Virologic failure during treatment with raltegravir, the first effective drug targeting HIV integrase, is associated with two exclusive pathways involving either Q148H/R/K, G140S/A or N155H mutations. We carried out a detailed analysis of the molecular and structural effects of these mutations. We observed no topological change in the integrase core domain, with conservation of a newly identified Omega-shaped hairpin containing the Q148 residue, in particular. In contrast, the mutations greatly altered the specificity of DNA recognition by integrase. The native residues displayed a clear preference for adenine, whereas the mutant residues strongly favored pyrimidines. Raltegravir may bind to N155 and/or Q148 residues as an adenine bioisoster. This may account for the selected mutations impairing raltegravir binding while allowing alternative DNA recognition by integrase. This study opens up new opportunities for the design of integrase inhibitors active against raltegravir-resistant viruses.
"Interaction with DNA is probably impaired owing to an unfavorable orientation when Y143 is mutated in R, an orientation which is more favorable when Y143 is mutated in C. This is in agreement with the work of Mouscadet et al  who propose that native residues in wild-type enzyme involved in resistance (N155 and Q148) have a clear preference for adenine recognition, while mutations of N155 in H or of Q148 in H, R or K give mutants that favor pyrimidines. It might be that RAL inhibits HIV replication by mimicking adenine. "
[Show abstract][Hide abstract] ABSTRACT: Resistance to HIV-1 integrase (IN) inhibitor raltegravir (RAL), is encoded by mutations in the IN region of the pol gene. The emergence of the N155H mutation was replaced by a pattern including the Y143R/C/H mutations in three patients with anti-HIV treatment failure. Cloning analysis of the IN gene showed an independent selection of the mutations at loci 155 and 143. Characterization of the phenotypic evolution showed that the switch from N155H to Y143C/R was linked to an increase in resistance to RAL. Wild-type (WT) IN and IN with mutations Y143C or Y143R were assayed in vitro in 3'end-processing, strand transfer and concerted integration assays. Activities of mutants were moderately impaired for 3'end-processing and severely affected for strand transfer. Concerted integration assay demonstrated a decrease in mutant activities using an uncleaved substrate. With 3'end-processing assay, IC(50) were 0.4 microM, 0.9 microM (FC = 2.25) and 1.2 microM (FC = 3) for WT, IN Y143C and IN Y143R, respectively. An FC of 2 was observed only for IN Y143R in the strand transfer assay. In concerted integration, integrases were less sensitive to RAL than in ST or 3'P but mutants were more resistant to RAL than WT.
PLoS ONE 04/2010; 5(4):e10311. DOI:10.1371/journal.pone.0010311 · 3.23 Impact Factor
"Only one IFD pose of raltegravir at the catalytic site of SIVmac251 (Fig. 7) came out from the IFD protocol, and it was similar to one of the two conformations of the drug at the HIV-1 IN catalytic site, as described in the previous IFD study . This IFD pose clearly showed raltegravir as an ideal prosecution of the 3' DNA strand of 3'processed viral DNA, consistently with the hypothesis  that this drug acts as a nucleotide mimic (Fig. 8A). The three pharmacophoric oxygens of the drug were engaged in bidentate chelation of the two Mg2+ ions within the catalytic cavity (Fig. 8B), while the substituted benzyl group deeply occupied a pocket mainly defined by IN residues Q148, E152 and H156, and viral nucleotides dG18, dC19, dA20, dG24 and dC25, as previously described in docking simulations at the HIV-1 IN CCD  [see Additional file 5]. "
[Show abstract][Hide abstract] ABSTRACT: In this study we successfully created a new approach to ART in SIVmac251 infected nonhuman primates. This drug regimen is entirely based on drugs affecting the pre-integration stages of replication and consists of only two nucleotidic/nucleosidic reverse transcriptase inhibitors (Nt/NRTIs) and raltegravir, a promising new drug belonging to the integrase strand transfer inhibitor (INSTI) class.
In acutely infected human lymphoid CD4+ T-cell lines MT-4 and CEMx174, SIVmac251 replication was efficiently inhibited by raltegravir, which showed an EC90 in the low nanomolar range. This result was confirmed in primary macaque PBMCs and enriched CD4+ T cell fractions. In vivo monotherapy with raltegravir for only ten days resulted in reproducible decreases in viral load in two different groups of animals. When emtricitabine (FTC) and tenofovir (PMPA) were added to treatment, undetectable viral load was reached in two weeks, and a parallel increase in CD4 counts was observed. In contrast, the levels of proviral DNA did not change significantly during the treatment period, thus showing persistence of this lentiviral reservoir during therapy.
In line with the high conservation of the three main amino acids Y143, Q148 and N155 (responsible for raltegravir binding) and molecular docking simulations showing similar binding modes of raltegravir at the SIVmac251 and HIV-1 IN active sites, raltegravir is capable of inhibiting SIVmac251 replication both in tissue culture and in vivo. This finding may help to develop effective ART regimens for the simian AIDS model entirely based on drugs adopted for treatment in humans. This ART-treated AIDS nonhuman primate model could be employed to find possible strategies for virus eradication from the body.
"However, molecular modeling demonstrated that the two primary pathways of resistance involving residues Q148 and N155 maintained all the structural features of the active site and catalytic loop. By contrast, the specific interactions between the mutated amino acids selected by raltegravir and DNA base pairs differed from those of the wild-type enzyme, accounting for the differences in efficacy between the mutant and wild-type integrases in vitro . Together with theoretical studies that have predicted that the Q146, Q148, and N144 residues of the loop form a DNA binding site , this result suggest that raltegravir acts by competing with DNA for residues N155 and/or Q148. "
[Show abstract][Hide abstract] ABSTRACT: Integration of the HIV-1 viral DNA generated by reverse transcription of the RNA genome into the host cell chromosomes is a key step of viral replication, catalyzed by the viral integrase. In October 2007, the first integrase inhibitor, raltegravir, was approved for clinical use under the name of Isentress superset. The results of the various clinical trials that have evaluated raltegravir have been very encouraging with regard to the immunological and virological efficacy and tolerance. However, as observed for other anti-retrovirals, specific resistance mutations have been identified in patients failing to respond to treatment with raltegravir. Although knowledge of the integrase structural biology remains fragmentary, the structures and modeling data available might provide relevant clues on the origin of the emergence of these resistance mutations. In this review, we describe the mechanism of action of this drug and the main data relating to its use in vivo, together with recent structural data important to our understanding of the origin of viral resistance.
European journal of medical research 11/2009; 14 Suppl 3(Suppl 3):5-16. DOI:10.1186/2047-783X-14-S3-5 · 1.50 Impact Factor
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