Synthesis and HIV-1 inhibitory activities of dicaffeoyl and digalloyl esters of quinic acid derivatives.

Twenty analogues of the anti-HIV-1 integrase (IN) inhibitors dicaffeoylquinic acids (DCQAs) were prepared. Their IC(50) values for 3'-end processing and strand transfer against recombinant HIV-1IN were determined in vitro, and their cell toxicities and EC(50) against HIV-1 were measured in cells (ex vivo). Acetylated or benzylated and/or with cyclohexylidene group compounds exhibited no inhibition of integration in biochemical assays or viral replication in HIV-infected cells, with the exception of 16 and 36. Removal of these groups, however, correlated with potent inhibition. Compounds 19, 31, and 38, all digalloyls, exhibited the most robust inhibitory performance in biochemical assays as well as in cell culture and less toxicity than other molecules in the current study.

Atomic force microscopy imaging of retroviruses: human immunodeficiency virus and murine leukemia virus.

Retroviruses are membrane-enveloped, RNA-containing viruses that produce a wide range of threatening diseases in higher animals. Among these are human immunodeficiency virus (HIV), which produces acquired immune deficiency syndrome (AIDS) in humans, and murine leukemia virus (MuLV), which produces leukemias in rodents. We have obtained the first atomic force microscopy (AFM) images of these two retroviruses, both isolated from culture media and emerging from infected cell surfaces. The HIV virions are 127 nm diameter on average, and those of MuLV are 145 nm, although there are wide distributions about the means. The AFM images show the arrangement of the envelope protein, responsible for host cell entry, on the surfaces of both virions. Disruption of the viruses using detergents or physical means allowed us to visualize interior structures, including the outer shells of both MuLV and HIV, the cores of MuLV, and the nucleic acid of HIV complexed with core proteins. Using immunolabeling techniques borrowed from electron microscopy, we were able to demonstrate the binding of gold-labeled antibodies directed against the envelope protein of MuLV. The AFM images are revealing, not only in terms of surface topology, but in terms of interior features as well, and they reveal the eccentricities and uniqueness of individual virus particles rather than yielding the average member of the population. Further application of AFM to viruses associated with other pathologies may ultimately have a significant impact on the diagnosis and treatment of virus-promoted diseases.

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.

The diketo acids are potent inhibitors of human immunodeficiency virus (HIV) integrase (IN). Mutations in IN, T66I, S153Y, and M154I, as well as T66I-S153Y and T66I-M154I double mutations, confer resistance to diketo acids (D. J. Hazuda et al., Science 287:646-650, 2000). The effects of these IN mutations on viral replication, enzymatic activity, and susceptibility to other HIV inhibitors are reported herein. By immunofluorescence assay and real-time PCR, all mutant viruses demonstrated a modest delay in viral spread compared to that of reference HIV. These viruses also showed a statistically significant defect in integration without defects in reverse transcription. Recombinant IN containing S153Y, T66I, and M154I-T66I mutations had an approximately twofold decrease in both disintegration and 3'-end-processing-strand transfer activities in vitro. In contrast, IN containing M154I demonstrated a greater than twofold increase in specific activity in both reactions. All mutant HIVs were resistant to l-chicoric acid, a dicaffeoyltartaric acid IN inhibitor, both in tissue culture and in biochemical assays, yet remained susceptible to the reverse transcriptase inhibitors zidovudine and nevirapine. Thus, IN mutations conferring resistance to the diketo acids can yield integration defects, attenuated catalysis in vitro, and cross-resistance to l-chicoric acid.

Atomic force microscopy investigation of human immunodeficiency virus (HIV) and HIV-infected lymphocytes.

