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

Abstract

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...

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

“…The precise orientation of each inhibitor in the active site therefore may be the result of establishing the most favorable interactions of the specific substituents and accommodating the increased rigidity and bulkiness of the naphthyridine relative to the more flexible diketo acid. The suggestion that affinity and specificity is driven by the pendant groups in these molecules is consistent with studies that have shown that diketone analogs, which lack a carboxylate, bind with relatively high affinity even though they do not inhibit integrase enzymatic activity (8), and with the observation that pharmacophores in which substituents are extended in both directions can exhibit enhanced affinity (data not shown). In addition, although the respective structure-activity relationships are quite distinct, the adaptable diketo acid pharmacophore can be exploited as a template for inhibitors of a variety of divalent metal-dependent phosphotransferases, such as HIV-1 RNase H (29) and hepatitis C virus polymerase (30).…”

“…The 8-hydroxy-(1,6)-naphthyridine carboxamide conserves the spatial orientation of the chelating moieties in the diketo acid that have been shown to be essential for activity, and this spacing is consistent with the distance between the two active-site metals observed in the crystal structure of ASV integrase and other divalent metal-dependent phosphotransferases (8). Although in some cases resistance may be mediated by mutations that can affect the metal-coordinating residues in integrase and alter the geometry of the active site (e.g., mutations at residues 155 or 66), other residues associated with resistance are more distal to the metal-binding site.…”

“…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). These observations are consistent with the proposed mechanism of action and provide further validation for integrase as the target of their antiviral effect (5).…”

Human Immunodeficiency Virus Type 1 (HIV-1) Integrase: Resistance to Diketo Acid Integrase Inhibitors Impairs HIV-1 Replication and Integration and Confers Cross-Resistance to l -Chicoric Acid

“…L-CA requires one free carboxylic acid for activity (37), while L-731,988 requires both its free carboxylic acid and adjacent carbonyl groups (29), although data have demonstrated that the active conformation for L-731,988 in vivo is the enol and not the diketo state (65). No cocrystals of either inhibitor with the catalytic core domain of IN have been reported; however, L-CA has been modeled into the active site of IN in several independent studies (51,56).…”

The Terminal (Catalytic) Adenosine of the HIV LTR Controls the Kinetics of Binding and Dissociation of HIV Integrase Strand Transfer Inhibitors

“…The assembly of IN onto the viral LTR stimulates the high affinity binding of STIs, suggesting that the binding pocket for these inhibitors is derived from a specific structural arrangement of the nucleoprotein complex (3)(4)(5)(14)(15)(16)(17)(18)(19)(20)(21). Published models of HIV IN (19,22) suggest that these specific STIs bind to the IN active site in a pocket flanked by the end of the LTR such that the diketoacid moiety, or its mimic, is positioned to coordinate with both of the catalytic magnesium atoms (23)(24)(25) (14,19,22,(26)(27)(28)(29). Amino acid substitutions which confer resistance to STIs cluster around the IN active site (30)(31)(32)(33)(34)(35)(36)(37), and changes to the 5′ end of the LTR reduce the affinity of a specific STI for its binding site (16).…”