anti-HIV Compounds Research Articles

In vitroanti-HIV and Cytotoxicological Evaluation of the Triple Combination: AZT and ddC with HIV Proteinase Inhibitor Saquinavir (Ro 31-8959)

A combination of three anti-HIV compounds (AZT, ddC and saquinavir) has been studied in vitro. In confirmation and extension of previous studies, a greater than additive effect was found against both HIV-1GB8and HIV-1N1T, when any two compounds were combined. Synergy of action was also found against both virus strains when all three compounds were employed together. Cytotoxicological studies indicated that a high therapeutic index could be maintained for this treatment. The potential benefits of improved anti-HIV surveillance, reduced risk of the development of resistance to the treatment and reduced requirement for drugs in quantities that produce toxic side-effects support the extension of clinical trials of saquinavir to investigate this combination.

HIV-sensitive transgenic rabbit

AbstractAn HIV-sensitive transgenic rabbit has been disclosed. This is a useful animal model for studying HIV infection physiology and in screening for anti-HIV compounds and treatments.

An Assay for HIV RNA in Infected Cell Lysates, and its use for the Rapid Evaluation of Antiviral Efficacy

A rapid, high-capacity assay for evaluating the potency of anti-HIV compounds was devised. This assay measures cell associated viral RNA levels 3 days after infection of susceptible T-cell lines grown in individual microtitre plate wells. Viral RNA was quantified by a sandwich hybridization assay, the first step of which was performed directly in crude infected cell lysates prepared in guanidinium isothio-cyanate. Levels of cell-associated viral RNA were shown to correlate with the yield of infectious virus and this correlation formed the basis of the test. Antiviral potencies of a large series of compounds tested in this RNA hybridization assay correlated closely with potency values determined by a sensitive but slower and more labour-intensive yield-reduction assay. Both laboratory strains and selected clinical isolates of HIV can be detected in this RNA hybridization assay.

Benzopurpurin and Related Compounds Inhibit the Binding of gp120 to Galactosyl Ceramide/Sulfatide and Infection of Human Immunodeficiency Virus

In an attempt to find compounds capable of inhibiting the binding and infection by human immunodeficiency virus type 1 (HIV-1) in neural cells, we studied the effect of benzopurpurin and related compounds on the binding of gp120 to galactosyl ceramide (GalCer) and sulfatide. In this report, we show that the binding of gp120 to GalCer and sulfatide is inhibited by benzopurpurin and related compounds. These compounds also inhibit the binding and entry of HIV-1 to neural cell line, SK-N-MC. Binding studies indicate that benzopurpurin and related compounds bind to gp120. Studies involving related compounds indicate that the minimal structure required for the inhibition is two naphthalene rings with amine or sulfonic acids attached to a central biphenyl molecule by an azo group. This approach will be useful for screening of potential anti-HIV compounds.

The inophyllums, novel inhibitors of HIV-1 reverse transcriptase isolated from the Malaysian tree, Calophyllum inophyllum Linn.

As part of a search for novel inhibitors of HIV-1 reverse transcriptase, the acetone extract of the giant African snail, Achatina fulica, was shown to be active. Fractionation of the extract yielded inophyllums A, B, C, and E and calophyllolide (1a, 2a, 3a, 3b, and 6), previously isolated from Calophyllum inophyllum Linn., a known source of nutrition for A. fulica. From a methanol/methylene chloride extract of C. inophyllum, the same natural products in considerably greater yield were isolated in addition to a novel enantiomer of soulattrolide (4), inophyllum P (2b), and two other novel compounds, inophyllums G-1 (7) and G-2 (8). The absolute stereochemistry of inophyllum A (1a) was determined to be 10(R), 11(S), 12(S) from a single-crystal X-ray analysis of its 4-bromobenzoate derivative, and the relative stereochemistries of the other inophyllums isolated from C. inophyllum were established by a comparison of their 1H NMR NOE values and coupling constants to those of inophyllum A (1a). Inophyllums B and P (2a and 2b) inhibited HIV reverse transcriptase with IC50 values of 38 and 130 nM, respectively, and both were active against HIV-1 in cell culture (IC50 of 1.4 and 1.6 microM). Closely related inophyllums A, C, D, and E, including calophyllic acids, were significantly less active or totally inactive, indicating certain structural requirements in the chromanol ring. Altogether, 11 compounds of the inophyllum class were isolated from C. inophyllum and are described together with the SAR of these novel anti-HIV compounds.

