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HIV integrase strand transfer inhibitors (InSTIs) represent an important class of antiviral therapeutics with proven efficacy and excellent tolerability for the treatment of HIV infections. In 2007, Raltegravir became the first marketed strand transfer inhibitor pioneering the way to a first-line therapy for treatment-naïve patients. Challenges with this class of therapeutics remain, including frequency of the dosing regimen and the genetic barrier to resistance. To address these issues, research towards next-generation integrase inhibitors has focused on imparting potency against RAL-resistent mutants and improving pharmacokinetic profiles. Herein, we detail medicinal chemistry efforts on a novel class of 2-pyridinone aminal InSTIs, inpsired by MK-0536, which led to the discovery of important lead molecules for our program. Systematic optimization carried out at the amide and aminal positions on the periphery of the core provided the necessary balance of antiviral activity and physiochemical properties. These efforts led to a novel aminal lead compound with the desired virological profile and preclinical pharmacokinetic profile to support a once-daily human dose prediction., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Three integrase strand transfer inhibitors have now been approved for the treatment of HIV infection, raltegravir, cobicistat-boosted elvitegravir, and dolutegravir. Each of these agents selects for unique signature mutations; however, there can be significant cross resistance among all three drugs when multiple mutations are present or are presented in the context of different genetic backgrounds such as non B-subtypes. Many of the mutations that are associated with integrase inhibitor resistance have a profound effect on integrase function and viral replication and thus, while only one or two mutations may be sufficient to impact susceptibility, virologic failure and treatment-associated resistance have been infrequent with all three drugs to date., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This review will summarize the role of integrase in HIV-1 infection, the mechanism of integrase inhibitors and resistance with an emphasis on raltegravir (RAL), the first integrase inhibitor licensed to treat HIV-1 infection.

MK-2048 represents a prototype second-generation integrase strand transfer inhibitor (INSTI) developed with the goal of retaining activity against viruses containing mutations associated with resistance to first-generation INSTIs, raltegravir (RAL) and elvitegravir (EVG). Here, we report the identification of mutations (G118R and E138K) which confer resistance to MK-2048 and not to RAL or EVG. These mutations were selected in vitro and confirmed by site-specific mutagenesis. G118R, which appeared first in cell culture, conferred low levels of resistance to MK-2048. G118R also reduced viral replication capacity to approximately 1% that of the isogenic wild-type (wt) virus. The subsequent selection of E138K partially restored replication capacity to approximately 13% of wt levels and increased resistance to MK-2048 to approximately 8-fold. Viruses containing G118R and E138K remained largely susceptible to both RAL and EVG, suggesting a unique interaction between this second-generation INSTI and the enzyme may be defined by these residues as a potential basis for the increased intrinsic affinity and longer "off" rate of MK-2048. In silico structural analysis suggests that the introduction of a positively charged arginine at position 118, near the catalytic amino acid 116, might decrease Mg(2+) binding, compromising enzyme function and thus leading to the significant reduction in both integration and viral replication capacity observed with these mutations.

Integramide A is a 16-amino acid peptide inhibitor of the enzyme HIV-1 integrase. We have recently reported that the absolute stereochemistries of the dipeptide sequence near the C terminus are L-Iva(14)-D-Iva(15). Herein, we describe the syntheses of the natural compound and its D-Iva(14)-L-Iva(15) diastereomer, and the results of their chromatographic/mass spectrometric analyses. We present the conformational analysis of the two compounds and some of their synthetic intermediates of different main-chain length in the crystal state (by X-ray diffraction) and in solvents of different polarities (using circular dichroism, FTIR absorption, and 2D NMR techniques). These data shed light on the mechanism of inhibition of HIV-1 integrase, which is an important target for anti-HIV therapy.

