ABSTRACT
Nonhuman primate models are needed for evaluations of proposed strategies targeting residual virus that persists in HIV-1-infected individuals receiving suppressive combination antiretroviral therapy (cART). However, relevant nonhuman primate (NHP) models of cART-mediated suppression have proven challenging to develop. We used a novel three-class, six-drug cART regimen to achieve durable 4.0- to 5.5-log reductions in plasma viremia levels and declines in cell-associated viral RNA and DNA in blood and tissues of simian immunodeficiency virus SIVmac239-infected Indian-origin rhesus macaques, then evaluated the impact of treatment with the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA; Vorinostat) on the residual virus pool. Ex vivo SAHA treatment of CD4(+) T cells obtained from cART-suppressed animals increased histone acetylation and viral RNA levels in culture supernatants. cART-suppressed animals each received 84 total doses of oral SAHA. We observed SAHA dose-dependent increases in acetylated histones with evidence for sustained modulation as well as refractoriness following prolonged administration. In vivo virologic activity was demonstrated based on the ratio of viral RNA to viral DNA in peripheral blood mononuclear cells, a presumptive measure of viral transcription, which significantly increased in SAHA-treated animals. However, residual virus was readily detected at the end of treatment, suggesting that SAHA alone may be insufficient for viral eradication in the setting of suppressive cART. The effects observed were similar to emerging data for repeat-dose SAHA treatment of HIV-infected individuals on cART, demonstrating the feasibility, utility, and relevance of NHP models of cART-mediated suppression for in vivo assessments of AIDS virus functional cure/eradication approaches.
Subject(s)
Anti-Retroviral Agents/therapeutic use , Histone Deacetylase Inhibitors/therapeutic use , Hydroxamic Acids/therapeutic use , Simian Acquired Immunodeficiency Syndrome/drug therapy , Simian Immunodeficiency Virus/drug effects , Acetylation/drug effects , Animals , CD4-Positive T-Lymphocytes/drug effects , Disease Models, Animal , Drug Therapy, Combination , Histones/metabolism , Macaca mulatta , RNA, Viral/blood , RNA, Viral/genetics , Simian Acquired Immunodeficiency Syndrome/virology , Viral Load/drug effects , VorinostatABSTRACT
Optimization studies using an HIV RNase H active site inhibitor containing a 1-hydroxy-1,8-naphthyridin-2(1H)-one core identified 4-position substituents that provided several potent and selective inhibitors. The best compound was potent and selective in biochemical assays (IC(50)=0.045 ĀµM, HIV RT RNase H; 13 ĀµM, HIV RT-polymerase; 24 ĀµM, HIV integrase) and showed antiviral efficacy in a single-cycle viral replication assay in P4-2 cells (IC(50)=0.19 ĀµM) with a modest window with respect to cytotoxicity (CC(50)=3.3 ĀµM).
Subject(s)
Anti-HIV Agents/pharmacology , Enzyme Inhibitors/pharmacology , HIV-1/enzymology , Ribonuclease H/antagonists & inhibitors , Anti-HIV Agents/chemistry , Enzyme Inhibitors/chemistry , HeLa Cells , Humans , Naphthyridines/chemistry , Naphthyridines/pharmacologyABSTRACT
A series of 10-hydroxy-7,8-dihydropyrazino[1',2':1,5]pyrrolo[2,3-d]pyridazine-1,9(2H,6H)-diones was synthesized and tested for their inhibition of HIV-1 replication in cell culture. Structure-activity studies indicated that high antiviral potency against wild-type virus as well as viruses containing integrase mutations that confer resistance to three different structural classes of integrase inhibitors could be achieved by incorporation of small aliphatic groups at certain positions on the core template. An optimal compound from this study, 16, inhibits integrase strand-transfer activity with an IC(50) value of 10 nM, inhibits HIV-1 replication in cell culture with an IC(95) value of 35 nM in the presence of 50% normal human serum, and displays modest pharmacokinetic properties in rats (i.v. t(1/2)=5.3 h, F=17%).
Subject(s)
Chemistry, Pharmaceutical/methods , HIV Integrase/chemical synthesis , HIV Integrase/pharmacology , Integrases/genetics , Mutation , Administration, Oral , Animals , Antiviral Agents/pharmacology , Biological Availability , Drug Design , Humans , Inhibitory Concentration 50 , Models, Chemical , Rats , Structure-Activity Relationship , Virus ReplicationABSTRACT
A series of potent novel 8-hydroxy-3,4-dihydropyrrolo[1,2-a]pyrazine-1(2H)-one HIV-1 integrase inhibitors was identified. These compounds inhibited the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 12 is active against replication of HIV-1 in cell culture with a CIC(95) of 0.31microM. Further SAR exploration led to the preparation of pseudosymmetrical tricyclic pyrrolopyrazine inhibitors 23 and 24 with further improvement in antiviral activity.
