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PubMed1998

Cobalamin inhibition of HIV-1 integrase and integration of HIV-1 DNA into cellular DNA.

Our prior studies showed that certain cobalamins inhibit productive HIV-1 infection of primary cultures of blood lymphocytes and monocytes. We demonstrate here that this antiviral activity may be mediated by an inhibition of HIV-1 integrase, an enzyme required for productive infection. Purified recombinant HIV-1 integrase activity was inhibited in vitro by hydroxocobalamin (OH-Cbl), methylcobalamin (Me-Cbl), adenosylcobalamin (Ado-Cbl), and dicyanocobinamide (CN2-Cbi) with IC50 values of approximately 17, 17, 17, and 4 microM, respectively. The agents inhibited HIV-1 infection of cultured monocytes (IC50 values for OH-Cbl, Me-Cbl, Ado-Cbl, and CN2-Cbi of 6, 7, 4, and 1 microM, respectively) and of cultured lymphocytes (IC50 values of 60, 50, 60, and 11 microM, respectively). Experiments using cultured monocytes or lymphocytes demonstrated that OH-Cbl inhibited integration of HIV-1 DNA into cellular DNA. Thus, cobalamins and cobinamides represent novel inhibitors of HIV-1 integrase. These or related agents may be useful as anti-viral treatments that target HIV-1 integrase.

Base Sequence↗
PubMed2004

2,6-Bis(3,4,5-trihydroxybenzylydene) derivatives of cyclohexanone: novel potent HIV-1 integrase inhibitors that prevent HIV-1 multiplication in cell-based assays.

A number of 2,6-bisbenzylidenecyclohexane-1-one derivatives have been synthesized and tested as HIV-1 integrase (IN) inhibitors with the aim of obtaining compounds capable to elicit antiviral activity at non-cytotoxic concentrations in cell-based assays. 3,5-Bis(3,4,5-trihydroxybenzylidene)-4-oxocyclohexaneacetic acid (20d) resulted one of the most potent and selective derivatives in acutely infected MT-4 cells (EC(50) and CC(50) values of 2 and 40 microM, respectively). In enzyme assays with recombinant HIV-1 integrase (rIN), this compound proved able to inhibit both 3'-processing and disintegration with IC(50) values of 0.2 and 0.5 microM, respectively. In order to develop a model capable to predict the anti HIV-IN activity and useful to design novel derivatives, we performed a comparative molecular field analysis (CoMFA) like 3-D-QSAR. In our model the ligands were described quantitatively in the GRID program, and the model was optimized by selecting only the most informative variables in the GOLPE program. We found the predictive ability of the model to increase significantly when the number of variables was reduced from 20,925 to 1327. A Q(2) of 0.73 was obtained with the final model, confirming the predictive ability of the model. By studying the PLS coefficients in informative 3-D contour plots, ideas for the synthesis of new compounds could be generated.

Anti-HIV Agents↗
PubMed1998

L-chicoric acid, an inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase, improves on the in vitro anti-HIV-1 effect of Zidovudine plus a protease inhibitor (AG1350).

Combinations of anti-human immunodeficiency virus (HIV) drugs, including reverse transcriptase inhibitors and protease inhibitors, have proven immensely potent in the therapy of acquired immune deficiency syndrome (AIDS). To determine whether HIV integrase is a suitable target for combination therapy, the ability of an HIV integrase inhibitor, L-chicoric acid, to work in combination with a protease inhibitor and Zidovudine was tested in vitro. The addition of L-chicoric acid to either Zidovudine or protease inhibitor improved upon the observed anti-HIV activity of either compound alone. When all three drugs were combined, the anti-HIV activity was substantially better than either of the three compounds alone or any combination of two inhibitors. Doses of both Zidovudine and protease inhibitor could be reduced by more than 33% for an equivalent anti-HIV effect if L-chicoric acid was added. The improved anti-HIV activity was observed with a tissue culture adapted strain of HIV (HIV(LAI)) and with limited passage clinical isolates of HIV (HIV(R19) and HIV(R45)). These data demonstrate that a first generation HIV integrase inhibitor, L-chicoric acid, is at least additive in combination with existing multi-drug regimens and suggest that HIV integrase will be an excellent target for combination therapy of HIV infection.

Anti-HIV Agents↗
PubMed1999

Activity of recombinant HIV-1 integrase on mini-HIV DNA.

