Anti-HIV-1 integrase potency of methylgallate from Alchornea cordifolia using in vitro and in silico approaches.

According to the 2018 report of the United Nations Programme on HIV/AIDS (UNAIDS), acquired immune deficiency syndrome (AIDS), a disease caused by the human immunodeficiency virus (HIV), remains a significant public health problem. The non-existence of a cure or effective vaccine for the disease and the associated emergence of resistant viral strains imply an urgent need for the discovery of novel anti-HIV drug candidates. The current study aimed to identify potential anti-retroviral compounds from Alchornea cordifolia. Bioactive compounds were identified using several chromatographic and spectroscopic techniques and subsequently evaluated for cytotoxicity and anti-HIV properties. Molecular modelling studies against HIV-1 integrase (HIV-1 IN) were performed to decipher the mode of action of methylgallate, the most potent compound (IC50 = 3.7 nM) and its analogues from ZINC database. Cytotoxicity assays showed that neither the isolated compounds nor the crude methanolic extract displayed cytotoxicity effects on the HeLa cell line. A strong correlation between the in vitro and in silico results was observed and important HIV-1 IN residues interacting with the different compounds were identified. These current results indicate that methylgallate is the main anti-HIV-1 compound in A. cordifolia stem bark, and could be a potential platform for the development of new HIV-1 IN inhibitors.

Anti-HIV-1 integrase compounds from Dioscorea bulbifera and molecular docking study.

CONTEXT: Dioscorea bulbifera L. (Dioscoreaceae) has been used in a traditional Thai longevity medicine preparation. Isolation of inhibitors from natural products is a potential source for continuous development of new HIV-1 integrase (IN) inhibitors. OBJECTIVE: The objective of this study is to isolate the compounds and evaluate their anti-HIV-1 IN activity, as well as to predict the potential interactions of the compounds with an IN. MATERIALS AND METHODS: The ethyl acetate and water fractions (1-100 µg/mL) of Dioscorea bulbifera bulbils were isolated and tested for their anti-HIV-1 IN activity using the multiplate integration assay (MIA). The interactions of the active compounds with IN were investigated using a molecular docking method. RESULTS AND DISCUSSIONS: The ethyl acetate and water fractions of Dioscorea bulbifera bulbils afforded seven compounds. Among these, allantoin (1), 2,4,3',5'-tetrahydroxybibenzyl (2), and 5,7,4'-trihydroxy-2-styrylchromone (5) were isolated for the first time from this plant. Myricetin (4) exhibited the most potent activity with an IC50 value of 3.15 µM, followed by 2,4,6,7-tetrahydroxy-9,10-dihydrophenanthrene (3, IC50 value= 14.20 µM), quercetin-3-O-ß-D-glucopyranoside (6, IC50 value = 19.39 µM) and quercetin-3-O-ß-D-galactopyranoside (7, IC50 value = 21.80 µM). Potential interactions of the active compounds (3, 4, 6, and 7) with the IN active site were additionally investigated. Compound 4 showed the best binding affinity to IN and formed strong interactions with various amino acid residues. These compounds interacted with Asp64, Thr66, His67, Glu92, Asp116, Gln148, Glu152, Asn155, and Lys159, which are involved in both the 3'-processing and strand transfer reactions of IN. In particular, galloyl, catechol, and sugar moieties were successful inhibitors for HIV-1 IN.

Anti-HIV-1 integrase effect of compounds from Aglaia andamanica leaves and molecular docking study with acute toxicity test in mice.

