Genetic Diversity on the Integrase Region of the pol Gene among HIV Type 1-Infected Patients Naive for Integrase Inhibitors in Sao Paulo City, Brazil

The presence of mutations associated with integrase inhibitor (INI) resistance among INI-naive patients may play an important clinical role in the use of those drugs Samples from 76 HIV-1-infected subjects naive to INIs were submitted to direct sequencing. No differences were found between naive (25%) subjects and subjects on HAART (75%). No primary mutation associated with raltegravir or elvitegravir resistance was found. However, 78% of sequences showed at least one accessory mutation associated with resistance. The analysis of the 76 IN sequences showed a high polymorphic level on this region among Brazilian HIV-1-infected subjects, including a high prevalence of aa substitutions related to INI resistance. The impact of these findings remains unclear and further studies are necessary to address these questions.

HIV-2 Integrase Polymorphisms and Longitudinal Genotypic Analysis of HIV-2 Infected Patients Failing a Raltegravir-Containing Regimen

To characterize the HIV-2 integrase gene polymorphisms and the pathways to resistance of HIV-2 patients failing a raltegravir-containing regimen, we studied 63 integrase strand transfer inhibitors (INSTI)-naïve patients, and 10 heavily pretreated patients exhibiting virological failure while receiving a salvage raltegravir-containing regimen. All patients were infected by HIV-2 group A. 61.4% of the integrase residues were conserved, including the catalytic motif residues. No INSTI-major resistance mutations were detected in the virus population from naïve patients, but two amino acids that are secondary resistance mutations to INSTIs in HIV-1 were observed. The 10 raltegravir-experienced patients exhibited resistance mutations via three main genetic pathways: N155H, Q148R, and eventually E92Q - T97A. The 155 pathway was preferentially used (7/10 patients). Other mutations associated to raltegravir resistance in HIV-1 were also observed in our HIV-2 population (V151I and D232N), along with several novel mutations previously unreported. Data retrieved from this study should help build a more robust HIV-2-specific algorithm for the genotypic interpretation of raltegravir resistance, and contribute to improve the clinical monitoring of HIV-2-infected patients.

Inibidores da HIV-integrase: potencial abordagem farmacológica para tratamento da AIDS

AIDS has the HIV as its etiological agent. Researches has been done to find new pharmacological agents to be used in therapy, because of problems of resistance and side effects. The HIV-integrase inhibitors are some of those new agents that are being studied. This updating focusses on the fundamental information about HIV and HIV-integrase and the main methods being used to develop these new drugs, with examples for each case.

Inibidores da HIV-integrase: Potencial abordagem farmacológica para tratamento da AIDS

AIDS has the HIV as its etiological agent. Researches has been done to find new pharmacological agents to be used in therapy, because of problems of resistance and side effects. The HIV-integrase inhibitors are some of those new agents that are being studied. This updating focusses on the fundamental information about HIV and HIV-integrase and the main methods being used to develop these new drugs, with examples for each case.

Avaliação da resistência primária aos inibidores de integrase em pacientes soropositivos para o HIV-1

Pós-graduação em Pesquisa e Desenvolvimento (Biotecnologia Médica) - FMB

Differential inhibition of HIV-1 preintegration complexes and purified integrase protein by small molecules.

To replicate, HIV-1 must integrate a cDNA copy of the viral RNA genome into a chromosome of the host. The integration system is a promising target for antiretroviral agents, but to date no clinically useful integration inhibitors have been identified. Previous screens for integrase inhibitors have assayed inhibition of reactions containing HIV-1 integrase purified from an Escherichia coli expression system. Here we compare action of inhibitors in vitro on purified integrase and on subviral preintegration complexes (PICs) isolated from lymphoid cells infected with HIV-1. We find that many inhibitors active against purified integrase are inactive against PICs. Using PIC assays as a primary screen, we have identified three new anthraquinone inhibitors active against PICs and also against purified integrase. We propose that PIC assays are the closest in vitro match to integration in vivo and, as such, are particularly appropriate for identifying promising integration inhibitors.

Nuclear import of HIV-1 integrase is inhibited in vitro by styrylquinoline derivatives

Nuclear import of HIV-1 preintegration complexes (PICs) allows the virus to infect nondividing cells. Integrase (IN), the PIC-associated viral enzyme responsible for the integration of the viral genome into the host cell DNA, displays karyophilic properties and has been proposed to participate to the nuclear import of the PIC. Styrylquinolines (SQs) have been shown to block viral replication at nontoxic concentrations and to inhibit IN 3'-processing activity in vitro by competing with the DNA substrate binding. However, several lines of evidence suggested that SQs could have a postentry, preintegrative antiviral effect in infected cells. To gain new insights on the mechanism of their antiviral activity, SQs were assayed for their ability to affect nuclear import of HIV-1 IN and compared with the effect of a specific strand transfer inhibitor. Using an in vitro transport assay, we have previously shown that IN import is a saturable mechanism, thus showing that a limiting cellular factor is involved in this process. We now demonstrate that SQs specifically and efficiently inhibit in vitro nuclear import of IN without affecting other import pathways, whereas a specific strand transfer inhibitor does not affect IN import. These data suggest that SQs not only inhibit IN-DNA interaction but would also inhibit the interaction between IN and the cellular factor required for its nuclear import.

