Human Immunodeficiency Virus Reverse Transcriptase Inhibitors Research Articles

Biocatalyst mediated green approach for 1,8-dioxo-octahydroxanthenes: SCXRD, Hirshfeld analysis and DFT studies as inhibitors of HIV reverse transcriptase

AIDS, caused by human immunodeficiency virus (HIV) is persistent on being one among the deadliest disease of all time. It destroys the immune system of the body and there is currently no effective cure for this. In this regard, in continuation to our studies on green catalysts, more efficient, constructive, and environmentally sustainable protocols for the synthesis of xanthene and its derivatives as HIV Reverse Transcriptase inhibitors were developed with the use of biocatalyst via baker's yeast that explores a viable approach on expanding environmental challenges and enlightens the superiority and exclusivity of this protocol. The current approach manifests numerous notable advantages that include ease of preparation, handling and benignity of the catalyst, low cost, green reaction conditions, facile workup, excellent yields (87–96 %) with extreme purity. The title compounds were docked on the crystal structure of a catalytic complex of HIV-1 reverse transcriptase (PDB: 1RTD). The C-score values achieved in the range 6.68–3.05 suggest that docking analysis was propitious signifying the review of all the forces of interaction between the ligand and the protein. The crystal studies and DFT analysis of compound 3d correlate the optimized geometrical features with empirically accessible XRD data.

Click Chemistry-Based Two-Component System for Efficient Inhibition of Human Immunodeficiency Virus (HIV) Reverse Transcriptase (RT).

We synthesized two dTTP analogues for copper-free “click” chemistry-coupling in the active sites of DNA polymerases. We found that in the presence of both analogues, human immunodeficiency virus (HIV) reverse transcriptase (RT) activity was suppressed by up to 93%. This inhibitory effect was not recovered by an excess amount of primer–template unlike that for a conventional HIV RT inhibitor, azidothymidine. This finding may become the basis for the development of efficient in vivo inhibitors of HIV RT and other DNA polymerases.

Synthesis, biological evaluation and molecular modeling of 2-Hydroxyisoquinoline-1,3-dione analogues as inhibitors of HIV reverse transcriptase associated ribonuclease H and polymerase

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not clinically validated as an antiviral target. 2-Hydroxyisoquinoline-1,3-dione (HID) is known to confer active site directed inhibition of divalent metal-dependent enzymatic functions, such as HIV RNase H, integrase (IN) and hepatitis C virus (HCV) NS5B polymerase. We report herein the synthesis and biochemical evaluation of a few C-5, C-6 or C-7 substituted HID subtypes as HIV RNase H inhibitors. Our data indicate that while some of these subtypes inhibited both the RNase H and polymerase (pol) functions of RT, potent and selective RNase H inhibition was achieved with subtypes 8–9 as exemplified with compounds 8c and 9c.

Computational prediction of human immunodeficiency resistance to reverse transcriptase inhibitors

Human immunodeficiency virus (HIV) causes acquired immunodeficiency syndrome (AIDS) and leads to over one million of deaths annually. Highly active antiretroviral treatment (HAART) is a gold standard in the HIV/AIDS therapy. Nucleoside and non-nucleoside inhibitors of HIV reverse transcriptase (RT) are important component of HAART, but their effect depends on the HIV susceptibility/resistance. HIV resistance mainly occurs due to mutations leading to conformational changes in the three-dimensional structure of HIV RT. The aim of our work was to develop and test a computational method for prediction of HIV resistance associated with the mutations in HIV RT. Earlier we have developed a method for prediction of HIV type 1 (HIV-1) resistance; it is based on the usage of position-specific descriptors. These descriptors are generated using the particular amino acid residue and its position; the position of certain residue is determined in a multiple alignment. The training set consisted of more than 1900 sequences of HIV RT from the Stanford HIV Drug Resistance database; for these HIV RT variants experimental data on their resistance to ten inhibitors are presented. Balanced accuracy of prediction varies from 80% to 99% depending on the method of classification (support vector machine, Naive Bayes, random forest, convolutional neural networks) and the drug, resistance to which is obtained. Maximal balanced accuracy was obtained for prediction of resistance to zidovudine, stavudine, didanosine and efavirenz by the random forest classifier. Average accuracy of prediction is 89%.

