Polygonumins A, a newly isolated compound from the stem of Polygonum minus Huds with potential medicinal activities.

Polygonumins A, a new compound, was isolated from the stem of Polygonum minus. Based on NMR results, the compound's structure is identical to that of vanicoside A, comprising four phenylpropanoid ester units and a sucrose unit. The structure differences were located at C-3″″'. The cytotoxic activity of polygonumins A was evaluated on several cancer cell lines by a cell viability assay using tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The compound showed the highest antiproliferative (p < 0.05) activities against K562 (Human Leukaemia Cell Line), MCF7 (Human breast adenocarcinoma cell line), and HCT116 (Colorectal cancer cells) cells. Cytotoxic studies against V79-4 cells were carried out and showed that polygonumins A was toxic at 50 µg/ml, suggesting that this compound may be used as an anticancer drug without affecting normal cells. Polygonumins A also showed promising activity as an HIV-1 protease inhibitor with 56% relative inhibition. Molecular docking results indicated that the compound possesses high binding affinity towards the HIV protease over the low binding free energy range of -10.5 to -11.3 kcal/mol. P. minus is used in Malaysian traditional medicine for the treatment of tumour cells. This is the first report on the use of P. minus as an HIV-1 protease inhibitor.

A novel central nervous system-penetrating protease inhibitor overcomes human immunodeficiency virus 1 resistance with unprecedented aM to pM potency.

Antiretroviral therapy for HIV-1 infection/AIDS has significantly extended the life expectancy of HIV-1-infected individuals and reduced HIV-1 transmission at very high rates. However, certain individuals who initially achieve viral suppression to undetectable levels may eventually suffer treatment failure mainly due to adverse effects and the emergence of drug-resistant HIV-1 variants. Here, we report GRL-142, a novel HIV-1 protease inhibitor containing an unprecedented 6-5-5-ring-fused crown-like tetrahydropyranofuran, which has extremely potent activity against all HIV-1 strains examined with IC50 values of attomolar-to-picomolar concentrations, virtually no effects on cellular growth, extremely high genetic barrier against the emergence of drug-resistant variants, and favorable intracellular and central nervous system penetration. GRL-142 forms optimum polar, van der Waals, and halogen bond interactions with HIV-1 protease and strongly blocks protease dimerization, demonstrating that combined multiple optimizing elements significantly enhance molecular and atomic interactions with a target protein and generate unprecedentedly potent and practically favorable agents.

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.

Inhibition Profiling of Retroviral Protease Inhibitors Using an HIV-2 Modular System.

Retroviral protease inhibitors (PIs) are fundamental pillars in the treatment of HIV infection and acquired immunodeficiency syndrome (AIDS). Currently used PIs are designed against HIV-1, and their effect on HIV-2 is understudied. Using a modular HIV-2 protease cassette system, inhibition profiling assays were carried out for protease inhibitors both in enzymatic and cell culture assays. Moreover, the treatment-associated resistance mutations (I54M, L90M) were introduced into the modular system, and comparative inhibition assays were performed to determine their effect on the susceptibility of the protease. Our results indicate that darunavir, saquinavir, indinavir and lopinavir were very effective HIV-2 protease inhibitors, while tipranavir, nelfinavir and amprenavir showed a decreased efficacy. I54M, L90M double mutation resulted in a significant reduction in the susceptibility to most of the inhibitors with the exception of tipranavir. To our knowledge, this modular system constitutes a novel approach in the field of HIV-2 protease characterization and susceptibility testing.

C-5-Modified Tetrahydropyrano-Tetrahydofuran-Derived Protease Inhibitors (PIs) Exert Potent Inhibition of the Replication of HIV-1 Variants Highly Resistant to Various PIs, including Darunavir.

