Garcinol from Garcinia indica inhibits HIV-1 reverse transcriptase-associated ribonuclease H.

The bioactive components of Garcinia indica, garcinol (camboginol), and isogarcinol (cambogin), are suitable drug candidates for the treatment of various human diseases. HIV-1-RNase H assay was used to study the RNase H inhibition by garcinol and isogarcinol. Docking of garcinol into the active site of the enzyme was carried out to rationalize the difference in activities between the two compounds. Garcinol showed higher HIV-1-RNase H inhibition than the known inhibitor RDS1759 and retained full potency against the RNase H of a drug-resistant HIV-1 reverse transcriptase form. Isogarcinol was distinctly less active than garcinol, indicating the importance of the enolizable ß-diketone moiety of garcinol for anti-RNase H activity. Docking calculations confirmed these findings and suggested this moiety to be involved in the chelation of metal ions of the active site. On the basis of its HIV-1 reverse transcriptase-associated RNase H inhibitory activity, garcinol is worth being further explored concerning its potential as a cost-effective treatment for HIV patients.

Flavonoids and Acid-Hydrolysis derivatives of Neo-Clerodane diterpenes from Teucrium flavum subsp. glaucum as inhibitors of the HIV-1 reverse transcriptase-associated RNase H function.

Bioassay-guided fractionation of the ethyl acetate extract from Teucrium flavum subsp. glaucum, endowed with inhibitory activity towards the HIV-1 reverse transcriptase-associated RNase H function, led to the isolation of salvigenin (1), cirsimaritin (2) and cirsiliol (3) along with the neo-clerodanes teuflavin (4) and teuflavoside (5). Acid hydrolysis of the inactive teuflavoside provided three undescribed neo-clerodanes, flavuglaucins A-C (7-9) and one known neo-clerodane (10). Among all neo-clerodanes, flavuglaucin B showed the highest inhibitory activity towards RNase H function with a IC50 value of 9.1 µM. Molecular modelling and site-directed mutagenesis analysis suggested that flavuglaucin B binds into an allosteric pocket close to RNase H catalytic site. This is the first report of clerodane diterpenoids endowed with anti-reverse transcriptase activity. Neo-clerodanes represent a valid scaffold for the development of a new class of HIV-1 RNase H inhibitors.

Bioactive tetrahydrofuran lignans from roots, stems, leaves and twigs of Anogeissus rivularis.

Four previously undescribed tetrahydrofuran lignans, named anorisols A-D (1-4) and fourteen known compounds (5-18) were isolated from the roots, stems, leaves and twigs of Anogeissus rivularis. The chemical structures were elucidated on the basis of their spectroscopic data and by comparison with the literature data. The absolute configurations of 1-4 were established by comparison of the experimental ECD spectra with the calculated ECD spectra. Some isolated compounds were evaluated for their cytotoxic activity as well as anti-HIV-1 activity employing reverse transcriptase (RT) and syncytium reduction assays using the ΔTat/RevMC99 virus in 1A2 cell line systems. Compound 6 displayed the most potent activity in syncytium inhibition assay with effective concentration at 50% (EC50) value of 13.3 µM (SI >3.0). In the reverse transcriptase assay, compound 1 exhibited moderate activity with IC50 value of 213.9 µM.

Bioactive daphnane diterpenes from Wikstroemia chuii with their potential anti-inflammatory effects and anti-HIV activities.

A phytochemical investigation on the stems and leaves of Wikstroemia chuii resulted in the isolation of three new daphnane diterpenes, wikstroechuins A-C (1-3), together with eight known analogues (4-11). The structures of new daphnane diterpenes (1-3) were determined on the basis of extensive spectroscopic methods and the known daphnane diterpenes (4-11) were identified by comparing their observable spectroscopic data with those reported spectral data in the literature. The anti-inflammatory effects as well as anti-HIV activities in vitro of all isolated daphnane diterpenes 1-11 were assessed. As a consequence, daphnane diterpenes 1-11 displayed remarkable inhibitory activities on NO (nitric oxide) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells showing IC50 values in the range of 0.12 ± 0.03 to 10.58 ± 0.16 µM. Meanwhile, daphnane diterpenes 1-11 displayed significant anti-HIV-1 reverse transcriptase (RT) effects showing EC50 values ranging from 0.09509 to 8.62356 µM. These research results indicated that the discovery of these new daphnane diterpenes with remarkable anti-inflammatory and anti-HIV activities from W. chuii, especially these new ones, could be extremely meaningful to the discovery of new anti-inflammatory agents and anti-HIV drugs as well as their potential practical values in the health and pharmaceutical products.

Validación de un método seguro y sencillo para la elaboración de secuencias consenso del virus de la inmunodeficiencia humana a partir de los datos de secuenciación masiva 454 / A safe an easy method for building consensus HIV sequences from 454 massively parallel sequencing data

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)

Identification of new potential HIV-1 reverse transcriptase inhibitors by QSAR modeling and structure-based virtual screening.

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.

Potent Inhibitor of Drug-Resistant HIV-1 Strains Identified from the Medicinal Plant Justicia gendarussa.

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.

Structure-enhanced methods in the development of non-nucleoside inhibitors targeting HIV reverse transcriptase variants.

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.

Polyoxygenated cyclohexene derivatives isolated from Dasymaschalon sootepense and their biological activities.

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.

Hepatitis B virus reverse transcriptase - Target of current antiviral therapy and future drug development.

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."

Purification and Characterization of a Lectin from Green Split Peas (Pisum sativum).

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.

Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening.

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.

Water-compatible molecularly imprinted polymer as a sorbent for the selective extraction and purification of adefovir from human serum and urine.

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.

Molecularly imprinted nanoparticles prepared by miniemulsion polymerization as a sorbent for selective extraction and purification of efavirenz from human serum and urine.

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.

Novel high-throughput screen identifies an HIV-1 reverse transcriptase inhibitor with a unique mechanism of action.

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.

First report on isolation of methyl gallate with antioxidant, anti-HIV-1 and HIV-1 enzyme inhibitory activities from a mushroom (Pholiota adiposa).

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.

Design and synthesis of novel distamycin-modified nucleoside analogues as HIV-1 reverse transcriptase inhibitors.

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.

A novel, cell-free PCR-based assay for evaluating the inhibitory activity of antiretroviral compounds against HIV reverse transcriptase.

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.

Phenylspirodrimanes with anti-HIV activity from the sponge-derived fungus Stachybotrys chartarum MXH-X73.

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.

Purification and characterization of a protein with antifungal, antiproliferative, and HIV-1 reverse transcriptase inhibitory activities from small brown-eyed cowpea seeds.

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.