Potent Reverse Transcriptase Inhibitors Research Articles

Anti‐HIV Agents: Design and Discovery of New Potent RT Inhibitors.

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Anti-HIV agents: design and discovery of new potent RT inhibitors

This paper reports our work in the field of nonnucleoside RT inhibitors (NNRTIs). On the basis of extensive studies on 1 H,3 H-thiazolo[3,4- a]benzimidazole derivatives (TBZs) followed by structure–activity relationship (SAR) considerations and molecular modeling, the design and synthesis of a series of 2,3-diaryl-1,3-thiazolidin-4-ones have been performed. Some derivatives proved to be highly effective in inhibiting human immunodeficiency virus type-1 (HIV-1) replication at nanomolar concentrations with minimal toxicity, acting as reverse transcriptase (RT) inhibitors. Computational studies were used in order to probe the binding of our ligands to HIV-1-RT.

ChemInform Abstract: Synthesis of Bis‐heteroaryl Piperazine Derivatives as Potential Reverse Transcriptase Inhibitors.

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Synthesis of bis‐heteroaryl piperazine derivatives as potential reverse transcriptase inhibitors

AbstractVinylogous analogs of non‐nucleoside reverse transcriptase inhibitors belonging to the bis‐(hetero‐aryl)piperazine (BHAP) series were synthesized by coupling a piperazine derivative with (E)‐propenoyl derivatives of heterocyclic moieties.

Macrophage are the principal reservoir and sustain high virus loads in rhesus macaques after the depletion of CD4+ T cells by a highly pathogenic simian immunodeficiency virus/HIV type 1 chimera (SHIV): Implications for HIV-1 infections of humans

The highly pathogenic simian immunodeficiency virus/HIV type 1 (SHIV) chimeric virus SHIV(DH12R) induces a systemic depletion of CD4(+) T lymphocytes in rhesus monkeys during the initial 3-4 weeks of infection. Nonetheless, high levels of viral RNA production continue unabated for an additional 2-5 months. In situ hybridization and immunohistochemical analyses revealed that tissue macrophage in the lymph nodes, spleen, gastrointestinal tract, liver, and kidney sustain high plasma virus loads in the absence of CD4(+) T cells. Quantitative confocal immunofluorescence analysis indicated that greater than 95% of the virus-producing cells in these tissues are macrophage and less than 2% are T lymphocytes. Interestingly, the administration of a potent reverse transcriptase inhibitor blocked virus production during the early T cell phase but not during the later macrophage phase of the SHIV(DH12R) infection. When interpreted in the context of HIV-1 infections, these results implicate tissue macrophage as an important reservoir of virus in vivo. They become infected during the acute infection, gradually increase in number over time, and can be a major contributor to total body virus burden during the symptomatic phase of the human infection.

Macrophage are the principal reservoir and sustain high virus loads in rhesus macaques after the depletion of CD4+ T cells by a highly pathogenic simian immunodeficiency virus/HIV type 1 chimera (SHIV): Implications for HIV-1 infections of humans.

The highly pathogenic simian immunodeficiency virus/HIV type 1 (SHIV) chimeric virus SHIV(DH12R) induces a systemic depletion of CD4(+) T lymphocytes in rhesus monkeys during the initial 3-4 weeks of infection. Nonetheless, high levels of viral RNA production continue unabated for an additional 2-5 months. In situ hybridization and immunohistochemical analyses revealed that tissue macrophage in the lymph nodes, spleen, gastrointestinal tract, liver, and kidney sustain high plasma virus loads in the absence of CD4(+) T cells. Quantitative confocal immunofluorescence analysis indicated that greater than 95% of the virus-producing cells in these tissues are macrophage and less than 2% are T lymphocytes. Interestingly, the administration of a potent reverse transcriptase inhibitor blocked virus production during the early T cell phase but not during the later macrophage phase of the SHIV(DH12R) infection. When interpreted in the context of HIV-1 infections, these results implicate tissue macrophage as an important reservoir of virus in vivo. They become infected during the acute infection, gradually increase in number over time, and can be a major contributor to total body virus burden during the symptomatic phase of the human infection.