UNLABELLED: We identified three nonpeptidic HIV-1 protease inhibitors (PIs), GRL-015, -085, and -097, containing tetrahydropyrano-tetrahydrofuran (Tp-THF) with a C-5 hydroxyl. The three compounds were potent against a wild-type laboratory HIV-1 strain (HIV-1(WT)), with 50% effective concentrations (EC50s) of 3.0 to 49 nM, and exhibited minimal cytotoxicity, with 50% cytotoxic concentrations (CC50) for GRL-015, -085, and -097 of 80, >100, and >100 µM, respectively. All the three compounds potently inhibited the replication of highly PI-resistant HIV-1 variants selected with each of the currently available PIs and recombinant clinical HIV-1 isolates obtained from patients harboring multidrug-resistant HIV-1 variants (HIVMDR). Importantly, darunavir (DRV) was >1,000 times less active against a highly DRV-resistant HIV-1 variant (HIV-1DRV(R) P51); the three compounds remained active against HIV-1DRV(R) P51 with only a 6.8- to 68-fold reduction. Moreover, the emergence of HIV-1 variants resistant to the three compounds was considerably delayed compared to the case of DRV. In particular, HIV-1 variants resistant to GRL-085 and -097 did not emerge even when two different highly DRV-resistant HIV-1 variants were used as a starting population. In the structural analyses, Tp-THF of GRL-015, -085, and -097 showed strong hydrogen bond interactions with the backbone atoms of active-site amino acid residues (Asp29 and Asp30) of HIV-1 protease. A strong hydrogen bonding formation between the hydroxyl moiety of Tp-THF and a carbonyl oxygen atom of Gly48 was newly identified. The present findings indicate that the three compounds warrant further study as possible therapeutic agents for treating individuals harboring wild-type HIV and/or HIVMDR. IMPORTANCE: Darunavir (DRV) inhibits the replication of most existing multidrug-resistant HIV-1 strains and has a high genetic barrier. However, the emergence of highly DRV-resistant HIV-1 strains (HIVDRV(R) ) has recently been observed in vivo and in vitro. Here, we identified three novel HIV-1 protease inhibitors (PIs) containing a tetrahydropyrano-tetrahydrofuran (Tp-THF) moiety with a C-5 hydroxyl (GRL-015, -085, and -097) which potently suppress the replication of HIVDRV(R) . Moreover, the emergence of HIV-1 strains resistant to the three compounds was considerably delayed compared to the case of DRV. The C-5 hydroxyl formed a strong hydrogen bonding interaction with the carbonyl oxygen atom of Gly48 of protease as examined in the structural analyses. Interestingly, a compound with Tp-THF lacking the hydroxyl moiety substantially decreased activity against HIVDRV(R) . The three novel compounds should be further developed as potential drugs for treating individuals harboring wild-type and multi-PI-resistant HIV variants as well as HIVDRV(R) .

[Chemical constituents from culture of Streptomyces sp. CPCC 202950].

Eleven compounds were isolated from the culture of Streptomyces sp. CPCC 202950 by a combination of various chromatographic techniques including column chromatography over macroporous resin HP-20, MCI, and reversed-phase HPLC. Their structures were identified as 1H-pyrrole-2-carboxamide(1),5'-deoxy-5'-methylthioinosine(2), vanillamide(3), trans-3-methylthioacrylamide(4), 1,2,3,4-Tetraydro-1H-pyrido[3,4-b]indole-3-carboxylic acid(5), cyclo(L-pro-L-tyr) (6), N-[2-(4-hydroxyphenyl)]ethylacetamide(7), benzamide (8), cyclo ('L-leucyl-trans-4-hydroxy-L-proline)(9), cyclo-(Phe-Gly) (10), and tryptophan (11). Among them, compounds 1 and 2 were new natural products. In the preliminary assays, none of the compounds exhibited obvious inhibition of HIV-1 protease activity (IC50 > 10 micromol x L(-1)).

Inhibition of HIV-1 enzymes, antioxidant and anti-inflammatory activities of Plectranthus barbatus.