Integration of the human immunodeficiency virus type 1 (HIV-1) cDNA into the genome of a human cell is an essential step in the viral replication cycle. Understanding of the integration process has been facilitated by the development of in vitro assays using specific oligonucleotides and recombinant integrase. However, understanding of the biology of retroviral integration will require in vitro and in vivo model systems using long DNA substrates that mimic the HIV cDNA. We have now studied the activity of recombinant HIV-1 integrase on a linear 4.7 kb double-stranded DNA, containing flanking regions of approximately 200 bp that represent the intact ends of the HIV-1 long terminal repeat (LTR) sequences (mini-HIV). The strand transfer products of the integration reaction can be directly visualized after separation in agarose gels by ethidium bromide staining. The most prominent reaction product resulted from integration of one LTR end into another LTR end (U5 into U5 and U5 into U3). Sequence analysis of the reaction products showed them to be products of legitimate integration preceded by correct processing of the viral LTR ends. Hotspots for integration were detected. Electron microscopy revealed the presence of a range of reaction products resulting from single or multiple integration events. The binding of HIV-1 integrase to mini-HIV DNA was visualized. Oligomers of integrase seem to induce DNA looping whereby the enzyme often appears to be bound to the DNA substrate that adopts the structure of a three-site synapsis that is reminiscent of the Mu phage transposase complex.

Base Sequence↗
PubMed2005

Synthesis and biological evaluation of geminal disulfones as HIV-1 integrase inhibitors.

Integration of HIV-1 viral DNA into the host genome is carried out by HIV-integrase (IN) and is a critical step in viral replication. Although several classes of compounds have been reported to inhibit IN in enzymatic assays, inhibition is not always correlated with antiviral activity. Moreover, potent antiviral IN inhibitors such as the chicoric acids do not act upon the intended enzymatic target but behave as entry inhibitors instead. The charged nature of the chicoric acids contributes to poor cellular uptake, and these compounds are further plagued by rapid ester hydrolysis in vivo. To address these critical deficiencies, we designed neutral, nonhydrolyzable analogues of the chicoric acids. Herein, we report the synthesis, enzyme inhibition studies, and cellular antiviral data for a series of geminal disulfones. Of the 10 compounds evaluated, 8 showed moderate to high inhibition of IN in purified enzyme assays. The purified enzyme data correlated with antiviral assays for all but two compounds, suggesting alternative modes of inhibition. Time-of-addition studies were performed on these analogues, and the results indicate that they inhibit an early stage in the replication process, perhaps entry. In contrast, the most potent member of the correlative group shows behavior consistent with IN being the cellular target.

Anti-HIV Agents↗
PubMed2005

Constructing HIV-1 integrase tetramer and exploring influences of metal ions on forming integrase-DNA complex.

HIV-1 integrase (IN) is essential for the replication of HIV-1 in human cells. At present, the complete structure of complex IN-DNA has not been resolved. In this paper, a HIV-1 IN tetramer model was built with homology modeling and molecular dynamics simulation approach, in which two Mg2+ ions were reasonably located in each catalytic core domain. Moreover, it was found that the AB and CD chains of HIV-1 IN tetramer were different in the structures and metal ions of HIV-1 IN tetramer might have great influences on DNA locating on IN. These findings may provide a more complete structural basis for guiding drug discovery and revealing integration mechanism.

Catalytic Domain↗
PubMed2004

Expression of HIV-1 integrase in CEM cells inhibits HIV-1 replication.

BACKGROUND: HIV-1 integrase (IN) is an interesting target for the gene therapy of AIDS. Although the in vivo functions are not well characterized, it is thought that IN has pleiotropic effects and plays a central role in the interplay between the virus and the host cell. Expression of IN in mammalian cells has proven difficult. We have previously established a 293T-derived cell line that stably expresses high levels of HIV-1 IN from a synthetic gene. We now have constructed CEM-derived cell lines stably expressing the enzyme or its different domains and studied the impact of IN expression on HIV-1 replication. METHODS: The CEM cell lines were selected following transduction with a retroviral vector encoding the full-length IN, the N-terminal domain, the catalytic core or the C-terminal domain. Stable IN expression in CEM cell lines was verified by Western blotting. The impact of IN expression on HIV-1 replication and HIV-1 vector transduction was studied. RESULTS: A marked inhibitory effect on HIV-1 replication was observed in CEM cells expressing IN. Expression of IN interfered with both particle production and integration. Expression of the N-terminal domain alone was sufficient for the inhibiting of HIV-1 replication. CONCLUSIONS: Expression of IN in CEM cells inhibits HIV-1 replication by a cumulative inhibitory effect on integration and particle production, in accord with the known pleiotropic interactions of IN. The inhibition of HIV-1 replication in CEM cells expressing the N-terminal domain of IN may lead to a novel approach for the gene therapy of AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Application of CoMFA and CoMSIA 3D-QSAR and docking studies in optimization of mercaptobenzenesulfonamides as HIV-1 integrase inhibitors.