Design, Synthesis, and Biological Evaluations of Hydroxypyridonecarboxylic Acids as Inhibitors of HIV Reverse Transcriptase Associated RNase H.

Targeting the clinically unvalidated reverse transcriptase (RT) associated ribonuclease H (RNase H) for human immunodeficiency virus (HIV) drug discovery generally entails chemotypes capable of chelating two divalent metal ions in the RNase H active site. The hydroxypyridonecarboxylic acid scaffold has been implicated in inhibiting homologous HIV integrase (IN) and influenza endonuclease via metal chelation. We report herein the design, synthesis, and biological evaluations of a novel variant of the hydroxypyridonecarboxylic acid scaffold featuring a crucial N-1 benzyl or biarylmethyl moiety. Biochemical studies show that most analogues consistently inhibited HIV RT-associated RNase H in the low micromolar range in the absence of significant inhibition of RT polymerase or IN. One compound showed reasonable cell-based antiviral activity (EC50 = 10 μM). Docking and crystallographic studies corroborate favorable binding to the active site of HIV RNase H, providing a basis for the design of more potent analogues.

Hyaluronic Acid-Based Biocompatible Supramolecular Assembly for Sustained Release of Antiretroviral Drug

Human immunodeficiency virus (HIV) infection and its associated diseases continue to increase despite the progress in our understanding of HIV biology and the availability of a number of antiretroviral drugs. Adherence is a significant factor in the success of HIV therapy and current HIV treatment regimens require a combination of antiviral drugs to be taken at least daily for the remainder of a patient’s life. A drug delivery system that allows sustained drug delivery could reduce the medical burden and costs associated with medication nonadherence. Here, we describe a novel supramolecular assembly or matrix that contains an anionic polymer hyaluronic acid, cationic polymer poly-l-lysine, and anionic oligosaccharide sulfobutylether-beta-cyclodextrin. HIV reverse transcriptase inhibitors Zidovudine and Lamivudine were successfully encapsulated into the polymer assembly in a noncovalent manner. The physicochemical properties and antiviral activity of the polymer assemblies were studied. The results of this study suggest that the supramolecular assemblies loaded with HIV drugs exert potent antiviral activity and allow sustained drug release. A novel drug delivery formulation such as the one described here could facilitate our efforts to reduce the morbidity and mortality associated with HIV infections and could be utilized in the design of therapeutic approaches for other diseases.

Role of Cyclic GMP-AMP Synthase in Sensing Human Immunodeficiency Virus

*Cyclic guanosine monophosphate adenosine monophosphate (cGAMP) synthase (cGAS) detects human immunodeficiency virus (HIV) and produces cGAMP to induce cytokines. Reverse transcribed DNA of HIV is critical for triggering innate immune responses as inhibitor of HIV reverse transcriptase blocked the induction of interferon-β by the virus. Furthermore, knockout of cGAS in human or mouse cell lines abrogated the production of cytokines by HIV infection highlighting the essential role of cGAS in detection of HIV and other retroviruses.

N-3 Hydroxylation of Pyrimidine-2,4-diones Yields Dual Inhibitors of HIV Reverse Transcriptase and Integrase

A new molecular scaffold featuring an N-hydroxyimide functionality and capable of inhibiting both reverse transcriptase (RT) and integrase (IN) of Human Immunodeficiency Virus (HIV) was rationally designed based on 1-[(2-hydroxyethoxy) methyl]-6-(phenylthio)-thymine (HEPT) non-nucleoside RT inhibitors (NNRTIs). The design involves a minimal 3-N hydroxylation of the pyrimidine ring of HEPT compound to yield a chelating triad which, along with the existing benzyl group, appeared to satisfy major structural requirements for IN binding. In the mean time, this chemical modification did not severely compromise the compound's ability to inhibit RT. A preliminary structure-activity-relationship (SAR) study reveals that this N-3 OH is essential for IN inhibition and that the benzyl group on N-1 side chain is more important for IN binding than the one on C-6.