Subject(s)
HIV Integrase Inhibitors/chemistry , HIV Integrase , Pyrazines/chemistry , Cell Line, Tumor , HIV Integrase/physiology , HIV Integrase Inhibitors/pharmacology , Humans , Pyrazines/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/enzymology , T-Lymphocytes/virologyABSTRACT
Developing new antiretroviral therapies for HIV-1 infection with potential for less frequent dosing represents an important goal within drug discovery. Herein, we present the discovery of ethylĆ¢ĀĀ (1-((4-((4-fluorobenzyl)carbamoyl)-1-methyl-2-(2-(5-methyl- 1,3,4-oxadiazole-2-carboxamido)propan-2-yl)-6-oxo-1,6-dihydropyrimidin-5-yl)oxy)ethyl) carbonate (MK-8970), a highly optimized prodrug of raltegravir (Isentress). Raltegravir is a small molecule HIV integrase strand-transfer inhibitor approved for the treatment of HIV infection with twice-daily administration. Two classes of prodrugs were designed to have enhanced colonic absorption, and derivatives were evaluated in pharmacokinetic studies, both in vitro and in vivo in different species, ultimately leading to the identification of MK-8970 as a suitable candidate for development as an HIV therapeutic with the potential to require less frequent administration while maintaining the favorable efficacy, tolerability, and minimal drug-drug interaction profile of raltegravir.
Subject(s)
HIV Integrase Inhibitors/chemistry , Oxadiazoles/chemistry , Prodrugs/chemistry , Pyrimidinones/chemistry , Pyrrolidinones/chemistry , Acetals/chemistry , Animals , Area Under Curve , Carbonates/chemistry , Dogs , Drug Evaluation, Preclinical , HIV Integrase/chemistry , HIV Integrase/metabolism , HIV Integrase Inhibitors/chemical synthesis , HIV Integrase Inhibitors/pharmacokinetics , HIV-1/enzymology , Half-Life , Hepatocytes/metabolism , Humans , Intestinal Mucosa/metabolism , Male , Oxadiazoles/chemical synthesis , Oxadiazoles/pharmacokinetics , Prodrugs/chemical synthesis , Prodrugs/pharmacokinetics , Pyrimidinones/chemical synthesis , Pyrimidinones/pharmacokinetics , ROC Curve , Raltegravir Potassium , Rats , Rats, Wistar , Structure-Activity RelationshipABSTRACT
Naphthyridine 7 inhibits the strand transfer of the integration process catalyzed by integrase with an IC50 of 10 nM and inhibits 95% of the spread of HIV-1 infection in cell culture at 0.39 microM. It does not exhibit cytotoxicity in cell culture at < or =12.5 microM and shows a good pharmacokinetic profile when dosed orally to rats. The antiviral activity of 7 and its effect on integration were confirmed using viruses with specific integrase mutations.
Subject(s)
Anti-HIV Agents/chemical synthesis , HIV Integrase Inhibitors/chemical synthesis , HIV-1/drug effects , Naphthyridines/chemical synthesis , Administration, Oral , Animals , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Cell Line , HIV Integrase Inhibitors/chemistry , HIV Integrase Inhibitors/pharmacology , Humans , Injections, Intravenous , Naphthyridines/chemistry , Naphthyridines/pharmacology , Rats , Structure-Activity RelationshipABSTRACT
A series of potent novel dihydroxypyridopyrazine-1,6-dione HIV-1 integrase inhibitors was identified. These compounds inhibited the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 6 is active against replication of HIV with a CIC(95) of 0.31 microM and exhibits no shift in potency in the presence of 50% normal human serum. It displays a good pharmacokinetic profile when dosed in rats and no covalent binding with microsomal proteins in both in vitro and in vivo models.
Subject(s)
HIV Integrase Inhibitors/chemistry , HIV Integrase Inhibitors/pharmacology , Pyrazines/chemistry , Pyrazines/pharmacology , Animals , Benzene/chemistry , Cell Line , HIV/drug effects , HIV/enzymology , HIV/physiology , HIV Integrase Inhibitors/chemical synthesis , HIV Integrase Inhibitors/pharmacokinetics , Humans , Microsomes, Liver/drug effects , Models, Molecular , Molecular Structure , Pyrazines/chemical synthesis , Pyrazines/pharmacokinetics , Rats , Structure-Activity Relationship , Virus Replication/drug effectsABSTRACT
A series of 5-amino derivatives of 8-hydroxy[1,6]-naphthyridine-7-carboxamide exhibiting sub-micromolar potency against replication of HIV-1 in cell culture was identified. One of these analogs, compound 12, displayed excellent pharmacokinetic properties when dosed orally in rats and in monkeys. This compound was demonstrated to be efficacious against replication of simian-human immunodeficiency virus (SHIV) 89.6P in infected rhesus macaques.