Isolated human immunodeficiency virus (HIV) and HIV-infected human lymphocytes in culture have been imaged for the first time by atomic force microscopy (AFM). Purified virus particles spread on glass substrates are roughly spherical, reasonably uniform, though pleomorphic in appearance, and have diameters of about 120 nm. Similar particles are also seen on infected cell surfaces, but morphologies and sizes are considerably more varied, possibly a reflection of the budding process. The surfaces of HIV particles exhibit "tufts" of protein, presumably gp120, which do not physically resemble spikes. The protein tufts, which number about 100 per particle, have average diameters of about 200 A, but with a large variance. They likely consist of arbitrary associations of small numbers of gp120 monomers on the surface. In examining several hundred virus particles, we found no evidence that the gp120 monomers form threefold symmetric trimers. Although >95% of HIV-infected H9 lymphocytic cells were producing HIV antigens by immunofluorescent assay, most lymphocytes displayed few or no virus on their surfaces, while others were almost covered by a hundred or more viruses, suggesting a dependence on cell cycle or physiology. HIV-infected cells treated with a viral protease inhibitor and their progeny viruses were also imaged by AFM and were indistinguishable from untreated virions. Isolated HIV virions were disrupted by exposure to mild neutral detergents (Tween 20 and CHAPS) at concentrations from 0.25 to 2.0%. Among the products observed were intact virions, the remnants of completely degraded virions, and partially disrupted particles that lacked sectors of surface proteins as well as virions that were split or broken open to reveal their empty interiors. Capsids containing nucleic acid were not seen, suggesting that the capsids were even more fragile than the envelope and were totally degraded and lost. From these images, a good estimate of the thickness of the envelope protein-membrane-matrix protein outer shell of the virion was obtained. Treatment with even low concentrations (<0.1%) of sodium dodecyl sulfate completely destroyed all virions but produced many interesting products, including aggregates of viral proteins with strands of nucleic acid.

Unprecedented forms of vanadium observed within the blood cells of Phallusia nigra using K-edge X-ray absorption spectroscopy.

Fits to the vanadium K-edge X-ray absorption spectra (XAS) of five whole blood cell samples from the tunicate Phallusia nigra revealed unprecedented forms of intracellular vanadium. Endogenous vanadium was divided between the V(III) ion (74.2+/-5.1% of total V) and the vanadyl ion [V(IV)=O](2+) (25.2+/-5.4% of total V). The V(III) fraction included both [V(H(2)O)(6)](3+) (36.7+/-5.5%) modeled as VCl(3) in 1 M HCl, and three previously unprecedented chelated V(III) forms (37.5+/-4.6%). Two of these could be represented by the model ligand environments V(acetylacetonate)(3) (17.9+/-3.2%) and K(3)V(catecholate)(3) (13.1+/-4.7%), implying DOPA-like complexation. The third chelated form was represented by the 7-coordinate N(2)O(5) complex Na[V(edta)(H(2)O)] (8.0+/-1.8%). This coordination array, suggestive of a novel mononuclear V(III) protein site, contributed only to fits to samples 1, 2, 3 and 5, which were prepared in the presence of DTT. Endogenous V(IV) (25.2+/-5.4%) was principally modeled as VOCl(2) in 1 M HCl. EPR spectra (averages: A(parallel)=(1.842+/-0.006)x10(-2) cm(-1); A( perpendicular)=(0.718+/-0.007)x10(-2) cm(-1); g(parallel)=1.936+/-0.002; g( perpendicular)=1.990+/-0.001) confirmed the predominance of the aquated vanadyl ion. Blood cell sample five uniquely required the XAS spectrum of VOSO(4) in 0.1 M H(2)SO(4) solution (13.0%) and of [OV(V)(pivalate)(3)] (3.1%) to successfully fit the XAS pre-edge energy region. This endogenous V(V) signal is also unprecedented. These results are compared with those of analogous fits to the blood cells of Ascidia ceratodes and may support assignment of P. nigra to a different genus.

Cadmium binding to a histidine-rich glycoprotein from marine mussel blood plasma: potentiometric titration and equilibrium speciation modeling.

Cadmium-binding parameters (conditional stability constants and carrying capacities) of Mytilus edulis blood plasma histidine-rich glycoprotein (HRG) were investigated by potentiometric titrations using a Cd ion-specific electrode. Titration data were applied to a single-component complexation model and expressed as Scatchard plots that were analyzed using the graphical curve peeling method and the algebraic statistical mechanical method. These sets of binding parameters, derived for the purified HRG, were subsequently entered into the geochemical speciation model MINTEQA2 and then used to simulate the experimental titration, thereby determining which set of log K and CL values best represented the titration data. The Cd binding to HRG was best described by a two-class model with log K values of 7.65 +/- 0.10 and 5.41 +/- 0.06 M-1 and carrying capacities of 6.0 +/- 1.2 and 9.5 +/- 0.4 sites/molecule, respectively. At concentration of total Cd measured in the blood plasma of field-collected mussels (< or = 2 x 10(-7) M), plasma Cd speciation would be dominated by the strong affinity sites of HRG (> 93.5% of total Cd binds to HRG), whereas HRG itself would only be 0.05% saturated with Cd, indicating a high-capacity, apparently nonsaturable Cd transport system.