Advances in pharmacotherapy: treatment of HIV infection

SUMMARY Current estimates indicate that at least one million people in the United States, 500 000 in Western Europe and perhaps six million individuals in sub-Saharan Africa are infected by the human immunodeficiency virus (HIV), the virus that causes the acquired immunodeficiency syndrome (AIDS). This article summarizes recent developments in the study and clinical application of agents directed against HIV. There are presently four primary targets in the life-cycle of HIV for interruption of replication. Of these, only the inhibition of reverse transcriptase by drugs such as zidovudine, didanosine and zalcitabine has been shown to be clinically beneficial. Zidovudine is the drug with which we have the greatest experience, and it remains the cornerstone of anti-HIV therapy. Even so, there is much that remains to be learned about zidovudine and other dideoxy-nucleoside agents regarding the clinical meaning of HIV-resistance, and the ideal time to initiate therapy. Putative anti-HIV compounds such as non-nucleoside reverse transcriptase inhibitors, for inhibitors and protease inhibitors are currently under investigation. The development of these compounds, with mechanisms of action different from dideoxynucleosides, represents exciting prospects for combination therapy.

A new procedure for the isolation of anti-HIV compounds (polysaccharides and polyphenols) from the marine alga Fucus vesiculosus.

Anti-HIV-active polysaccharides and polyphenols were isolated from the brown seaweed Fucus vesiculosus by hot H2O extraction of both the intact and the homogenized algae. This was followed by XAD2 chromatography and by sequential precipitation of the non-adsorbed compounds with glacial HOAc and thereafter with EtOH. The precipitate was solubilized, dialyzed against distilled H2O, and chromatographed on SP-Sephadex C25 and on QAE-Sephadex A25. This was followed by gel filtration on Sephadex G50 and Sephadex G100 and finally by hplc on a Shodex Ionpak S-804 column. For comparison, the commercial product fucoidan, a sulfated algal polysaccharide, was also further purified by the chromatographic techniques mentioned above. The isolated freeze-dried fractions obtained by these procedures were tested for inhibition of both HIV-induced syncytium formation and HIV reverse transcriptase enzyme activity. Some of these fractions inhibited both of these activities at concentrations that were not cytotoxic.

A novel coculture system for evaluating anti-HIV drugs.

To develop a useful system for evaluating novel anti-HIV drugs. The activity of most antiviral compounds in cell-free HIV infection systems has been evaluated. However, the inhibitory effects on both the process of HIV induction and viral dissemination to uninfected cells have not been fully investigated. We have therefore developed a new cocultivation system using chronically HIV-infected monocytes and CD4+ T-lymphocytes in the presence of tumor necrosis factor (TNF). We designed a cocultivation system using flow cytometry with U1 cells and Molt-4 cells in the presence of TNF. The antiviral activities of several compounds in the cocultivation system and other assay systems were compared. Only pradimicin A and glycyrrhizin showed strong inhibitory activity in the cocultivation system in the presence of TNF, whereas dextran sulfate, curdlan sulfate and N-acetylcysteine exhibited moderate or weak inhibitory activity in the system. 3'-azido-2',3'-dideoxythymidine and 2',3'-dideoxyadenosine were completely ineffective in the system. These results support the suggestion that our cocultivation system includes HIV induction in chronically infected monocytes, and the resulting cell-to-cell infection between HIV-infected monocytes and Molt-4 cells or Molt-4 cells and their HIV-converted counterparts. Our new cocultivation system may constitute a useful tool in the identification of novel anti-HIV compounds.

Sulfonic acid polymers as a new class of human immunodeficiency virus inhibitors

Four sulfonic acid polymers [poly(4-styrenesulfonic acid)(PSS), poly(anetholesulfonic acid)(PAS), poly(vinylsulfonic acid)(PVS), poly(2-acrylamido-2-methyl-1-propanesulfonic acid)(PAMPS)] have been found to inhibit the cytopathicity of HIV-1 and HIV-2 in MT-4 cells at concentrations that are not toxic to the host cells. The sulfonic acid polymers also inhibited syncytium formation in co-cultures of MOLT-4 cells with HIV-1- or HIV-2-infected HUT-78 cells. They also inhibited binding of anti-gp120 mAb to HIV-1 gp120 and blocked adsorption of HIV-1 virions to MT-4 cells. PSS and PAS, but not PVS and PAMPS, interfered with the binding of OKT4A/Leu3a to the CD4 receptor. The anti-HIV activity of these polyanionic compounds can be ascribed to inhibition of the gp120-CD4 interaction. Sulfonic acid polymers represent a lead of anti-HIV compounds that warrant further evaluation of their therapeutic potential.

Interpretation of the roles of adenylosuccinate lyase and of AMP deaminase in the anti-HIV activity of 2′,3′-dideoxyadenosine and 2′,3′-dideoxyinosine

Some 2',3'-dideoxynucleotides, of importance in the enzymology of the anti-HIV compounds, ddA and ddI, have been synthesized and purified by ion-exchange chromatography. 2',3'-Dideoxyadenylosuccinate, an intermediate in the pathway of ddI to ddATP, is converted to ddAMP by AMPS lyase at 1.85% of the efficiency of the natural substrate, adenylosuccinate. Interestingly, ddAMP and other 2',3'-dideoxygenated nucleotides are not substrates for AMP deaminase, another relevant enzyme in the conversion of ddA to ddATP via ddI.

