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PubMed1998

Dicaffeoylquinic and dicaffeoyltartaric acids are selective inhibitors of human immunodeficiency virus type 1 integrase.

Current pharmacological agents for human immunodeficiency virus (HIV) infection include drugs targeted against HIV reverse transcriptase and HIV protease. An understudied therapeutic target is HIV integrase, an essential enzyme that mediates integration of the HIV genome into the host chromosome. The dicaffeoylquinic acids (DCQAs) and the dicaffeoyltartaric acids (DCTAs) have potent activity against HIV integrase in vitro and prevent HIV replication in tissue culture. However, their specificity against HIV integrase in cell culture has been questioned. Thus, the ability of the DCQAs and DCTAs to inhibit binding of HIV type 1 (HIV-1) gp120 to CD4 and their activities against HIV-1 reverse transcriptase and HIV RNase H were studied. The DCQAs and DCTAs inhibited HIV-1 integrase at concentrations between 150 and 840 nM. They inhibited HIV replication at concentrations between 2 and 12 microM. Their activity against reverse transcriptase ranged from 7 microM to greater than 100 microM. Concentrations that inhibited gp120 binding to CD4 exceeded 80 microM. None of the compounds blocked HIV-1 RNase H by 50% at concentrations exceeding 80 microM. Furthermore, when the effects of the DCTAs on reverse transcription in acutely infected cells were measured, they were found to have no activity. Therefore, the DCQAs and DCTAs exhibit > 10- to > 100-fold specificity for HIV integrase, and their activity against integrase in biochemical assays is consistent with their observed anti-HIV activity in tissue culture. Thus, the DCQAs and DCTAs are a potentially important class of HIV inhibitors that act at a site distinct from that of current HIV therapeutic agents.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Inhibition of human immunodeficiency virus type 1 integrase by a hydrophobic cation: the phenanthroline-cuprous complex.

The human immunodeficiency virus type 1 integrase (HIV-1 integrase) is required for integration of a double-stranded DNA copy of the viral RNA genome into a host chromosome and for HIV replication. We have examined the effects of 2:1 1,10-phenanthroline-cuprous complexes on purified HIV-1 integrase. Although the uncomplexed phenanthrolines are not active below 100 microM, four of the cuprous complexes (neocuproine, 4-phenyl neocuproine, 2,3,4,7,8,9-hexamethyl phenanthroline, and 2,3,4,7,8-pentamethyl phenanthroline) have a 50% inhibitory concentration (IC50) for integration ranging between 1 and 10 microM. Disintegration is also inhibited by these phenanthroline-cuprous complexes at slightly higher concentrations (between 10 and 40 microM). Dialysis experiments showed that the inhibition is reversible and kinetic analyses revealed that the mode of inhibition by these cuprous complexes appears to be noncompetitive with respect to the substrate DNA. Consistent with these findings, binding assays demonstrate that, although these complexes can inhibit binding to DNA at high concentrations, they do not inhibit binding of integrase to the DNA substrate at their IC50 values. Because these complexes do not bind to B-DNA below 50 microM, inhibition via binding to a specific region on the enzyme was examined. Using deletion mutants of integrase, it was determined that neither the amino-terminal (zinc finger) nor the carboxy-terminal (DNA-binding) integrase domain is required for inhibition by the phenanthroline-cuprous complexes. Therefore, inhibition via binding to the enzyme catalytic core or to the interface between the enzyme and a noncanonical DNA structure generated during the enzymatic reaction is the probable mechanism. These results suggest the utility of neocuproine-cuprous complexes in developing inhibitors of HIV-1 integrase as well as probes for drug-binding sites and enzymatic reaction mechanism.

Base Sequence↗
PubMed2002

CoMFA and CoMSIA 3D QSAR and docking studies on conformationally-restrained cinnamoyl HIV-1 integrase inhibitors: exploration of a binding mode at the active site.

