Drug discovery and basic science of HBV replication

I have been studying HBV replication in 1991. HBV replicates by “reverse transcription”, where an RNA copy of the viral genome is made and then copied back into DNA, which in turn supports production of new RNA. Almost all of my work has been with the HBV polymerase protein. The polymerase is the only enzyme (a protein that causes chemical reactions happen) that HBV makes. It can do two things—make new viral DNA, and destroy the viral RNA after it has been copied into RNA so that DNA synthesis can finish. Stopping either of those functions stops viral replication. That was done a long time ago for the DNA synthesis activity, resulting in the dominant drugs against HBV such as Entecavir and Tenofovir. Most of my lab’s current focus is on the activity that destroys the viral RNA, called the “RNase H”.

We are conducting two projects with the RNase H: Basic science to understand how it works, and drug discovery to develop inhibitors that may eventually become drugs to treat people. We are also trying to understand how the shape of the full polymerase protein affects its functions. The basic science and drug discovery efforts are mutually supportive, with the more we learn in one project helping us to move faster on the other.

The basic science studies with the RNase H involve making the enzyme by itself (a “recombinant enzyme”) so we can purify it and figure out how it works. This is really, hard, and it took me 19 years of trying before I finally made active RNase H. We still only get usable enzyme only about 10% of the times we try to make it. Despite this, we’ve learned the types of DNAs or RNAs it can work on, that the enzyme can both cut at the end and in the middle of an RNA strand, what conditions (pH, salt, etc.) it likes to use, and how some inhibitors we discovered inhibit the RNaseH.

Our drug discovery efforts against the RNase H have yielded >300 compounds that can block HBV replication. The best of them are in 3 different chemical families, and their activity is better than the approved HBV drug Lamivudine. We are currently working with chemists around the world to make the inhibitors even better and to convert the inhibitors into drugs. This is because the current compounds are not safe enough to use in people, and we have to build “drug like properties” into them. Other highlights of this project are that we found that HBV’s high variability (ie, genotypes etc) is unlikely to cause problems for RNase H drugs, that RNase H drugs and nucleoside analogs (ie, Lamivudine, and by extension Entecavir, Tenofovir…) are “synergistic”, meaning that using them together makes both of them work better than they do by themselves (similar to 1 +1 = 3), and that they can inhibit HBV replication in a mouse model.

Finally, we are really excited about our recent identification of the overall 3-dimensional structure of the HBV polymerase protein. People have been trying for > 30 years to figure out its shape because the shape can tell you a lot about how it works. The structure is at Predicted structure of the hepatitis B virus polymerase reveals an ancient conserved protein fold | bioRxiv. This structure is not as good as some types of protein structures, but we have demonstrated that the overall shape is right and that it can be used to help advance drug discovery. We are already starting new basic science and drug discovery projects using the structure to guide us.