The structure of HIV-1 reverse transcriptase complexed with an RNA pseudoknot inhibitor.

Small RNA pseudoknots, selected to bind human immunodeficiency virus type 1 (HIV-1) reverse transcriptase tightly, are potent inhibitors of reverse transcriptase. The co-crystal structure of reverse transcriptase complexed with a 33 nucleotide RNA pseudoknot has been determined by fitting the ligand into a high quality, 4-fold averaged 4.8 A resolution electron density map. The RNA is kinked between stems S1 and S2, thereby optimizing its contacts with subunits of the heterodimer. Its binding site extends along the cleft that lies between the polymerase and RNase H active sites, partially overlaps with that observed for duplex DNA and presumably overlaps some portion of the tRNA site. Stem S2 and loop L1 stabilize the 'closed' conformation of the polymerase through extensive electrostatic interactions with several basic residues in helix I of the p66 thumb and in the p66 fingers domain. Presumably, this RNA ligand inhibits reverse transcriptase by binding to a site that partly overlaps the primer-template binding site.

A comparison of human immunodeficiency virus type 1 inhibition by partially purified aqueous extracts of chinese medicinal herbs

A multiple screening approach to detect compounds inhibitory to various aspects of the human immunodeficiency virus type 1 (HIV-1) life-cycle has been applied to aqueous extracts of 19 herbs traditionally used in Chinese medicine as anti-viral agents. The extracts were tested for their ability to inhibit HIV-1 in a series of in vitro assays. The extracts were tested for inhibition of the interaction between HIV-1 gp 120 and immobilized CD4 receptor, inhibition of recombinant HIV-1 reverse transcriptase and for inhibition of three glycohydrolase enzymes that contribute to viral protein glycosylation. Six of the herb extracts (30%) were potent inhibitors of the interaction between HIV-1 gp 120 and the CD4 receptor (ID 50 5.6–79.4 μg/ml), two extracts (10%) contained potent reverse transcriptase inhibitors (ID 5016.9–26.0 μg/ml) and 14 extracts (75%) were able to inhibit at least one of the glycohydrolase enzymes.

Structural analogues of the calanolide anti-HIV agents. Modification of the trans-10,11-dimethyldihydropyran-12-ol ring (ring C).

(+)-Calanolide A is a potent inhibitor of reverse transcriptase from human immunodeficiency virus type 1 (HIV-1), which was isolated from an extract of Calophyllum lanigerum, along with seven related compounds. In order to examine the structure-activity relationships of the trans-10,11-dimethyldihydropyran-12-ol ring (designated ring C), a series of structural analogues were prepared and evaluated using a whole cell cytopathicity assay (XTT). Removal of the 10-methyl group resulted in decreased activity, with only one epimer exhibiting anti-HIV activity. Substituting the 10-methyl group with an ethyl chain maintained anti-HIV activity, with only a 4-fold reduction in potency relative to racemic calanolide A. Substitution of the 10-methyl group with an isopropyl moiety completely eliminated the anti-HIV activity. Addition of an extra methyl group at either the 10- or 11-position maintained the basic stereochemical features of the parent calanolide system while removing the chirality at the respective carbon, but resulted in decreased activity relative to calanolide A. In all the above examples, analogues containing a cis relationship between the 10- and 11-alkyl moieties were completely devoid of activity. Synthetic intermediates in which the 12-hydroxyl group was in the ketone oxidation state exhibited suppressing anti-HIV activity, with EC50 values only 5-fold less potent than that of calanolide A for both the 10,11-cis (6) and -trans (5) series. These ketones represent the first derivatives in the calanolide series to exhibit anti-HIV activity while not containing a 12-hydroxyl group. Likewise, ketone derivative 6 was the first example of a compound in the calanolide series having a cis relationship between the 10- and 11-methyl groups found to exhibit anti-HIV activity. Analogues which showed anti-HIV activity in the CEM-SS cytoprotection assay were further confirmed to be inhibitors of HIV-1 reverse transcriptase.