An essential step in the HIV life cycle is integration of the viral DNA into the host chromosome. This step is catalyzed by a 32-kDa viral enzyme HIV integrase (IN). HIV-1 IN is an important and validated target, and the drugs that selectively inhibit this enzyme, when used in combination with reverse transcriptase (RT) and protease (PR) inhibitors, are believed to be highly effective in suppressing the viral replication. IN catalyzes two discrete enzymatic processes referred to as 3' processing and DNA strand transfer. As a part of a study to optimize new lead molecules we previously identified from a series of 2-mercaptobenzenesulfonamides (MBSAs), we applied three-dimensional quantitative structure-activity relationship methods, comparative molecular field analysis (CoMFA), and comparative molecular similarity indices analysis (CoMSIA) to training sets of up to 66 compounds. Two different conformational templates were used: Conf-d, obtained from docking into the HIV-1 IN active site and Conf-s obtained by a systematic conformational search, using lead compounds 1 and 14, respectively. Reliable models of good predictive power were obtained after removal of compounds with high residuals. The Conf-s models tended to perform better than the Conf-d models. Cross-validated coefficients (q(2)) of up to 0.719 (strand transfer CoMSIA, Conf-s) regression coefficients (r(2)) of up to 0.932 (strand transfer CoMSIA, Conf-d) were obtained, with the number of partial least squares (PLS) components varying from 3 to 6, and the number of outliers being 4 in most of the models. Because all biological data were determined under exactly the same conditions using the same enzyme preparation, our predictive models are promising for drug optimization. Therefore, these results combined with docking studies were used to guide the rational design of new inhibitors. Further synthesis of 12 new analogues was undertaken, and these were used as a test set for validation of the quantitative structure-activity relationship (QSAR) models. For compounds with closely related structures, binding energies given by the FlexX scoring function correlated with HIV-1 IN inhibitory activity.

HIV Integrase↗
PubMed2006

Binding mode prediction of strand transfer HIV-1 integrase inhibitors using Tn5 transposase as a plausible surrogate model for HIV-1 integrase.

The crystal structure of Tn5 transposase-DNA complex was used in docking experiments to predict binding modes of HIV-1 integrase strand transfer inhibitors (INSTIs). In fact, the identification of HIV-1 integrase inhibitors from an in vitro screen using Tn5 transposase as the target has been recently reported. Our results suggest the utility of this protein as a useful surrogate model for IN and also for in silico screening, in the search for new potential INSTIs.

HIV Integrase↗
PubMed1998

Crystal structures of the catalytic domain of HIV-1 integrase free and complexed with its metal cofactor: high level of similarity of the active site with other viral integrases.

Human immunodeficiency virus (HIV) integrase is the enzyme responsible for insertion of a DNA copy of the viral genome into host DNA, an essential step in the replication cycle of HIV. HIV-1 integrase comprises three functional and structural domains: an N-terminal zinc-binding domain, a catalytic core domain and a C-terminal DNA-binding domain. The catalytic core domain with the F185H mutation has been crystallized without sodium cacodylate in a new crystal form, free and complexed with the catalytic metal Mg2+. The structures have been determined and refined to about 2.2 A. Unlike the previously reported structures, the three active-site carboxylate residues (D,D-35-E motif) are well ordered and both aspartate residues delineate a proper metal-binding site. Comparison of the active binding site of this domain with that of other members from the polynucleotidyl transferases superfamily shows a high level of similarity, providing a confident template for the design of antiviral agents.

Binding Sites↗
PubMed2000

Identification of HIV-1 integrase inhibitors via three-dimensional database searching using ASV and HIV-1 integrases as targets.