ETHNOPHARMACOLOGICAL RELEVANCE: Plectranthus barbatus is widely used in African countries as an herbal remedy to manage HIV/AIDS and related conditions. AIM OF THE STUDY: To investigate the HIV-1 inhibitory, anti-inflammatory and antioxidant properties of P. barbatus and thereby provide empirical evidence for the apparent anecdotal success of the extracts. MATERIALS AND METHODS: Ethanolic extract of P. barbatus's leaves was screened against two HIV-1 enzymes: protease (PR) and reverse transcriptase (RT). Cytotoxicity of the extract was determined through measuring tetrazolium dye uptake of peripheral blood mononuclear cells (PBMCs) and the TZM-bl cell line. Confirmatory assays for cytotoxicity were performed using flow cytometry and real-time cell electronic sensing (RT-CES). The free radical scavenging activity of the extract was investigated with 2,2-diphenyl-1-picrylhydrazyl while the anti-inflammatory properties of the plant extract were investigated using a Th1/Th2/Th17 cytometric bead array technique. RESULTS: P. barbatus extract inhibited HIV-1PR and the 50% inhibitory concentration (IC50) was 62.0 µg/ml. The extract demonstrated poor inhibition of HIV-1 RT. Cytotoxicity testing presented CC50 values of 83.7 and 50.4 µg/ml in PBMCs and TZM-bl respectively. In addition, the extract stimulated proliferation in HIV negative and positive PBMCs treated. RT-CES also registered substantial TZM-bl proliferation after extract treatment. The extract exhibited strong antioxidant activity with an IC50 of 16 µg/ml and reduced the production of pro-inflammatory cytokines indicating anti-inflammatory potential. CONCLUSION: This is the first demonstration of the in vitro anti HIV-1 potential of P. barbatus including direct activity as well as through the stimulation of protective immune and inflammation responses. The low cytotoxicity of the extract is also in agreement with the vast anecdotal use of this plant in treating various ailments with no reported side-effects.

Enhancement of probe signal for screening of HIV-1 protease inhibitors in living cells.

The global human immunodeficiency virus infection/acquired immuno-deficiency syndrome (HIV/AIDS) epidemic is one of the biggest threats to human life. Mutation of the virus and toxicity of the existing drugs necessitate the development of new drugs for effective AIDS treatment. Previously, we developed a molecular probe that utilizes the Förster resonance energy transfer (FRET) principle to visualize HIV-1 protease inhibition within living cells for drug screening. We explored using AcGFP1 (a fluorescent mutant of the wild-type green fluorescent protein) as a donor and mCherry (a mutant of red fluorescent protein) as an acceptor for FRET microscopy imaging measurement of HIV-1 protease activity within living cells and demonstrated that the molecular probe is suitable for the High-Content Screening (HCS) of anti-HIV drugs through an automated FRET microscopy imaging measurement. In this study, we genetically engineered a probe with a tandem acceptor protein structure to enhance the probe's signal. Both in vitro and in vivo studies revealed that the novel structure of the molecular probe exhibits a significant enhancement of FRET signals, reaching a probe FRET efficiency of 34%, as measured by fluorescence lifetime imaging microscopy (FLIM) measurement. The probe developed herein would enable high-content screening of new anti-HIV agents.

A drug discovery platform: a simplified immunoassay for analyzing HIV protease activity.

Although numerous methods for the determination of HIV protease (HIV-PR) activity have been described, new high-throughput assays are required for clinical and pharmaceutical applications due to the occurrence of resistant strains. In this study, a simple enzymatic immunoassay to identify HIV-PR activity was developed based on a Ni(2+)-immobilized His(6)-Matrix-Capsid substrate (H(6)MA-CA) is cleaved by HIV protease-His(6) (HIV-PRH(6)) which removes the CA domain and exposes the free C terminus of MA. Following this cleavage, two monoclonal antibodies specific for either the free C-terminal MA or CA epitope are used to quantify the proteolytic activity using a standard ELISA-based system. Specificity for detection of the HIV-PRH(6) activity was confirmed with addition of protease inhibitor (PI), lopinavir. In addition, the assay was able to detect an HIV-PR variant activity indicating that this assay is capable of assessing viral mutation affect HIV-PR activity. The efficacy of commercially available PIs and their 50% inhibitory concentration (IC(50)) were determined. This assay provides a high-throughput method for both validating the efficiency of new drugs in vitro and facilitating the discovery of new PIs. In addition, it could serve as a method for examining the influence of various mutations in HIV-PRs isolated from drug-resistant strains.