CONTEXT: Acquired immunodeficiency syndrome (AIDS) is a serious health problem worldwide. It has been reported that Aglaia andamanica Hiern (Meliaceae) leaves possessed an antiviral effect. Therefore, a search of anti-HIV-1 integrase (HIV-1 IN) agents from A. andamanica is a promising target. OBJECTIVE: The objective of this study is to evaluate anti-HIV-1 IN activity of isolated compounds from A. andamanica using an in vitro assay and molecular docking study as well as testing acute toxicity in mice using the up and down method. MATERIALS AND METHODS: The leaves and compounds (3-100 µg/mL) from A. andamanica were determined for the anti-HIV-1 IN effect using the multiplate integration assay (MIA) by detection the absorbance of the final product, p-nitrophenol, at 405 nm. The molecular docking with the HIV-1 IN of the active compound N-methyl-trans-4-hydroxy-l-proline (10) was also studied. The Swiss albino mice were used for an acute toxicity test. RESULTS AND DISCUSSION: Among the isolated compounds, 10 showed marked anti-HIV-1 IN effect with an IC50 value of 11.8 µg/mL, whereas other compounds were inactive (IC50 value > 100 µg/mL). The molecular docking of compound 10 with an HIV-1 IN enzyme was also studied. The result revealed that this compound formed the hydrogen bonding with the Thr66, Asn155, and Lys159 of the HIV-1 IN binding site. The acute toxicity of the A. andamanica extract was not observed at the dose 2000 mg/kg mice. This is the first report of A. andamanica for anti-HIV-1 IN activity.

Anti-HIV-1 integrase activity and molecular docking of compounds from Albizia procera bark.

CONTEXT: Albizia procera (Roxb.) Benth. (Mimosaceae) has been traditionally used in Thai longevity preparations. Thus, searching for HIV-1 integrase (HIV-1 IN) agents from natural sources is of interest. OBJECTIVE: The objective of this study is to examine the inhibitory activity against HIV-1 IN of compounds isolated from the stem bark of Albizia procera. MATERIALS AND METHODS: The EtOH extract and isolated compounds of Albizia procera bark were examined for anti-HIV-1 IN activity at various concentrations (10-100 µg/mL and 10-100 µM) using the multiplate integration assay and molecular docking. RESULTS AND DISCUSSIONS: The results showed that the ethanol extract had good anti-HIV-1 IN activity with an IC50 value of 19.5 µg/mL, whereas ethyl acetate fraction exhibited the most potent with an IC50 value of 19.1 µg/mL, followed by water fraction (IC50 value = 21.3 µg/mL), hexane and chloroform fractions (IC50 value > 100 µg/mL), respectively. From bioassay-guided isolation, the ethyl acetate fraction was further separated to give two compounds which are (+)-catechin (1) and protocatechuic acid (2), respectively. Of the tested samples, (+)-catechin (1) exhibited appreciable activity against HIV-1 IN with an IC50 value of 46.3 µM, whereas protocatechuic acid (2) showed mild activity with 46.0% inhibition at concentration of 100 µM. (+)-Catechin (1) could interact with Thr66, Gly148, and Glu152 in the core domain of IN enzyme, whereas protocatechuic acid (2) could bind with Thr66, His67, Glu152, Asn155, and Lys159. This is the first report on anti-HIV-1 IN activity of Albizia procera bark. These results may suggest that Albizia procera bark has potential as anti-HIV-1 IN agent.

Bioactive glycosides from the African medicinal plant Boerhavia erecta L.

Phytochemical studies of the previously unexplored stem of Boerhavia erecta from Burkina Faso, resulted in the isolation of an unreported glycoside 4, 2,3-dihydroxypropylbenzoate-3-O-ß-[4″-methoxy] glucuronide as well as seven known glycosides (1-3, 5-8). The major isolate 5 and 8 indicated a significant inhibition against HIV integrase (IC50 10 and 22 µg/mL, respectively). The extracts and isolates were also tested for anti-malarial activity, but insignificant activity was observed.

Natural stilbenoids isolated from grapevine exhibiting inhibitory effects against HIV-1 integrase and eukaryote MOS1 transposase in vitro activities.