Anti-HIV (human immunodeficiency virus) drug discovery has been increasingly focusing on HIV integrase (IN) as a potential therapeutic target. This enzyme is required for the integration of reverse transcribed proviral DNA into the host cell's genome and is essential for the propagation of the HIV life cycle. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) three-dimensional quantitative structure-activity relationship (3D QSAR) studies and docking simulations were conducted on a series of potent conformationally restrained cinnamoyl inhibitors of HIV-1 IN (Artico; et al. J. Med. Chem. 1998, 41, 3948-3960). Predictive 3D QSAR models were established using SYBYL multifit molecular alignment rule, which had conventional r(2) and cross-validated coeffiecient (q(2)) values up to 0.981 and 0.721 for CoMFA and 0.975 and 0.804 for CoMSIA, respectively. These models were validated by an external test set (Burke; et al. J. Med. Chem. 1995, 38, 4171-4178). CoMFA and CoMSIA 3D QSAR models were also derived using a molecular alignment obtained by docking the compounds into the active site of HIV IN. These latter models were comparable to multifit-derived models in terms of relative descriptor field contributions and the partial least squares (PLS) contour maps. The CoMSIA 3D QSAR models performed better than the CoMFA models. The superior performance of CoMSIA was attributed to the large contribution of hydrogen-bonding interactions to the inhibitory activity differences among the compounds. This was supported by FlexX binding energy scores that correlated well with the inhibitory activity differences between hydroxylated compounds and their corresponding methoxy or deoxy counterparts. The CoMFA and CoMSIA PLS contour maps and MOLCAD-generated active site electrostatic, lipophilicity, and hydrogen-bonding potential surface maps, as well as the docking results, were integrated to propose a binding mode for the cinnamoyl inhibitors at the active site of HIV-1 IN.

Binding Sites↗
PubMed2005

Targeting HIV-1 integrase with aptamers selected against the purified RNase H domain of HIV-1 RT.

Several in vitro strategies have been developed to selectively screen for nucleic acid sequences that bind to specific proteins. We previously used the SELEX procedure to search for aptamers against HIV-1 RNase H activity associated with reverse transcriptase (RT) and human RNase H1. Aptamers containing G-rich sequences were selected in both cases. To investigate whether the interaction with G-rich oligonucleotides (ODNs) was a characteristic of these enzymes, a second in vitro selection was performed with an isolated RNase H domain of HIV-1 RT (p15) as a target and a new DNA library. In this work we found that the second SELEX led again to the isolation of G-rich aptamers. But in contrast to the first selection, these latter ODNs were not able to inhibit the RNase H activity of either the p15 domain or the RNase H embedded in the complete RT. On the other hand, the aptamers from the first SELEX that were inhibitors of the RT-associated RNase H did not inhibit the activity of the isolated p15 domain. This suggests that the active conformation of both RNase H domains is different according to the presence or absence of the DNA polymerase domain. HIV-1 RNase H and integrase both belong to the phosphotransferase family and share structural similarities. An interesting result was obtained when the DNA aptamers initially raised against p15 RNase H were assayed against HIV-1 integrase. In contrast to RNase H, the HIV-1 integrase was inhibited by these aptamers. Our results point out that prototype structures can be exploited to develop inhibitors of two related enzymes.

Aptamers, Nucleotide↗
PubMed2006

A potent and orally active HIV-1 integrase inhibitor.

A 1,6-naphthyridine inhibitor of HIV-1 integrase has been discovered with excellent inhibitory activity in cells, good pharmacokinetics, and an excellent ability to inhibit virus with mutant enzyme.

Administration, Oral↗
PubMed2000

Anti-HIV-1 integrase drugs: how far from the shelf?

Chemotherapy of HIV-1 infection/AIDS currently employs inhibitors of two products of the viral pol gene, the reverse transcriptase and protease enzymes. However, a third product of the pol gene is essential for retroviral multiplication, the integrase. As no cellular homologue of HIV integrase has been described, potential inhibitors could be relatively nontoxic. Development of HIV-1 integrase inhibitors could have favorable implication for combination therapy, including potential synergy with currently available inhibitors, as well as prevention of the chronic carrier state and the emergence of resistant mutants. Although several classes of putative integrase inhibitors that been described, still no clinically useful anti-integration drugs are available. It is the structural and functional complexity of the integration process together with the limitations of the available in vitro assays that has made it problematic to develop inhibitors of the HIV integrase. In this review we summarize current knowledge concerning the biology of this enzyme and of the integration process, and discuss major classes representatives of integrase inhibitors considering the obstacles to the development of true anti-integrase drugs.