Visualization of human immunodeficiency virus protease inhibition using a novel Förster resonance energy transfer molecular probe.

The in vivo high-throughput screening (HTS) of human immunodeficiency virus (HIV) protease inhibitors is a significant challenge because of the lack of reliable assays that allow the visualization of HIV targets within living cells. In this study, we developed a new molecular probe that utilizes the principles of Förster resonance energy transfer (FRET) to visualize HIV-1 protease inhibition within living cells. The probe is constructed by linking two fluorescent proteins: AcGFP1 (a mutant green fluorescent protein) and mCherry (a red fluorescent protein) with an HIV-1 protease cleavable p2/p7 peptide. The cleavage of the linker peptide by HIV-1 protease leads to separation of AcGFP1 from mCherry, quenching FRET between AcGFP1 and mCherry. Conversely, the addition of a protease inhibitor prevents the cleavage of the linker peptide by the protease, allowing FRET from AcGFP1 to mCherry. Thus, HIV-1 protease inhibition can be determined by measuring the FRET signal's change generated from the probe. Both in vitro and in vivo studies demonstrated the feasibility of applying the probe for quantitative analyses of HIV-1 protease inhibition. By cotransfecting HIV-1 protease and the probe expression plasmids into 293T cells, we showed that the inhibition of HIV-1 protease by inhibitors can be visualized or quantitatively determined within living cells through ratiometric FRET microscopy imaging measurement. It is expected that this new probe will allow high-content screening (HCS) of new anti-HIV drugs.

Isolation of adenosine, iso-sinensetin and dimethylguanosine with antioxidant and HIV-1 protease inhibiting activities from fruiting bodies of Cordyceps militaris.

According to previous studies, a close relationship between oxidative stress and AIDS suggests that antioxidants might play an important role in the treatment of AIDS. Cordyceps militaris was selected from nine edible mushrooms by assay of inhibition of erythrocyte hemolysis. Macroporous adsorption resin and HPLC were used to purify three micromolecular compounds named L3a, L3b and L3c. L3a was identified to be adenosine with the molecular formula C(10)H(13)N(5)O(4); L3b was 6,7,2',4',5'-pentamethoxyflavone with the molecular formula C(20)H(20)O(7), and L3c was dimethylguanosine with the molecular formula C(12)H(17)N(5)O(5). The compound 6,7,2',4',5'-pentamethoxyflavone was first isolated from C. militaris. The assay of inhibition of HIV-1 protease (HIV-1 PR) was based on the fact that the expression of this enzyme can inhibit the growth of E. coli. This is a new screening system for HIV-1 PR inhibitors. Both L3a and L3b showed high inhibition to HIV-1 PR. These compounds could be new anti-HIV-1 PR drugs.

Bioactive triterpenoids from Kadsura heteroclita.

A phytochemical study of Kadsura heteroclita led to the isolation of eight triterpenoids, including two new compounds, named kadheterilactone A (1) and kadheterilactone B (2), as well as six known compounds, longipedlactone H (3), longipedlactone A (4), longipedlactone F (5), kadsuranic acid A (6), nigranoic acid (7), and schisandronic acid (8). Their structures were elucidated on the basis of spectroscopic methods, including 2D NMR techniques. The cytotoxic activities of 1-8 were tested against several tumor cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium hydrobromide (MTT) assay in vitro. As a result, 4 and 5 turned out to be significantly cytotoxic against Hep-G2 and Bel-7402 tumor cell lines. All compounds were also tested for inhibition on HIV-1 protease (PR) and reverse transcriptase (RT). Compounds 6 and 7 showed strong inhibition on HIV-1 PR, while 8 exhibited moderate activity, others were only weakly active. No compounds were active against HIV-1 RT.

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.

A bioactive isoprenylated xanthone and other constituents of Garcinia edulis.

