherwise have suffered a lifetime of liver problems. Published studies reported profound responses to treatment with either of these drugs when used in combination with pegylated interferon (PegIFN) and ribavirin—the current standard, but often ineffective, therapy (J.G. McHutchison et al., NEJM, 360:1827-38, 2009 and P.Y. Kwo et al., Lancet, 376:705-16, 2010).

After that pivotal call from Feinstone, Rice began studying the biology of hepatitis C, a task that attracted little interest in his laboratory at the time. Like a lone archaeologist mapping a forgotten city, Rice charted the structure and function of the virus's various components. His lab group, then at Washington University in St. Louis, found that the HCV polyprotein produced at least ten polypeptides upon cleavage. Rice also identified structural and nonstructural proteins at the polyprotein's N-terminus, several of which would eventually become drug-target candidates. Researchers had already noted the prominent role of serine proteases in flavivirus replication, so Rice and others identified the serine protease in HCV and began to characterize its functional requirements. They found that optimal protease activity depended upon binding to a small viral protein located immediately downstream in viral polypeptide (A. Grakoui et al., J Virol, 67:1385-95, 1993).

Rice was invited in 2000 to lead the newly established Center for the Study of Hepatitis C at Rockefeller University in New York City where he continued to elucidate the molecular biology of the viral serine protease. Because the HCV protease cleaves the polyprotein responsible for viral RNA synthesis at multiple sites, Rice reasoned that the virus probably couldn’t survive if the enzyme's function was blocked. Still, making the leap from the benchtop to an effective therapy required first knowing the exact structure of the protease molecule.

Enter Vertex Pharmaceuticals. After the mid 1990s, when protease inhibitors revolutionized HIV treatment, the company was eager to investigate employing a similar strategy to thwart HCV. Vertex had been keeping tabs on Rice’s HCV work and invited him to collaborate on determining the crystal structure of the protease to aid in drug development.

Rice and Vertex quickly achieved several milestones: development of a biochemical assay, determination of the protease cleavage site specificity, and identification of the residues in the downstream protease cofactor required for optimal activity. Using X-ray crystallography, they determined the crystal structure of the serine protease/cofactor complex. For cell-based studies, Rice built on a landmark study from Ralf Bartenschlager's lab, then at the University of Mainz, Germany, to create highly efficient HCV “replicon” cell culture systems to study the effect of drug candidates on protease activity. “As a result of the development of HCV replicons, scientists now had the ability to screen vast numbers of molecules to test their ability to inhibit HCV replication,” says Ira Jacobson of Weill Cornell Medical College, in New York City.

But knowing the protease’s structure presented a thorny problem—it turned out that the enzyme contained a flat active site. Designing a structure-based drug was going to be like rock-climbing a smooth mountain face. Prepared for this obstacle by some preliminary images, Vertex and Rice were confident that some nook or cranny could be found.

Scientists at Vertex spent months combing the crystal structure for any shred of texture and finally managed to find a few small footholds within the protease’s active site. Vertex’s protease inhibitor, VX-950 (now known as telaprevir), has emerged as a leading agent for the treatment of HCV patients with the genotype-1 strain of the disease. A second compound, boceprevir, developed by Schering-Plough, proved equally effective in those patients. Doctors and HCV patients alike are anxiously awaiting FDA approval of these drugs, expected in 2011.

Still, more work is needed. Although the genotype-1 variant is the most common HCV strain in the United States, other genotypes prevail across Europe, Asia, and Africa. In addition, clinicians are hoping to phase out the use of PegIFN and ribavirin, both of which have toxic side effects. As second-generation HCV protease inhibitors are being developed, and research on the optimal combination regimen continues, Rice’s discoveries remain integral to the future of HCV research and treatment. “There can always be surprises when you’re dealing with a virus in 100 to 200 million people and generating [new] variants every day,” says Rice. “The game is not over at all.”

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