From Bench to Bedside
NS4B plays an important role in establishing the HCV replication complex. Several key domains have been genetically validated as essential for NS4B’s function in HCV replication. This in turn has led to the development of small molecule approaches to targeting NS4B, including a new role for an old antihistamine.
Jeffrey S. Glenn, M.D., Ph.D., associate professor of medicine, department of gastroenterology and hepatology at Stanford University School of Medicine, notes that HCV poses a big clinical problem. “It’s no secret that clinical therapies right now don’t work very well,” he says. “There is a lot of room for improvement.”
And it’s quite a challenge—one of the first protease inhibitors to hit the clinic failed because of concerns about cardiotoxicity. Some compounds work well in vitro, but when tested in vivo, almost all have breakthrough resistance within a few weeks of use as monotherapy. They do have the potential to work well as a part of combination therapy, however.
Dr. Glenn works with a number of viral-specific agents. “We’re trying to increase the number of targets that can form the basis for future drug cocktails designed to maximize efficacy and decrease the emergence of resistance. We work with and have discovered a number of such novel targets, and we’re focusing on a few outside the normal range, having successfully genetically validated them.”
For example, NS4B is a protein that Dr. Glenn’s group has studied pretty extensively. “We don’t know for sure all it’s functions,” Dr. Glenn explains, “but we do know that it plays a central role in membrane-associated RNA replication—both in establishing the HCV membranous replication platform and in bringing the replicase complex components to the sites of replication. We also discovered that NS4B can specifically bind a key segment of the viral RNA and that this RNA binding is essential for HCV genome replication.”
NS4B has multiple transmembrane domain segments, threading itself in and out of the ER membrane four times, presenting a challenge to successful large-scale purification for mechanistic studies dependent on maintaining a proper topology. In vitro translation reactions with microsomal membranes can yield properly folded NS4B, but the yields are typically small.
Fortunately, however, the amounts made are sufficient for analysis using microfluidics. Dr. Glenn has initiated a collaboration with an expert in microfluidics technology to study NS4B RNA binding on the nanoliter scale. As described in their recent Nature Biotechnology paper, by screening a library enriched in previously used drugs, the colleagues identified clemizole, an antihistamine used in the 1960s, as being quite potent at specifically inhibiting NS4B RNA binding and efficacious at inhibiting HCV replication.
“Because of the extensive use in humans, we know that clemizole was extremely well tolerated in patients,” says Dr. Glenn. “We have also found in the literature a lot of the data needed for an IND package, although of course these studies were done in the 1950s and 1960s and grandfathered in, leaving some holes for what typically is found today in a modern IND filing.”
Dr. Glenn notes that for an antiviral, most researchers like to see at least 2 logs of inhibition—clemizole has only one. “But as we studied it, we noted its ability to dramatically synergize with other molecules in advanced clinical development, which makes it ideal as a future cocktail component.”
And with so few compounds able to make the final cut to drug status, there is real potential value in repurposing drugs. “There’s already been a lot published with regard to safety, we don’t have human PK data yet,” Dr. Glenn adds. “The problem is that we can’t just pull it off the shelves—it has to be made again—and as soon as we have it available, we forsee clinical trials within the year.”