Polynucleotidyl transferases are enzymes involved in several DNA mobility mechanisms in prokaryotes and eukaryotes. Some of them such as retroviral integrases are crucial for pathogenous processes and are therefore good candidates for therapeutic approaches. To identify new therapeutic compounds and new tools for investigating the common functional features of these proteins, we addressed the inhibition properties of natural stilbenoids deriving from resveratrol on two models: the HIV-1 integrase and the eukaryote MOS-1 transposase. Two resveratrol dimers, leachianol F and G, were isolated for the first time in Vitis along with fourteen known stilbenoids: E-resveratrol, E-piceid, E-pterostilbene, E-piceatannol, (+)-E-ε-viniferin, E-ε-viniferinglucoside, E-scirpusin A, quadragularin A, ampelopsin A, pallidol, E-miyabenol C, E-vitisin B, hopeaphenol, and isohopeaphenol and were purified from stalks of Vitis vinifera (Vitaceae), and moracin M from stem bark of Milliciaexelsa (Moraceae). These compounds were tested in in vitro and in vivo assays reproducing the activity of both enzymes. Several molecules presented significant inhibition on both systems. Some of the molecules were found to be active against both proteins while others were specific for one of the two models. Comparison of the differential effects of the molecules suggested that the compounds could target specific intermediate nucleocomplexes of the reactions. Additionally E-pterostilbene was found active on the early lentiviral replication steps in lentiviruses transduced cells. Consequently, in addition to representing new original lead compounds for further modelling of new active agents against HIV-1 integrase, these molecules could be good tools for identifying such reaction intermediates in DNA mobility processes.

Anti-HIV-1 integrase compound from Pometia pinnata leaves.

CONTEXT: HIV-1 integrase (HIV-1 IN) is a key enzyme involved in the replication cycle of the retrovirus. Any new knowledge on inhibitors of this enzyme could provide essential clues for the development of anti-HIV drugs. OBJECTIVE: To evaluate anti-HIV-1 IN activity of some Thai medicinal plant extracts, and the extract that possessed the strongest anti-HIV-1 IN activity was subjected to isolation of the active compounds. MATERIALS AND METHODS: Ethanol extracts of eight Thai medicinal plants were evaluated for their inhibitory effect against HIV-1 IN. An extract of Pometia pinnata J. R. Forst. & G. Forst (Sapindaceae) leaves that possessed the strongest anti-HIV-1 IN activity was fractionated to isolate the active compounds by anti-HIV-1 IN assay-guided isolation process. RESULTS AND DISCUSSION: The leaf extract from P. pinnata had the strongest anti-HIV-1 IN activity with an IC50 value of 8.8 µg/mL. An anti-HIV-1 IN assay-guided isolation of the active compounds from a leaf extract of P. pinnata resulted in the isolation of one active compound, identified as proanthocyanidin A2. Proanthocyanidin A2 showed satisfactory anti-HIV-1 IN activity with an IC50 value of 30.1 µM. Three flavonoids, epicatechin, kaempferol-3-O-rhamnoside, quercetin-3-O-rhamnoside; a glycolipid, 1-O-palmitoyl-3-O-[α-.-galactopyranosyl-(1 → 6)-ß-.-galactopyranosyl]-sn-glycerol; a steroidal glycoside; stigmasterol-3-O-glucoside; and a pentacyclic triterpenoid saponin, 3-O-α-.-arabinofuranosyl-(1 → 3)-[α-.-rhamnopyranosyl-(1 → 2)]-α-.-arabinopyranosyl hederagenin were also isolated but were inactive at a concentration of 100 µM.

Reporter gene expression from LTR-circles as tool to identify HIV-1 integrase inhibitors.

