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.

A novel polysaccharide with antioxidant, HIV protease inhibiting and HIV integrase inhibiting activities from Fomitiporia punctata (P. karst.) murrill (Basidiomycota, hymenochaetales).

A novel polysaccharide fraction (G1) was obtained from the fungus Fomitiporia punctata (P. Karst.) Murrill. G1 exhibited a molecular weight of approximately 151kDa. The FT-IR results suggested that the monosaccharide components of G1 possessed furanoid rings and there were ß-glycosidic bonds between the sugar units. The1H NMR results showed that G1 was composed of arabinose, fructose, galactose and glucose in the molar ratio of 1.6:3.8:19.7:19.7, as determined by gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC). G1 produced significant antioxidant effects as evidenced by its potency in inhibiting erythrocyte hemolysis, and in scavenging hydroxyl radicals and superoxide radicals. The highest rates of inhibition achieved were 73.58%, 36.55% and 50.98% respectively. In addition, G1 brought about 19.6% inhibition of HIV-1 protease activity at the concentration of 50µg/mL. G1 displayed inhibitory activity toward HIV-1 integrase in the concentration range of 100-1000µg/mL. The present study indicates that G1 from Fomitiporia punctate (P. Karst.) Murrill is a novel natural antioxidant.

A novel assay for screening inhibitors targeting HIV-1 integrase dimerization based on Ni-NTA magnetic agarose beads.

Human immunodeficiency virus (HIV)-1 integrase (IN), which mediates integration of viral cDNA into the cellular chromosome, is a validated antiviral drug target. Three IN inhibitors, raltegravir, elvitegravir and dolutegravir, have been clinically approved since 2008. However, drug resistance have emerged in infected patients receiving treatment using these drugs which share the same mechanism of action and have a low genetic barrier for resistance. Therefore, there is an urgent need to develop drugs with novel mechanism. IN requires a precise and dynamic equilibrium between several oligomeric species for its activities. The modulation of the process which is termed as IN oligomerization, presents an interesting allosteric target for drug development. In this research, we developed a magnetic beads based approach to assay the IN dimerization. Then, using the assay we screened a library of 1000 Food and Drug Administration (FDA)-approved drugs for IN dimerization inhibitors and identified dexlansoprazole as a potential IN dimerization inhibitor. In conclusion, the assay presented here has been proven to be sensitive and specific for the detection of IN dimerization as well as for the identification of antiviral drugs targeting IN dimerization. Moreover, a FDA-approved proton-pump inhibitors, dexlansoprazole, was identified as a potential inhibitor for IN dimerization.

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.

New Lignans and Iridoid Glycosides from Dipsacus asper Wall.

Six new compounds, including four new lignans, dipsalignan A (1), B-D (3-5), and two new bis-iridoid glycoside dimmers, dipsanosides M (7) and N (8), together with two known compounds (2) and (6), have been isolated from the roots of Dipsacus asper Wall. Their structures were established on the basis of spectroscopic data (MS, 1D, 2D NMR, and CD) and chemical methods. All the isolated compounds were tested against human immunodeficiency virus-1 (HIV-1) integrase inhibition activities, and only compounds 1, 2, 7, and 8 displayed weak activities.

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.

Development of a fluorescence-based HIV-1 integrase DNA binding assay for identification of novel HIV-1 integrase inhibitors.

Human immunodeficiency virus integrase (HIV-1 IN) inhibitors that are currently approved or are in advanced clinical trials specifically target the strand transfer step of integration. However, considerable cross-resistance exists among some members of this class of IN inhibitors. Intriguingly, though, HIV-1 IN possesses multiple sites, distinct from those involved in the strand transfer step, that could be targeted to develop new HIV-1 IN inhibitors. We have developed a fluorescent HIV-1 IN DNA binding assay that can identify small molecules termed IN binding inhibitors (INBIs) that inhibit IN binding to viral DNA. This assay has been optimized with respect to concentrations of each protein, long terminal repeat (LTR) DNA substrate, salt, and time, and has been used successfully to measure the HIV-1 IN DNA binding activity of a well-characterized INBI termed FZ41. In addition, we have used the assay to screen a small library of natural products, resulting in the identification of nigranoic acid as a new INBI. The proposed fluorescence assay is easy and inexpensive, and provides a high-throughput detection method for determination of HIV-1 IN DNA binding activity, monitoring of enzyme kinetics, and high-throughput screening for the identification of new INBIs.

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.

Thiazoline peptides and a tris-phenethyl urea from Didemnum molle with anti-HIV activity.