Subject(s)
HIV Integrase Inhibitors/chemical synthesis , HIV Integrase Inhibitors/pharmacology , Naphthyridines/chemistry , Naphthyridines/pharmacology , Amination , HIV Integrase Inhibitors/chemistry , Molecular Structure , Naphthyridines/chemical synthesis , Structure-Activity RelationshipABSTRACT
Introduction of a 5,6-dihydrouracil functionality in the 5-position of N-(4-fluorobenzyl)-8-hydroxy-[1,6]naphthyridine-7-carboxamide 1 led to a series of highly active HIV-1 integrase inhibitors. These compounds displayed low nanomolar activity in inhibiting both the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 11 is a 150-fold more potent antiviral agent than 1, with a CIC(95) of 40 nM in the presence of human serum. It displays good pharmacokinetics when dosed in rats and dogs.
Subject(s)
Benzyl Compounds/pharmacology , HIV Integrase Inhibitors/pharmacology , HIV-1/drug effects , Naphthyridines/pharmacology , Uracil/analogs & derivatives , Virus Replication/drug effects , Animals , Benzyl Compounds/chemistry , Benzyl Compounds/pharmacokinetics , Biological Availability , Crystallography, X-Ray , HIV Integrase Inhibitors/chemistry , HIV Integrase Inhibitors/pharmacokinetics , HIV-1/physiology , Naphthyridines/chemistry , Naphthyridines/pharmacokinetics , Rats , Uracil/chemistryABSTRACT
Human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase (RT) coordinates DNA polymerization and ribonuclease H (RNase H) activities using two discrete active sites embedded within a single heterodimeric polyprotein. We have identified a novel thiophene diketo acid, 4-[5-(benzoylamino)thien-2-yl]-2,4-dioxobutanoic acid, that selectively inhibits polymerase-independent RNase H cleavage (IC(50) = 3.2 microm) but has no effect on DNA polymerization (IC(50) > 50 microm). The activity profile of the diketo acid is shown to be distinct from previously described compounds, including the polymerase inhibitor foscarnet and the putative RNase H inhibitor 4-chlorophenylhydrazone. Both foscarnet and the hydrazone inhibit RNase H cleavage and DNA polymerization activities of RT, yet neither inhibits the RNase H activity of RT containing a mutation in the polymerase active site (D185N) or an isolated HIV-1 RNase H domain chimera containing the alpha-C helix from Escherichia coli RNase HI, suggesting these compounds affect RNase H indirectly. In contrast, the diketo acid inhibits the RNase H activity of the isolated RNase H domain as well as full-length RT, and inhibition is not affected by the polymerase active site mutation. In isothermal titration calorimetry studies using the isolated RNase H domain, binding of the diketo acid is independent of nucleic acid but strictly requires Mn(2+) implying a direct interaction between the inhibitor and the RNase H active site. These studies demonstrate that inhibition of HIV-1 RNase H may occur by either direct or indirect mechanisms, and they provide a framework for identifying novel agents such as 4-[5-(benzoylamino)thien- 2-yl]-2,4-dioxobutanoic acid that specifically targets RNase H.
Subject(s)
Butyrates/pharmacology , Enzyme Inhibitors/pharmacology , HIV-1/enzymology , Ribonuclease H/antagonists & inhibitors , Thiophenes/pharmacology , Butyrates/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Foscarnet/pharmacology , Kinetics , RNA-Directed DNA Polymerase/metabolism , Reverse Transcriptase Inhibitors/pharmacology , Ribonuclease H/chemistry , Ribonuclease H/genetics , Structure-Activity Relationship , Substrate Specificity , Thiophenes/chemical synthesisABSTRACT
The process of integrating the reverse-transcribed HIV-1 DNA into the host chromosomal DNA is catalyzed by the virally encoded enzyme integrase (IN). Integration requires two metal-dependent reactions, 3' end processing and strand transfer. Compounds that contain a diketo acid moiety have been shown to selectively inhibit the strand transfer reaction of IN in vitro and in infected cells and are effective as inhibitors of HIV-1 replication. To characterize the molecular basis of inhibition, we used functional assays and binding assays to evaluate a series of structurally related analogs. These studies focused on investigating the role of the conserved carboxylate and metal binding. We demonstrate that an acidic moiety such as a carboxylate or isosteric heterocycle is not required for binding to the enzyme complex but is essential for inhibition and confers distinct metal-dependent properties on the inhibitor. Binding requires divalent metal and resistance is metal dependent with active site mutants displaying resistance only when the enzymes are evaluated in the context of Mg(2+). The mechanism of action of these inhibitors is therefore likely a consequence of the interaction between the acid moiety and metal ion(s) in the IN active site, resulting in a functional sequestration of the critical metal cofactor(s). These studies thus have implications for modeling active site inhibitors of IN, designing and evaluating analogs with improved efficacy, and identifying inhibitors of other metal-dependent phosphotransferases.