Dyslipidaemia and Intima-Media Thickness of Carotid Arteries in Thirty-Five HIV/AIDS Patients Receiving Highly Active Antiretroviral Therapy

Objectives: Highly active antiretroviral therapy (HAART) has being impacted significantly therapies for natural human immunodeficiency virus (HIV) infection, which leads to a remarkable decrease in its morbidity and mortality but it is frequently associated with metabolic complications such as dyslipidaemia and cardiovascular complications. HIV reverse transcriptase (RT) inhibitors can be classified into nucleoside and non-nucleoside types. Mitochondrial dysfunction due to the depletion of mt-DNA is partly responsible for various nucleoside RT inhibitors-associated adverse effects including dyslipidaemia. Efavirenz (EFV) is metabolized primarily by cytochrome P450 2B6 (CYP2B6) and the metabolic effects of EFV have been described previously. All patients in this study received the same HAART treatment regime (Stavudine (d4T) + Lamivudine (3TC) + Efavirenz (EFV)). This study aims to assess incidences for dyslipidaemia and atherosclerosis. Methods: This retrospective study was conducted within outpatients of Shanghai Public Health Center. We selected thirty-five HIV-1 infected patients who receiving highly active antiretroviral therapy. Their mean CD4 cell count was 69.5 (±34.6) copies per micro liter before therapy. Fasting total cholesterol, triglyceride, high-density lipoprotein (HDL) cholesterol and low-density lipoprotein (LDL) cholesterol values were respectively compared among patients at present and before therapy. Then the data was statistically analyzed. Twenty-two patients had the intima-media thickness (IMT) of their carotid arteries measured by Philips 5000 Color-Doppler ultrasound tests. Results: After therapy, patients achieved significant changes in levels of triglycerides (1.44 ± 0.35mmol/L Vs. 2.07 ± 0.54mmol/L) (P<0.001), total cholesterol (4.96 ± 0.46 mmol/L Vs. 6.15 ± 0.83mmol/L) (P<0.001) and LDL cholesterol (2.29 ± 0.33 Vs. 3.11 ± 0.29 mmol/L) (P<0.001). In contrast, the level of HDL cholesterol did not significantly change (1.06 ± 0.01 mmol/L Vs. 1.04 ± 0.01 mmol/L) (P>0.5). The mean IMT of twenty-two patients was (0.86 ± 0.14) mm after HAART, which is higher than the norm age-matched value of (0.7 ± 0.2) mm (P<0.05). Conclusion: These data suggest that HAART is potentially dangerous for hyperlipidaemia and maybe an increase in atherosclerosis.

Novel Aptamer Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase

Primer-template-based double-stranded nucleic acids capable of binding human immunodeficiency virus reverse transcriptase (HIV-RT) with high affinity were used as starting material to develop small single-stranded loop-back DNA aptamers. The original primer-templates were selected using a SELEX (Systematic Evolution of Ligands by EXponential enrichment) approach and consisted of 46- and 50-nt primer and template strands, respectively. The major determinant of the approximately 10-fold tighter binding in selected sequences relative to control primer-templates was a run of 6.8 G residues at the 3' primer end. Sixty, thirty-seven, twenty-seven, and twenty-two nucleotide loop-back single-stranded versions that retained the base pairs near the 3' primer terminus were constructed. Both the 60- and 37-nt versions retained high affinity for RT with K(d) values of approximately 0.44 nM and 0.66 nM, respectively. Random sequence primer-templates of the same length had K(d)s of approximately 20 nM and approximately 161 nM. The shorter 27- and 22-nt aptamers bound with reduced affinity. Several modifications of the 37-nt aptamer were also tested including changes to the terminal 3' G nucleotide and internal bases in the G run, replacement of specific nucleotides with phosphothioates, and alterations to the 5' overhang. Optimal binding required a 4- to 5-nt overhang, and internal changes within the G run had a pronounced negative effect on binding. Phosphothioate nucleotides or the presence of a 3' dideoxy G residue did not alter affinity. The 37-nt aptamer was a potent inhibitor of HIV-RT in vitro and functioned by blocking binding of other primer-templates.

