Like HIV-1 reverse transcriptase, HIV-1 integrase (IN) is an important drug target; however, as is the case for all anti-HIV drugs, treatment with INSTIs leads to resistance. We are making good progress on two fronts: understanding how mutations in IN confer resistance to the currently available compounds, and developing new INSTIs that are effective against the common drug-resistance mutations. Dr. Terrence Burke is synthesizing new anti-IN compounds; Dr. Yves Pommier is using biochemical assays to test Dr. Burke's anti-IN compounds in vitro (using purified recombinant IN); and we are testing how the new inhibitors affect viral replication and measuring their toxicity in cultured cells. Until relatively recently, we had no structural information to guide the development of IN inhibitors. However, Dr. Peter Cherepanov has obtained high-resolution structures of full-length foamy virus (FV) IN in complexes with both DNA substrates and anti-IN drugs. Dr. Cherepanov has joined our collaborative effort and has solved the structures of FV IN in complex with some of the more promising compounds developed by Dr. Burke. The active site of FV IN is similar, but not identical, to the active site of HIV-1 IN, and we used Dr. Cherepanov's data to develop models of HIV-1 IN (both WT and mutant). These models have helped us understand how resistance arises and have been useful in the design of more effective compounds. Dr. Burke has recently synthesized several novel compounds that have IC50s in the low nanomolar range in a one-round replication assay, and that effectively inhibit both WT IN and most of the common drug-resistant variants. Based on tests done in cultured cells, these compounds have excellent therapeutic indexes (the CC50s are more than 3 logs higher than the IC50s). _____We also have three projects that involve studies of HIV-1 integration. In the first project, we are working with Drs. Alan Engelman and Vineet KewalRamani to define the host factors that are involved in transporting the preintegration complex (PIC) and to determine the exact roles they play in this process. In the second project, we are taking advantage of the fact that it is possible to redirect where HIV-1 DNA preferentially integrates. Redirecting HIV-1 DNA integration has the potential to make gene therapy safer because it may help solve the problems associated with the insertional activation of oncogenes; in addition, the technology can be used to determine where in the genome proteins/domains bind to chromatin. Different retroviruses have different integration-site preferences. There are good reasons to believe that such preferences are based on which host factor(s) the PIC interacts with; we now have information about the host factors that are targeted by HIV-1 IN and murine leukemia virus (MLV) IN. HIV-1 IN is known to bind to lens epithelium-derived growth factor (LEDGF); the distribution of LEDGF on chromatin is the major factor that determines the local sites where HIV-1 DNA integrates. However, working with Dr. KewalRamani, we showed that the host factor CPSF6, which binds to the viral CA protein, helps direct the PIC to nuclear speckles, which are associated with regions of the genome that are enriched in highly expressed genes. Thus, the interaction of CA and CPSF6 directs the PIC to broad regions of the genome, and the interaction of LEDGF and IN determines the exact sites where the HIV DNA is integrated. _____We, and others, showed that replacing the N-terminus of LEDGF with chromatin-binding domains (CBDs) from other proteins changes the specificity of HIV-1 DNA integration. The initial experiments were done either with single CBDs or, in one case, with two linked domains taken from a larger protein. These analyses showed that the binding sites for CBD can be accurately determined by mapping redirected HIV-1 integration sites and that the distance between the CBD binding site and the integration site(s) is relatively small. We alsoshowed that the binding sites for multiple-domain modules reflect the combinatorial interactions of the individual domains and chromatin and that the structural relationship of the domains helps define binding specificity. We have recently moved from an analysis of isolated CBDs to intact proteins. Although not every protein we have tried has worked, we have been able to get excellent data with a number of relatively large proteins. Last year we published a paper that explored how TAF3 interacts with its binding sites on chromatin using mutants with altered binding specificities (the TAF3 experiments are part of a collaboration with Dr. Robert Roeder). This year we have published two additional papers in which we mapped the genome-wide distribution of the binding sites for the proteins in the integrator complex (with Drs. Jeffrey Skaar and Michele Pagano) and WT and mutant DNMTs (with Drs. Kyung-Min Noh and C. David Allis). _____In the third project, we are working with Drs. Xiaolin Wu, Frank Maldarelli, John Coffin, John Mellors, and Mary Kearney to determine the distribution of integration sites in HIV-infected patients. Last year we showed that there is extensive clonal expansion of HIV-infected cells in patients, and that, in some cases, integration of HIV DNA in specific oncogenes (MKL2 and BACH2) can contribute to this clonal expansion. However, there was a question of whether any of the clonally expanded cells carried an infectious provirus. This year, we have been able to show not only that a highly expanded clone carries an infectious provirus, but also that cells of this clone released detectable amounts of virus into the blood of the patient. We are currently asking whether all of the cells in the clone are making a small amount of virus, or whether (as we suspect) only a fraction of the cells are making virus at any one time. We are analyzing additional infected clones to see what fraction carry replication-competent proviruses. We are also looking at the distribution of integration sites in acutely infected patients, and have begun a collaboration with Dr. Jeffrey Lifson and his colleagues (Leidos, Frederick) to develop a monkey model for integration site analysis. _____PATENTS LINKED TO THIS PROJECT: (1) Zhao XZ, Burke TR, Hughes SH, Johnson B, Marchand CR, Metifiot MA, Pommier Y, Smith SJ: Hydroxylamide-containing Compounds With Improved Efficacy Against Raltegravir-resistant Strains Of HIV-1 Integrase. Tracking number: E-093-2013/0-US-01 (US application), submitted in 2013. (2) Zhao XZ, Burke TZ, Hughes SZ, Johnson BZ, Marchand CZ, Metifiot MZ, Pommier YZ, Smith SZ: Substituted 4-amino-n-(2,4-difluorobenzyl)-1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides With Improved Efficacy Against Viral Constructs Harboring Mutant Strains Of HIV-1 Integrase. Tracking number: E-093-2013/1-US-01 (US application), submitted in 2013. (3) Burke TR, Hughes SH, Johnson B, Pommier Y, Smith SJ, Vu B, Zhao XZ (submitted in 2011): HIV Integrase Inhibitory Oxoisoindoline Sulfonamides. Patent pending: PCT/US2012/048169 (PC application).
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