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1.
Enferm. infecc. microbiol. clín. (Ed. impr.) ; 36(2): 91-94, feb. 2018. ilus, tab, graf
Artigo em Espanhol | IBECS | ID: ibc-170696

RESUMO

Objetivo: Generar una secuencia consenso a partir de los datos de secuenciación masiva obtenidos en estudios de resistencias a antiretrovirales, que sea representativa de la secuencia Sanger y que sirva para estudios de epidemiología molecular. Material y métodos: En 62 pacientes se obtuvo la secuencia de transcriptasa reversa-proteasa, mediante Sanger (Trugene-Siemens), y NGS (454GSJunior-Roche). Las secuencias consenso NGS se generaron con Mesquite, seleccionando umbrales 10%, 15% y 20%. Para el estudio filogenético se empleó MEGA. Resultados: Utilizando el umbral 10%, 17/62 pacientes presentaron secuencias pareadas NGS-Sanger, con una mediana de bootstrap del 88% (IQR83,5-95,5). La asociación aumenta a 36/62 pacientes y el bootstrap, a 94% (IQR85,5-98), y alcanza el máximo al 20% en 61/62 pacientes, bootstrap 99% (IQR98-100). Conclusión: Mostramos un método seguro para generar secuencias consenso NGS para su uso en estudios de epidemiología molecular procesadas con umbral 20%, de fácil uso y aplicación en los servicios de microbiología clínica (AU)


Objective: To show how to generate a consensus sequence from the information of massive parallel sequences data obtained from routine HIV anti-retroviral resistance studies, and that may be suitable for molecular epidemiology studies. Material and methods: Paired Sanger (Trugene-Siemens) and next-generation sequencing (NGS) (454 GSJunior-Roche) HIV RT and protease sequences from 62 patients were studied. NGS consensus sequences were generated using Mesquite, using 10%, 15%, and 20% thresholds. Molecular evolutionary genetics analysis (MEGA) was used for phylogenetic studies. Results: At a 10% threshold, NGS-Sanger sequences from 17/62 patients were phylogenetically related, with a median bootstrap-value of 88% (IQR83.5-95.5). Association increased to 36/62 sequences, median bootstrap 94% (IQR85.5-98)], using a 15% threshold. Maximum association was at the 20% threshold, with 61/62 sequences associated, and a median bootstrap value of 99% (IQR98-100). Conclusion: A safe method is presented to generate consensus sequences from HIV-NGS data at 20% threshold, which will prove useful for molecular epidemiological studies (AU)


Assuntos
Humanos , Adulto , HIV , Infecções por HIV/epidemiologia , Análise de Sequência/métodos , Epidemiologia Molecular/métodos , Resistência a Medicamentos , Epidemiologia Molecular/estatística & dados numéricos , Inibidores da Transcriptase Reversa/análise , Inibidores da Transcriptase Reversa/isolamento & purificação , Filogenia , Antirretrovirais
2.
J Recept Signal Transduct Res ; 38(1): 37-47, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29254400

RESUMO

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have gained a definitive place due to their unique antiviral potency, high specificity and low toxicity in antiretroviral combination therapies which are used to treat HIV. To design more specific HIV-1 inhibitors, 218 diverse non-nucleoside reverse transcriptase inhibitors with their EC50 values were collected. Then, different types of molecular descriptors were calculated. Also, genetic algorithm (GA) and enhanced replacement methods (ERM) were used as the variable selection approaches to choose more relevant features. Based on selected descriptors, a classification support vector machine (SVM) model was constructed to categorize compounds into two groups of active and inactive ones. The most active compound in the set was docked and was used as the input to the Pharmit server to screen the Molport and PubChem libraries by constructing a structure-based pharmacophore model. Shape filters for the protein and ligand as well as Lipinski's rule of five have been applied to filter out the output of virtual screening from pharmacophore search. Three hundred and thirty-four compounds were finally retrieved from the virtual screening and were fed to the previously constructed SVM model. Among them, the SVM model rendered seven active compounds and they were also analyzed by docking calculations and ADME/Tox parameters.