Genotypic resistance testing in HIV by arrayed primer extension

The analysis of mutations that are associated with the occurrence of drug resistance is important for monitoring the antiretroviral therapy of patients infected with human immunodeficiency virus (HIV). Here, we describe the establishment and successful application of Arrayed Primer Extension (APEX) for genotypic resistance testing in HIV as a rapid and economical alternative to standard sequencing. The assay is based on an array of oligonucleotide primers that are immobilised via their 5′-ends. Upon hybridisation of template DNA, a primer extension reaction is performed in the presence of the four dideoxynucleotides, each labelled with a distinct fluorophore. The inserted label immediately indicates the sequence at the respective position. Any mutation changes the colour pattern. We designed a microarray for the analysis of 26 and 33 codons in the HIV protease and reverse transcriptase, respectively, which are of special interest with respect to drug resistance. The enormous genome variability of HIV represents a big challenge for genotypic resistance tests, which include a hybridisation step, both in terms of specificity and probe numbers. The use of degenerated oligonucleotides resulted in a significant reduction in the number of primers needed. For validation, DNA of 94 and 48 patients that exhibited resistance to inhibitors of HIV protease and reverse transcriptase, respectively, were analysed. The validation included HIV subtype B, prevalent in industrialised countries, as well as non-subtype B samples that are more common elsewhere.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-007-1775-0) contains supplementary material, which is available to authorized users.

Coumarins as Inhibitors of HIV Reverse Transcriptase

Acquired immunodeficiency syndrome (AIDS), a degenerative disease of the immune and central nervous systems, is an enormous world-wide health threat. No cure has been found, and research is aimed at developing chemotherapy against the causative agent, human immunodeficiency virus (HIV). Chemotherapy for AIDS has progressed steadily in the past decade. However, new, effective, and less toxic chemotherapeutic agents are still needed. Plants, particularly anti-infective or immunomodulating herbal medicines, can serve as sources of new active leads to be further developed as anti-AIDS drug candidates. A lot of structurally different natural coumarins were found to display potent anti-HIV activity and continued progress is anticipated in the discovery of new leads and in the development of these agents as potential anti-AIDS drug candidates. Recent studies based on the account of various coumarins from plant sources and their analogs--synthetic coumarins, indicate that some of them serve as potent nonnucleoside RT-inhibitors, another as inhibitors of HIV-integrase or HIV-protease. The current review demonstrates the variety of coumarins of natural plant origin and synthetic coumarins having unique mechanism of action to one of the most important stage of HIV replication (RT-inhibition). The merits of selecting potential anti-HIV agents to be used in rational combination drugs design and structure-activity relationships are discussed. The scientific community is looking actively for new drugs and combinations for treatment of HIV infection effective for first-line treatment, as well as against drug-resistant mutants.

Circulating Metabolites of the Human Immunodeficiency Virus Protease Inhibitor Nelfinavir in Humans: Structural Identification, Levels in Plasma, and Antiviral Activities

Nelfinavir mesylate (Viracept, formally AG1343) is a potent and orally bioavailable human immunodeficiency virus (HIV) type 1 (HIV-1) protease inhibitor (K(i) = 2 nM) and is being widely prescribed in combination with HIV reverse transcriptase inhibitors for the treatment of HIV infection. The current studies evaluated the presence of metabolites circulating in plasma following the oral administration of nelfinavir to healthy volunteers and HIV-infected patients, as well as the levels in plasma and antiviral activities of these metabolites. The results showed that the parent drug was the major circulating chemical species, followed in decreasing abundance by its hydroxy-t-butylamide metabolite (M8) and 3'-methoxy-4'-hydroxynelfinavir (M1). Antiviral assays with HIV-1 strain RF-infected CEM-SS cells showed that the 50% effective concentrations (EC50) of nelfinavir, M8, and M1 were 30, 34, and 151 nM, respectively, and that the corresponding EC50 against another HIV-1 strain, IIIB, in MT-2 cells were 60, 86, and 653 nM. Therefore, apparently similar in vitro antiviral activities were demonstrated for nelfinavir and M8, whereas an approximately 5- to 11-fold-lower level of antiviral activity was observed for M1. The active metabolite, M8, showed a degree of binding to human plasma proteins similar to that of nelfinavir (ca. 98%). Concentrations in plasma of nelfinavir and its metabolites in 10 HIV-positive patients receiving nelfinavir therapy (750 mg three times per day) were determined by a liquid chromatography tandem mass spectrometry assay. At steady state (day 28), the mean plasma nelfinavir concentrations ranged from 1.73 to 4.96 microM and the M8 concentrations ranged from 0.55 to 1.96 microM, whereas the M1 concentrations were low and ranged from 0.09 to 0.19 microM. In conclusion, the findings from the current studies suggest that, in humans, nelfinavir forms an active metabolite circulating at appreciable levels in plasma. The active metabolite M8 may account for some of the antiviral activity associated with nelfinavir in the treatment of HIV disease.