Mismatched double-stranded RNA (polyI-polyC(12)U) is synergistic with multiple anti-HIV drugs and is active against drug-sensitive and drug-resistant HIV-1 in vitro.

Although highly active anti-retroviral therapy (HAART) is successful in the treatment of HIV infection, problems with toxicity, drug-resistant variants, and therapeutic failures have compromised the long-term utility of existing combination regimens. Mismatched double-stranded RNA (polyI-polyC(12)U) is an immune modulator with inherent anti-HIV activity. Cell toxicities and anti-HIV activities of fourteen anti-HIV agents were determined alone and in combination with polyI-polyC(12)U. Combination analyses for anti-HIV activity were performed at three drug ratios. Using Mixed Dose Effect analyses and the CalcuSyn for Windows software package, combination indeces were determined for all drug combinations. In general, polyI-polyC(12)U was synergistic in combination with abacavir, zidovudine, zalcitabine, didanosine, stavudine, efavirenz, indinavir, ritonavir, nelfinavir, and amprenavir. It was synergistic to antagonistic with lamivudine, delavirdine, nevirapine, and saquinavir. Thus, polyI-polyC(12)U is synergistic with most anti-HIV agents at most drug ratios and across most effective concentrations in vitro, although, certain members of each class were exceptions. PolyI-polyC(12)U alone was equally active against wild-type HIV and HIV resistant to nevirapine, protease inhibitors, or nucleoside analogue reverse transcriptase inhibitors. These results suggest that polyI-polyC(12)U should be re-evaluated as a potential adjunct therapy in patients who have failed current anti-retroviral therapeutic regimens.

Generation of replication-defective helper-free vectors based on simian immunodeficiency virus.

A systematic study on generating simian immunodeficiency virus (SIV)-based vectors was carried out. The goal was to generate helper-free, replication-defective SIVmac-based vectors at high titers. The general approach was to cotransfect into human 293T cells a plasmid carrying the vector construct along with two helper plasmids that together expressed the SIVmac virion proteins. Initial vectors carried the bacterial beta-galactosidase gene (beta-gal). These vectors had a technical difficulty: "pseudotransduction" of beta-gal protein produced during the 293T cell transfections. As a result, infection of cultures with these vector stocks also resulted in passive transfer into, and X-gal staining of, cells that had not actually been infected by the vector. A second generation of vectors expressing the enhanced jellyfish green fluorescence protein (EGFP) was not subject to this artifact. A systematic study of the SIVmac-based EGFP vectors was carried out. Helper-free vector stocks were obtained when helper plasmids lacking the SIVmac packaging signals were used. By employing envelope helper plasmids derived from different SIVmac isolates, it was possible to generate SIVmac-based vectors pseudotyped with envelope proteins of different cell tropism. Optimization of vector and helper plasmid structures, transfection conditions, and infection procedures ultimately yielded vector titers in excess of 10(6)/ml.

Histidine--rich glycoprotein in the blood of the bivalve Mytilus edulis: role in cadmium speciation and cadmium transfer to the kidney.

Cadmium transfer from M. edulis L. blood plasma to tissues was investigated in relation to its chemical speciation in the blood. 109Cd was injected into the posterior adductor muscle along with synthetic chelators oxine, ethylenediaminetetraacetic acid (EDTA) or cyclohexanediaminetetraacetic acid (CDTA). Chelator concentrations were chosen to bind up from 0 to 98.7% of the total blood Cd, based on speciation calculations using the geochemical speciation model MINTEQA2. Increases in synthetically chelated Cd were accompanied by linear decreases in the calculated percentages of Histidine-rich Glycoprotein (HRG)-bound Cd, and nonlinear decreases in Cd(2+) and Cd chloro-complexes. Cadmium uptake by the kidneys decreased with increasing percentage synthetic chelation, while uptake by other tissues was not affected by chelation. Results indicate that there is at least one mechanism of Cd uptake common to all M. edulis tissues, and an additional, more rapid uptake mechanism in the kidneys that is mediated by CdHRG.