Early HIV-1 integrase inhibitors, such as compounds containing a ß-diketo acid moiety, were identified by extensive high-throughput screening campaigns. Traditionally, in vitro biochemical assays, measuring the catalytic activities of integrase, have been used for this purpose. However, these assays are confounded by the absence of cellular processes or cofactors that play a role in the integration of HIV-1 DNA in the cellular genome. In contrast to regular cell-based virus inhibition assays, which targets all steps of the viral replication cycle, a novel cellular screening assays was developed to enable the specific identification of integrase inhibitors, employing a readout that is linked with the inhibition of integrase activity. Therefore, a HIV-1 lentiviral vector equipped with the enhanced green fluorescent protein (eGFP) reporter gene was used to detect expression from extrachromosomal viral DNA (1- or 2-long terminal repeat circles), formed when integration of vector DNA into the cellular genome is prevented by an integrase inhibitor. In this assay, eGFP expression from the low residual level of transcriptional activity of extrachromosomal DNA was measured via high-throughput flow cytometry. An algorithm for analysis of eGFP expression histograms enabled the specific identification of integrase inhibitors. This assay is amenable for high throughput screening to identify inhibitors of HIV-1 integrase.

Azaphilone and isocoumarin derivatives from the endophytic fungus Penicillium sclerotiorum PSU-A13.

Two new azaphilone derivatives, penicilazaphilones A (1) and B (2), and one new isocoumarin, penicilisorin (3), together with six known compounds were isolated from the endophytic fungus Penicillium sclerotiorum PSU-A13. Their structures were identified by analysis of spectroscopic data. The antimicrobial activity against Staphylococcus aureus, Candida albicans and Cryptococcus neoformans as well as the inhibitory effect on human immunodeficiency virus (HIV)-1 integrase and protease were examined.

Screening for antiviral inhibitors of the HIV integrase-LEDGF/p75 interaction using the AlphaScreen luminescent proximity assay.

Small-molecule inhibitors of HIV integrase (HIV IN) have emerged as a promising new class of antivirals for the treatment of HIV/AIDS. The compounds currently approved or in clinical development specifically target HIV DNA integration and were identified using strand-transfer assays targeting the HIV IN/viral DNA complex. The authors have developed a second biochemical assay for identification of HIV integrase inhibitors, targeting the interaction between HIV IN and the cellular cofactor LEDGF/p75. They developed a luminescent proximity assay (AlphaScreen) designed to measure the association of the 80-amino-acid integrase binding domain of LEDGF/p75 with the 163-amino-acid catalytic core domain of HIV IN. This assay proved to be quite robust (with a Z' factor of 0.84 in screening libraries arrayed as orthogonal mixtures) and successfully identified several compounds specific for this protein-protein interaction.

HIV-1 protease and HIV-1 integrase inhibitory substances from Eclipta prostrata.

The bioassay-guided fractionation for anti-HIV-1 integrase activity led to the isolation of six compounds from the whole plant extract of Eclipta prostrata extract. They were identified as 5-hydroxymethyl-(2,2':5',2'')-terthienyl tiglate (1), 5-hydroxymethyl-(2,2':5',2'')-terthienyl agelate (2), 5-hydroxymethyl-(2,2':5',2'')-terthienyl acetate (3), ecliptal (4), orobol (5) and wedelolactone (6). Of these, compound 6 showed the highest activity against HIV-1 integrase (IN) with an IC50 value of 4.0+/-0.2 microm, followed by compound 5 (IC50=8.1+/-0.5 microm), whereas the four terthiophene compounds (1-4) were inactive (IC50>100 microm). Regarding HIV-1 protease (PR) inhibitory activity, compound 1 exhibited appreciable activity against HIV-1 PR with an IC50 of 58.3+/-0.8 microm, followed by compound 4 (IC50=83.3+/-1.6 microm) and compound 3 (IC50=93.7+/-0.8 microm), while compounds 2, 5 and 6 were inactive against HIV-1 PR (IC50>100 microm). This is the first report of anti-HIV-1 IN activities for wedelolactone (6), a coumarin derivative, and orobol (5), an isoflavone derivative. This study supports the use of E. prostrata in AIDS patients, which is in accord with its traditional use by Thai traditional doctors for curing blood related diseases.

Vanillic acid glycoside and quinic acid derivatives from Gardeniae Fructus.