The early events of HIV-1 replication are highlighted by reverse transcription and integration, and the reverse transcriptase and integrase enzymes are important therapeutic targets. Integration proceeds through a series of steps including assembly of integrase on the viral donor DNA ends, 3'-processing, and DNA strand transfer. First generation integrase assays typically included all biochemical reagents in solution where excess donor substrate could serve as the target for DNA strand transfer. These conditions, though valuable for understanding mechanistic aspects of HIV-1 integration, fell short of critical pharmacological designs as most early inhibitors were found to block assembly instead of enzyme function. Second generation designs, which decoupled assembly from DNA strand transfer, afforded the specificity required to identify clinically relevant compounds. Here, we describe versatile scintillation proximity-based assays whereby integrase is assembled onto donor DNA that is immobilized onto the surface of beads. Immobilization and subsequent washing of excess donor DNA eliminates its potential to serve as target DNA, allowing investigation of the DNA strand transfer reaction in isolation. Assembled complexes can be used in high-throughput DNA strand transfer assays if radio labeled target DNA is employed or in integrase binding assays using a suitable radioligand.

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.

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%).

Background: We evaluated the efficacy of raltegravir and the development of viral resistance in two identical trials involving patients who were infected with human immunodeficiency virus type 1 (HIV-1) with triple-class drug resistance and in whom antiretroviral therapy had failed., Methods: We conducted subgroup analyses of the data from week 48 in both studies according to baseline prognostic factors. Genotyping of the integrase gene was performed in raltegravir recipients who had virologic failure., Results: Virologic responses to raltegravir were consistently superior to responses to placebo, regardless of the baseline values of HIV-1 RNA level; CD4 cell count; genotypic or phenotypic sensitivity score; use or nonuse of darunavir, enfuvirtide, or both in optimized background therapy; or demographic characteristics. Among patients in the two studies combined who were using both enfuvirtide and darunavir for the first time, HIV-1 RNA levels of less than 50 copies per milliliter were achieved in 89% of raltegravir recipients and 68% of placebo recipients. HIV-1 RNA levels of less than 50 copies per milliliter were achieved in 69% and 80% of the raltegravir recipients and in 47% and 57% of the placebo recipients using either darunavir or enfuvirtide for the first time, respectively. At 48 weeks, 105 of the 462 raltegravir recipients (23%) had virologic failure. Genotyping was performed in 94 raltegravir recipients with virologic failure. Integrase mutations known to be associated with phenotypic resistance to raltegravir arose during treatment in 64 patients (68%). Forty-eight of these 64 patients (75%) had two or more resistance-associated mutations., Conclusions: When combined with an optimized background regimen in both studies, a consistently favorable treatment effect of raltegravir over placebo was shown in clinically relevant subgroups of patients, including those with baseline characteristics that typically predict a poor response to antiretroviral therapy: a high HIV-1 RNA level, low CD4 cell count, and low genotypic or phenotypic sensitivity score. (ClinicalTrials.gov numbers, NCT00293267 and NCT00293254.), (2008 Massachusetts Medical Society)

A series of 4-oxo-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine-2-carboxamides was synthesized and tested for their inhibition of HIV-1 integrase catalytic activity and HIV-1 replication in cells. Structure-activity studies around lead compound 5 indicated that a coplanar relationship of metal-binding heteroatoms provides optimal binding to the integrase active site. Identification of potency-enhancing substituents and adjustments in lipophilicity provided 17b which inhibits integrase-catalyzed strand transfer with an IC(50) value of 74 nM and inhibits HIV-1 replication in cell culture in the presence of 50% normal human serum with an IC(95) value of 63 nM.

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.

HIV-1 integrase is a critical enzyme for replication of HIV, and its inhibition is one of the most promising new drug targets for anti-retroviral therapy with potentially significant advantages over existing therapies. In this Note, the isolation, structure elucidation, and absolute stereochemistry of integrasone, a novel polyketide, derived from an unidentified sterile mycelium have been described. This bicyclic dihydroxy epoxide lactone inhibited the strand transfer reaction of HIV-1 integrase with an IC(50) of 41 microM.

