Anti-HIV-1 protease activity of the crude extracts and isolated compounds from Auricularia polytricha.

BACKGROUND: Acquired immunodeficiency syndrome (AIDS) is caused by the Human immunodeficiency virus type-1 (HIV-1). HIV-1 protease (HIV-1 PR) is an essential enzyme for the HIV replication, and therefore, it is an important target for antiretroviral drugs development, particularly from natural products. Auricularia polytricha (AP) is an edible mushroom with several important therapeutic properties. These properties will be investigated as HIV-1 PR inhibitors. METHODS: The sequential hexane (APH), ethanol (APE) and water (APW) extracts from AP were screened for inhibitory activity against HIV-1 PR. The extract that consistently showed the strong HIV-1 PR inhibition was further investigated for its phytochemical constituents. The compounds were purified by column chromatography. The isolated compounds were structurally elucidated using 1D and 2D NMR, HRMS, FTIR, and GC/MS techniques. Each compound was screened against HIV-1 PR to determine its inhibitory activity and to provide an explanation for the activity found in the extract. RESULTS: Hexane crude extract of AP (APH) exhibited significant inhibition on HIV-1 PR activity. Four major compounds isolated from APH fraction were identified to be two triacylglycerols, linoleic acid and ergosterol. Moreover, all four compounds showed significant inhibition of HIV-1 PR activity. CONCLUSION: The findings from this study suggest that AP is a good source of fatty esters, fatty acids and ergosterol. These natural products exhibit anti-HIV-1 properties by blocking HIV-1 PR. These important biological results warrant further development of AP as an alternative antiretroviral drug.

In vitro anti-HIV and antioxidant activity of Hoodia gordonii (Apocynaceae), a commercial plant product.

BACKGROUND: Hoodia gordonii products are widely commercialized for anti-obesity purposes; however, minimal research is available on the other health properties demonstrated by this popular herbal plant. METHODS: H. gordonii crude extracts (ethanol and ethyl acetate) were assayed for in vitro anti-HIV-1 protease (PR), reverse transcriptase (RT) and integrase activity. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and reducing power assays were used for the antioxidant analysis. In addition, qualitative and quantitative phytochemical analyses of the extracts were determined using standard methods. RESULTS: H. gordonii extract demonstrated good inhibition against HIV RT with IC50 values of 73.55 ± 0.04 and 69.81 ± 9.45 µg/mL for ethanol and ethyl acetate extracts, respectively. Both extracts also demonstrated inhibitory activity against HIV PR with IC50 values of 97.29 ± 0.01 and 63.76 ± 9.01 µg/mL for ethanol and ethyl acetate extracts. In addition, H. gordonii also showed good antioxidant activity with IC50 values of 124.6 ± 11.3 and 126.2 ± 3.15 µg/mL obtained for ethanol and ethyl acetate extracts, respectively. The reducing power of H. gordonii extracts increased as the concentration increased which confirmed the presence of antioxidants (reductants) in the extracts. Phytochemical screening of H. gordonii revealed the presence of phenolics, alkaloids, terpenes, steroids, cardiac glycosides and tannins in the ethanolic extract, while the ethyl acetate extract only showed the presence of phenolics, cardiac glycosides and steroids. The total phenolic content was 420 ± 0.17 and 319.9 ± 0.2 mg GAE/g for the ethanol and ethyl acetate extracts, respectively. The ethanol extract, which revealed the presence of tannins, had a tannin content of 330 ± 0.2 mg TAE/g extract. CONCLUSION: This data suggests that H. gordonii has good in vitro inhibition against selected HIV-1 enzymes as well as antioxidant properties, suggesting new potential uses for this commercial plant.

Complex patterns of protease inhibitor resistance among antiretroviral treatment-experienced HIV-2 patients from Senegal: implications for second-line therapy.