Assuntos
Antivirais/química , Transcriptase Reversa do HIV/química , HIV-1/química , Inibidores da Transcriptase Reversa/química , Antivirais/isolamento & purificação , Antivirais/uso terapêutico , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Humanos , Simulação de Acoplamento Molecular , Conformação Proteica/efeitos dos fármacos , Relação Quantitativa Estrutura-Atividade , Inibidores da Transcriptase Reversa/isolamento & purificação , Inibidores da Transcriptase Reversa/uso terapêutico , Máquina de Vetores de Suporte , Interface Usuário-Computador
3.
J Nat Prod ; 80(6): 1798-1807, 2017 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-28613071

RESUMO

Justicia gendarussa, a medicinal plant collected in Vietnam, was identified as a potent anti-HIV-1 active lead from the evaluation of over 4500 plant extracts. Bioassay-guided separation of the extracts of the stems and roots of this plant led to the isolation of an anti-HIV arylnaphthalene lignan (ANL) glycoside, patentiflorin A (1). Evaluation of the compound against both the M- and T-tropic HIV-1 isolates showed it to possess a significantly higher inhibition effect than the clinically used anti-HIV drug AZT. Patentiflorin A and two congeners were synthesized, de novo, as an efficient strategy for resupply as well as for further structural modification of the anti-HIV ANL glycosides in the search for drug leads. Subsequently, it was determined that the presence of a quinovopyranosyloxy group in the structure is likely essential to retain the high degree of anti-HIV activity of this type of compounds. Patentiflorin A was further investigated against the HIV-1 gene expression of the R/U5 and U5/gag transcripts, and the data showed that the compound acts as a potential inhibitor of HIV-1 reverse transcription. Importantly, the compound displayed potent inhibitory activity against drug-resistant HIV-1 isolates of both the nucleotide analogue (AZT) and non-nucleotide analogue (nevaripine). Thus, the ANL glycosides have the potential to be developed as novel anti-HIV drugs.


Assuntos
Fármacos Anti-HIV/isolamento & purificação , Fármacos Anti-HIV/farmacologia , Glicosídeos/isolamento & purificação , Glicosídeos/farmacologia , HIV-1/efeitos dos fármacos , Adhatoda/química , Lignanas/isolamento & purificação , Lignanas/farmacologia , Plantas Medicinais/química , Inibidores da Transcriptase Reversa/isolamento & purificação , Inibidores da Transcriptase Reversa/farmacologia , Fármacos Anti-HIV/química , Glicosídeos/química , HIV-1/genética , Humanos , Lignanas/química , Estrutura Molecular , Raízes de Plantas/química , Caules de Planta/química , Inibidores da Transcriptase Reversa/química , Vietnã , Zidovudina/farmacologia
4.
Future Microbiol ; 10(11): 1767-72, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26517310

RESUMO

Resistance continues to emerge as a leading cause for antiretroviral treatment failure. Several mutations in HIV reverse transcriptase (RT) confer resistance to non-nucleoside inhibitors (NNRTIs), vital components of antiretroviral combination therapies. Since the majority of mutations are located in the NNRTI binding pocket, crystal structures of RT variants in complex with NNRTIs have provided ideas for new drug design strategies. This article reviews the impact of RT crystal structures on the multidisciplinary design and development of new inhibitors with improved resistance profiles.


Assuntos
Fármacos Anti-HIV/isolamento & purificação , Fármacos Anti-HIV/farmacologia , Descoberta de Drogas/métodos , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV/efeitos dos fármacos , Inibidores da Transcriptase Reversa/isolamento & purificação , Inibidores da Transcriptase Reversa/farmacologia , Cristalografia por Raios X , Descoberta de Drogas/tendências , Farmacorresistência Viral , HIV/enzimologia , Transcriptase Reversa do HIV/química , Humanos , Simulação de Acoplamento Molecular , Mutação , Conformação Proteica , Seleção Genética
5.
Antiviral Res ; 123: 132-7, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26408354

RESUMO

Hepatitis B virus (HBV) infections rely on the proper functioning of the viral polymerase enzyme, a specialized reverse transcriptase (RT) with multiple activities. All currently approved antiviral drugs for the treatment of chronic HBV infection, except for interferon, target the RT and belong to the same chemical class - they are all nucleoside analogs. Viral DNA synthesis is carried out by the RT enzyme in several different steps, each with distinct RT conformational requirements. In principle, each stage may be targeted by distinct antiviral drugs. In particular, the HBV RT has the unique ability to initiate viral DNA synthesis using itself as a protein primer in a novel protein priming reaction. In order to help identify RT inhibitors and study their mechanisms of action, a number of experimental systems have been developed, each varying in its ability to dissect the protein priming stage and subsequent stages of viral DNA synthesis at the molecular level. Two of the most effective drugs to date, entecavir and tenofovir, can inhibit both the protein priming and the subsequent DNA elongation stages of HBV DNA synthesis. Interestingly, clevudine, a thymidine analog, can inhibit both protein priming and DNA elongation in a non-competitive manner and without being incorporated into the viral DNA. Thus, a nucleoside RT inhibitor (NRTI) can functionally mimic a non-NRTI (NNRTI) in its inhibition of the HBV RT. Therefore, novel NRTIs as well as NNRTIs may be developed to inhibit the DNA synthesis activity of the HBV RT. Furthermore, additional activities of the RT that are also essential to HBV replication, including specific recognition of the viral RNA and its packaging into viral nucleocapsids, may be exploited for antiviral development. To achieve a more potent inhibition of viral replication and ultimately cure chronic HBV infection, the next generation of anti-HBV therapies will likely need to include NRTIs, NNRTIs, and other agents that target the viral RT as well as other viral and host factors in various combinations. This article forms part of a symposium in Antiviral Research on "An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B."


