Human immunodeficiency virus type-1 (HIV-1) integrase is one of the three virally encoded enzymes required for replication and therefore a rational target for chemotherapeutic intervention in the ...treatment of HIV-1 infection. We report here the discovery of Raltegravir, the first HIV-integrase inhibitor approved by FDA for the treatment of HIV infection. It derives from the evolution of 5,6-dihydroxypyrimidine-4-carboxamides and N-methyl-4-hydroxypyrimidinone-carboxamides, which exhibited potent inhibition of the HIV-integrase catalyzed strand transfer process. Structural modifications on these molecules were made in order to maximize potency as HIV-integrase inhibitors against the wild type virus, a selection of mutants, and optimize the selectivity, pharmacokinetic, and metabolic profiles in preclinical species. The good profile of Raltegravir has enabled its progression toward the end of phase III clinical trials for the treatment of HIV-1 infection and culminated with the FDA approval as the first HIV-integrase inhibitor for the treatment of HIV-1 infection.
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 Mg2+. 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.
Diketo acids such as L-731,988 are potent inhibitors of HIV-1 integrase that inhibit integration and viral replication in cells. These compounds exhibit the unique ability to inhibit the strand ...transfer activity of integrase in the absence of an effect on 3′end processing. To understand the reasons for this distinct inhibitory profile, we developed a scintillation proximity assay that permits analysis of radiolabeled inhibitor binding and integrase function. High-affinity binding of L-731,988 is shown to require the assembly of a specific complex on the HIV-1 long terminal repeat. The interaction of L-731,988 with the complex and the efficacy of L-731,988 in strand transfer can be abrogated by the interaction with target substrates, suggesting competition between the inhibitor and the target DNA. The L-731,988 binding site and that of the target substrate are thus distinct from that of the donor substrate and are defined by a conformation of integrase that is only adopted after assembly with the viral end. These results elucidate the basis for diketo acid inhibition of strand transfer and have implications for integrase-directed HIV-1 drug discovery efforts.
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 (IC50=0.045μM, HIV RT RNase H; 13μM, HIV RT-polymerase; 24μM, HIV integrase) and showed antiviral efficacy in a single-cycle viral replication assay in P4-2 cells (IC50=0.19μM) with a modest window with respect to cytotoxicity (CC50=3.3μM).
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 μ m ) but has no effect on DNA polymerization (IC 50 > 50 μ m ). 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 α-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.
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.
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 μM. It ...does not exhibit cytotoxicity in cell culture at ≤12.5 μM 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.
HIV-1 integrase catalyzes the insertion of viral DNA into the genome of the host cell. Integrase inhibitor
N-(4-fluorobenzyl)-8-hydroxy-1,6-naphthyridine-7-carboxamide selectively inhibits the strand ...transfer process of integration. 4-Substituted pyrrolidinones possessing various groups on the pyrrolidinone nitrogen were introduced at the 5-position of the naphthyridine scaffold. These analogs exhibit excellent activity against viral replication in a cell-based assay. The preparation of these compounds was enabled by a three-step, two-pot reaction sequence from a common butenolide intermediate.
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