Animals↗
PubMed1998

Three new structures of the core domain of HIV-1 integrase: an active site that binds magnesium.

HIV-1 integrase is an essential enzyme in the life cycle of the virus, responsible for catalyzing the insertion of the viral genome into the host cell chromosome; it provides an attractive target for antiviral drug design. The previously reported crystal structure of the HIV-1 integrase core domain revealed that this domain belongs to the superfamily of polynucleotidyltransferases. However, the position of the conserved catalytic carboxylic acids differed from those observed in other enzymes of the class, and attempts to crystallize in the presence of the cofactor, Mg2+, were unsuccessful. We report here three additional crystal structures of the core domain of HIV-1 integrase mutants, crystallized in the presence and absence of cacodylate, as well as complexed with Mg2+. These three crystal forms, containing between them seven independent core domain structures, demonstrate the unambiguous extension of the previously disordered helix alpha4 toward the amino terminus from residue M154 and show that the catalytic E152 points in the general direction of the two catalytic aspartates, D64 and D116. In the vicinity of the active site, the structure of the protein in the absence of cacodylate exhibits significant deviations from the previously reported structures. These differences can be attributed to the modification of C65 and C130 by cacodylate, which was an essential component of the original crystallization mixture. We also demonstrate that in the absence of cacodylate this protein will bind to Mg2+, and could provide a satisfactory platform for binding of inhibitors.

Binding Sites↗
PubMed1997

The solution structure of the amino-terminal HHCC domain of HIV-2 integrase: a three-helix bundle stabilized by zinc.

BACKGROUND: Integrase mediates a crucial step in the life cycle of the human immunodeficiency virus (HIV). The enzyme cleaves the viral DNA ends in a sequence-dependent manner and couples the newly generated hydroxyl groups to phosphates in the target DNA. Three domains have been identified in HIV integrase: an amino-terminal domain, a central catalytic core and a carboxy-terminal DNA-binding domain. The amino-terminal region is the only domain with unknown structure thus far. This domain, which is known to bind zinc, contains a HHCC motif that is conserved in retroviral integrases. Although the exact function of this domain is unknown, it is required for cleavage and integration. RESULTS: The three-dimensional structure of the amino-terminal domain of HIV-2 integrase has been determined using two-dimensional and three-dimensional nuclear magnetic resonance data. We obtained 20 final structures, calculated using 693 nuclear Overhauser effects, which display a backbone root-mean square deviation versus the average of 0.25 A for the well defined region. The structure consists of three alpha helices and a helical turn. The zinc is coordinated with His 12 via the N epsilon 2 atom, with His16 via the N delta 1 atom and with the sulfur atoms of Cys40 and Cys43. The alpha helices form a three-helix bundle that is stabilized by this zinc-binding unit. The helical arrangement is similar to that found in the DNA-binding domains of the trp repressor, the prd paired domain and Tc3A transposase. CONCLUSION: The amino-terminal domain of HIV-2 integrase has a remarkable hybrid structure combining features of a three-helix bundle fold with a zinc-binding HHCC motif. This structure shows no similarity with any of the known zinc-finger structures. The strictly conserved residues of the HHCC motif of retroviral integrases are involved in metal coordination, whereas many other well conserved hydrophobic residues are part of the protein core.

Amino Acid Sequence↗
PubMed2006

Anti-HIV therapy: Current and future directions.

Although combinations of drugs that target the HIV reverse transcriptase and protease enzymes have clearly revolutionized the treatment of HIV/AIDS, problems with these agents, such as viral escape mutants, persistence of viral reservoirs, poor patient compliance due to complicated regimens, and toxic side effects, have emphasized the need for development of new drugs with novel mechanisms of action, as well as an HIV vaccine. Recently two new classes of drugs have been identified that interfere with the membrane fusion reaction required for HIV entry of target cells. Two such agents, T-20 (enfuvirtide) and T-1249, which have been approved by the Food and Drug Administration (FDA), block the action of the fusogenic envelope glycoprotein gp41. Others target the HIV coreceptors CCR5 and CXCR4, and are now in clinical trials. Also under development are novel agents that target the HIV integrase and HIV regulatory gene products as well as immunomodulators such as IL-12 and IL-2. This article will focus on these and other novel approaches to HIV therapeutics.