Optimization of the isolation and effective use of mRNA from rat mast cells

To define the molecular regulation of mast cell phenotype and function optimized procedures must be available to study mRNA from mast cells freshly isolated from tissues. However, rat peritoneal mast cells (PMC) contain large amounts of the proteoglycan heparin, and unfortunately, this molecule which is a potent inhibitor of reverse transcriptase (RT) and Taq polymerase and thus RT-PCR, copurifies with RNA. Here we describe an optimized protocol for extracting and amplifying RNA from rat PMC. Mast cells were isolated from rat peritoneum and a method modified from that of Chomczynski and Sacchi (1987)was used to extract the RNA. Following the removal of heparin by heparinase digestion, first strand cDNA synthesis was primed with oligo-dT and the resulting cDNA was quantified by rapid paper chromatography. The use of a detection system for the reverse transcription reaction ensured that the production of cDNA had occurred and allowed subsequent PCR testing to be optimal. cDNA thus produced can be used to detect relatively specific (histidine decarboxylase) and non-specific ( β-actin) mast cell products. Our PCR studies have shown a 300-fold increase in sensitivity over RNA processed by other methods.

(s 4dU) 35: a novel, highly potent oligonucleotide inhibitor of the human immunodeficiency virus type 1 reverse transcriptase

Oligodeoxycytidylates were converted to s 4dUMP-containing oligomers by treatment with liquid H 2S. The inhibitory potency of the modified oligonucleotides on human immunodeficiency virus type 1 reverse transcriptase depended on the chain length and on the percentage of modification. The most potent reverse transcriptase inhibitor was (s 4dU) 35. The inhibitory pattern was competitive, when either poly(A)·(dT) 16 or poly(C)·(dG) 16 was used as template-primer (variable substrate), suggesting that the free enzyme interacts with (s 4dU) 35. The K i values were 3.0 and 2.2 nM in the presence of poly(A)·(dT) 16 and poly(C)·(dG) 16, respectively.

Potent activity of 2'-beta-fluoro-2',3'-dideoxyadenosine against human immunodeficiency virus type 1 infection in hu-PBL-SCID mice.

A new antiretroviral agent, 2'-beta-fluoro-2',3'-dideoxyadenosine (FddA), is an acid-stable compound whose triphosphate form is a potent reverse transcriptase inhibitor with in vitro anti-human immunodeficiency virus (HIV) activity and a favorable pharmacokinetic profile. Severe combined immunodeficiency (SCID) mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID mice) provide a useful small-animal model for HIV research. In the present study we utilized this experimental system for the in vivo evaluation of the anti-HIV activity of this new compound when administered prior to infection. Initial studies revealed that, following a challenge with 50 100% tissue culture infective doses of HIV type 1 lymphadenopathy-associated virus, 39 of 42 (93%) control mice developed HIV infection, as evidenced by positive coculture or positive PCR. Administration of zidovudine decreased the infection rate to 5 of 16 (31%), while administration of FddA decreased the infection rate to 0 of 44 (0%). In follow-up controlled studies, the anti-HIV activity of FddA was confirmed, with 18 of 20 control mice showing evidence of HIV infection, compared with 4 of 20 FddA-treated mice. In addition to having direct anti-HIV effects, FddA was found to have a protective effect on human CD4+ T cells in the face of HIV infection. Mice treated with FddA were found to have a significantly higher percentage of CD4+ T cells than controls (10.3% +/- 3.4% versus 0.27% +/- 0.21%; P = 0.01). Thus, FddA, with its potent anti-HIV activity in vivo, high oral bioavailability, long intracellular half-life, and ability to preserve CD4+ cells in the presence of HIV, appears to be a promising agent for clinical investigation.

Novel non-nucleoside inhibitors of HIV-1 reverse transcriptase. 3. Dipyrido[2,3-b:2',3'-e]diazepinones.

We have explored the potential of derivatives of the dipyrido[2,3-b:2',3'-e][1,4]diazepinone ring system as inhibitors of HIV-1 reverse transcriptase (RT). These compounds are isomeric to the potent RT inhibitor nevirapine and are available via a novel Smiles rearrangement on intermediates used for the synthesis of nevirapine analogs. Derivatives of this isomeric series are weaker inhibitors of RT than corresponding nevirapine analogs, although with appropriate substitution of the A- and C-pyridine rings activity can be improved.