Integration of viral DNA into the host cell genome is a critical step in the life cycle of HIV. This essential reaction is catalyzed by integrase (IN) through two steps, 3'-processing and DNA strand transfer. Integrase is an attractive target for drug design because there is no known cellular analogue and integration is essential for successful replication of HIV. A computational three-dimensional (3-D) database search was used to identify novel HIV-1 integrase inhibitors. Starting from the previously identified Y3 (4-acetylamino-5-hydroxynaphthalene-2,7-disulfonic acid) binding site on the avian sarcoma virus integrase (ASV IN), a preliminary search of all compounds in the nonproprietary, open part of the National Cancer Institute 3-D database yielded a collection of 3100 compounds. A more rigorous scoring method was used to rescreen the 3100 compounds against both ASV IN and HIV-1 IN. Twenty-two of those compounds were selected for inhibition assays against HIV-1 IN. Thirteen of the 22 showed inhibitory activity against HIV-1 IN at concentrations less than 200 microM and three of them showed antiviral activities in HIV-1 infected CEM cells with effective concentrations (EC50) ranging from 0.8 to 200 microM. Analysis of the computer-generated binding modes of the active compounds to HIV-1 IN showed that simultaneous interaction with the Y3 site and the catalytic site is possible. In addition, interactions between the active compounds and the flexible loop involved in the binding of DNA by IN are indicated to occur. The structural details and the unique binding motif between the HIV-1 IN and its inhibitors identified in the present work may contribute to the future development of IN inhibitors.

Avian Sarcoma Viruses↗
PubMed2004

L-chicoric acid inhibits human immunodeficiency virus type 1 integration in vivo and is a noncompetitive but reversible inhibitor of HIV-1 integrase in vitro.

The human immunodeficiency virus (HIV) integrase (IN) must covalently join the viral cDNA into a host chromosome for productive HIV infection. l-Chicoric acid (l-CA) enters cells poorly but is a potent inhibitor of IN in vitro. Using quantitative real-time polymerase chain reaction (PCR), l-CA inhibits integration at concentrations from 500 nM to 10 microM but also inhibits entry at concentrations above 1 microM. Using recombinant HIV IN, steady-state kinetic analyses with l-CA were consistent with a noncompetitive or irreversible mechanism of inhibition. IN, in the presence or absence of l-CA, was successively washed. Inhibition of IN diminished, demonstrating that l-CA was reversibly bound to the protein. These data demonstrate that l-CA is a noncompetitive but reversible inhibitor of IN in vitro and of HIV integration in vivo. Thus, l-CA likely interacts with amino acids other than those which bind substrate.

Acetoacetates↗
PubMed2005

Comparison of multiple molecular dynamics trajectories calculated for the drug-resistant HIV-1 integrase T66I/M154I catalytic domain.

HIV-1 integrase (IN) is an essential enzyme for the viral replication and an interesting target for the design of new pharmaceuticals for multidrug therapy of AIDS. Single and multiple mutations of IN at residues T66, S153, or M154 confer degrees of resistance to several inhibitors that prevent the enzyme from performing its normal strand transfer activity. Four different conformations of IN were chosen from a prior molecular dynamics (MD) simulation on the modeled IN T66I/M154I catalytic core domain as starting points for additional MD studies. The aim of this article is to understand the dynamic features that may play roles in the catalytic activity of the double mutant enzyme in the absence of any inhibitor. Moreover, we want to verify the influence of using different starting points on the MD trajectories and associated dynamical properties. By comparison of the trajectories obtained from these MD simulations we have demonstrated that the starting point does not affect the conformational space explored by this protein and that the time of the simulation is long enough to achieve convergence for this system.

Biophysics↗
PubMed2002

Thalassiolins A-C: new marine-derived inhibitors of HIV cDNA integrase.

Human immunodeficiency virus (HIV) replication requires integration of viral cDNA into the host genome, a process mediated by the viral enzyme integrase. We describe a new series of HIV integrase inhibitors, thalassiolins A-C (1-3), isolated from the Caribbean sea grass Thalassia testudinum. The thalassiolins are distinguished from other flavones previously studied by the substitution of a sulfated beta-D-glucose at the 7-position, a substituent that imparts increased potency against integrase in biochemical assays. The most active of these molecules, thalassiolin A (1), displays in vitro inhibition of the integrase catalyzed strand transfer reaction (IC50=0.4 microM) and an antiviral IC50 of 30 microM. Molecular modeling studies indicate a favorable binding mode is probable at the catalytic core domain of HIV-1 integrase.

Binding Sites↗
PubMed2006

Design, synthesis, and biological evaluation of chicoric acid analogs as inhibitors of HIV-1 integrase.