Cell disposition of raltegravir and newer antiretrovirals in HIV-infected patients: high inter-individual variability in raltegravir cellular penetration.

Objectives The site of pharmacological activity of raltegravir is intracellular. Our aim was to determine the extent of raltegravir cellular penetration and whether raltegravir total plasma concentration (C(tot)) predicts cellular concentration (C(cell)). Methods Open-label, prospective, pharmacokinetic study on HIV-infected patients on a stable raltegravir-containing regimen. Plasma and peripheral blood mononuclear cells were simultaneously collected during a 12 h dosing interval after drug intake. C(tot) and C(cell) of raltegravir, darunavir, etravirine, maraviroc and ritonavir were measured by liquid chromatography coupled to tandem mass spectrometry after protein precipitation. Longitudinal mixed effects analysis was applied to the C(cell)/C(tot) ratio. Results Ten HIV-infected patients were included. The geometric mean (GM) raltegravir total plasma maximum concentration (C(max)), minimum concentration (C(min)) and area under the time-concentration curve from 0-12 h (AUC(0-12)) were 1068 ng/mL, 51.1 ng/mL and 4171 ng·h/mL, respectively. GM raltegravir cellular C(max), C(min) and AUC(0-12) were 27.5 ng/mL, 2.9 ng/mL and 165 ng·h/mL, respectively. Raltegravir C(cell) corresponded to 5.3% of C(tot) measured simultaneously. Both concentrations fluctuate in parallel, with C(cell)/C(tot) ratios remaining fairly constant for each patient without a significant time-related trend over the dosing interval. The AUC(cell)/AUC(tot) GM ratios for raltegravir, darunavir and etravirine were 0.039, 0.14 and 1.55, respectively. Conclusions Raltegravir C(cell) correlated with C(tot) (r = 0.86). Raltegravir penetration into cells is low overall (∼5% of plasma levels), with distinct raltegravir cellular penetration varying by as much as 15-fold between patients. The importance of this finding in the context of development of resistance to integrase inhibitors needs to be further investigated.

Drogas anti-VIH: passado, presente e perspectivas futuras

Currently available anti-HIV drugs can be classified into three categories: nucleoside analogue reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs). In addition to the reverse transcriptase (RT) and protease reaction, various other events in the HIV replicative cycle can be considered as potential targets for chemotherapeutic intervention: (1) viral adsorption, through binding to the viral envelope glycoprotein gp120; (2) viral entry, through blockage of the viral coreceptors CXCR4 and CCR5; (3) virus-cell fusion, through binding to the viral envelope glycoprotein gp 41; (4) viral assembly and disassembly through NCp7 zinc finger-targeted agents; (5) proviral DNA integration, through integrase inhibitors and (6) viral mRNA transcription, through inhibitors of the transcription (transactivation) process. Also, various new NRTIs, NNRTIs and PIs have been developed, possessing different improved characteristics.

HIV - recentes avanços na pesquisa de fármacos

The development of new antiretroviral drugs is a dynamic process that is continuously fueled by identification of new molecular targets and new compounds for know targets. The current available drugs can be classified into five categories: nucleoside analogues reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors and entry inhibitors (fusion inhibitors and CCR5 antagonist). In addition, the maturation inhibitors may be considered as potential target for chemotherapeutic intervention. This review presents some anti-HIV agents that have already gone through the advance development process for final approval for the treatment of AIDS.