Subject(s)
Anti-HIV Agents/chemistry , HIV Integrase Inhibitors/chemistry , HIV Integrase/chemistry , HIV-1/enzymology , Anti-HIV Agents/pharmacology , Binding, Competitive , HIV Integrase/drug effects , HIV Integrase Inhibitors/pharmacology , HIV Long Terminal Repeat , Humans , Ligands , Magnesium , Manganese , Models, Chemical , Molecular Structure , Phosphotransferases/chemistry , Streptavidin , StyrenesABSTRACT
We describe the efficacy of L-870812, an inhibitor of HIV-1 and SIV integrase, in rhesus macaques infected with the simian-human immunodeficiency virus (SHIV) 89.6P. When initiated before CD4 cell depletion, L-870812 therapy mediated a sustained suppression of viremia, preserving CD4 levels and permitting the induction of virus-specific cellular immunity. L-870812 was also active in chronic infection; however, the magnitude and durability of the effect varied in conjunction with the pretreatment immune response and viral load. These studies demonstrate integrase inhibitor activity in vivo and suggest that cellular immunity facilitates chemotherapeutic efficacy in retroviral infections.
Subject(s)
Acquired Immunodeficiency Syndrome/drug therapy , Acquired Immunodeficiency Syndrome/immunology , HIV-1/physiology , Integrase Inhibitors/therapeutic use , Naphthyridines/therapeutic use , Simian Acquired Immunodeficiency Syndrome/drug therapy , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/physiology , Acquired Immunodeficiency Syndrome/virology , Animals , Anti-HIV Agents/administration & dosage , Anti-HIV Agents/blood , Anti-HIV Agents/pharmacology , Anti-HIV Agents/therapeutic use , CD4 Lymphocyte Count , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cells, Cultured , Drug Resistance, Viral , HIV Integrase/genetics , HIV Integrase/metabolism , HIV Integrase Inhibitors/administration & dosage , HIV Integrase Inhibitors/blood , HIV Integrase Inhibitors/pharmacology , HIV Integrase Inhibitors/therapeutic use , HIV-1/drug effects , HIV-1/enzymology , HIV-1/genetics , Immunity, Cellular , Integrase Inhibitors/administration & dosage , Integrase Inhibitors/blood , Integrase Inhibitors/pharmacology , Integrases/genetics , Integrases/metabolism , Leukocytes, Mononuclear/virology , Macaca mulatta , Mutation , Naphthyridines/administration & dosage , Naphthyridines/blood , Naphthyridines/pharmacology , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/drug effects , Simian Immunodeficiency Virus/enzymology , Simian Immunodeficiency Virus/genetics , Viral Load , Viremia/drug therapy , Virus Replication/drug effectsABSTRACT
The increasing incidence of resistance to current HIV-1 therapy underscores the need to develop antiretroviral agents with new mechanisms of action. Integrase, one of three viral enzymes essential for HIV-1 replication, presents an important yet unexploited opportunity for drug development. We describe here the identification and characterization of L-870,810, a small-molecule inhibitor of HIV-1 integrase with potent antiviral activity in cell culture and good pharmacokinetic properties. L-870,810 is an inhibitor with an 8-hydroxy-(1,6)-naphthyridine-7-carboxamide pharmacophore. The compound inhibits HIV-1 integrase-mediated strand transfer, and its antiviral activity in vitro is a direct consequence of this ascribed effect on integration. L-870,810 is mechanistically identical to previously described inhibitors from the diketo acid series; however, viruses selected for resistance to L-870,810 contain mutations (integrase residues 72, 121, and 125) that uniquely confer resistance to the naphthyridine. Conversely, mutations associated with resistance to the diketo acid do not engender naphthyridine resistance. Importantly, the mutations associated with resistance to each of these inhibitors map to distinct regions within the integrase active site. Therefore, we propose a model of the two inhibitors that is consistent with this observation and suggests specific interactions with discrete binding sites for each ligand. These studies provide a structural basis and rationale for developing integrase inhibitors with the potential for unique and nonoverlapping resistance profiles.