Phytochemical investigation on the root bark of Garcinia edulis (Clusiaceae) gave a new isoprenylated xanthone, 1,4,6-trihydroxy-3-methoxy-2-(3-methyl-2-butenyl)-5-(1,1-dimethyl-prop-2-enyl)xanthone (1), a known xanthone, forbexanthone (2) together with three known pentacyclic triterpenoids, friedelin, lupeol and lupeol acetate. The structure of the new compound was fully characterised by NMR spectroscopic analysis. Compound 1 showed significant anti-HIV-1 protease activity with IC(50) value of 11.3 microg/mL. Furthermore, compound 1 showed potent cytotoxic activity with LC(50) value of 2.36 microg/mL against brine shrimp larva in vitro.

Lignan, sesquilignans and dilignans, novel HIV-1 protease and cytopathic effect inhibitors purified from the rhizomes of Saururus chinensis.

Five lignans were isolated from the ethyl acetate extracts of Saururus chinensis rhizomes and evaluated for anti-HIV-1 activity. Their structures were elucidated as two dilignans, manassantin A (1), manassantin B (2), two sesquilignans, saucerneol B (3) and saucerneol C (4), and a new lignan, saururin B (5) by spectroscopic analysis. Of these components, manassantin A (1) and saururin B (5) showed dose-dependent inhibitory activities on HIV-1 protease with IC(50) values of 38.9 and 5.6 microM. In addition, manassantins A (1), B (2) and saucerneol B (3) inhibited HIV-1-induced cytopathic effects in a human T lymphoblastoid cell line with IC(100) values of 1.0, 1.0 and 0.2 microM, respectively. Of these active constituents, saucerneol B (3) showed the most potent and selective anti-HIV-1 activity (IC(100) of 0.2 microM, CC(0) of >125.0 microM, and SI of >520.8).

Inhibition of the dimerization and active site of HIV-1 protease by secondary metabolites from the Vietnamese mushroom Ganoderma colossum.

A new farnesyl hydroquinone, ganomycin I (1), was isolated along with ganomycin B (2) from the chloroform extract of the fruiting bodies of the Vietnamese mushroom Ganoderma colossum. These compounds inhibited HIV-1 protease with IC50 values of 7.5 and 1.0 microg/mL, respectively. Kinetic studies using Zhang-Poorman and Lineweaver plots revealed that compound 2 competitively inhibited the active site of the enzyme, whereas the tetracyclic triterpene schisanlactone A, previously isolated from the same fungus, was a dimerization inhibitor, with an IC50 value of 5.0 microg/mL. The previous findings were also confirmed by the virtual docking of both compounds with HIV-1 protease crystal structure.

Anti-human immunodeficiency virus-1 protease activity of new lanostane-type triterpenoids from Ganoderma sinense.

Five new highly oxygenated lanostane-type triterpenoids [ganoderic acid GS-1 (1), ganoderic acid GS-2 (2), ganoderic acid GS-3 (3), 20(21)-dehydrolucidenic acid N (4) and 20-hydroxylucidenic acid A (5)] were isolated from the fruiting body of Ganoderma sinense, together with known compounds including 6 triterpenoids and 3 sterols. The structures of the new triterpenoids determined by spectroscopic means including 2D NMR were 7beta-hydroxy-3,11,15-trioxo-lanosta-8,24(E)-dien-26-oic acid (1), 7beta,15alpha-dihydroxy-3,11-dioxo-lanosta-8,24(E)-dien-26-oic acid (2), 12beta-acetoxy-3beta,7beta-dihydroxy-11,15-dioxo-lanosta-8,24(E)-dien-26-oic acid (3), 3beta,7beta-dihydroxy-11,15-dioxo-25,26,27-trinorlanosta-8,20-dien-24-oic acid (4), and 7beta,20xi-dihydroxy-3,11,15-trioxo-25,26,27-trinorlanost-8-en-24-oic acid (5), respectively. Among these, ganoderic acid GS-2, 20-hydroxylucidenic acid N, 20(21)-dehydrolucidenic acid N and ganoderiol F inhibited human immunodeficiency virus-1 protease with IC(50) values of 20-40 microM.