Bioassay-directed chromatographic fractionation of an ethyl acetate extract of Gardenia jasminoides (Gardeniae Fructus) afforded a new vanillic acid 4-O-beta-d-(6'-sinapoyl)glucopyranoside (1) and five new quinic acid derivatives, methyl 5-O-caffeoyl-3-O-sinapoylquinate (2), ethyl 5-O-caffeoyl-3-O-sinapoylquinate (3), methyl 5-O-caffeoyl-4-O-sinapoylquinate (4), ethyl 5-O-caffeoyl-4-O-sinapoylquinate (5), and methyl 3,5-di-O-caffeoyl-4-O-(3-hydroxy-3-methyl)glutaroylquinate (6), together with three known quinic acid derivatives, two flavonoids, two iridoids, and two phenolic compounds. The structures of new compounds were elucidated by the aid of spectroscopic methods. These compounds were assessed for antioxidant activity using three different cell-free bioassay systems and for HIV-1 integrase inhibitory activity. Five new quinic acid derivatives showed potent DPPH radical scavenging, superoxide anion scavenging, and lipid peroxidation inhibition activities. These new quinic acid derivatives also exhibited HIV-1 integrase inhibitory activity.

Evaluation of selected South African medicinal plants for inhibitory properties against human immunodeficiency virus type 1 reverse transcriptase and integrase.

Seventeen aqueous and methanol extracts from nine South African medicinal plants, ethnobotanically selected, were screened for inhibitory properties against HIV-1 reverse transcriptase (RT). Isolated compounds were additionally evaluated on HIV-1 integrase (IN). The strongest inhibition against the RNA-dependent-DNA polymerase (RDDP) activity of RT was observed with the methanol extract of the stem-bark of Peltophorum africanum Sond. (Fabaceae) (IC(50) 3.5 microg/ml), while the methanol extract of the roots of Combretum molle R.Br. ex G. Don (Combretaceae) was the most inhibitory on the ribonuclease H (RNase H) activity (IC(50) 9.7 microg/ml). The known compounds bergenin and catechin, and a red coloured gallotannin composed of meta-depside chains of gallic and protocatechuic acids esterified to a 1-O-isobutyroly-beta-D-glucopyranose core, were isolated from the methanol extract of the roots and stem-bark of Peltophorum africanum. The gallotannin inhibited the RDDP and RNase H functions of RT with IC(50) values of 6.0 and 5.0 microM, respectively, and abolished the 3'-end processing activity of IN at 100 microM. Catechin showed no effect on RT but had a moderate activity on HIV-1 IN. Bergenin was inactive on both enzymes. The aqueous and methanol extracts were non-toxic in a HeLaP4 cell line at a concentration of 400 microg/ml.

Sesquiterpenes and alkaloids from Lindera chunii and their inhibitory activities against HIV-1 integrase.

Three new eudesmane type sesquiterpenoid lindenanolides E (1), F (2) and G (3), and two new aporphine alkaloid lindechunines A (18) and B (20) were isolated from roots of Lindera chunii MERR., together with seven known sesquiterpenes including a new naturally-occurring lindenanolide H (4) and eight known aporphine alkaloids. The structures of these compounds were determined by spectroscopic means. Of the isolated compounds, hernandonine (14), laurolistine (16), 7-oxohernangerine (17) and lindechunine A (18) showed significant anti-human immunodeficiency virus type 1 (HIV-1) integrase activity with IC(50) values of 16.3, 7.7, 18.2 and 21.1 microM, respectively. The major alkaloids presented in the roots of L. chunii were quantitatively analyzed by an HPLC method.

A new caffeoyl quinic acid from aster scaber and its inhibitory activity against human immunodeficiency virus-1 (HIV-1) integrase.

The phytochemical study of the aerial parts of Aster scaber Thunb. (Asteraceae) yielded a new caffeoyl quinic acid, (-) 3,5-dicaffeoyl-muco-quinic acid (2) and three known compounds, (-) 3,5-dicaffeoyl quinic acid (1), (-) 4,5-dicaffeoyl quinic acid (3), (-) 5-caffeoyl quinic acid (4). The structures were established by high resolution spectroscopic methods. The antiviral effects against HIV-1 integrase of the compounds was evaluated. (-) 3,5-Dicaffeoyl-muco-quinic acid (2) exhibited potent antiviral activity with an IC50 value of 7.0 +/- 1.3 microg/ml.