Anti-HIV Agents↗
PubMed2002

Structural determinants for HIV-1 integrase inhibition by beta-diketo acids.

Among all the HIV-1 integrase inhibitors, the beta-diketo acids (DKAs) represent a major lead in anti-HIV-1 integrase drug design. These derivatives inhibit the integration reaction in vitro with a strong specificity for the 3'-end joining step. They are also antiviral and inhibit integration in vivo. The aim of the present study has been to investigate the molecular interactions between DKAs and HIV-1 integrase. We have compared 5CITEP with one of the most potent DKAs reported by the Merck group (L-708,906) and found that 5CITEP inhibits 3'-processing at concentrations where L-708,906 is only active on strand transfer. We also report a novel bifunctional DKA derivative that inhibits 3'-processing even more effectively than 5CITEP. The interactions of these inhibitors with the viral DNA donor ends have been studied by performing experiments with oligonucleotides containing defined modifications. We propose that the bifunctional DKA derivative binds to both the acceptor and donor sites of HIV-1 integrase, whereas the monofunctional L-708,906 derivative binds selectively to the acceptor site.

Acetoacetates↗
PubMed2002

[Studies on the inhibition of polyhydroxylated aromatic compounds against HIV-1 integrase].

AIM: Three major enzymes of HIV-1, reverse transcriptase (RT), protease (PR), and integrase (IN), are important targets for anti-HIV drugs. Nine RT and five PR inhibitors have been effectively used in treatment of AIDS patients. In order to find active integrase inhibitors, twenty polyhydroxylated aromatic compounds were tested. METHODS: ELISA method was used to test the integrase activity. The synthesized donor substrate oligonucleotide representing the HIV-1 U5LTR was immobilized onto Covalink polystyrene microtiter plates, and a synthesized biotinlated 20 bp oligonucleotide was used as the target substrate. The products were detected and quantified by a colorimetric avidin-linked alkaline phosphatase reporter system. RESULTS: Compound NQ-2 was found to inhibit HIV-1 integrase with the IC50 of 78.5 mumol.L-1 by ELISA method. Its novel analogue NQ-3 was found to be 2 fold more potent on HIV intrgrase than NQ-2, IC50 was 37.2 mumol.L-1. The IC50s of NQ-2 and NQ-3 to inhibit the 3'-pro + assembly activity of integrase were 96.94 mumol.L-1 and 8.48 mumol.L-1; to inhibit assembly activity were 168 and 6.9 mumol.L-1 and to inhibit strand-transfer activity were 49.8 and 1.1 mumol.L-1, respectively. Compound NQ-2 mostly inhibited the strand transfer activity of HIV-1 integrase. Compound NQ-3 inhibited both the assembly and strand-transfer with high activities. CONCLUSION: Naphthoquinone compound NQ-3 was found to be a novel HIV integrase inhibitor which warrants further study. Uncoupled ELISA HIV integrase assay is shown to be useful to screen HIV-1 integrase inhibitors.

Coumarins↗
PubMed2004

HIV-1 integrase: a target for new AIDS chemotherapeutics.

Since the beginning of the HIV epidemic almost 70 million people have been infected with HIV. It is estimated that 42 million people are currently living with HIV/AIDS. The spread of HIV continues throughout the world and current estimates indicate that in 2002, 5 million people were newly infected with HIV and 3 million people died. Current treatments employ a combination of therapeutic agents that target the viral reverse transcriptase and protease enzymes and viral entry. However the clinical benefit of these agents is often limited due to issues of regimen compliance, significant side effects, and the emergence of viral strains that are drug resistant. The introduction of novel agents that interfere with alternate stages in the viral life cycle represent potential solutions to these problems. The integration of the HIV genome into the cellular chromosome, a process catalyzed by the viral enzyme integrase, has been shown to be essential for viral replication. Since HIV integrase has no direct cellular counterpart it presents itself as an attractive target for therapeutic intervention. This review summarizes recent and promising developments both in the HIV integrase field and the global quest for therapeutically useful inhibitors of HIV integrase.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Linker-modified quinoline derivatives targeting HIV-1 integrase: synthesis and biological activity.