Rapid, simultaneous determination of lopinavir and ritonavir in human plasma by stacking protein precipitations and salting-out assisted liquid/liquid extraction, and ultrafast LC-MS/MS.

Lopinavir and ritonavir are co-formulated in Kaletra approved for the treatment of human immunodeficiency virus infection. A validated analytical method is mandatory for clinical development and therapeutic drug monitoring. Here we are reporting a method for rapid, simultaneous determination of lopinavir and ritonavir in human plasma with stacked protein precipitations and salting-out assisted extraction (SALLE), and ultrafast LC-MS/MS detection. With stacked protein precipitations and SALLE, the sample preparation for a 96-well plate can be completed within 20 min by an automated pipette. Due to the unique cleanliness of SALLE extracts post double protein precipitations, the extracts were injected into an ultrafast liquid chromatography and tandem mass spectrometry system (LC-MS/MS) after simple dilution. An Agilent Zobax Extend-C18 Rapid resolution HT column (1.8 microm, 2.1 mm x 30 mm) was used for the separation. A mixture of acetonitrile:water (55:45, v/v) with 0.1% formic acid was used as the mobile phase. LC ran for approximately 48 s at a flow rate of 0.5 mL min(-1), tandem mass spectrometric data collection started at 15 s and lasts for 30 s. The method was validated with reference to Industry Guidance for Bioanalytical Method Validation and then used for clinical samples. The method is ultrafast, and robust. Results of incurred samples demonstrated excellent method of reproducibility. This ultrafast analysis speed did not compromise with the data quality. To our knowledge, this is the fastest analytical method for simultaneous determination of lopinavir and ritonavir.

Synthesis of dammarane-type triterpene derivatives and their ability to inhibit HIV and HCV proteases.

We synthesized dammarane-type triterpene derivatives and evaluated their ability to inhibit HIV-1 and HCV proteases to understand their structure-activity relationships. All of the mono- and di-succinyl derivatives (5a-5f) were powerful inhibitors of HIV-1 protease (IC(50)<10 microM). However, only di-succinyl (5e) and 2,3-seco-2,3-dioic acid (3b) derivatives similarly inhibited HCV protease (IC(50)<10 microM). A-nor dammarane-type triterpenes (4a and 4b, IC(50) 10.0 and 29.9 microM, respectively) inhibited HIV-1 protease moderately or strongly, but were inactive against HCV protease. All compounds that powerfully inhibited HIV-1 or HCV protease did not appreciably inhibit the general human proteases, renin and trypsin (IC(50)>1000 microM). These findings indicated that the mono-succinyl dammarane type derivatives (5a-5d) selectively inhibited HIV-1 protease and that the di-succinyl (5e, 5f) as well as 2,3-seco-2,3-dioic acid (3b) derivatives preferably inhibited both viral proteases.

Prenylisoflavonoids from Erythrina senegalensis as novel HIV-1 protease inhibitors.

Eight compounds were isolated from the CH (2)Cl (2) extracts of ERYTHRINA SENEGALENSIS to assess HIV-1 protease (PR) activity inhibition. The prenylated isoflavone structures, identified by spectroscopic analysis, were 8-prenylluteone ( 1), auriculatin ( 2), erysenegalensein O ( 3), erysenegalensein D ( 4), erysenegalensein N ( 5), derrone ( 6), alpinumisoflavone ( 7), and 6,8-diprenylgenistein ( 8). The constituents showed dose-dependent inhibitory activities on HIV-1 PR with IC (50) values from 0.5 to 30.0 muM. Compounds 1 - 5 possessing two hydroxy groups in the 2' and 4' positions of the B ring, potently inhibited HIV-1 PR activity. In addition, 6,8-diprenylgenistein ( 8) with two prenyl groups in the 6 and 8 positions of the A ring and one hydroxy group in the 4' position of B-ring was the most potent HIV-1 PR inhibitor.