Assuntos
Antivirais/isolamento & purificação , Antivirais/farmacologia , Vírus da Hepatite B/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Inibidores da Transcriptase Reversa/isolamento & purificação , Inibidores da Transcriptase Reversa/farmacologia , Descoberta de Drogas/métodos , Descoberta de Drogas/tendências , Humanos
6.
Fitoterapia ; 106: 158-66, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26347951

RESUMO

Six new naturally occurring polyoxygenated cyclohexene derivatives together with eight related known derivatives, two known alkaloids, and two known flavonoid derivatives were isolated from bioassay-guided fractionation of the ethyl acetate extract of the leaves and twigs of Dasymaschalon sootepense. The structure elucidation and determination of absolute configurations were established by various spectroscopic methods, X-ray diffraction techniques as well as comparison with the literature data. Several isolated compounds were evaluated for their cytotoxic, anti-HIV-1 RT and anti-inflammatory activities.


Assuntos
Alcaloides/química , Annonaceae/química , Fármacos Anti-HIV/química , Anti-Inflamatórios/química , Cicloexenos/química , Alcaloides/isolamento & purificação , Animais , Fármacos Anti-HIV/isolamento & purificação , Anti-Inflamatórios/isolamento & purificação , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Linhagem Celular Tumoral , Cicloexenos/isolamento & purificação , HIV-1/efeitos dos fármacos , Humanos , Masculino , Camundongos , Estrutura Molecular , Folhas de Planta/química , Ratos , Ratos Sprague-Dawley , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/isolamento & purificação
7.
Appl Biochem Biotechnol ; 177(6): 1374-85, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26304129

RESUMO

Lectins have captured the attention of a large number of researchers on account of their various exploitable activities, including antitumor, immunomodulatory, antifungal, as well as HIV reverse transcriptase inhibitory activities. A mannose/glucose-specific lectin was isolated from green split peas (a variety of Pisum sativum) and characterized. The purification step involved anion-exchange chromatography on a DEAE-cellulose column, cation-exchange chromatography on an SP-Sepharose column, and gel filtration by fast protein liquid chromatography (FPLC) on Superdex 200. The purified lectin had a native molecular mass of around 50 kDa as determined by size exclusion chromatography. It appeared as a heterotetramer, composed of two distinct polypeptide bands with a molecular mass of 6 and 19 kDa, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The N-terminal sequence of green split pea lectin shows some degree of homology compared to lectins from other legume species. Its hemagglutinating activity was inhibited by glucose, mannose, and sucrose, and attenuated at pH values higher than 12 or lower than 3. Hemagglutinating activity was preserved at temperatures lower than 80 °C. The lectin did not show antifungal activity toward fungi including Fusarium oxysporum, Botrytis cinerea, and Mycosphaerella arachidicola. Green split pea lectin showed a mitogenic effect toward murine splenocytes and could inhibit the activity of HIV-1 reverse transcriptase.


Assuntos
Mitógenos , Ervilhas/química , Lectinas de Plantas , Inibidores da Transcriptase Reversa , Animais , Proliferação de Células/efeitos dos fármacos , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/enzimologia , Camundongos , Mitógenos/química , Mitógenos/isolamento & purificação , Mitógenos/farmacologia , Lectinas de Plantas/química , Lectinas de Plantas/isolamento & purificação , Lectinas de Plantas/farmacologia , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/isolamento & purificação , Inibidores da Transcriptase Reversa/farmacologia , Baço/metabolismo
8.
Proc Natl Acad Sci U S A ; 112(22): 6979-84, 2015 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-26038551