As part of our screening for anti-HIV agents from marine invertebrates, the MeOH extract of Didemnum molle was tested and showed moderate in vitro anti-HIV activity. Bioassay-guided fractionation of a large-scale extract allowed the identification of two new cyclopeptides, mollamides E and F (1 and 2), and one new tris-phenethyl urea, molleurea A (3). The absolute configurations were established using the advanced Marfey's method. The three compounds were evaluated for anti-HIV activity in both an HIV integrase inhibition assay and a cytoprotective cell-based assay. Compound 2 was active in both assays with IC(50) values of 39 and 78 µM, respectively. Compound 3 was active only in the cytoprotective cell-based assay, with an IC(50) value of 60 µM.

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.

Brassiparin, an antifungal peptide from Brassica parachinensis seeds.

AIMS: To isolate and characterize an antifungal peptide from the seeds of Brassica parachinensis L.H.Bailey. METHODS AND RESULTS: An antifungal peptide designated as brassiparin was isolated. It exhibited a molecular mass of 5716 Da. It potently inhibited mycelial growth in a number of fungal species including Fusarium oxysporum, Helminthosporium maydis, Mycosphaerella arachidicola and Valsa mali. The antifungal activity of brassiparin toward M. arachidicola exhibited pronounced thermostability and pH stability. It inhibited proliferation of hepatoma (HepG2) and breast cancer (MCF7) cells and the activity of HIV-1 reverse transcriptase. Its N-terminal sequence differed from those of antifungal proteins which have been reported to date. CONCLUSIONS: Brassiparin can be purified by using a protocol involving ion exchange chromatography, affinity chromatography and gel filtration. It manifests potent, thermostable and pH-stable antifungal activity. It demonstrates antiproliferative activity toward tumour cells, and inhibitory activity toward HIV-1 reverse transcriptase. Thus, brassiparin is a defense protein. SIGNIFICANCE AND IMPACT OF THE STUDY: Brassiparin represents one of the few antifungal proteins reported to date from Brassica species. Its antifungal activity has pronounced pH stability and thermostability. Brassiparin exhibits other exploitable activities such as antiproliferative activity toward hepatoma and breast cancer cells and inhibitory activity toward HIV-reverse transcriptase.

Recent advances in lamellarin alkaloids: isolation, synthesis and activity.

Lamellarins are a large family of marine alkaloids with potential anticancer activity that have been isolated from diverse marine organisms, mainly ascidians and sponges. All lamellarins feature a 3,4-diarylpyrrole system. Pentacyclic lamellarins, whose polyheterocyclic system has a pyrrole core, are the most active compounds. Some of these alkaloids are potently cytotoxic to various tumor cell lines. To date, Lam-D and Lam-H have been identified as lead compounds for the inhibition of topoisomerase I and HIV-1 integrase, respectively-nuclear enzymes which are over-expressed in deregulation disorders. Moreover, these compounds have been reported for their efficacy in treatment of multi-drug resistant (MDR) tumors cells without mediated drug efflux, as well as their immunomodulatory activity and selectivity towards melanoma cell lines. This article is an overview of recent literature on lamellarins, encompassing their isolation, recent synthetic strategies for their total synthesis, the preparation of their analogs, studies on their mechanisms of action, and their structure-activity relationships (SAR).

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.

A potent anti-HIV polyphenol from Salvia yunnanensis.

A new polyphenol, designated as salvianolic acid N, was isolated from the aqueous extracts of the roots of Salvia yunnanensis. Its chemical structure was elucidated as 3-(3,4-dihydroxylphenyl)-2-[(E)-3-(1,8,9-trihydroxyl-dibenzo[b,f]oxpin-3-yl)acryloxloxy]propanoic acid (1) on the basis of NMR and MS spectral analyses. The new polyphenol inhibited both HIV-1 IN in vitro and also reduced HIV-1 p24 antigen in MT-4 cell lines.

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.

Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol: Part I. fusion [corrected] inhibition.

We have identified oleuropein (Ole) and hydroxytyrosol (HT) as a unique class of HIV-1 inhibitors from olive leaf extracts effective against viral fusion and integration. We used molecular docking simulation to study the interactions of Ole and HT with viral targets. We find that Ole and HT bind to the conserved hydrophobic pocket on the surface of the HIV-gp41 fusion domain by hydrogen bonds with Q577 and hydrophobic interactions with I573, G572, and L568 on the gp41 N-terminal heptad repeat peptide N36, interfering with formation of the gp41 fusion-active core. To test and confirm modeling predications, we examined the effect of Ole and HT on HIV-1 fusion complex formation using native polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Ole and HT exhibit dose-dependent inhibition on HIV-1 fusion core formation with EC(50)s of 66-58nM, with no detectable toxicity. Our findings on effects of HIV-1 integrase are reported in the subsequent article.