Avarol restores the altered prostaglandin and leukotriene metabolism in monocytes infected with human immunodeficiency virus type 1.

Infection of monocytes with human immunodeficiency virus type 1 (HIV-1) (strain Ada-M) caused increased levels of leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) in vitro. These two products result from the activities of the two enzymes cyclooxygenase and 5-lipoxygenase. The addition of the sesquiterpenoid hydroquinone Avarol, an HIV inhibitor, strongly reduced the levels of LTB4 and PGE2 via inhibition of both cyclooxygenase and lipoxygenase in monocytes. The 50% inhibition concentrations (IC50) for the enzymes were determined to be 2.26 microM (cyclooxygenase) and 1.97 microM (lipoxygenase). A 50% reduction of the extent of PGE2 and LTB4 production in HIV-infected monocytes was measured at a concentration of 0.9 microM Avarol, a dose which caused an 80% anti-HIV effect in vitro (50% inhibition of virus release from infected cells: 0.3 microM). We conclude that Avarol inhibits the enzymes cyclooxygenase and lipoxygenase and suggest that, in general, inhibitors of these enzymes are promising anti-HIV compounds.

Novel dideoxynucleoside isosteres

Novel 5-aza-7-deaza isosteres of the anti-HIV compounds, dideoxyinosine (ddI) and dideoxyguanosine (ddG), have been prepared by glycosylation of the corresponding isosteric bases with appropriately tailored dideoxyribose components. The glycosylation reactions were regiospecific and the position of glycosylation was established by UV and high-field NMR data. However, attempted preparation of the 3- deaza-5-aza isostere of dideoxyinosine led to a different regioisomer, a ring-extended dideoxycytidine analogue, whose structure was established by X-ray crystallographic data. The 5-aza-7-deaza isosteres of ddI and ddG were more stable with respect to hydrolytic cleavage than ddI and ddG.

Modified tetrazolium-based colorimetric method for determining the activities of anti-HIV compounds

A modified tetrazolium-based colorimetric assay was used to determine the anti-HIV activities of ddAzThd, ddCyd, ddIno, and PFA. In this assay, poly-1-lysine-coated plates were used to attach the MT-2 cells to the bottom of the plates. A fixed amount of virus (50 TCID 50) was used in each well. A modified version of the formula published by Pauwels et al. (1988) was used for calculating the percentage cell protection from virus infection. Using CC10/ EC90 to calculate the selective indices, the decreasing order of selectivity against HIV-1 strain A87SF, was: ddAzThd > PFA > ddCyd > ddIno. Against HIV-1 strain A79SK-1 the decreasing order of selectivity was: PFA > ddIno > AzThd > ddCyd. The modified formula showed lack of antiHIV activity for thymidine at non-toxic concentrations.

A semiautomated multiparameter approach for anti-HIV drug screening

We are implementing a series of complementary assays for initial follow-up confirmation and prioritization of new active anti-HIV compounds identified by the U.S. National Cancer Institute's large-scale in vitro primary anti-HIV screen. Two different kinds of cellular viability assays, in addition to specific assays for total cellular DNA content, supernatant reverse transcriptase activity, p24 core antigen production and the synthesis of infectious HIV virions are all performed from a single well of a 96-well microtiter plate containing human host cells infected with HIV. Antiviral activities of several known prototype HIV inhibitors including 3'-azido,3'-deoxythymidine, 2',3'-dideoxycytidine, dextran sulfate and phorbol myristate acetate were compared in these multiparameter assays as a means of validation. Procedures to automate the method optimally, as well as to maximize the safety of the technicians working with HIV and HIV-infected cells have been emphasized. The resulting semiautomated, highly reproducible battery of assays yields a maximum amount of antiviral and cytotoxicity information from a minimum amount of sample. This is especially crucial when analyzing new synthetic compounds and natural product extracts or fractions where the available amounts of sample may be very limited.

Hybrid pharmacokinetic models to describe anti-HIV nucleoside brain disposition following parent and prodrug administration in mice.

Brain delivery of active anti-HIV compounds is important for successful treatment of the AIDS patient. As an initial step in predicting human brain drug concentrations, hybrid pharmacokinetic models were developed to characterize the disposition of anti-HIV nucleosides following parent and prodrug administrations in mice. Mouse data were obtained following intravenous administration of 3'-azido-2',3'-dideoxyuridine (AZddU or AZDU), 3'-azido-3'-deoxythymidine (AZT), and their dihydropyridine prodrugs (AZddU-DHP and AZT-DHP). Exponential equations were fitted to the serum concentration-time data for each species, including the pyridinium ion moieties, and subsequently used in differential mass balance equations describing the brain dynamics of each compound. Model parameters for the mass balance equations were estimated by various techniques, including the utilization of in vitro data. In general, model-predicted brain concentrations agreed with the observed data. Similar data in larger animals will permit scale-up of the current model to predict human brain drug concentrations.

