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.

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.

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.

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.

Diaryltriazine non-nucleoside reverse transcriptase inhibitors are potent candidates for pre-exposure prophylaxis in the prevention of sexual HIV transmission.

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.

[Shizukaol F: a new structural type inhibitor of HIV-1 reverse transcriptase RNase H].

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.

Identification of anti-HIV and anti-reverse transcriptase activity from Tetracera scandens.

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].

Purification and characterization of a novel laccase from the edible mushroom Hericium coralloides.

A novel laccase from the edible mushroom Hericium coralloides was purified by ion exchange chromatography on diethylaminoethyl (DEAE) cellulose, carboxymethyl (CM) cellulose, and Q-Sepharose columns followed by fast protein liquid chromatography gel filtration on a Superdex 75 column. Analysis by gel filtration and SDS-PAGE indicated that the protein is a monomer in solution with a molecular mass of 65 kDa. Its N-terminal amino acid sequence was AVGDDTPQLY, which exhibits partial sequence homology to previously isolated laccases. Optimum activity was observed at pH 2.2 and at 40°C. The enzyme showed activity toward a variety of substrates, the most sensitive of which was 2,2'-azinobis [3-ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS). The degradation activity toward substrates was ABTS > N,N-dimethyl-1,4-phenylenediamine > catechol > 2-methylcatechol > pyrogallol. The laccase did not exert any antiproliferative activity against Hep G2 or MCF 7 tumor cell lines at a concentration of 60 µM, unlike some previously reported mushroom proteins, but showed significant activity toward human immunodeficiency virus-1 (HIV-1) reverse transcriptase with an IC(50) of 0.06 µM.

Inhibition of HIV-1 and M-MLV reverse transcriptases by a major polyphenol (3,4,5 tri-O-galloylquinic acid) present in the leaves of the South African resurrection plant, Myrothamnus flabellifolia.

A polyphenol-rich extract of the medicinal resurrection plant Myrothamnus flabellifolia was shown to inhibit viral (M-MLV and HIV-1) reverse transcriptases. Fractionation and purification of this extract yielded the major polyphenol, 3,4,5 tri-O-galloylquinic acid, as the main active compound. A sensitive, ethidium bromide based fluorescent assay, was developed and used to monitor the kinetics of M-MLV and HIV-1 reverse transcriptases in the presence and absence of 3,4,5 tri-O-galloylquinic acid. Kinetic monitoring of these enzymes in the presence of 3,4,5 tri-O-galloylquinic acid revealed non-competitive inhibition with IC(50) values of 5 µM and 34 µM for the M-MLV and HIV-1 enzymes, respectively. We propose that 3,4,5 tri-O-galloylquinic acid and related polymers have potential as indigenous drugs for anti-viral therapy.

[The influence of compound aITEL1296 on telomerase activity and the growth of cancer cells].

Telomerase is a ribonucleoprotein that synthesizes telomeric repeats and identified as a promising target for anticancer therapy. Here we describe a new compound aITEL1296 as a potent telomerase inhibitor. Its inhibitory activity was a bit higher (IC50 = 0,19 +/- 0,02 ng/ml) than that of BIBR1532, one of the most potent telomerase inhibitors known to date. Besides telomerase inhibition aITEL1296 activated apoptotic mechanisms and effectively suppressed proliferation of tumor cell lines (GI50 = 5,0 +/- 0,2 ng/ml for most sensitive cell line LnCap) but not normal fibroblast cell line.

A novel lectin with antiproliferative and HIV-1 reverse transcriptase inhibitory activities from dried fruiting bodies of the monkey head mushroom Hericium erinaceum.

A lectin designated as Hericium erinaceum agglutinin (HEA) was isolated from dried fruiting bodies of the mushroom Hericium erinaceum with a chromatographic procedure which entailed DEAE-cellulose, CM-cellulose, Q-Sepharose, and FPLC Superdex 75. Its molecular mass was estimated to be 51 kDa and its N-terminal amino acid sequences was distinctly different from those of other isolated mushroom lectins. The hemagglutinating activity of HEA was inhibited at the minimum concentration of 12.5 mM by inulin. The lectin was stable at pH 1.9-12.1 and at temperatures up to 70 degrees C, but was inhibited by Hg(2+), Cu(2+), and Fe(3+) ions. The lectin exhibited potent mitogenic activity toward mouse splenocytes, and demonstrated antiproliferative activity toward hepatoma (HepG2) and breast cancer (MCF7) cells with an IC(50) of 56.1 microM and 76.5 microM, respectively. It manifested HIV-1 reverse transcriptase inhibitory activity with an IC(50) of 31.7 microM. The lectin exhibited potent mitogenic activity toward murine splenocytes but was devoid of antifungal activity.

Chromatographic separation of antiviral/anticancer nucleoside reverse transcriptase inhibitor drugs.

This paper discusses the current methods used for quantitative determination of analogues of nucleotide reverse transcriptase inhibitors (NtRTIs) in body fluids, cells, and tissues. Nucleoside reverse transcriptase inhibitors (NRTIs) prodrugs given to AIDS/herpes/cancer patients conjugate with phosphates at the site of their action. Separation of phosphorylated NRTIs is generally performed by reversed-phase chromatography. After separation, plasma NRTIs can be detected using a variety of methods, including immunoassay through monitoring of UV absorbance, fluorescence, and mass spectrometry. The most recent development in the field of detection of plasma NtRTIs shows a tendency toward the use double- or triple-focusing mass spectrometry, the most specific and sensitive monitoring technique.

Bauhinia variegata var. variegata trypsin inhibitor: from isolation to potential medicinal applications.