Mécanismes de résistance aux inhibiteurs de CCR5

Le travail décrit dans ce manuscrit vise à caractériser les voies de résistance aux inhibiteurs de CCR5. Lors d’une première étape, nous avons développé un test phénotypique clonal nous permettant d’une part d’identifier le tropisme viral et d’autre part de mesurer la résistance aux inhibiteurs des CCR5. Des virus à tropisme R5 ou X4 représentant aussi peu que 0,4% d’un mélange de populations virales sont détectables par ce test, démontrant ainsi sa sensibilité. De plus, grâce à son approche clonale, cette technique permet de différencier les virus à tropisme double de populations virales mixtes. Par la suite, nous avons étudié l’impact des mutations dans les régions variables de la protéine gp120 de l’enveloppe du virus VIH-1 sur la résistance aux inhibiteurs de CCR5. Pour ce faire, nous avons généré des virus résistants par passage des isolats CC1/85 et BAL, en présence de concentrations sous-inhibitrices de maraviroc (MVC) et vicriviroc (VCV). Après quelques passages du virus CC1/85 en présence de MVC, certaines sont apparues dans differentes régions de la gp120. Par la suite, nous avons sélectionné trois mutations dans les domaines variables de la gp 120, V169M en V2, L317W en V3 et I408T en V4 pour construire des virus contenant des mutations simples, doubles et triples afin d’évaluer la contribution des mutations individuelles ou combinées au phénotype de résistance. Nous avons déterminé la sensibilité de chaque mutant à MVC et VCV, le pourcentage d’infectivité et le tropisme viral par rapport au phénotype sauvage. Tous les mutants ont conservé le tropisme R5 et ont montré une diminution d’infectivité par rapport au contrôle. Nos résultats ont montré que les mutants qui portent des mutations en V4 (I408T) ont eu le plus d'impact sur la susceptibilité au MVC. Finalement, nous avons voulu évaluer l’activité antivirale d’un nouvel inhibiteur de CCR5, VCH-286 avec d’autres inhibiteurs de CCR5 tels que MVC et VVC ainsi que ses interactions avec des médicaments représentatifs de différentes classes d’antirétroviraux ARV employés en clinique pour traiter le HIV/SIDA., afin d’évaluer si ces médicaments pourraient être utilisés dans un même régime thérapeutique. Nous avons tout d’abord évalué indépendamment l’activité antivirale des trois inhibiteurs de CCR5 : VCH-286, MVC et VVC. Par la suite nous avons évalué les interactions de VCH-286 avec MVC et VVC. Finalement nous avons évalué les interactions de VCH-286 avec d’autres médicaments antirétroviraux. Ces études ont montré que VCH-286 est un inhibiteur puissant de CCR5 avec une activité antivirale in vitro de l’ordre du nanomolaire et des interactions médicamenteuses favorables avec la majorité des ARV tels que les inhibiteurs de transcriptase inverse, de protéase, d’intégrase, et de fusion employés en clinique pour traiter le VIH/SIDA et des interactions allant de synergie à l'antagonisme avec les inhibiteurs de CCR5. Nos résultats montrent que la plasticité de l’enveloppe virale du VIH-1 a des répercussions sur la résistance aux inhibiteurs de CCR5, le tropisme et la possible utilisation de ces molécules en combinaison avec d’autres molécules appartenant à la même classe.

Targeting the Transposase Domain of the DNA Repair Component Metnase to Enhance Chemotherapy

Previous studies have shown that the DNA repair component Metnase (SETMAR) mediates resistance to DNA damaging cancer chemotherapy. Metnase has a nuclease domain that shares homology with the Transposase family. We therefore virtually screened the tertiary Metnase structure against the 550,000 compound ChemDiv library to identify small molecules that might dock in the active site of the transposase nuclease domain of Metnase. We identified eight compounds as possible Metnase inhibitors. Interestingly, among these candidate inhibitors were quinolone antibiotics and HIV integrase inhibitors, which share common structural features. Previous reports have described possible activity of quinolones as antineoplastic agents. Therefore, we chose the quinolone ciprofloxacin for further study, based on its wide clinical availability and low toxicity. We found that ciprofloxacin inhibits the ability of Metnase to cleave DNA and inhibits Metnase-dependent DNA repair. Ciprofloxacin on its own did not induce DNA damage, but it did reduce repair of chemotherapy-induced DNA damage. Ciprofloxacin increased the sensitivity of cancer cell lines and a xenograft tumor model to clinically relevant chemotherapy. These studies provide a mechanism for the previously postulated antineoplastic activity of quinolones, and suggest that ciprofloxacin might be a simple yet effective adjunct to cancer chemotherapy. Cancer Res; 72(23); 6200-8. (C) 2012 AACR.

Analysis of Transmitted Resistance to Raltegravir and Selective Pressure among HIV-1-Infected Patients on a Failing HAART in Sao Paulo, Brazil

We studied the presence of primary resistance to raltegravir (RAL), natural polymorphisms, and selection pressure on HIV-1 integrase. We found a high frequency of integrase polymorphisms related to the resistance to RAL and sequence stability. Further studies are needed to determine the importance of these polymorphisms to RAL resistance.

Inhibitors of HIV-1 replication that inhibit HIV integrase.

HIV-1 replication depends on the viral enzyme integrase that mediates integration of a DNA copy of the virus into the host cell genome. This enzyme represents a novel target to which antiviral agents might be directed. Three compounds, 3,5-dicaffeoylquinic acid, 1-methoxyoxalyl-3,5-dicaffeoylquinic acid, and L-chicoric acid, inhibit HIV-1 integrase in biochemical assays at concentrations ranging from 0.06-0.66 microgram/ml; furthermore, these compounds inhibit HIV-1 replication in tissue culture at 1-4 microgram/ml. The toxic concentrations of these compounds are fully 100-fold greater than their antiviral concentrations. These compounds represent a potentially important new class of antiviral agents that may contribute to our understanding of the molecular mechanisms of viral integration. Thus, the dicaffeoylquinic acids are promising leads to new anti-HIV therapeutics and offer a significant advance in the search for new HIV enzyme targets as they are both specific for HIV-1 integrase and active against HIV-1 in tissue culture.