HIV-1 integrase inhibitors that compete with the target DNA substrate define a unique strand transfer conformation for integrase

Espeseth, Amy S.; Felock, Peter J.; Wolfe, Abigail; Witmer, Marc; Grobler, Jay A.; Anthony, Neville J.; Egbertson, Melissa S.; Melamed, Jeffrey Y.; Young, Steve; Hamill, Terence G.; Cole, James L.; Hazuda, Daria J.

doi:10.1073/pnas.200139397

Cited by 299 publications

(290 citation statements)

References 35 publications

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“…L-870,810 is also the prototype of a structural class of 8-hydroxy-1,6-naphthyridine-7-carboxamides, which provide a previously uncharacterized pharmacophore for inhibitor design. Like previously described diketo acids, the 8-hydroxy-1,6-naphthyridine-7-carboxamides selectively inhibit integrasemediated strand transfer (5,7). Although these inhibitors are mechanistically indistinguishable, we have shown that distinct resistance profiles can be obtained with homologous naphthyridine and diketo acid analogs, providing the proof of concept and rationale for designing integrase inhibitors with the potential for unique and nonoverlapping resistance.…”

Section: Discussionsupporting

confidence: 59%

“…1) that prevent integration and viral replication in cell culture provided the first proof of concept for HIV-1 integrase inhibitors as antiviral agents. These compounds were shown to act specifically as inhibitors of the integrase strand transfer reaction by virtue of their ability to bind selectively to the enzyme complexed with the viral (or donor) DNA and compete with the host (or target) DNA substrate (7,8). The critical diketo carboxylate pharmacophore interacts with critical divalent metal ions in the active site (8) and mutations that engender resistance to these prototype inhibitors (5,9), and closely related analogs map to the integrase active site proximal to residues that coordinate divalent metals (D66, D116, and E152).…”

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confidence: 99%

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A naphthyridine carboxamide provides evidence for discordant resistance between mechanistically identical inhibitors of HIV-1 integrase

Hazuda

Anthony

Gomez

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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319

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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.A gents for the treatment of HIV-1 infection target two of the three virally encoded enzymes and belong to three mechanistic classes known as nucleoside reverse transcriptase, nonnucleoside reverse transcriptase (NNRTI), and protease inhibitors. Although treatment regimens comprising combinations of these agents have significantly reduced AIDS-related morbidity and mortality, it is estimated that 78% of treatment-naive patients harbor viruses that have evolved resistance to at least one of these drug classes (1, 2). The emergence of HIV-1 strains resistant to reverse transcriptase and protease inhibitors highlights the need to develop antiviral agents with novel mechanisms of action.Integrase (3, 4), one of the three virally encoded enzymes required for HIV-1 replication, catalyses the integration of viral DNA into the genome of the host cell. The integration reaction requires three discrete steps: assembly of a stable preintegration complex at the termini of the viral DNA and two sequential transesterification reactions. In the first reaction, 3Ј-end processing, endonucleolytic cleavage of the two 3Ј nucleotides at each DNA end generates 3Ј-hydroxyl groups that function as nucleophiles in the second reaction. The strand breakage of the cellular DNA and concomitant covalent linkage to the viral DNA is a consequence of the second transesterification reaction, strand transfer.The discovery of a series of diketo acids containing HIV-1 integrase i...

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Section: Discussionsupporting

confidence: 59%

mentioning

confidence: 99%

A naphthyridine carboxamide provides evidence for discordant resistance between mechanistically identical inhibitors of HIV-1 integrase

Hazuda

Anthony

Gomez

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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319

292

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“…Binding of each compound was saturable, not cooperative (Hill coefficent Ϸ1), and required both IN and DNA. As reported (20), binding correlated with activity of the strand transfer complex: (i) binding was detected with full-length IN and not with truncated enzymes competent to catalyze disintegration but not strand transfer, and (ii) binding was detected when IN was assembled onto viral DNA ends and not nonspecific sequence or single-stranded DNAs (data not shown). In saturation binding studies using the U5 long terminal repeat DNA sequence, dissociation constants of 15 and 5 nM were determined for compounds I and II, respectively (Fig.…”