A novel series of HIV-1 integrase inhibitors was synthesized and tested in both in vitro and ex vivo assays. These inhibitors are featured by the presence of a quinoline subunit and an ancillary aromatic ring linked by functionalized spacers such as amide, hydrazide, urea and 1-hydroxyprop-1-en-3-one moiety. Amide derivatives are the most promising ones and could serve as leads for further developments.

Cell Line↗
PubMed2006

Biotinylated biphenyl ketone-containing 2,4-dioxobutanoic acids designed as HIV-1 integrase photoaffinity ligands.

The diketo acid (DKA) class of HIV-1 integrase inhibitors are thought to function by chelating divalent metal ions within the enzyme catalytic center. However, differences in mutations conferring resistance among sub-families of DKA inhibitors suggest that multiple binding orientations may exist. In order to facilitate identification of DKA-binding sites, biotin-tagged biphenyl ketone-containing 2,4-dioxobutanoic acids were prepared as DKA photoaffinity probes. Introduction of biotin was obtained by means of Huisgen [3+2] cycloaddition 'click chemistry.' Two photoprobes, 5a and 5b, were prepared bearing short and long linker segments, respectively, between the biotin and DKA nucleus. The greatest inhibitory potency was shown by 5b, which inhibited 3'-processing and strand transfer reactions with IC50 values of > 333 microM and 12.4 microM, respectively. In cross-linking assays designed to measure disruption of substrate DNA binding, the photoprobes behaved similarly to a reference DKA inhibitor. Analogues 5a and 5b represent novel photoaffinity ligands, which may be useful in clarifying the HIV-1 binding interactions of DKA inhibitors.

Acetoacetates↗
PubMed2004

Isolation, structure, and HIV-1-integrase inhibitory activity of structurally diverse fungal metabolites.

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.

Alkenes↗
PubMed2006

Effect of substitution on novel tricyclic HIV-1 integrase inhibitors.

A series of novel tricyclic inhibitors of HIV-1 integrase enzyme was prepared. The effect of substitution at C-6 of the 9-hydroxy-6,7-dihydropyrrolo[3,4-g]quinolin-8-one compounds was studied in vitro. Inhibitors with small side chains at C-6 were generally well tolerated by the enzyme, and the physicochemical properties of the inhibitors were improved by substitution of a small alkyl group at this position. A second series of analogs bearing a sulfamate at the C-5 position with various C-6 substituents were prepared to explore the interplay between the two groups. The SAR of the two classes are not parallel; modification at C-5 impacts the effect of substitutions at C-6.

HIV Integrase↗
PubMed2004

Rational design and synthesis of novel dimeric diketoacid-containing inhibitors of HIV-1 integrase: implication for binding to two metal ions on the active site of integrase.

Discovery of diketoacid-containing compounds as HIV-1 integrase (IN) inhibitors played a major role in validating this enzyme as an important target for the development of therapeutics against HIV infection. In fact, S-1360, the first clinically used IN inhibitor containing a triazole ring as a bioisostere of a carboxylic acid moiety belongs to this class of compounds. To understand the role of divalent metal-chelating in the inhibition of IN (J. Med. Chem. 2002, 45, 5661-5670), we designed and synthesized a series of novel dimeric diketo-containing compounds with the notion that such dimeric compounds may simultaneously bind to two divalent metal ions on the active site of IN. We rationalized that the two diketo subunits separated by uniquely designed linkers can potentially chelate two metal ions that are either provided from one IN active site or two active sites juxtaposed together in a higher order tetramer. Herein, we show that all the new compounds are highly potent against purified IN with varied selectivity for strand transfer, and that some of the analogues exert potent inhibition of the cytopathic effect of HIV-1 in infected CEM cells. This study represents the first attempt to rationally target two divalent metal ions on the active site of IN and may have potential implications for the design of second generation diketoacid-containing class of inhibitors.

Binding Sites↗