Combinations of reverse transcriptase, protease, and integrase inhibitors can be synergistic in vitro against drug-sensitive and RT inhibitor-resistant molecular clones of HIV-1.

Combinations of anti-HIV agents including one or two reverse transcriptase inhibitors with a protease inhibitor are potent and effective. However, toxicities, costs and the emergence of drug-resistant organisms have compromised their long-term efficacy in people. A next, likely, target for anti-HIV therapy is HIV-1 integrase. Viral integration, catalyzed by integrase, is absolutely required for HIV replication. L-chicoric acid is a potent and selective inhibitor of HIV-1 integrase that also inhibits HIV-1 replication in cell culture. As a first step in understanding the potential role for integrase inhibitors in clinical medicine, the activities of L-chicoric acid alone and in combination with 2', 3'-dideoxycytidine, zidovudine, and a protease inhibitor, nelfinavir, were tested in vitro against molecular clones of HIV-1 resistant to reverse transcriptase inhibitors. L-chicoric acid was equally effective against a wild-type clone of HIV-1, HIV(NL4-3), or against HIV-1 resistant to either zidovudine or dideoxycytidine. L-chicoric acid was largely synergistic with zidovudine and synergistic with both dideoxycytidine and nelfinavir.

Cadmium speciation and transport in the blood of the bivalve Mytilus edulis.

Cadmium (Cd) speciation in the blood plasma of Mytilus edulis was investigated using the metal speciation model MINTEQA2. In the presence of inorganic ions alone, Cd-chloro complexes dominated the speciation (97% of total Cd), with 3% as Cd2+. Inclusion of a novel Cd-binding histidine-rich glycoprotein (HRG) purified from mussel blood plasma decreased the contribution of chloro-complexes to 11.9%, with 86.8% of the Cd bound to the HRG and 1.3% present as Cd2+. Cd transfer from the blood plasma to the kidneys in vivo was studied by injecting 109Cd (both with and without additional chelation) into mussels. Oxine and EDTA complexed a significant amount of blood-borne Cd (23.7% Cd Oxine; 57.1% CdEDTA). In the presence of each chelator, plasma retained significantly more Cd, although there was no significant difference in Cd uptake by tissues (kidney, gill-mantle, and remaining viscera).

Cadmium turnover in the hemocytes of Mercenaria mercenaria (L.) in relation to hemocyte turnover.

The turnover of cadmium (Cd) in the hemocytes of the quahog Mercenaria mercenaria L. was investigated using 109Cd radiolabeling, and compared to the rate of hemocyte turnover as estimated following [3H]thymidine incorporation. Quahogs were injected with 5 microl of 0.8 microM 109Cd, and the hemocytes sampled over a 60-day depuration period. Additional organisms were injected with 15 microl of 4.7 microM [3H]thymidine and radioactivity monitored over a 60-day period. Assuming that the loss of 3H from the hemocytes during the first 27 days of the experiment is due to hemocyte turnover, results indicate that Cd turnover rate (t 0.5 = 25.3 +/- 6.3 days) was similar to hemocyte turnover rate (t 0.5 = 28.5 +/- 14.3 days). This suggests that hemocytes retain Cd internally throughout their circulatory lifespan.

Purification and characterization of a histidine-rich glycoprotein that binds cadmium from the blood plasma of the bivalve Mytilus edulis.

An unusual cadmium-binding protein was purified for the first time from the blood plasma of the blue mussel, Mytilus edulis. The protein was isolated and purified to homogeneity using ammonium sulfate precipitation and immobilized metal-ion affinity chromatography. It was identified as a glycoprotein with an apparent Mr of 63 kDa and a pI of 4.8. Electrophoresis of the protein under denaturing conditions on polyacrylamide gels produced four bands of 35, 37, 39 and 29 kDa. Isoelectric focusing under denaturing conditions produced 12 closely spaced bands with pIs of 4.2 to 5.8, revealing charge microheterogeneity. Molecular proterties (Mr and pI), carbohydrate content (11.6%) and composition, high histidine content (13.7%), as well cadmium-binding property of the protein (approximate log K >/= 5.4) indicated that it is similar to the mammalian histidine-rich glycoprotein, hitherto unreported in aquatic invertebrates. The cadmium-binding ability of the protein was retained even after heat denaturation and polyacrylamide gel electrophoresis.