Acute Infection of Cynomolgus Monkeys with Simian Immunodeficiency Virus (SIVSM) as a Model to Evaluate Antiviral Compounds. Effects of 3′-Azido, 3′-Deoxythymidine, Foscarnet and 2′,3′-Dideoxycytidine

Screening of compounds with potential use in the treatment of human immunodeficiency virus (HIV) infections and acquired immunodeficiency syndrome (AIDS) can currently be made in cell culture systems, but there is a need for relevant animal models. We have infected cynomolgus monkeys with simian immunodeficiency virus of sooty mangabey origin (SIVSM) and investigated the influence of multiplicity of infection (MOI) and the effects of three different anti-HIV compounds, 3′-azido-3′-deoxythymidine (AZT), foscarnet (PFA) and 2′,3′-dideoxycytidine (ddC) on the acute infection. To secure a maximal effect of treatment this was started 8h before challenge with SIVSMand the drugs were given subsequently every 8h for 7–9 days. The appearance of viral antigen and antibodies in serum was determined. In control animals the mean day for SIV antigen appearance was Day 5.9 post-infection, whereas in animals treated with 3 × 25 mg kg−1day−1of AZT and 3 × 65 mg kg−1day−1of PFA there were significant delays in SIV antigen appearance of 1.0 and 3.6 days, respectively. Some delay in antigen appearance was indicated in animals treated with 3 × 0.2 mg kg−1day−1of ddC. A delayed antibody response was only seen in animals treated with PFA. Viral infection was not prevented at the multiplicity used with any of the drugs, despite treatment prior to virus inoculation. The animal model described offers attractive features for in vivo evaluation of potential anti-HIV compounds.

Novel phosphate derivatives of zidovudine as anti-HIV compounds

Novel phosphate derivatives of zidovudine as anti-HIV compounds

Synthesis and Evaluation of Some Novel Phosphoramidate Derivatives of 3′-Azido-3′-Deoxythymidine (AZT) as Anti-HIV Compounds

A series of monophosphate triester derivatives of 3′-azido-3′-deoxythymidine (AZT), designed as membrane-soluble pro-drugs of the nucleotide (AZTMP), have been tested for activity against the human immunodeficiency virus (HIV-1). It has been found that when carboxyl-protected, amino-linked amino acids, and alkyl chains, are asymmetrically substituted on the 5′-phosphate a significant antiviral effect is observed. Moreover, the activity of the compounds is profoundly dependent on the structure of the phosphate moiety, and in particular on the nature of the amino acid.

Dextran sulfate and other polyanionic anti-HIV compounds specifically interact with the viral gp120 glycoprotein expressed by T-cells persistently infected with HIV-1

Eighty to 100% of persistently HIV-1-infected HUT-78 cells express the viral glycoprotein gp120 as demonstrated with anti-gp120 monoclonal antibody (mAb) and fluorescence-activated cell sorter (FACS) analysis. Several polyanionic anti-HIV compounds, i.e., dextran sulfate, pentosan polysulfate, heparin, aurintricarboxylic acid (ATA), suramin, and Evans blue, which are known to inhibit the adsorption of HIV particles to CD4 + cells, prevented the binding of anti-gp120 mAb to the persistently HIV-1 infected HUT-78 cells. This effect was dose-dependent and reversible. Except for ATA, the polyanionic compounds did not interfere with the binding of Leu3a/OKT4A mAB, indicating that they do not directly bind to the CD4 receptor. Thus, the inhibitory effect of dextran sulfate and its congeners on the interaction of the HIV gp120 with the cellular CD4 receptor can be ascribed to a specific binding (“shielding”) of gp120.

Synthesis of Indole 2′,3′-Dideoxyribonucleosides

The synthese of 1,3,7-trideaza-2',3'-dideoxyadenosine and its 2',3'-didehydroderivative as potential anti-HIV compounds are described. Nucleobase anion glycosylation of 4-nitroindole (4) with 1-dichloro-2-deoxy-3,(-bis-O-(4-methylbenzoyl)-α-D-erythro-pentofuranose yields 1-[2-deoxy-3,5-bis-O-(4-methylbenzoyl)-β-D-erythro-pentofuranosyl]-4-nitro-1H-indole stereoselectively. Upon detoluoylation the 3'-hydroxyl group was removed by two different routes using a mesylation/elimination process