HIV-1 integrase is a critical enzyme for replication of HIV, and its inhibition is one of the most promising new drug strategies for anti-retroviral therapy, with potentially significant advantages over existing therapies. In this report, a series of HIV-1 inhibitors isolated from the organic extract of fermentations from terrestrial fungi is described. These fungal species, belonging to a variety of genera, were collected from throughout the world following the strict guidelines of Rio Convention on Biodiversity. The polyketide- and terpenoid-derived inhibitors are represented by two naphthoquinones, a biphenyl and two triphenyls, a benzophenone, four aromatics with or without catechol units, a linear aliphatic terpenoid, a diterpenoid, and a sesterterpenoid. These compounds inhibited the coupled and strand-transfer reaction of HIV-1 integrase with an IC(50) value of 0.5-120 micro M. The bioassay-directed isolation, structure elucidation, and HIV-1 inhibitory activity of these compounds are described.

Integracides, 4,4-dimethylergostane triterpenoids, are inhibitors of HIV-1 integrase, a critical enzyme in replication of HIV-1. The chemistry and structure-activity relationship of integracide B and related natural products are described. A charged group, e.g., a sulfate, carboxyl, or amino, is required for the HIV-1 integrase activity. These compounds showed HIV-1 integrase activity with IC(50) values in the range 4.8-15 muM and exhibited antiviral activity in a viral spread assay, but with only a small or no therapeutic window.

HIV-1 integrase is a critical enzyme in the replication of HIV-1. It is absent in the host cells and therefore is a good target for treatment of HIV-1 infections. Integracides are members of the tetracyclic triterpenoids family that were isolated from the fermentation broth of a Fusarium sp. Integracide A, a sulfated ester, exhibited significant inhibitory activity against strand transfer reaction of HIV-1 integrase. The discovery, structure elucidation including single crystal X-ray structure and HIV-1 inhibitory activity of these compounds are described.

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.

[structure: see text]. Integramides A and B are two novel 16-mer linear peptides rich in C(alpha)-methyl amino acids that were isolated from fungal extracts of Dendrodochium sp. by employing a bioassay-guided isolation procedure using recombinant HIV-1 integrase. The structure and stereochemistry were elucidated by a combination of 2D NMR and ESI- and FAB-MS including MS/MS studies and by Marfey's method. Integramides A and B inhibited the coupled reaction of HIV-1 integrase with IC50 values of 17 and 10 microM, respectively.

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.

Integrase is essential for human immunodeficiency virus-type 1 (HIV-1) replication; however, potent inhibition of the isolated enzyme in biochemical assays has not readily translated into antiviral activity in a manner consistent with inhibition of integration. In this report, we describe diketo acid inhibitors of HIV-1 integrase that manifest antiviral activity as a consequence of their effect on integration. The antiviral activity of these compounds is due exclusively to inhibition of one of the two catalytic functions of integrase, strand transfer.

Previous in vitro analyses have shown that the human immunodeficiency virus type 1 (HIV-1) integrase uses either manganese or magnesium to assemble as a stable complex on the donor substrate and to catalyze strand transfer. We now demonstrate that subsequent to assembly, catalysis of both 3' end processing and strand transfer requires a divalent cation cofactor and that the divalent cation requirements for assembly and catalysis can be functionally distinguished based on the ability to utilize calcium and cobalt, respectively. The different divalent cation requirements manifest by these processes are exploited to uncouple assembly and catalysis, thus staging the reaction. Staged 3' end processing and strand transfer assays are then used in conjunction with exonuclease III protection analysis to investigate the effects of integrase inhibitors on each step in the reaction. Analysis of a series of related inhibitors demonstrates that these types of compounds affect assembly and not either catalytic process, therefore reconciling the apparent disparate results obtained for such inhibitors in assays using isolated preintegration complexes. These studies provide evidence for a distinct role of the divalent cation cofactor in assembly and catalysis and have implications for both the identification and characterization of integrase inhibitors.