Section: Dka Binding Correlates With Assembly Of An In Complex Competentsupporting

confidence: 69%

“…The inhibitor 5CITEP [1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-propenone] also binds within the IN active site (19). In the crystal structure of the core domain of IN (amino acids 50-212) with 5CITEP, the inhibitor contacts IN residues that are identical or close to those predicted to be important for the DKAs on the basis of resistance.Previous studies have shown that the binding of DKA inhibitors requires that IN be assembled into a nucleoprotein complex competent to catalyze strand transfer and that binding of the inhibitor and the target DNA substrate are mutually exclusive (20). These studies suggest a biochemical basis for the strand transfer selectivity of these inhibitors.…”

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confidence: 86%

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Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes

Grobler

Stillmock²,

Hu³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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377

325

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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 metaldependent 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.A n essential step in HIV replication is the integration of the reverse-transcribed viral genome into host chromosomal DNA by the virally encoded integrase (IN) protein (1-3). Integration is required for efficient long terminal repeat-driven transcription of the provirus for the production of viral proteins and RNA progeny. IN represents an important chemotherapeutic target, as its inactivation, either by mutagenesis or inhibition, blocks productive infection by HIV-1 (4-7).Integration is carried out in the cell in a series of distinct steps (8-10). First, IN cleaves the two terminal nucleotides from each 3Ј end of the viral DNA. The 3Ј processing reaction is carried out concurrently with or soon after reverse transcription in the cytoplasm. In the second step, strand transfer, IN catalyzes staggered nicking of the target chromosomal DNA and joining of each 3Ј end of the viral DNA to the 5Ј ends of the host DNA. Strand transfer is temporally and spatially separated from 3Ј processing and occurs after transport of the preintegration complex from the cytoplasm into the nucleus.Divalent metals such as Mg 2ϩ or Mn 2ϩ are required for both 3Ј processing and strand transfer and for the assembly of IN onto specific viral donor DNA to form a complex competent to carry out either function (11)(12)(13) (18), L-731,988 and related DKAs inhibit integration and viral replication in cell culture (7). Mutations that confer resistance to DKAs have been identified and map to IN residues adjacent to D64 and E152. The in...

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A targeted DNA substrate mechanism for the inhibition of HIV‐1 integrase by inhibitors with antiretroviral activity

et al. 2016

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We recently reported that viral DNA could be the primary target of raltegravir ( RAL ), an efficient anti‐ HIV ‐1 drug, which acts by inhibiting integrase. To elucidate this mechanism, we conducted a comparative analysis of RAL and TB 11, a diketoacid abandoned as an anti‐ HIV ‐1 drug for its weak efficiency and marked toxicity, and tested the effects of the catalytic cofactor Mg 2+ (5 m m ) on drug‐binding properties. We used circular dichroism and fluorescence to determine drug affinities for viral DNA long terminal repeats ( LTR s) and peptides derived from the integrase active site and DNA retardation assays to assess drug intercalation into DNA base pairs. We found that RAL bound more tightly to LTR ends than did TB 11 (a diketo acid bearing an azido group) and that Mg 2+ significantly increased the affinity of both RAL and TB 11. We also observed a good relationship between drug binding with processed LTR and strand transfer inhibition. This unusual type of inhibition was caused by Mg 2+ ‐assisted binding of drugs to DNA substrate, rather than to enzyme. Notably, while RAL bound exclusively to the cleavable/cleaved site, TB 11 further intercalated into DNA base pairs and interacted with the integrase‐derived peptides. These unwanted binding sites explain the weaker bioavailability and higher toxicity of TB 11 compared with the more effective RAL .

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HIV-1 integrase inhibitors that compete with the target DNA substrate define a unique strand transfer conformation for integrase

Cited by 299 publications

References 35 publications

A naphthyridine carboxamide provides evidence for discordant resistance between mechanistically identical inhibitors of HIV-1 integrase

A naphthyridine carboxamide provides evidence for discordant resistance between mechanistically identical inhibitors of HIV-1 integrase

Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes

A targeted DNA substrate mechanism for the inhibition of HIV‐1 integrase by inhibitors with antiretroviral activity

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