structures revealing the binding mode of these compounds with a full length prototype foamy virus IN and synthetic viral DNA ends. ZD6474 Earlier docking studies relied on omplete structures and did not lude the contribution of the viral DNA to inhibitor binding. Using the structure of PFV IN as the starting point, we generated a model of the corresponding HIV 1 complex and developed a molecular dynamics based approach that correlates with the in vitro activities of novel compounds. Four well characterized compounds were used as a training set, and the data for their in vitro activity against the Y143R, N155H, and G140S/Q148H mutants were used in addition to the wild type IN data. Three additional compounds were docked into the IN DNA complex model and subjected toMDsimulations.
All three gave interaction potentials within 1 standard Hedgehog Pathway deviation of values estimated from the training set, and the most active compound was identified. AdditionalMDanalysis of the raltegravir and dolutegravir bound complexes gave internal and interaction energy values that closely match the experimental binding energy of a compound related to raltegravir that has similar activity. These approaches can be used to gain a deeper understanding of the interactions of the inhibitors with the HIV 1 intasome and to identify promising scaffolds for novel integrase inhibitors, in particular, compounds that retain activity against a range of drug resistant mutants, making it possible to streamline synthesis and testing.Retroviruses are distinguished by their ability to reverse transcribe a single stranded RNA genome into a linear doublestranded DNA.
The viral integrase then inserts this linear viral DNA into the genome of the target cell to establish a stable infection. Integration catalyzed by IN follows two distt steps . First, IN cleaves a GT dinucleotide from the 3= end of both strands of the viral DNA . This leaves a conserved nonpositivist CA sequence with a free hydroxyl on each of the 3= ends of the viral DNA and a 2 nucleotide overhang on each of the 5= ends. In the second reaction, IN uses the newly created 3= hydroxyl to attack the phosphodiester backbone of the host genome . Depending on the retrovirus, the two viral ends are inserted 4 to 6 bases apart, creating a small duplication in the target DNA flanking the provirus . Cellular enzymes repair the gaps, leaving the double stranded proviral DNA stably integrated into the genome of the infected cell.
Integrases consist of three functionally distt domains that have been characterized by biochemical and mutational analyses. The N terminal domain contains a zbinding HHCC motif and contributes to multimer formation . The C terminal domain nonspecifically binds DNA . The catalytic core domain contains the catalytic triad DD35E motif that is well conserved among the retroviral integrase superfamily . This active site coordinates two metal ions and binds one viral DNA end. Although IN complexes exist in solution in different multimeric states, recent crystallographic data confirmed that the functional intasome is a tetrameric protein, with the two inner INs forming a head to tail dimer . This arrangement allows all three domains of each of the inner subunits to participate in multimerization and DNA binding.