Combating Hepatitis B Virus by Targeting DNA Polymerase Computational Insights into Resistance Development and Search for Novel Inhibitors by Virtual Screening

Hepatitis B virus is among the top ten infectious diseases in the world. Hundreds of thousands of people are chronically infected with hepatitis B virus (HBV), a major cause of liver cirrhosis and cancer. Availability of vaccine has certainly helped to control the increasing number of HBV infected patients. However, it has no role in treating already infected patients. Currently seven drugs have been approved for the treatment of HBV infections, out of which two are interferons and five are small molecule therapies. Certain limitations of the currently available therapies necessitate development of novel and robust drug therapy against HBV infections.;There are 5 marketed nucleotide/nucleoside analogs for the treatment of HBV infections which inhibit HBV DNA polymerase (HDP), but resistant HBV strains have developed in most cases. We built a three-dimensional comparative model of HDP based on an HIV-1 RT X-ray structure using multiple alignment followed by minimization, validation and molecular dynamics (MD) simulation. The resultant model demonstrates reasonable stereochemical properties. Our homology model of HDP differed from the previously reported homology model in two aspects. Our sequence alignment made sure to match properly a conserved K residue found in HIV-1 RT; we believe that analogous to the role of K65 in HIV-1 RT, rtK32 plays a considerable role in the binding of nucleotides. Secondly, we further used a higher resolution 3D-structure template (1T05) compared to the previously used template (1RTD), which is also more appropriate since it contains a co-crystallized inhibitor, tenofovir.;Conformational changes in amino acid side chains during a subsequent MD simulation led to the formation of a small pocket lined by hydrophobic residues including rtM204 near the nucleotide binding site. The exocyclic alkene moiety of entecavir and the sulfur atom of lamivudine occupied this pocket, explaining the better binding affinity of the inhibitors compared to natural substrates. Furthermore, we used the model to predict that mutation of rtM204 to a beta-branched amino acid would cause steric hindrance, thus leading to the reduced activity of the inhibitors, as has been reported.;Binding free energy calculations were carried out for one of the substrates (dCTP) of HDP and two known inhibitors (3TC and FTC) with wild-type HDP and with one of the most common mutants of HDP, using molecular dynamics simulations. Our MD simulations and binding energy calculations support the available experimental data that the mutation rtM204I causes decreased binding affinity of the inhibitors because of steric hindrance, which results in decreased activity of the inhibitors against the mutant HDP.;We further used the equilibrated wild-type HDP-CTP complex to build a structure-based pharmacophore model, which was further employed to search for novel active-site inhibitors. This exercise resulted in seven non-nucleoside screening hits with probable binding towards the HDP active site. However, the biological results available thus far did not show any anti-HBV activity for these small molecules.;All the small molecule inhibitors available in the market to inhibit HBV DNA polymerase are nucleoside/nucleotide analogs. We have carried out structure-based virtual screening to find novel non-nucleoside HBV DNA polymerase inhibitors. This screening approach required a different homology model of HDP. Another homology model was built using HIV-1 RT, complexed with a non-nucleoside RT inhibitor. Through a series of filtering procedures and visual inspections, we selected ∼40 small molecules for biological testing. None of the selected molecules showed any anti-HBV activity. The molecules were also tested for anti-HCV activity. Two molecules showed moderate anti-HCV activity. To our knowledge, this is the first of its kind study to seek non-nucleoside small-molecule inhibitors of HDP and will serve as a starting point for further investigations.