A series of analogs of the potent HIV-1 integrase (HIV IN) inhibitor chicoric acid (CA) was designed with the intention of ameliorating some of the parent natural product's undesirable properties, in particular its toxicity, instability, and poor membrane permeability. More than 70 analogs were synthesized and assayed for three types of activity: (1) the ability to inhibit 3'-end processing and strand transfer reactions using recombinant HIV IN in vitro, (2) toxicity against the CD4+ lymphoblastoid cell line, MT2, and (3) anti-HIV activity against HIV(LAI). CA analogs lacking one of the carboxyl groups of CA and with 3,4,5-trihydroxycinnamoyl sidechains in place of the caffeoyl group of CA exhibited the most potent inhibition of HIV replication and end-processing activity. Galloyl-substituted derivatives also displayed very potent in vitro and in vivo activities, in most cases exceeding the inhibitory effects of CA itself. Conversely, analogous monocarboxy caffeoyl analogs exhibited only modest inhibition, while the corresponding 3,4-dihydroxybenzoyl-substituted compounds were devoid of activity.

Anti-HIV Agents↗
PubMed1999

Molecular dynamics studies on the HIV-1 integrase catalytic domain.

The HIV-1 integrase, which is essential for viral replication, catalyzes the insertion of viral DNA into the host chromosome, thereby recruiting host cell machinery into making viral proteins. It represents the third main HIV enzyme target for inhibitor design, the first two being the reverse transcriptase and the protease. Two 1-ns molecular dynamics simulations have been carried out on completely hydrated models of the HIV-1 integrase catalytic domain, one with no metal ions and another with one magnesium ion in the catalytic site. The simulations predict that the region of the active site that is missing in the published crystal structures has (at the time of this work) more secondary structure than previously thought. The flexibility of this region has been discussed with respect to the mechanistic function of the enzyme. The results of these simulations will be used as part of inhibitor design projects directed against the catalytic domain of the enzyme.

Amino Acid Sequence↗
PubMed2003

Four novel bis-(naphtho-gamma-pyrones) isolated from Fusarium species as inhibitors of HIV-1 integrase.

Integration of viral DNA into host cell DNA is an essential step in retroviral (HIV-1) replication and is catalyzed by HIV-1 integrase. HIV-1 integrase is a novel therapeutic target and is the focus of efforts to identify effective inhibitors that will prevent/or cure HIV infections. Four novel naphtho-gamma-pyrones, belonging to the chaetochromin and ustilaginoidin family, were discovered as inhibitors of HIV-1 integrase from the screening of fungal extracts using a recombinant in vitro assay. These compounds inhibit both the coupled and strand transfer activity of HIV-1 integrase with IC(50) values of 1-3 and 4-12 microM, respectively. The discovery, structure elucidation, chemical modification and the structure-activity relationship of these compounds are described.

Anti-HIV Agents↗
PubMed2005

A structural study of model peptides derived from HIV-1 integrase central domain.

The HIV-1 integrase (IN) catalyzes the integration of viral DNA in the human genome. In vitro the enzyme displays an equilibrium of monomers, dimers, tetramers and larger oligomers. However, its functional oligomeric form in vivo is not known. We report a study of the auto-associative properties of three peptides denoted K156, E156 and E159. These derive from the alpha4 helix of the IN catalytic core. The alpha4 helix is an amphipatic helix exposed at the surface of the protein and could be involved in the oligomerization process through its hydrophobic face. The peptides were obtained from the replacement of several amino acid residues by more helicogenic ones in the alpha4 helix peptide. K156 carries the basic residues Lys156 and Lys159, which have been shown important for the binding of IN to viral DNA. In E156 and E159 they are replaced with the acidic residue Glu. A fourth peptide K(E)156 obtained from the replacement of hydrophobic residues with Glu in K156 in order to abolish the auto-associative properties is used as a negative control. The capacity shown by peptides for alpha-helical formation is demonstrated by circular dichroism (CD) analysis performed in aqueous solution and in aqueous trifluoroethanol (TFE) mixtures. Both electrospray ionization mass spectrometry (ESI-MS) and glutaraldehyde chemical cross-linking show that peptides adopt different solvent-dependent equilibriums of monomers, dimers, trimers and tetramers. Oligomerization of peptides in aqueous solution is related to their ability to form helical structures. Addition of a small amount of TFE (<10%) stimulates helix stabilization and the interhelical hydrophobic contacts. Higher amounts of TFE alter the hydrophobic contacts and disrupt the oligomeric species. In addition to hydrophobic interactions, the patterns indicate that the biologically important Lys156 and Lys159 residues also participate in helix association. K(E)156 despite its ability to adopt a helical structure is unable to associate into oligomers, demonstrating the importance of hydrophobic contacts for oligomerization. Thus, the designed peptides provide us information on the functional properties of the alpha4 IN that seems to hold a dual role in DNA recognition and protein oligomerization.

Circular Dichroism↗