t-30177 and HIV-Infections

Current strategies for the treatment of HIV infection are based on cocktails of drugs that target the viral reverse transcriptase or protease enzymes. At present, the clinical benefit of this combination therapy for HIV-infected patients is considerable, although it is not clear how long this effect will last taking into account the emergence of multiple drug-resistant viral strains. Addition of new anti-HIV drugs targeting additional steps of the viral replication cycle may increase the potency of inhibition and prevent resistance development. During HIV replication, integration of the viral genome into the cellular chromosome is an essential step catalysed by the viral integrase. Although HIV integrase is an attractive target for antiviral therapy, so far all research efforts have led to the identification of only one series of compounds that selectively inhibit the integration step during HIV replication, namely the diketo acids. In this review we try to address the question why it has proven so difficult to find potent and selective integrase inhibitors. We point to potential pitfalls in defining an inhibitor as an authentic integrase inhibitor, and propose new strategies and technologies for the discovery of authentic HIV integration inhibitors.

Topics: Anti-HIV Agents; Caffeic Acids; DNA, Viral; Drug Design; HIV Infections; HIV Integrase; HIV Integrase Inhibitors; HIV-1; Humans; Oligonucleotides; Succinates; Virus Integration

Resistance to raltegravir (RAL), the first HIV-1 integrase (IN) inhibitor approved by the FDA, involves three genetic pathways: IN mutations N155H, Q148H/R/K, and Y143H/R/C. Those mutations are generally associated with secondary point mutations. The resulting mutant viruses show a high degree of resistance against RAL but somehow are affected in their replication capacity. Clinical and virological data indicate the high relevance of the combination G140S + Q148H because of its limited impact on HIV replication and very high resistance to RAL. Here, we report how mutations at the amino acid residues 140, 148, and 155 affect IN enzymatic activity and RAL resistance. We show that single mutations at position 140 have limited impact on 3'-processing (3'-P) but severely inactivate strand transfer (ST). On the other hand, single mutations at position 148 have a more profound effect and inactivate both 3'-P and ST. By examining systematically all of the double mutants at the 140 and 148 positions, we demonstrate that only the combination G140S + Q148H is able to restore the catalytic properties of IN. This rescue only operates in cis when both the 140S and 148H mutations are in the same IN polypeptide flexible loop. Finally, we show that the G140S-Q148H double mutant exhibits the highest resistance to RAL. It also confers cross-resistance to elvitegravir but less to G-quadraduplex inhibitors such as zintevir. Our results demonstrate that IN mutations at positions 140 and 148 in the IN flexible loop can account for the phenotype of RAL-resistant viruses.

Topics: Amino Acid Sequence; Amino Acid Substitution; Drug Resistance, Viral; HIV Infections; HIV Integrase; HIV Integrase Inhibitors; HIV-1; Humans; Molecular Sequence Data; Oligonucleotides; Point Mutation; Pyrrolidinones; Raltegravir Potassium; Sequence Homology, Amino Acid; Virus Replication

5' GTGGTGGGTGGGTGGGT-3' (AR177) is a 17-mer oligonucleotide with anti-human immunodeficiency virus (HIV) activity that is composed of a phosphodiester backbone and single phosphorothioate linkages at the 3' and 5' ends. A hemodynamic toxicity study was conducted in which cynomolgus monkeys were infused i.v. over a 10-minute period with single doses of 5, 20 or 50 mg AR177/kg or saline. Blood pressure, ECG, clinical chemistry, hematology, complement factors, coagulation parameters and the AR177 plasma concentration were determined. AR177 did not cause any mortality in this study, nor did it cause changes in blood pressure, ECG, clinical chemistry or hematology parameters at any dose. There was a minimal, dose-dependent increase in the levels of complement split product Bb and total hemolytic complement. There was a significant dose-dependent and reversible inhibition of coagulation with the 20- and 50-mg/kg doses that lasted up to several hours after infusion. The time course of the inhibition of coagulation closely matched the plasma levels of AR177. There was a no-effect plasma AR177 concentration vs. activated partial thromboplastin time of approximately 60 to 100 micrograms AR177/ml, above which there was prolongation of activated partial thromboplastin time. These data demonstrate that AR177 does not cause significant hemodynamic toxicity at the doses studied and that this drug could be administered as a rapid infusion without any acute, life-threatening effects at doses that produce plasma concentrations that have shown anti-HIV activity in vitro.

Topics: Animals; Anti-HIV Agents; Blood Coagulation; Complement Activation; Dose-Response Relationship, Drug; Hemodynamics; HIV Infections; HIV Integrase Inhibitors; Macaca fascicularis; Oligonucleotides; Partial Thromboplastin Time

T30177, an oligonucleotide composed of only deoxyguanosine and thymidine, is 17 nucleotides in length and contains single phosphorothioate internucleoside linkages at its 5' and 3' ends for stability. This oligonucleotide does not share significant primary sequence homology with or possess any complementary (antisense) sequence motifs to the human immunodeficiency virus type 1 (HIV-1) genome. T30177 inhibited replication of multiple laboratory strains of HIV-1 in human T-cell lines, peripheral blood lymphocytes, and macrophages. T30177 was also found to be capable of inhibiting multiple clinical isolates of HIV-1 and preventing the cytopathic effect of HIV-1 in primary CD4+ T lymphocytes. In assays with human peripheral blood lymphocytes there was no observable toxicity associated with T30177 at the highest concentration tested (100 microM), while the median inhibitory concentration was determined to be in the range of 0.1 to 1.0 microM for the clinical isolates tested, resulting in a high therapeutic index for this drug. In temporal studies, the kinetics of addition of T30177 to infected cell cultures indicated that, like the known viral adsorption blocking agents dextran sulfate and Chicago sky blue, T30177 needed to be added to cells during or very soon after viral infection. However, analysis of nucleic acids extracted at 12 h postinfection from cells treated with T30177 at the time of virus infection established the presence of unintegrated viral cDNA, including circular proviral DNA, in the treated cells. In vitro analysis of viral enzymes revealed that T30177 was a potent inhibitor of HIV-1 integrase, reducing enzymatic activity by 50% at concentrations in the range of 0.050 to 0.09 microM. T30177 was also able to inhibit viral reverse transcriptase activity; however, the 50% inhibitory value obtained was in the range of 1 to 10 microM, depending on the template used in the enzymatic assay. No observable inhibition of viral protease was detected at the highest concentration of T30177 used (10 microM). In experiments in which T30177 was removed from infected cell cultures at 4 days post-HIV-1 infection, total suppression of virus production was observed for more than 27 days. PCR analysis of DNA extracted from cells treated in this fashion was unable to detect the presence of viral DNA 11 days after removal of the drug from the infected cell cultures. The ability of T30177 to inhibit both laboratory and clinical isolates of HIV-1 and the experimenta

Topics: Antiviral Agents; Base Sequence; Cell Fusion; Cell Line; Cell Survival; Cells, Cultured; DNA Nucleotidyltransferases; DNA, Viral; Flow Cytometry; HIV Infections; HIV-1; Humans; Integrases; Lymphocytes; Macrophages; Molecular Sequence Data; Oligonucleotides; Reverse Transcriptase Inhibitors; T-Lymphocyte Subsets; Virus Replication