Inhibitory effect of human immunodeficiency virus protease inhibitors on multidrug resistance transporter P-glycoproteins.

The objective of this study was to determine whether human immunodeficiency virus (HIV) protease inhibitors (saquinavir, ritonavir and nelfinavir) interact with other HIV protease inhibitors and/or HIV reverse transcriptase inhibitors (zidovudine, didanosine, lamivudine, zalcitabine and sanilvudine). We measured transport of nelfinavir, an HIV protease inhibitor which is known as a substrate for the multidrug resistance transporter P-glycoprotein (P-gp), in an epithelial monolayer model and Ki for P-gp of some drugs by a calcein flux assay. Transport in a basal to apical direction was 2-fold greater than apical to basal flux for nelfinavir, Ki for P-gp of a potent P-gp inhibitor cyclosporin A was 1.09 microM and those of ritonavir and nelfinavir were 111 microM and 28.6 microM, whereas all HIV reverse transcriptase inhibitors gave high K1 values. These data show that nelfinavir, which is a substrate for P-gp, inhibits a P-gp function as a drug efflux pump and that HIV reverse transcriptase inhibitors do not inhibit P-gp.

Anti-human immunodeficiency virus (anti-HIV) natural products with special emphasis on HIV reverse transcriptase inhibitors

This review article aims at summarizing research findings concerning natural products which are endowed with the ability to inhibit human immunodeficiency virus (HIV). An emphasis is placed on HIV reverse transcriptase inhibitors because the bulk of the literature is focused on these compounds. It was found that a spectacular diversity of chemical structures encompassing proteins, terpenoids, coumarins, xanthones, alkaloids, flavonoids, polyphenols, and polysaccharides, which are elaborated by plant species as phylogenetically remote as the algae, gymnosperms and angiosperms, were capable of rendering the retroviral enzyme less active. The literature pertaining to natural products with HIV protease and integrase inhibitory activites is less voluminous.

Genetic Selection inEscherichia colifor Active Human Immunodeficiency Virus Reverse Transcriptase Mutants

Most catalytically active human immunodeficiency virus (HIV) reverse transcriptase (RT) mutants characterized to date have been isolated from the virus after treatment with HIV RT inhibitors such as nucleoside analogs. However, detailed understanding of structure–function relationships, and of the roles of the several catalytic activities of HIV RT in viral replication, requires characterization of a greater diversity of mutant enzymes than has been obtained from viral variants. Coupling of a bacterial genetic selection system for functional HIV RT with random mutagenesis has yielded a large number of active mutant enzymes, most of which have not been found in viral variants. The genetic selection system, combined with biochemical characterization of active mutant proteins, affords three major benefits. First, we can increase our understanding of the roles of individual amino acids in catalysis. Second, the mutational spectrum observed among active HIV RT variants can identify amino acids that are intolerant, or relatively intolerant, of substitution. Third, this system provides us with HIV RT variants with altered biochemical properties, such as replicational fidelity and processivity. Characterization of HIV harboring these mutant RTs with defined structural and functional alterations will contribute to elucidation of the roles of each catalytic activity of HIV RT in viral replication.

Inhibitory Effects of Polyphenolic Catechins from Chinese Green Tea on HIV Reverse Transcriptase Activity.