Here we report for the first time of a new Kunitz-type trypsin inhibitor (termed BvvTI) from seeds of the Camel's foot tree, Bauhinia variegata var. variegata. BvvTI shares the same reactive site residues (Arg, Ser) and exhibits a homology of N-terminal amino acid sequence to other Bauhinia protease inhibitors. The trypsin inhibitory activity (K(i), 0.1 x 10(-9)M) of BvvTI ranks the highest among them. Besides anti-HIV-1 reverse transcriptase activity, BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way. This may partially be contributed by its induction of cytokines and apoptotic bodies. These results unveil potential medicinal applications of BvvTI.

Characterisation of a haemagglutinin from Hokkaido red bean (Phaseolus vulgaris cv. Hokkaido red bean).

BACKGROUND: A haemagglutinin was purified from Japanese Hokkaido red beans (Phaseolus vulgaris cv. Hokkaido red bean) with a procedure that included three chromatographic media. RESULTS: Haemagglutinating activity was adsorbed on DEAE cellulose, Affi-gel blue gel and Mono S. The pure haemagglutinin was a homodimer and each subunit was around 30 kDa in molecular mass. The haemagglutinating activity of this agglutinin could not be inhibited by a variety of simple sugars at 200 mmol L(-1) concentration including alpha-L-fucose, D(+)-galactose, D(+)-glucose, D(+)-glucosamine, D(-)galactosamine, galacturonic acid, (+)-lactose, D(+)-melibose, L(-)-mannose, D(+)-mannose, D-mannosamine, D(+)-raffinose, L-rhamnose, (+)-xylose and galacturonic acid. The haemagglutinating activity was fully retained at pH 4-11 and at 0-80 degrees C, but was completely lost at extreme pH values (0-2 and 13-14) and at very high temperatures (90 degrees C and 100 degrees C). The haemagglutinin exhibited a weak mitogenic activity toward mouse splenocytes, a stronger anti-proliferative activity than Con A toward HepG2 (human hepatoma) cells and inhibited >80% of HIV-1 reverse transcriptase inhibitory activity at 3.3 micromol L(-1). It was devoid of anti-fungal activity. CONCLUSION: Hokkaido red bean haemagglutinin possesses a potent anti-proliferative effect on HepG2 cells.

A novel lectin with highly potent antiproliferative and HIV-1 reverse transcriptase inhibitory activities from cicada (Cicada flammata).

A dimeric lectin with a molecular weight of 60 kDa and high hemagglutinating activity was isolated from dried cicadas. It was adsorbed on Q-Sepharose and unadsorbed on Affi-Gel Blue gel. Its hemagglutinating activity was stable up to 55 degrees C and between pH 2 and 13. The activity was inhibited by glucuronic acid and raffinose, K(+) ions, and Mg(2+) ions. Cicada lectin potently inhibited proliferation of HepG2 hepatoma and MCF 7 breast cancer cells, with an IC(50) value of 0.76 and 0.49 microM, respectively. It potently inhibited HIV-1 reverse transcriptase activity with an IC(50) of 0.36 microM but was devoid of mitogenic activity on spleen cells. Its N-terminal sequence exhibited slight similarity to a conserved hypothetical protein from Culex quinquefasciatus and a gene product from transcript GH19834-RA of Drosophila grimshawi, but there was no resemblance to lectins from other insects, including Drosophila, Sarcophaga, Glossina, and Aedes species.

Isolation and characterization of a French bean hemagglutinin with antitumor, antifungal, and anti-HIV-1 reverse transcriptase activities and an exceptionally high yield.

A dimeric 64-kDa hemagglutinin was isolated with a high yield from dried Phaseolus vulgaris cultivar "French bean number 35" seeds using a chromatographic protocol that involved Blue-Sepharose, Q-Sepharose, and Superdex 75. The yield was exceptionally high (1.1g hemagglutinin per 100g seed), which is around 10-85 times higher than other Phaseolus cultivars. Its N-terminal sequence resembled those of other Phaseolus hemagglutinins. The hemagglutinating activity of the hemagglutinin was stable in the pH range 6-8, and in the temperature range 0 degrees C-50 degrees C. It inhibited HIV-1 reverse transcriptase with an IC50 of 2microM. It suppressed mycelial growth in Valsa mali with an IC50 of 10microM. It inhibited proliferation of hepatoma HepG2 cells and breast cancer MCF-7 cells with an IC50 of 100 and 2microM, respectively. It had no antiproliferative effect on normal embryonic liver WRL68 cells.

Purification and characterization of a Laccase with inhibitory activity toward HIV-1 reverse transcriptase and tumor cells from an edible mushroom (Pleurotus cornucopiae).

A 66-kDa laccase, with an N-terminal sequence different from those of other mushroom laccases, was purified from fresh fruiting bodies of the edible mushroom Pleurotus cornucopiae by using affinity chromatography on Affi-gel blue gel, ion exchange chromatography on Mono Q and gel filtration on Superdex 75. The procedure resulted in a 16-fold purification and a specific enzyme activity of 17.3 U mg(-1). The optimum pH and temperature for the purified laccase were pH 4 and 40 degrees C, respectively. This laccase inhibited proliferation of murine leukemia cell line L1210 and human hepatoma cell line HepG2, and reduced the activity of HIV-1 reverse transcriptase with an IC50 of 22 microM. There was neither mitogenic activity toward mouse splenocytes, nor hemagglutinating/hemolytic activity toward rabbit erythrocytes. This study yielded information about the potentially exploitable activities of P. cornucopiae laccase.