RESUMO

Fragment-based screening methods can be used to discover novel active site or allosteric inhibitors for therapeutic intervention. Using saturation transfer difference (STD) NMR and in vitro activity assays, we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase (RT) with distinct chemical scaffolds and mechanisms compared to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit RNA- and DNA-dependent DNA polymerase activity of HIV-1 RT in the micromolar range while retaining potency against RT variants carrying one of three major NNRTI resistance mutations: K103N, Y181C, or G190A. These compounds also inhibit Moloney murine leukemia virus RT but not the Klenow fragment of Escherichia coli DNA polymerase I. Steady-state kinetic analyses demonstrate that one of these fragments is a competitive inhibitor of HIV-1 RT with respect to deoxyribonucleoside triphosphate (dNTP) substrate, whereas a second compound is a competitive inhibitor of RT polymerase activity with respect to the DNA template/primer (T/P), and consequently also inhibits RNase H activity. The dNTP competing RT inhibitor retains activity against the NRTI-resistant mutants K65R and M184V, demonstrating a drug resistance profile distinct from the nucleotide competing RT inhibitors indolopyridone-1 (INDOPY-1) and 4-dimethylamino-6-vinylpyrimidine-1 (DAVP-1). In antiviral assays, the T/P competing compound inhibits HIV-1 replication at a step consistent with an RT inhibitor. Screening of additional structurally related compounds to the three fragments led to the discovery of molecules with improved potency against HIV-1 RT. These fragment inhibitors represent previously unidentified scaffolds for development of novel drugs for HIV-1 prevention or treatment.


Assuntos
Descoberta de Drogas/métodos , HIV-1/enzimologia , Pró-Fármacos/isolamento & purificação , Inibidores da Transcriptase Reversa/isolamento & purificação , Inibidores da Transcriptase Reversa/farmacologia , Primers do DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Espectroscopia de Ressonância Magnética , Pró-Fármacos/análise , Inibidores da Transcriptase Reversa/análise , Ribonuclease H/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas , Replicação Viral/efeitos dos fármacos
9.
J Sep Sci ; 38(10): 1755-62, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25763883

RESUMO

A molecularly imprinted polymer has been synthesized to specifically extract adefovir, an antiviral drug, from serum and urine by dispersive solid-phase extraction before high-performance liquid chromatography with UV analysis. The imprinted polymers were prepared by bulk polymerization by a noncovalent imprinting method that involved the use of adefovir (template molecule) and functional monomer (methacrylic acid) complex prior to polymerization, ethylene glycol dimethacrylate as cross-linker, and chloroform as porogen. Molecular recognition properties, binding capacity, and selectivity of the molecularly imprinted polymers were evaluated and the results show that the obtained polymers have high specific retention and enrichment for adefovir in aqueous medium. The new imprinted polymer was utilized as a molecular sorbent for the separation of adefovir from human serum and urine. The serum and urine extraction of adefovir by the molecularly imprinted polymer followed by high-performance liquid chromatography showed a linear calibration curve in the range of 20-100 µg/L with excellent precisions (2.5 and 2.8% for 50 µg/L), respectively. The limit of detection and limit of quantization were determined in serum (7.62 and 15.1 µg/L), and urine (5.45 and 16 µg/L). The recoveries for serum and urine samples were found to be 88.2-93.5 and 84.3-90.2%, respectively.


Assuntos
Adenina/análogos & derivados , Impressão Molecular , Organofosfonatos/isolamento & purificação , Polímeros/química , Inibidores da Transcriptase Reversa/isolamento & purificação , Água/química , Adenina/sangue , Adenina/isolamento & purificação , Adenina/urina , Humanos , Concentração de Íons de Hidrogênio , Organofosfonatos/sangue , Organofosfonatos/urina , Inibidores da Transcriptase Reversa/sangue , Inibidores da Transcriptase Reversa/urina , Espectrofotometria Infravermelho , Termodinâmica
10.
Artigo em Inglês | MEDLINE | ID: mdl-25463191

RESUMO

A molecularly imprinted polymer (MIP) has been synthesized in order to specifically extract efavirenz from serum and urine by dispersive solid-phase extraction following by HPLC-UV analysis. The imprinted nanoparticles were prepared by miniemulsion polymerization method using efavirenz as template molecule and methacrylic acid as functional monomer. Molecular recognition properties, binding capacity and selectivity of the MIPs were evaluated and the results revealed that the obtained MIPs had high specific retention for efavirenz in aqueous medium. The MIP was used as a molecular sorbent for the separation of efavirenz from human serum and urine. The extraction of efavirenz by MIP coupled with HPLC analysis showed a linear calibration curve in the range of 50-300 µg/L with exellent precisions (3.66% and 4.6% for 100 and 300 µg/L respectively). The limit of detection (LOD) and limit of quantification (LOQ) were determind in serum (17.3 and 57.5 µg/L) and urine (10.6 and 36.2 µg/L). The maximum recoveries for serum and urine samples were found to be 95.2% and 92.7% respectively. Due to the high precision and accuracy, this method may be the UV-HPLC choice with MIP extraction for bioequivalence analysis of efavirenz in serum and urine.