Three polyphenolic catechins, epigallocatechin (1), epicatechin-3-O-gallate (2) and epigallocatechin-3-O-gallate (3), were isolated from Chinese green tea, Ti-Kaun-Yin (Camellia sinensis) and demonstrated as a new class of human immunodeficiency virus-reverse transcriptase (HIV-RT) inhibitor. The concentrations required for 50% inhibition for the compounds (1), (2) and (3) were 7.80, 0.32 and 0.68 &mgr;M, respectively. The polyphenolic catechins with a galloyl group at the 3 position were potent inhibitors of HIV-RT. Kinetic analysis indicated that the polyphenolic catechins were competitive inhibitors with respect to the template-primer (rA)(n)(dT)(12-18) and noncompetitive inhibitors to dTTP. Copyright 1994 S. Karger AG, Basel

Combinative interactions of a human immunodeficiency virus (HIV) Tat antagonist with HIV reverse transcriptase inhibitors and an HIV protease inhibitor.

Combinations of the human immunodeficiency virus (HIV) Tat protein antagonist Ro 24-7429 with either the HIV protease inhibitor Ro 31-8959 or the HIV reverse transcriptase inhibitors AZT (3'-azido-3'-deoxythymidine), ddC (2',3'-dideoxycytidine), ddI (2',3'-dideoxyinosine), and nevirapine were synergistic or additive in reducing HIV type 1 p24 antigen production in CEM cells or inhibiting HIV type 1-induced syncytium formation in HT4-6C cells.

Phosphorylation of carbovir enantiomers by cellular enzymes determines the stereoselectivity of antiviral activity.

Two enantiomers of carbovir, a carbocyclic analog of 2',3'-dideoxyguanosine, were compared with respect to their phosphorylation and the phosphorylation of their nucleotides by mammalian enzymes. 5'-Nucleotidase catalyzed the phosphorylation of (-)-carbovir, which is active against HIV (human immunodeficiency virus), but did not phosphorylate (+)-carbovir. (-)-Carbovir monophosphate was 7,000 times more efficient as a substrate for GMP kinase than was (+)-carbovir monophosphate. Pyruvate kinase, phosphoglycerate kinase, and creatine kinase phosphorylated both enantiomers of carbovir diphosphate at similar rates. Nucleoside-diphosphate kinase preferentially phosphorylated the (-)-enantiomer. Both enantiomers of carbovir triphosphate were substrates and alternative substrate inhibitors of HIV reverse transcriptase. Thus, the contrasting HIV-inhibitory activities of carbovir enantiomers were due to differential phosphorylation by cellular enzymes and not due to enantioselectivity of HIV reverse transcriptase.

Pharmacodynamics of 2′,3′-dideoxycytidine: An inhibitor of human immunodeficiency virus

The incidence of acquired immunodeficiency syndrome and the number of people infected with the human immunodeficiency virus (HIV) is likely to increase into the 1990s and perhaps beyond. Zidovudine, a 2′,3′-dideoxynucleoside approved for the treatment of acquired immunodeficiency syndrome, provides immunologic, virologic, and survival benefits. However, because its hematologic toxicity can be dose-limiting, investigations are ongoing with other 2′,3′-dideoxynucleosides. After zidovudine, the first of these agents to be tested was 2′,3′-dideoxycytidine (ddC), the most potent inhibitor of HIV reverse transcriptase among the dideoxynucleosides tested thus far. Concentrations of ddC as low as 0.5 μM provide protection against HIV in cultured T cells (and monocytes), even at high multiplicities of infection. Like the other dideoxynucleosides, activation of ddC is dependent on intracellular phosphorylation to its 5′-triphosphate form. Efforts are under way to alter enzymatically the intracellular ratio of ddC-5′-triphosphate to deoxycytidine-5′-triphosphate, its endogenous counterpart. ddC has relatively straightforward pharmacokinetics; it has a plasma half-life of about 1.2 hours and an oral bioavailability of about 87 percent. Approximately 75 percent of the drug is excreted unchanged in the urine. Patients treated with ddC have experienced both immunologic and virologic benefit, although long-term high doses are limited by the development of painful peripheral neuropathy. Significant hematologic toxicity is not evident in most patients; low-dose regimens of ddC alone, as well as alternating or in combination with zidovudine, are being tested in an effort to retain antiviral activity while minimizing treatment toxicities.