Protease inhibitor (PI)-based antiretroviral therapy (ART) can effectively suppress HIV-2 plasma load and increase CD4 counts; however, not all PIs are equally active against HIV-2, and few data exist to support second-line therapy decisions. To identify therapeutic options for HIV-2 patients failing ART, we evaluated the frequency of PI resistance-associated amino acid changes in HIV-2 sequences from a cohort of 43 Senegalese individuals receiving unboosted indinavir (n = 18 subjects)-, lopinavir/ritonavir (n = 4)-, or indinavir and then lopinavir/ritonavir (n = 21)-containing ART. Common protease substitutions included V10I, V47A, I54M, V71I, I82F, I84V, L90M, and L99F, and most patients harbored viruses containing multiple changes. Based on genotypic data, we constructed a panel of 15 site-directed mutants of HIV-2ROD9 containing single- or multiple-treatment-associated amino acid changes in the protease-encoding region of pol. We then quantified the susceptibilities of the mutants to the HIV-2 "active" PIs saquinavir, lopinavir, and darunavir using a single-cycle assay. Relative to wild-type HIV-2, the V47A mutant was resistant to lopinavir (6.3-fold increase in the mean 50% effective concentration [EC50]), the I54M variant was resistant to darunavir and lopinavir (6.2- and 2.7-fold increases, respectively), and the L90M mutant was resistant to saquinavir (3.6-fold increase). In addition, the triple mutant that included I54M plus I84V plus L90M was resistant to all three PIs (31-, 10-, and 3.8-fold increases in the mean EC50 for darunavir, saquinavir, and lopinavir, respectively). Taken together, our data demonstrate that PI-treated HIV-2 patients frequently harbor viruses that exhibit complex patterns of PI cross-resistance. These findings suggest that sequential PI-based regimens for HIV-2 treatment may be ineffective.

[Effective components against HIV-1 replicative enzymes isolated from plants].

Plant active components characterized of many different structures and activities on multiple targets, have made them to be the important sources of inhibitors on HIV-1. For finding leading compounds with new structure against HIV-1, three key HIV-1 replicative enzymes (reverse transcriptase, protease and integrase) were used as screening models. The in vitro activities of 45 plant derived components isolated from Schisandraceae, Rutaceae and Ranunculaceae were reported. Within twelve triterpene components isolated, eight compounds were found to inhibit HIV-1 protease, in these eight active compounds, kadsuranic acid A (7) and nigranoic acid (8), inhibited both HIV-1 protease and integrase; Among fifteen lignans, meso-dihydroguaiaretic acid (15) and kadsurarin (16) were active on HIV-1 reverse transcriptase, and 4, 4-di(4-hydroxy-3-methoxyphenly)-2, 3-dimethylbutanol (13) active on HIV-1 integrase. All of the six alkaloids, seven flavones, and five others compounds were not active or only with low activities against HIV-1 replicative enzymes. Further studies of the triterpene components showing strong inhibitory activities on HIV-1 were warranted.

Synthesis and activity of tetrapeptidic HTLV-I protease inhibitors possessing different P3-cap moieties.

The causative agent behind adult T-cell leukemia and tropical spastic paraparesis/HTLV-I-associated myelopathy is the human T-cell leukemia virus type 1 (HTLV-I). Tetrapeptidic HTLV-I protease inhibitors were designed on a previously reported potent inhibitor KNI-10516, with modifications at the P(3)-cap moieties. All the inhibitors showed high HIV-1 protease inhibitory activity (over 98% inhibition at 50nM) and most exhibited highly potent inhibition against HTLV-I protease (IC(50) values were less than 100nM).

Factors associated with the selection of mutations conferring resistance to protease inhibitors (PIs) in PI-experienced patients displaying treatment failure on darunavir.

The objective of this study was to characterize the mutations selected by darunavir (DRV) use in protease inhibitor (PI)-experienced patients and the associated factors. We analyzed treatment failure in 54 PI-experienced human immunodeficiency virus (HIV)-infected patients on a DRV- and ritonavir-containing regimen. Viral genotyping was carried out at the baseline, at between 1 and 3 months of treatment, and at between 3 and 6 months of treatment to search for the selection of mutations conferring resistance to PIs. The median baseline HIV RNA level was 4.9 log(10) copies/ml, and the median CD4 count was 87 cells/mm(3). At the baseline, the median numbers of resistance mutations were as follows: 3 DRV resistance mutations, 4 major PI resistance mutations, and 10 minor PI resistance mutations. The most common mutations that emerged at rebound included V32I (44%), I54M/L (24%), L33F (25%), I84V (21%), and L89V (12%). Multivariate analysis showed that higher baseline HIV RNA levels and smaller numbers of nucleoside reverse transcriptase inhibitor simultaneously used with DRV were associated with a higher risk of DRV resistance mutation selection. By contrast, L76V, a known DRV resistance mutation, was found to decrease the risk of selection of another DRV resistance mutation. The occurrence of virological failure while a patient was on DRV was associated with the selection of mutations that increased the level of DRV resistance without affecting susceptibility to tipranavir (TPV). In these PI-treated patients who displayed treatment failure while they were on a DRV-containing regimen, we confirmed the set of emerging mutations associated with DRV failure and identified the factors associated with the selection of these mutations. TPV susceptibility does not seem to be affected by the selection of a DRV resistance mutation.

