Fragment-based lead discovery of novel inhibitors of HIV-1 RT

The emergence of drug-resistant strains of human immunodeficiency virus (HIV) threatens the efficacy of current therapeutics. Novel drugs are required that are active against these mutants to be successful as a preventative agent and to provide therapeutic options. In this study we have focused on the HIV type 1 (HIV-1) reverse transcriptase (RT) enzyme, which was the first successful anti- acquired immunodeficiency syndrome (AIDS) target. RT remains as relevant as it was 25 years ago, with half of the anti-AIDS drugs approved by the Food and Drug Administration (FDA) being directed at this enzyme in addition to many ongoing studies to identify new RT inhibitors. The purpose of this study was to identify novel inhibitors of HIV-1 RT using a fragment- based drug design approach. The hypothesis was that since HIV RT is a multifunctional enzyme highly dependent on conformational changes for activity, there may be a number of allosteric sites in HIV-1 RT that could be exploited other than those already identified in the literature, and that these sites could be found by probing the HIV-1 RT using fragments. Molecular modelling studies identified 29 novel pockets in HIV-1 RT that could be targeted by small molecule inhibitors. A screen was conducted using a library of fragments and saturation transfer difference (STD) nuclear magnetic resonance (NMR), to identify new binding sites in RT for the design of novel inhibitors, which led to the identification of seven fragment inhibitors of HIV-1 RT. These hits were characterised using an in vitro RT inhibition assay. Steady state kinetic assays showed that two of these fragments, V89 and 2C4, compete with the dNTP substrate and a third, 4A2, competes with DNA substrate. These fragments were tested for inhibitory activity against common drug resistant mutants of HIV-1 RT, K103N, Y181C, G190A as well as against murine moloney leukemia virus (MoMLV) RT and klenow DNA polymerase. In an RNase H activity assay, fragment 4A2 was shown to inhibit RNase H activity of HIV-1 RT. Fragment 4A2 is also an active inhibitor when tested in an anti-HIV assay using TZMbl cells. The fragments identified in this screen were found to possess distinct mechanisms of action from currently available drugs and are promising leads for the development of novel HIV-1 RT inhibitors.