Rapid microtiter assays for poxvirus topoisomerase, mammalian type IB topoisomerase and HIV-1 integrase: application to inhibitor isolation.

We have developed microtiter assays for detecting catalysis by type IB topoisomerases and retroviral integrases. Each assay employs model DNA substrates containing biotin in one strand and digoxigenin in another. In each case action of the enzyme results in the formation of a single DNA strand containing both groups. This allows the reaction product to be quantified by capturing biotinylated product DNA on avidin-coated plates followed by detection using an anti-digoxigenin ELISA. The order of addition of reactants and inhibitors can be varied to distinguish effects of test compounds on different steps in the reaction. These assays were used to screen compound libraries for inhibitors active against mammalian topoisomerase or HIV integrase. We identified (-)-epigallocatechin 3-O:-gallate, as a potent inhibitor of religation by mammalian topoisomerase (IC(50) of 26 nM), potentially explaining the anti-cancer properties previously attributed to this compound. New integrase inhibitors were also identified. A similar strategy may be used to develop microtiter assays for many further DNA modifying enzymes.

HIV integrase inhibitory activity of Agastache rugosa.

We have been screening anti-HIV integrase compounds from Korean medicinal plants by using an in vitro assay system which is mainly composed of recombinant human immunodeficiency virus type 1 integrase and radiolabeled oligonucleotides. From the above screening, the aqueous methanolic extract of the roots of Agastache rugosa exhibited a significant activity. Bioactivity-guided chromatographic fractionation of the methanolic extract resulted in the isolation of rosmarinic acid. The structure of the compound was determined by spectroscopic data and by the comparison with the reported values. The IC50 of the rosmarinic acid was approximately 10 microg/ml against HIV integrase.

Identification of HIV-1 integrase inhibitors based on a four-point pharmacophore.

The rapid emergence of human immunodeficiency virus (HIV) strains resistant to available drugs implies that effective treatment modalities will require the use of a combination of drugs targeting different sites of the HIV life cycle. Because the virus cannot replicate without integration into a host chromosome, HIV-1 integrase (IN) is an attractive therapeutic target. Thus, an effective IN inhibitor should provide additional benefit in combination chemotherapy. A four-point pharmacophore has been identified based on the structures of quinalizarin and purpurin, which were found to be potent IN inhibitors using both a preintegration complex assay and a purified enzyme assay in vitro. Searching with this four-point pharmacophore in the 'open' part of the National Cancer Institute three-dimensional structure database produced 234 compounds containing the pharmacophore. Sixty of these compounds were tested for their inhibitory activity against IN using the purified enzyme; 19 were found to be active against IN with IC50 values of less than 100 microM, among which 10 had IC50 values of less than 10 microM. These inhibitors can further serve as leads, and studies are in progress to design novel inhibitors based on the results presented in this study.

A new flavonol glycoside gallate ester from Acer okamotoanum and its inhibitory activity against human immunodeficiency virus-1 (HIV-1) integrase.

Bioassay-directed chromatographic fractionation of an ethyl acetate extract of the leaves of Acer okamotoanum using HIV-1 integrase afforded a new acylated flavonol glycoside, quercetin 3-O-(2",6"-O-digalloyl)-beta-D-galactopyranoside (1), together with six known flavonol glycosides and three known phenolic compounds. The structure of the new compound was determined by spectroscopic methods. The most active compounds were quercetin 3-O-(2"-galloyl)-alpha-L-arabinopyranoside (6) and 1, which exhibited IC50 values of 18.1 +/- 1.3 and 24.2 +/- 6.6 micrograms/mL, respectively, against HIV-1 integrase.