Evaluation of Natural Products as Inhibitors of Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcriptase

Inhibition of human immunodeficiency virus reverse transcriptase is currently considered a useful approach in the prophylaxis and intervention of acquired immunodeficiency syndrome (AIDS), and natural products have not been extensively explored as inhibitors of this enzyme. We currently report that the reverse transcriptase assay developed for the detection of the enzyme in virions involving polyadenylic acid.oligodeoxythymidylic acid (poly rA.oligo dT) and radiolabeled thymidine 5'-triphosphate (TTP), can be applied as a simple method for screening the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) inhibitory potential of natural products. As reported herein, 156 pure natural products have been examined in this system. Benzophenanthridine alkaloids such as faragaronine chloride [1] and nitidine chloride, which are known inhibitors of avian myeloblastosis virus reverse transcriptase, demonstrated potent activity in the HIV-1 RT system, and 1 (IC50 10 micrograms/ml) was adopted as a positive-control substance. Additional inhibitors found were columbamine iodide [2] and other protoberberine alkaloids, the isoquinoline alkaloid O-methylpsychotrine sulfate [3], and the iridoid fulvoplumierin [4]. A number of indolizidine, pyrrolizidine, quinolizidine, indole, and other alkaloids, as well as compounds of many other structural classes, were tested and found to be inactive. A total of 100 plant extracts have also been evaluated, and 15 of these extracts showed significant inhibitory activity. Because tannins and other polyphenolic compounds are potent reverse transcriptase inhibitors, methods were evaluated for the removal of these from plant extracts prior to testing. Polyphenolic compounds were found to be responsible for the activity demonstrated by the majority of plant extracts. After appropriate tannin removal procedures were established, the bioassay system was shown to be generally applicable to both pure natural products and plant extracts. The method also proved useful in directing an isolation procedure with Plumeria rubra to yield fulvoplumierin [4] as an active compound (IC50 45 micrograms/ml).

Inhibition of duck hepatitis B virus replication by 2′,3′-dideoxycytidine: A potent inhibitor of reverse transcriptase

The effect of 2′,3′-dideoxycytidine, a potent antiviral agent, which, following anabolic phosphorylation, inhibits the reverse transcriptase of the human immunodeficiency virus in vitro, was assessed in 16 Pekin ducks chronically infected with the duck hepatitis B virus. Nine ducks were given 11 mg/m2 of dideoxycytidine intravenously every 6 h, and 7 ducks received no treatment. Serum duck hepatitis B virus deoxyribonucleic acid and deoxyribonucleic acid polymerase activity decreased in every duck treated with dideoxycytidine. The mean inhibition of deoxyribonucleic acid polymerase and duck hepatitis B virus deoxyribonucleic acid on the third day of treatment measured 64% (p < 0.01) and 73% (p < 0.01), respectively. The inhibition of deoxyribonucleic acid polymerase persisted after treatment was stopped, and 4 ducks continued to show >50% inhibition 12 days after stopping treatment. Duck hepatitis B virus deoxyribonucleic acid, which was measured in total cellular deoxyribonucleic acid extracted from liver biopsy specimens obtained before and on the last day of treatment with dideoxycytidine, showed an average inhibition of 96% in 3 ducks treated with dideoxycytidine, but showed no decrease in the remaining 5 ducks. Thus, dideoxycytidine has potent antiviral activity against duck hepatitis B virus and warrants further evaluation as an antiviral agent in the treatment of chronic hepatitis B virus infection in humans.