Assuntos
Benzoxazinas/química , Nanopartículas/química , Inibidores da Transcriptase Reversa/química , Extração em Fase Sólida/instrumentação , Extração em Fase Sólida/métodos , Adsorção , Benzoxazinas/sangue , Benzoxazinas/isolamento & purificação , Benzoxazinas/urina , Cromatografia Líquida de Alta Pressão , Humanos , Impressão Molecular , Polimerização , Polímeros/síntese química , Inibidores da Transcriptase Reversa/sangue , Inibidores da Transcriptase Reversa/isolamento & purificação , Inibidores da Transcriptase Reversa/urina
11.
Biochem J ; 462(3): 425-32, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-24969820

RESUMO

HIV-1 resistance to zidovudine [AZT (azidothymidine)] is associated with selection of the mutations M41L, D67N, K70R, L210W, T215F/Y and K219Q/E in RT (reverse transcriptase). These mutations decrease HIV-1 susceptibility to AZT by augmenting RT's ability to excise the chain-terminating AZT-MP (AZT-monophosphate) moiety from the chain-terminated DNA primer. Although AZT-MP excision occurs at the enzyme's polymerase active site, it is mechanistically distinct from the DNA polymerase reaction. Consequently, this activity represents a novel target for drug discovery, and inhibitors that target this activity may increase the efficacy of nucleoside/nucleotide analogues, and may help to delay the onset of drug resistance. In the present study, we have developed a FRET (Förster resonance energy transfer)-based high-throughput screening assay for the AZT-MP excision activity of RT. This assay is sensitive and robust, and demonstrates a signal-to-noise ratio of 3.3 and a Z' factor of 0.69. We screened three chemical libraries (7265 compounds) using this assay, and identified APEX57219 {3,3'-[(3-carboxy-4-oxo-2,5-cyclohexadien-1-ylidene)methylene]bis[6-hydroxybenzoic acid]} as the most promising hit. APEX57219 displays a unique activity profile against wild-type and drug-resistant HIV-1 RT, and was found to inhibit virus replication at the level of reverse transcription. Mechanistic analyses revealed that APEX57219 blocked the interaction between RT and the nucleic acid substrate.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Inibidores da Transcriptase Reversa/isolamento & purificação , Salicilatos/isolamento & purificação , Transferência Ressonante de Energia de Fluorescência , Transcriptase Reversa do HIV/efeitos dos fármacos , Transcriptase Reversa do HIV/metabolismo , Cinética , Inibidores da Transcriptase Reversa/farmacologia , Salicilatos/farmacologia , Replicação Viral/efeitos dos fármacos
12.
Environ Toxicol Pharmacol ; 37(2): 626-37, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24572641

RESUMO

In this study, a compound with antioxidant and anti-HIV activities designated as HEB was first isolated from the edible mushroom Pholiota adiposa by extraction with ethanol and ethyl acetate. HEB was then purified by high performance liquid chromatography (HPLC) and identified to be methyl gallate (C8H8O5, 184.1 Da) based on data from its mass spectrum (MS) and nuclear magnetic resonance (NMR) spectrum. HEB displayed strong antioxidant potency in inhibiting, at 1.36 mM concentration, erythrocyte hemolysis and scavenging DPPH radicals and superoxide anion (O2(-)) by 82.4%, 85.6% and 71.4%, respectively. Besides exhibiting a low cytotoxicity, compound HEB demonstrated significant anti-HIV activity in that it inhibited HIV-1 replication in TZM-BL cells infected by pseudovirus with an IC50 value of 11.9 µM. Further study disclosed that HEB inhibited the viral entry process and activities of key enzymes essential for the HIV-1 life cycle. HEB inhibited HIV-1 reverse transcriptase and integrase activities with an IC50 value of 80.1 µM and 228.5 µM, respectively, and at 10 mM concentration inhibited HIV-1 protease activity by 17.1% which was higher than that achieved by the positive control pepstatin A. Interestingly, this study first revealed that H2O2 stimulation not only activated cell oxidative stress responses, but also accelerated HIV-1 long terminal repeat (LTR) promotion in TZM-BL cells, which was significantly reduced by HEB from 18.2% to about 2%. It implied a direct relationship between the antioxidant and anti-HIV activities of the natural active constituent HEB. Nuclear transcription factor kappa B (NF-κB) signal pathways plays an important role in oxidative stress responses. Meanwhile, there is κB target sequence in HIV promoter LTR which is significant for virus replication and gene expression. In this study, Western Blot assay showed that HEB could inhibit the activation of NF-κB signal pathway stimulated by H2O2 in mouse spleen cells through suppressing NF-κB (p65) translocation into nucleus and NF-kappa-B inhibitor (IκB) degradation in cytoplasm. In summary, the antioxidant HEB from P. adiposa could inhibit HIV-1 replication through multiple target sites. The data suggest that natural antioxidant compounds might have a potential for treatment of AIDS.