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.

Screening for HIV protease inhibitors by protection against activity-mediated cytotoxicity in Escherichia coli.

Expressed retroviral proteases are often cytotoxic to the hosts. The cytotoxicity of a tethered dimer HIV protease described previously is particularly severe that transformed Escherichia coli cells could not survive the bactericidal activity of the low-level protease produced under uninduced conditions. The presence of HIV protease inhibitors protected the transformed cells from cytotoxic effects and allowed the growth of these cells on plates and in broth. A high throughput screening method was developed to seek compounds that served as "growth factors" for the HIV protease restricted cells. Several compounds identified by this screening supported the growth of these cells, preserved their viability, and inhibited HIV protease. This assay could be used as a general method for screening for inhibitors of recombinant enzymes that produce a cytotoxic phenotype in host cells.

Anti-HIV-1 protease- and HIV-1 integrase activities of Thai medicinal plants known as Hua-Khao-Yen.

Ethanolic- and water extracts from five species of Thai medicinal plants known as Hua-Khao-Yen were tested for their inhibitory effects against HIV-1 protease (HIV-PR) and HIV-1 integrase (HIV-1 IN). The result revealed that the ethanolic (EtOH) extract of Smilax corbularia exhibited anti-HIV-1 IN activity with an IC50 value of 1.9 microg/ml, followed by the water extract of Dioscorea birmanica (IC50 = 4.5 microg/ml), the EtOH extract of Dioscorea birmanica (IC50 = 4.7 microg/ml), the water extract of Smilax corbularia (IC50 = 5.4 microg/ml), the EtOH extract of Smilax glabra (IC50 = 6.7 microg/ml) and the water extract of Smilax glabra (IC50 = 8.5 microg/ml). The extracts of Pygmaeopremna herbacea and Dioscorea membranacea were apparently inactive (IC50 > 100 microg/ml). Interestingly, only the EtOH extract of Dioscorea membranacea showed appreciable activity (IC50 = 48 microg/ml) against HIV-1 PR, while the other extracts possessed mild activity. This result strongly supported the basis for the use of Smilax corbularia and Dioscorea membranacea for AIDS treatment by Thai traditional doctors.

Anti-HIV-1 protease activity of compounds from Boesenbergia pandurata.

Searching for anti-HIV-1 protease (PR) inhibitors of Thai medicinal plants led to the isolation of a new cyclohexenyl chalcone named panduratin C (1) and chalcone derivatives (2-6) from the methanol extract of Boesenbergia pandurata rhizomes. The known compounds were identified to be panduratin A (2), hydroxypanduratin A (3), helichrysetin (4), 2',4',6'-trihydroxyhydrochalcone (5), and uvangoletin (6). The structures of all compounds were elucidated on the basis of chemical and spectroscopic methods. It was found that 3 possessed the most potent anti-HIV-1 PR activity with an IC50 value of 5.6 microM, followed by 2 (IC50 = 18.7 microM), whereas other compounds exhibited only mild activity. Structure-activity relationships of these compounds on anti-HIV-1 PR activity are summarized as follows: (1) hydroxyl moiety at position 4 conferred higher activity than methoxyl group; (2) prenylation of dihydrochalcone was essential for activity; (3) hydroxylation at position 4''' reduced activity; and (4) introduction of double bond at C1' and C6' of chalcone gave higher activity. As regards active constituents contained in B. pandurata rhizomes, hydroxypanduratin A (3) and panduratin A (2) are active principles against HIV-1 PR.

Anti-HIV protease activity from rosa family plant extracts and rosamultin from Rosa rugosa.