Irreversible inhibition of human immunodeficiency virus type 1 integrase by dicaffeoylquinic acids.

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses require integration of a double-stranded DNA copy of the RNA genome into the host cell chromosome for productive infection. The viral enzyme, integrase, catalyzes the integration of retroviral DNA and represents an attractive target for developing antiretroviral agents. We identified several derivatives of dicaffeoylquinic acids (DCQAs) that inhibit HIV-1 replication in tissue culture and catalytic activities of HIV-1 integrase in vitro. The specific step at which DCQAs inhibit the integration in vitro and the mechanism of inhibition were examined in the present study. Titration experiments with different concentrations of HIV-1 integrase or DNA substrate found that the effect of DCQAs was exerted on the enzyme and not the DNA. In addition to HIV-1, DCQAs also inhibited the in vitro activities of MLV integrase and truncated variants of feline immunodeficiency virus integrase, suggesting that these compounds interacted with the central core domain of integrase. The inhibition on retroviral integrases was relatively specific, and DCQAs had no effect on several other DNA-modifying enzymes and phosphoryltransferases. Kinetic analysis and dialysis experiments showed that the inhibition of integrase by DCQAs was irreversible. The inhibition did not require the presence of a divalent cation and was unaffected by preassembling integrase onto viral DNA. The results suggest that the irreversible inhibition by DCQAs on integrase is directed toward conserved amino acid residues in the central core domain during catalysis.

Structure-activity relationships: analogues of the dicaffeoylquinic and dicaffeoyltartaric acids as potent inhibitors of human immunodeficiency virus type 1 integrase and replication.

The dicaffeoylquinic acids (DCQAs) and dicaffeoyltartaric acids (DCTAs) are potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase. They also inhibit HIV-1 replication at nontoxic concentrations. Since integrase is an excellent target for anti-HIV therapy, structure-activity relationships were employed to synthesize compounds with: (1) improved potency against HIV-1 integrase, (2) improved anti-HIV effect in tissue culture, and (3) increased selectivity as indicated by low cellular toxicity. Thirty-four analogues of the DCTAs and DCQAs were synthesized and tested for cell toxicity, anti-HIV activity, and inhibition of HIV-1 integrase. Seventeen of the 34 analogues had potent activity against HIV-1 integrase ranging from 0. 07 to >10 microM. Seventeen analogues that were synthesized or purchased had no inhibitory activity against integrase at concentrations of 25 microM. Of the biologically active analogues, 7 of the 17 inhibited HIV replication at nontoxic concentrations. The most potent compounds were D-chicoric acid, meso-chicoric acid, bis(3,4-dihydroxydihydrocinnamoyl)-L-tartaric acid, digalloyl-L-tartaric acid, bis(3,4-dihydroxybenzoyl)-L-tartaric acid, dicaffeoylglyceric acid, and bis(3, 4-dihydroxyphenylacetyl)-L-tartaric acid. Anti-HIV activity of the active compounds in tissue culture ranged from 35 to 0.66 microM. Structure-activity relationships demonstrated that biscatechol moieties were absolutely required for inhibition of integrase, while at least one free carboxyl group was required for anti-HIV activity. These data demonstrate that analogues of the DCTAs and the DCQAs can be synthesized which have improved activity against HIV integrase.

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.

Calcium speciation and exchange between blood and extrapallial fluid of the quahog Mercenaria mercenaria (L.).