Assuntos
Fármacos Anti-HIV/farmacologia , Antioxidantes/farmacologia , Ácido Gálico/análogos & derivados , Pholiota , Inibidores da Transcriptase Reversa/farmacologia , Animais , Fármacos Anti-HIV/isolamento & purificação , Antioxidantes/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Compostos de Bifenilo/metabolismo , Eritrócitos/efeitos dos fármacos , Ácido Gálico/isolamento & purificação , Ácido Gálico/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Hemólise/efeitos dos fármacos , Masculino , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Picratos/metabolismo , Inibidores da Transcriptase Reversa/isolamento & purificação , Superóxidos/metabolismo
13.
J Med Virol ; 86(1): 1-7, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24068579

RESUMO

This study describes a novel, PCR-based assay that evaluates the ability of compounds to inhibit cDNA generation by HIV reverse transcriptase (RT), of both commercial and viral lysate origin, from a known RNA template. The template consisted of RNA from stable transfectants ectopically expressing the US6 gene of herpes simplex virus-1, coding for glycoprotein D. Controls were carried out to demonstrate that no residual DNA polymerase activity or DNA contamination was responsible for the amplified DNA in the tested, control samples. In this assay, 0.1 µM nevirapine totally inhibited the RT activity of 0.5 U commercial HIV RT, while 10 nM inhibited it by only 10%. Conversely, 10 pM efavirenz completely inhibited 0.5 U HIV RT. Similar results were obtained when a self-prepared viral lysate was used as a source of HIV RT. A reference commercial kit directly measuring HIV RT activity, without amplification, was less sensitive than the new assay. As a consequence, the HIV RT 50% inhibitory concentration of nevirapine and efavirenz in the newly described assay was 8 and 5 × 10(3) times lower, respectively, than in the commercial assay. In conclusion, this novel method was sensitive, reproducible, and sufficiently rapid for screening in vitro the functional activity of known or potential antiretroviral compounds against HIV RT.


Assuntos
Fármacos Anti-HIV/isolamento & purificação , Fármacos Anti-HIV/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Transcriptase Reversa do HIV/antagonistas & inibidores , Reação em Cadeia da Polimerase/métodos , Inibidores da Transcriptase Reversa/isolamento & purificação , Inibidores da Transcriptase Reversa/farmacologia , Benzoxazinas/farmacologia , DNA Complementar/biossíntese , Humanos , Testes de Sensibilidade Microbiana/métodos , Nevirapina/farmacologia , Reprodutibilidade dos Testes , Transcrição Reversa/efeitos dos fármacos , Sensibilidade e Especificidade
14.
Antiviral Res ; 102: 54-60, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24342709

RESUMO

Design and synthesis of nucleoside analogues have persistently attracted extensive interest because of their potential application in the field of antiviral therapy, and its study also receives additional impetus for improvement in the ProTide technology. Previous studies have made great strides in the design and discovery of monophosphorylated nucleoside analogues as potential kinase-independent antiretrovirals. In this work, a series of nucleoside phosphoramidates modified by distamycin analogues was synthesized and evaluated as nucleoside reverse transcriptase inhibitors (NRTIs) in HIV-1-infected MT-4 and CEM cells, including variations in nucleoside, alkyl moiety, and the structure of distamycin analogues. These compounds exhibited modest potency with the EC50 value in the range of 1.3- to 6.5-fold lower than their corresponding parent drugs in MT-4 cells, which may be attributed to increasing intracellular availability due to the existence of distamycin analogue with favorable hydrophilic-lipophilic equilibrium. Meanwhile, the length of distamycin analogue was considered and assessed as an important factor that could affect antiviral activity and cytotoxicity. Enzymatic and metabolic stability studies have been performed in order to better understand the antiviral behavior of these compounds. The present work revealed the compounds to have a favorable and selective anti-HIV-1 activity in MT-4 and CEM cells, and helped to develop strategies for design and synthesis of effective monophosphorylated nucleoside analogues, which may be applied to antiretroviral research as NRTIs.


Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/isolamento & purificação , Transcriptase Reversa do HIV/antagonistas & inibidores , Nucleosídeos/química , Nucleosídeos/isolamento & purificação , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/isolamento & purificação , Linhagem Celular , Distamicinas/química , HIV-1/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana
15.
J Nat Prod ; 76(12): 2298-306, 2013 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-24308675

RESUMO

Seven new phenylspirodrimanes, named stachybotrins D-F (1, 3, 4), stachybocins E and F (5, 6), and stachybosides A and B (7, 8), and four known compounds (2, 9-11), were isolated from the sponge-derived fungus Stachybotrys chartarum MXH-X73. Their structures were determined by detailed analysis of spectroscopic data. The absolute configurations of 1-8 were determined by chemical hydrolysis and modified Mosher's and Marfey's methods. All compounds were tested in an anti-HIV activity assay, and compound 1 showed an inhibitory effect on HIV-1 replication by targeting reverse transcriptase. Further study exhibited that 1 could block NNRTIs-resistant strains (HIV-1RT-K103N, HIV-1RT-L100I,K103N, HIV-1RT-K103N,V108I, HIV-1RT-K103N,G190A, and HIV-1RT-K103N,P225H) as well as wild-type HIV-1 (HIV-1wt) with EC50 values of 7.0, 23.8, 13.3, 14.2, 6.2, and 8.4 µM, respectively.


Assuntos
Fármacos Anti-HIV/isolamento & purificação , Fármacos Anti-HIV/farmacologia , Poríferos/microbiologia , Inibidores da Transcriptase Reversa/isolamento & purificação , Inibidores da Transcriptase Reversa/farmacologia , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/farmacologia , Compostos de Espiro/isolamento & purificação , Compostos de Espiro/farmacologia , Stachybotrys/química , Animais , Fármacos Anti-HIV/química , HIV-1/efeitos dos fármacos , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Sesquiterpenos Policíclicos , Inibidores da Transcriptase Reversa/química , Sesquiterpenos/química , Compostos de Espiro/química , Relação Estrutura-Atividade
16.
Biotechnol Appl Biochem ; 60(4): 393-8, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24033593

RESUMO

A 36-kDa protein, with an N-terminal sequence highly homologous to polygalacturonase (PG) inhibiting proteins, was isolated from small brown-eyed cowpea seeds. The protein was unadsorbed on diethylaminoethyl cellulose but adsorbed on both Affi-gel blue gel and SP-sepharose. It inhibited mycelial growth in the fungus Mycosphaerella arachidicola with an half-maximal (50%) inhibitory concentration (IC50 ) of 3.3 µM. It reduced [methyl-(3) H] thymidine incorporation into MBL2 lymphoma and L1210 leukemia cells with an IC50 of 7.4 and 5.4 µM, respectively. It inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase with an IC50 of 12.9 µM. However, it did not inhibit PG. The potent antifungal and antitumor activities of the protein suggest that it can be developed into an antifungal agent for combating M. arachidicola invasion in crops and an agent for cancer therapy in humans.


Assuntos
Antifúngicos/isolamento & purificação , Fabaceae/química , Transcriptase Reversa do HIV/antagonistas & inibidores , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Inibidores da Transcriptase Reversa/isolamento & purificação , Sementes/química , Sequência de Aminoácidos , Antifúngicos/química , Antifúngicos/farmacologia , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fungos/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Proteínas de Plantas/química , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia
17.
J Antimicrob Chemother ; 68(9): 2038-47, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23645585

RESUMO

OBJECTIVES: Pre-exposure prophylaxis and topical microbicides are important strategies in the prevention of sexual HIV transmission, especially since partial protection has been shown in proof-of-concept studies. In search of new candidate drugs with an improved toxicity profile and with activity against common non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV, we have synthesized and investigated a library of 60 new diaryltriazine analogues. METHODS: From this library, 15 compounds were evaluated in depth using a broad armamentarium of in vitro assays that are part of a preclinical testing algorithm for microbicide development. Antiviral activity was assessed in a cell line, and in primary human cells, against both subtype B and subtype C HIV-1 and against viruses resistant to therapeutic NNRTIs and the candidate NNRTI microbicide dapivirine. Toxicity towards primary blood-derived cells, cell lines originating from the female reproductive tract and female genital microflora was also studied. RESULTS AND CONCLUSIONS: We identified several compounds with highly potent antiviral activity and toxicity profiles that are superior to that of dapivirine. In particular, compound UAMC01398 is an interesting new candidate that warrants further investigation because of its superior toxicity profile and potent activity against dapivirine-resistant viruses.