To identify substances with anti-human immunodeficiency virus (HIV) activity from plant sources, 12 extracts of Rosa family plants were screened for their inhibitory effects against HIV-1 protease. Of the extracts tested, the strongest inhibitory effects were observed in the root of Rosa rugosa and the leaves of Prunus sargentii, at a concentration of 100 microg/mL. Rosamultin isolated from the root of R. rugosa inhibited HIV-1 protease by 53% at a concentration of 100 microM.

Lysine derivatives as potent HIV protease inhibitors. Discovery, synthesis and structure-activity relationship studies.

A screening assay program on HIV-protease was carried out on more than fifty commercially available N-protected amino acids and has revealed that those with a long side chain such as lysine, ornithine and arginine exhibited significant inhibition of HIV protease enzyme. The presence of an Fmoc group was found to be essential to obtain micromolar inhibitors and the addition of an alkyl group at the Nalpha-position resulted in the discovery of the lead compound 11 displaying a 5 nM inhibition constant. Although this new inhibitor series is not categorized among those mimicking the substrate with a non-hydrolyzable transition-state isoster, it was found very specific to inhibit HIV protease enzyme in comparison to the mammalian aspartyl proteases pepsin, renin and cathepsin. Furthermore, these inhibitors did not show any cytotoxicity at a concentration below 75 microM.

Inhibitory effects of (-)-epigallocatechin gallate on the life cycle of human immunodeficiency virus type 1 (HIV-1).

Epigallocatechin gallate (EGCg), the major tea catechin, is known as a potent anti-bacterial agent. In addition, anti-tumor promoting, anti-inflammatory, anti-oxidative and antiviral activities have been reported. In the present study, we investigated possible anti-human immunodeficiency virus type-1 (HIV-1) activity of EGCg and its mechanisms of action in the viral life cycle. EGCg impinges on each step of the HIV life cycle. Thus, destruction of the viral particles, viral attachment to cells, post-adsorption entry into cells, reverse transcription (RT), viral production from chronically-infected cells, and the level of expression of viral mRNA, were analyzed using T-lymphoid (H9) and monocytoid (THP-1) cell systems, and antiviral protease activity was measured using a cell-free assay. Inhibitory effects of EGCg on specific binding of the virions to the cellular surfaces and changes in the steady state viral regulation (mRNA expression) due to EGCg were not observed. However, EGCg had a destructive effect on the viral particles, and post-adsorption entry and RT in acutely infected monocytoid cells were significantly inhibited at concentrations of EGCg greater than 1 microM, and protease kinetics were suppressed at a concentration higher than 10 microM in the cell-free study. Viral production by THP-1 cells chronically-infected with HIV-1 was also inhibited in a dose-dependent manner and the inhibitory effect was enhanced by liposome modification of EGCg. As expected, increased viral mRNA production was observed in lipopolysaccharide (LPS)-activated chronically HIV-1-infected cells. This production was significantly inhibited by EGCg treatment of THP-1 cells. In contrast, production of HIV-1 viral mRNA in unstimulated or LPS-stimulated T-lymphoid cells (H9) was not inhibited by EGCg. Anti-HIV viral activity of EGCg may thus result from an interaction with several steps in the HIV-1 life cycle.

Design and synthesis of new inhibitors of HIV-1 protease dimerization with conformationally constrained templates.

To inhibit the HIV-1 protease dimerization necessary to exhibit enzymatic activity, we synthesized and evaluated a new beta-sheet peptide (compound 1), containing 4-(2-aminoethyl)-6-dibenzofuranpropionic acid as a conformationally restricted linker and a non-peptidic beta-strand mimetic, 2-[3-([2-[(9-fluorenylmethoxy)carbonyl]hydrazino]carbonyl)-4-methoxyanilino]-2-oxoacetic acid (Fmoc-Hao-OH, compound 2). Kinetic analysis showed that compound 1 inhibited the dimerization of HIV-1 protease by a dissociative mechanism with a K(id) value of 5.4 microM at 37 degrees C (pH 5.0). However, compound 2 showed a small shift in the slope of the lines in the Zhang-Poorman plot (K(id)=9.1 microM), suggesting that compound 2 inhibits the dimerization of HIV-1 PR not only through a dissociative mechanism but also through an active-site directed mechanism partly. This is the first study of a non-peptidic inhibitor of HIV-1 protease dimerization.