Calcium and small organic molecules (e.g., tyrosine, MW 181 Da) introduced into the extrapallial fluid (EPF) of the quahog Mercenaria mercenaria exhibit rapid fluxes across the outer mantle epithelium and are distributed throughout the circulatory system within 3 h. Larger molecules (e.g., bovine serum albumin, MW 66,000 Da) are less readily exchanged between EPF and blood. The protein compositions of blood plasma and EPF are different, with at least seven protein bands expressed more prominently in the EPF. Equilibrium dialysis experiments reveal that Ca2+ constitutes only 2% of the total Ca in plasma; most of the Ca (85%) is bound to macromolecules, and the remaining 13% is present as dialyzable low molecular weight moieties. This distribution cannot be explained by speciation of inorganic Ca alone, since the MINTEQA2 equilibrium speciation model predicts that 79%-86% of the Ca should be present as Ca2+, with the remainder as CaSO4 (20%-13%). However, inclusion of a weakly Ca-binding organic molecule (log10 Ka approximately 2 M-1) into MINTEQA2 could fully reconcile modeling with experimental measurements. Results suggest that calcium transport in blood plasma and EPF is mediated by a suite of proteins and small organic ligands with a low affinity for Ca.

Vanadium K-edge x-ray absorption spectroscopy reveals species differences within the same ascidian genera. A comparison of whole blood from Ascidia nigra and Ascidia ceratodes.

Vanadium K-edge x-ray absorption spectroscopy (XAS) was used to examine whole blood preparations from the tunicates Ascidia nigra and Ascidia ceratodes. Each XAS spectrum exhibits a rising edge inflection near 5480 eV characteristic of vanadium(III) and an intensity maximum at 5484.0 eV. In A. ceratodes blood cells, intrinsic aquo-VSO4+ complex ion is indicated by an inflection feature at 5476 eV in the first derivative of the vanadium K-edge XAS spectrum, but this feature is notably absent from the first derivative of the vanadium K-edge spectrum of blood cells from A. nigra. A strong pre-edge feature at 5468.6 eV also uniquely distinguishes the vanadium K-edge XAS spectrum of A. nigra blood cells, implying that vanadyl ion represents approximately 25% of the endogenous vanadium. However, the energy position of the rising edge inflection of the vanadium K-edge XAS spectrum of A. nigra (5479.5 eV) is 1 eV lower than that of A. ceratodes (5480.5 eV), the reverse of any expected shift arising from the endogenous vanadyl ion. Thus, in contrast to A. ceratodes, a significant fraction of the blood cell vanadium(III) in A. nigra is apparently in a ligation environment substantially different from that provided by water. These novel species-related differences may have taxonomic significance.

Human immunodeficiency virus type 1 cDNA integration: new aromatic hydroxylated inhibitors and studies of the inhibition mechanism.

Integration of the human immunodeficiency virus type 1 (HIV-1) cDNA is a required step for viral replication. Integrase, the virus-encoded enzyme important for integration, has not yet been exploited as a target for clinically useful inhibitors. Here we report on the identification of new polyhydroxylated aromatic inhibitors of integrase including ellagic acid, purpurogallin, 4,8, 12-trioxatricornan, and hypericin, the last of which is known to inhibit viral replication. These compounds and others were characterized in assays with subviral preintegration complexes (PICs) isolated from HIV-1-infected cells. Hypericin was found to inhibit PIC assays, while the other compounds tested were inactive. Counterscreening of these and other integrase inhibitors against additional DNA-modifying enzymes revealed that none of the polyhydroxylated aromatic compounds are active against enzymes that do not require metals (methylases, a pox virus topoisomerase). However, all were cross-reactive with metal-requiring enzymes (restriction enzymes, a reverse transcriptase), implicating metal atoms in the inhibitory mechanism. In mechanistic studies, we localized binding of some inhibitors to the catalytic domain of integrase by assaying competition of binding by labeled nucleotides. These findings help elucidate the mechanism of action of the polyhydroxylated aromatic inhibitors and provide practical guidance for further inhibitor development.

Resistance to the anti-human immunodeficiency virus type 1 compound L-chicoric acid results from a single mutation at amino acid 140 of integrase.

L-Chicoric acid is an inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase in vitro and of HIV-1 replication in tissue culture. Following 3 months of selection in the presence of increasing concentrations of L-chicoric acid, HIV-1 was completely resistant to the compound. Introduction of the mutant integrase containing a single glycine-to-serine amino acid change at position 140 into the native, L-chicoric acid-sensitive virus demonstrated that this change was sufficient to confer resistance to L-chicoric acid. These results confirm through natural selection previous biochemical studies showing that L-chicoric acid inhibits integrase and that the drug is likely to interact at residues near the catalytic triad in the integrase active site.