Structural characterization of HIV reverse transcriptase: a target for the design of specific virus inhibitors

The reverse transcriptase (RT) of HIV is an important target for chemotherapy as demonstrated by the effective treatment of AIDS patients with zidovudine, a potent inhibitor of RT. Structural studies of HIV RT were therefore undertaken with a view to designing more effective inhibitors. To obtain sufficient quantities of enzyme for these studies the reverse transcriptase gene of HIV was cloned into a high level expression plasmid yielding reverse transcriptase at a level of 10% of the total Escherichia coli proteins. Monoclonal antibodies to RT were raised in mice and have been used to purify the enzyme by immunoaffinity chromatography. Crystallization of the enzyme has been achieved and studies are underway to determine its three-dimensional structure. In addition, carboxy-terminal truncated mutants were prepared by inserting stop codons into the gene at appropriate sites. The proteins expressed were analysed for RT and RNase H activity and used for mapping RT epitopes. This, together with previous data on site-directed mutagenesis of conserved regions of HIV RT has helped to map some of the structural and functional regions of the enzyme.

Inhibition of murine leukaemia virus reverse transcriptase by 2-halogenated polyadenylic acids.

Several new analogues of polyadenylic acid [(A)n], i.e. poly(2-fluoroadenylic acid) [(fl2A)n], poly(2-chloroadenylic acid [(cl2A)n], poly(2-bromoadenylic acid) [(br2A)n] and poly(2-iodoadenylic acid) [(io2A)n] have been synthesized and evaluated for their effects on the RNA-directed DNA polymerase (reverse transcriptase) activity of Moloney murine leukaemia virus. All (A)n analogues were found to be potent inhibitors of reverse transcriptase, the order of (decreasing) potency being (fl2A)n greater than (io2A)n greater than (br2A)n greater than (cl2A)n. For all four (A)n analogues the inhibition of reverse transcriptase was competitive with respect to the template-primer. (A)n . oligo(dT). The K1 values were 0.02 microgram/ml for (fl2A)n, 0.1 microgram/ml for (io2A)n, 0.5 microgram/ml for (br2A)n and 8 microgram/ml for (cl2A)n. With a Ki of 0.02 microgram/ml (approx. 0.04 microM), (fl2A)n can be considered as one of the most, if not the most, potent polynucleotide inhibitor of reverse transcriptase that has been described so far.

Inhibition of reverse transcriptase activity of avian myeloblastosis virus by pyrophosphate analogues

Several pyrophosphate analogues have been studied for their effects on avian myeloblastosis virus reverse transcriptase and on cellular DNA polymerase α. Examination of structure-activity relationships for these compounds revealed that two acidic groups connected by a short bridge were necessary, but not sufficient, for inhibition of the enzyme activities. Foscarnet sodium (trisodium phosphonoformate) was the most potent inhibitor of reverse transcriptase, giving non-competitive inhibition of reactions primed by (rA) n · (dT) 12–18, (rC) n · (dG) 12–18, (dC) n · (dG) 12–18, and activated DNA. Carbonyldiphosphonate and 2-hydroxyphosphonoacetate also caused non-competitive inhibition patterns, whereas hypophosphate and imidodiphosphonate inhibited AMV reverse transcriptase in a competitive, non-linear manner. The reverse transcriptase reactions directed by (rA) n · (dT) 12–18 and activated DNA were most affected by the non-competitive inhibitors. Hypophosphate and imidodiphosphonate inhibited preferentially reactions primed by (dC) n · (dG) 12–18 and activated DNA. In all cases the (rC) n · (dG) 12–18 directed reaction was the least affected.

Antiviral properties of polyinosinic acids containing thio and methyl substitutions.

Polyinosinic acids containing methyl and sulphur substitutions are potent inhibitors of reverse transcriptase. Substitution of sulphur for oxygen at the 6 position produces significant effects on the properties of polyinosinic acid: the kinetics of inhibition change from competitive to mixed-type and the inhibition constant falls by three orders of magnitude. In contrast, 1-methyl substitution produces no such effects. Poly(1-methyl-6-thioinosinic acid) or poly(m1s6I) inhibits irreversibly, inhibiting all ten reverse transcriptases tested under a variety of assay conditions. In cell culture test systems, poly(m1s6I) is capable of blocking both infection by non-transforming viruses and transformation by a sarcoma virus. The presence of poly(m1s6I) in a preinfected culture results in the production of non-infectious virus particles lacking reverse transcriptase activity.