Assuntos
Anti-Infecciosos Locais/farmacologia , HIV-1/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Triazinas/farmacologia , Animais , Anti-Infecciosos Locais/isolamento & purificação , Anti-Infecciosos Locais/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quimioprevenção/métodos , Avaliação Pré-Clínica de Medicamentos , Feminino , Infecções por HIV/prevenção & controle , Infecções por HIV/transmissão , Humanos , Inibidores da Transcriptase Reversa/isolamento & purificação , Inibidores da Transcriptase Reversa/toxicidade , Triazinas/síntese química , Triazinas/toxicidade
18.
Yao Xue Xue Bao ; 47(8): 1011-6, 2012 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-23162897

RESUMO

This study is to investigate the mechanism of action of lindenane disesquiterpenoid shizukaol F on HIV-1 replication. Real time quantity PCR, ELISA assay and fluorescence methods were used to test HIV-1 reverse transcription process, RNA-dependent DNA polymerase activity, and RNase H activity, respectively. It showed that shizukaol F inhibited LTR/Gag production of HIV-1 reverse transcription with an IC50 of 9.11 micromol x L(-1). This result is consistent with its inhibitory effect on HIV-1 replication (IC50 of 6.12 micromol x L(-1)). Mechanism studies showed that compound shizukaol F inhibited HIV-1 RT-RNase H with IC50 of 26.4 micromol x L(-1), but had no effect on HIV-1 RT RNA-dependent DNA polymerase activity. In conclusion, shizukaol F is a new structural type HIV-1 RNase H inhibitor. This discovery will provide a clue for new type of reverse transcriptase inhibitors development.


Assuntos
Magnoliopsida/química , Inibidores da Transcriptase Reversa/farmacologia , Ribonuclease H/antagonistas & inibidores , Sesquiterpenos/farmacologia , Replicação Viral/efeitos dos fármacos , Linhagem Celular Tumoral , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Células HEK293 , Transcriptase Reversa do HIV/metabolismo , HIV-1/fisiologia , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Plantas Medicinais/química , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/isolamento & purificação , Ribonuclease H/metabolismo , Sesquiterpenos/química , Sesquiterpenos/isolamento & purificação
19.
J Biomed Biotechnol ; 2012: 536725, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22536022

RESUMO

A 59 kDa laccase with inhibitory activity against HIV-1 reverse transcriptase (IC(50) = 2.4 µM) was isolated from the broth of mycelial culture of the mushroom Lentinus tigrinus. The isolation procedure involved ion exchange chromatography on DEAE-cellulose and CM-cellulose, and gel filtration by fast protein liquid chromatography on Superdex 75. The laccase was adsorbed on both types of ion exchangers. About 95-fold purification was achieved with a 25.9% yield of the enzyme. The procedure resulted in a specific enzyme activity of 76.6 U/mg. Its N-terminal amino acid sequence was GIPDLHDLTV, which showed little similarity to other mushroom laccase and other Lentinus tigrinus strain laccase. Its characteristics were different from previously reported laccase of other Lentinus tigrinus strain. Maximal laccase activity was observed at a pH of 4 and at a temperature of 60°C, respectively. This study yielded the information about the potentially exploitable activities of Lentinus tigrinus laccase.


Assuntos
Proteínas Fúngicas/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Lacase/farmacologia , Lentinula/enzimologia , Inibidores da Transcriptase Reversa/farmacologia , Sequência de Aminoácidos , Cromatografia por Troca Iônica , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Lacase/isolamento & purificação , Lacase/metabolismo , Lentinula/química , Dados de Sequência Molecular , Micélio/química , Micélio/enzimologia , Inibidores da Transcriptase Reversa/isolamento & purificação , Inibidores da Transcriptase Reversa/metabolismo , Temperatura
20.
BMB Rep ; 45(3): 165-70, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22449703

RESUMO

We report here that an ethanol extract of Tetracera scandens, a Vietnamese medicinal plant, has anti-HIV activity and possesses strong inhibitory activity against HIV-1 reverse transcriptase (RTase). Using a MT-4 cell-based assay, we found that the T. scandens extract inhibited effectively HIV virus replication with an IC(50) value in the range of 2.0-2.5 µg/ml while the cellular toxicity value (CC50) was more than 40-50 µg/ml concentration, thus yielding a minimum specificity index of 20-fold. Moreover, the anti-HIV efficacy of the T. scandens extract was determined to be due, in part, to its potent inhibitory activity against HIV-1 RTase activity in vitro. The inhibitory activity against the RTase was further confirmed by probing viral cDNA production, an intermediate of viral reverse transcription, in virus-infected cells using quantitative DNA-PCR analysis. Thus, these results suggest that T. scandens can be a useful source for the isolation and development of new anti- HIV-1 inhibitor(s). [BMB reports 2012; 45(3): 165-170].


Assuntos
Fármacos Anti-HIV/farmacologia , Dilleniaceae/química , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Extratos Vegetais/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Fármacos Anti-HIV/química , Fármacos Anti-HIV/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Extratos Vegetais/química , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/isolamento & purificação , Relação Estrutura-Atividade , Células Tumorais Cultivadas
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