Integrase is encoded by the Pol gene from the HIV provirus and can be expressed as an active recombinant protein. Our laboratory has pioneered the integrase inhibitors research field (PNAS 1993), discovered several families of lead inhibitors (Nature Rev Drug Discovery 2005;Current Topics in Medicinal Chemistry 2009;Viruses 2010) and patented some with the aim of therapeutic development. Our current studies are focused on the discovery of novel chemotype integrase inhibitors to overcome resistance to raltegravir and target novel site of integrase. In the past year we have reported novel chemotypes derived from Vpr peptides. These short peptides represent a novel kind of inhibitors. We have shown they could serve as therapeutics by the addition of a poly-arginine tail that confers cellular uptake and antiviral activity. They can also been used to build non-peptidic derivatives. We have also reported novel synthetic chemotypes affiliated with the diketo acid strand transfer inhibitors (phtalimide and quinolinonyl diketo acid derivatives) (collaborations with Dr. Terrence Burke, Laboratory of Medicinal Chemistry, CCR, NCI, with Dr. ZhengQuiang Wang and Dr. Robert Vince at the Center for Drug Design, University of Minnesota, and with Dr. Roberto DiSanto, University of Rome, Italy). We have developed a panel of recombinant integrase proteins bearing the mutations observed in patients that develop resistance to raltegravir and elvitegravir. Using our set of raltegravir-resistant IN mutants, we have characterized the molecular pharmacology of elvitegravir, dolutegravir and MK-0536, comparing them in parallel to raltegravir. We have shown that both raltegravir and elvitegravir are highly selective for the strand transfer reaction, while being more than 100-fold less potent against the 3-processing reaction, and almost inactive against the disintegration reaction mediated by integrase. The selective activity of raltegravir and elvitegravir against strand transfer (one of the 3 reactions mediated by integrase) demonstrates the very high specificity of the clinically developed strand transfer inhibitors. It is consistent with our pharmacological hypothesis (TIPS 2005) that the strand transfer inhibitors trap the integrase-viral DNA complex by chelating the divalent metals in the enzyme catalytic site following 3-processing of the viral DNA. We recently characterized in several publications the biochemical enzymatic activities and drug sensitivities of the integrase mutants that confer clinical drug resistance. Those same mutants are now available to test our novel inhibitors. We are expanding these studies to double-mutants in the integrase flexible loop that commonly arise in raltegravir-resistant patients. The working hypothesis is that the second mutation acts as gain of function to rescue the biochemical activity of integrase after it had become defective by the presence of the first mutation. One of aims is to understand the molecular mechanisms of such complementation and the structural connections between the flexible loop, the viral and host DNAs, and the inhibitors. Notably, we found that the flexible loop double-mutant 140S-148H is cross-resistant to both raltegravir and elvitegravir. On the other hand, the 143Y mutant is primarily resistant to raltegravir and minimally resistant to elvitegravir. These results provide a rationale for using elvitegravir in patients that develop resistance to raltegravir due to mutation 143Y (but not in the case of mutations 140S-148H). Having established the assays with a battery of recombinant mutant integrase, we are in a position to discover novel chemotypes to overcome raltegravir and elvitegravir resistance. In an upcoming publication (Hare et al. 2011), we have studied dolutegravir, the novel INSTI in clinical trials. Our results demonstrate that dolutegravir effectively inhibits a panel of HIV-1 IN variants resistant to raltegravir. To elucidate the structural basis for the increased potency of DTG against raltegravir-resistant INs, we determined crystal structures of wild type and mutant prototype foamy virus intasomes bound to the drug. This work was done in collaboration with Dr. Peter Cherepanov at the Imperial College in London. The overall IN binding mode of dolutegravir is strikingly similar to that of the tricyclic hydroxypyrrole MK-2048 (Merck &Co.). Both second-generation INSTIs occupy the same physical space within the IN active site and make contacts with the beta-4-alpha-2 loop of the catalytic core domain. Furthermore, the structures indicate that DTG displays considerable flexibility, particularly in the linker connecting the metal chelating core and the fluorophenyl group, allowing it to adjust to the structural changes in the active sites of the mutant integrases. The ability to structurally adapt to the structural changes associated with drug resistance appears to be a desirable characteristic that could be used in the development of new INSTIs. More recently, we reported the biochemical and antiviral properties of MK-0536 (see Mtifiot 2011). We synthesized this compound in collaboration with Dr. Terrence Burke (Chemical Biology Laboratory, CCR-NCI). MK-0536 was evaluated in the laboratory using our panel of recombinant mutant integrases while the antiviral assays where conducted in Dr. Steven Hughes laboratory (HIV Drug Resistance Program, CCR-NCI). We demonstrated that, like RAL, MK-0536 is highly potent against recombinant IN and viral replication (see Table 1). It is also effective against integrases that carry the three main raltegravir-resistance mutations (Y143R, N155H and to a lesser extent G140S-Q148H) and against the G118R mutant. Molecular models of integrases developed from the recent PFV structures, were generated in collaboration with Dr. Steven Hughes (Barry et al., AAC, in revision). These models allow us to rationalize the differences between RAL and MK-0536 in the context of mutant integrases. We are continuing our long-term collaboration with Dr. Terrence Burke (Chemical Biology Laboratory, CCR-NCI). We recently developed a new pharmacophore model for second generation INSTIs. A patent has been filed and we recently published our most recent series of compounds (see Zhao et al. 2011). With Dr. Stephen Hughes, also at the NCI-Frederick (HIV Drug Resistance Program), we are testing the antiviral activity of those compounds and with Dr. Peter Cherepanov at the Imperial College in London we have recently obtained co-crystal structures. Our aim is to optimize our novel chemical series to obtain potent novel inhibitors active against clinically used INSTIs.