Synthesis and biological evaluation of first N-alkyl syn dimeric 4-aryl-1,4-dihydropyridines as competitive HIV-1 protease inhibitors.

A first series of novel N-alkyl substituted syn dimeric 4-aryl-1,4-dihydropyridines 12--17 have been synthesised and evaluated as HIV-1 protease inhibitors in in vitro assays. While the N-methyl derivatives 12 and 13 were almost inactive, with IC(50)-values of about 225 microM, the N-benzyl compounds with varied ester groups all exhibited stronger activities, with IC(50)-values of 11--12 microM for the presently best compounds 16 and 17 with ethyl ester functions. The type of HIV-1 protease inhibition of the novel inhibitors was characterised as competitive. With the increase of observed activity from N-methyl derivatives to N-benzyl compounds the binding mode may correspond to that of cyclic ureas with hydrophobic interactions of the four aromatic residues to the S1/S1' and S2/S2' regions of HIV-1 protease.

Screening of Korean plants against human immunodeficiency virus type 1 protease.

With the aim of finding novel anti-human immunodeficiency virus agents from natural products, 93 MeOH extracts of Korean plants were screened for their inhibitory activities against HIV-1 protease. The most potent inhibition was shown by the root of Rodiola rosea with 70.4% inhibition at a concentration of 100 microg/mL.

A search for anti-viral properties in Panamanian medicinal plants. The effects on HIV and its essential enzymes.

Aqueous and methanolic extracts of 39 Panamanian medicinal plants were tested for anti-human immunodeficiency virus (HIV) effects. The extracts were tested for the inhibition of HIV-induced cytopathic effects in cultured cells, HIV-reverse transcriptase (RT) and HIV-protease (PR) enzymes. The water extract of the branches of Jatropha curcas (Euphorbiaceae) inhibited strongly the HIV-induced cytopathic effects with low cytotoxicity. On the other hand, the water extracts of the whole plant of Chamaesyce hyssopifolia (Euphorbiaceae), the leaves of Cordia spinescens (Boraginaceae) and the aerial parts of Hyptis lantanifolia (Labiatae), and the methanol extract of the aerial parts of Tetrapteris macrocarpa (Malpighiaceae) were potent inhibitors of HIV-RT (IC50: 6-8 microg/ml). Seven out of 39 plants were found to be moderate inhibitors of HIV-PR (IC50: 43-100 microg/ml). Furthermore, we report on the respective inhibitory substances of J. curcas, C. hyssopifolia and C. spinescens, and their possible mechanism of action.

Correlation of response to treatment and HIV genotypic changes during phase III trials with saquinavir and reverse transcriptase inhibitor combination therapy.

OBJECTIVES: Assessment of genotypic change in HIV protease during treatment with saquinavir (SQV) in combination with zidovudine (ZDV) and/or zalcitabine (ddC), to determine the influence of such changes on viral phenotype and response to treatment. DESIGN: Virologic substudies of Phase III clinical trials NV14256 and SV14604. METHODS: Population sequencing of HIV protease genes amplified from pre- and post-treatment plasma. Phenotyping of peripheral blood mononuclear cell (PBMC)-derived virus isolates, and genotyping of proviral DNA clones amplified from PBMC used in the expansion of virus isolates. RESULTS: In both trials the incidence of Met90 remained at < or = 20% in subjects receiving SQV in combination with ddC (with or without ZDV) for 1 year. A Val48 substitution was observed in two out of 81 subjects after 24 weeks and in two out of 75 subjects after 48 weeks. In 12 out of 13 NV14256 subjects with viral load rebound during SQV monotherapy these substitutions were associated with the rebound. In subjects treated with SQV plus ddC, rebound was associated with SQV resistance in six out of 22 cases and ddC resistance in five out of 22 cases. The incidences of non-BRU residues at positions 10, 63 and 71 were increased significantly (P < 0.05, Fisher's exact test) after SQV treatment with or without ZDV. However, comparison of genotypic and phenotypic data showed that these changes were not associated with reduced sensitivity to SQV. CONCLUSIONS: Virological failure during combination therapy can be due to resistance to either treatment drug, emphasising the need to change both the reverse transcriptase inhibitor and the protease inhibitor. Only Val48 and Met90 correlated directly with the development of reduced drug sensitivity during treatment with SQV in vivo.