Anti-inflammatory cyclohexenyl chalcone derivatives in Boesenbergia pandurata.

The cyclohexenyl chalcone derivative [(-)-hydroxypanduratin A], together with the previously known panduratin A, sakuranetin, pinostrobin, pinocembrin, and dihydro-5,6-dehydrokawain were isolated from the chloroform extract of the red rhizome variety of Boesenbergia pandurata (Robx.) Schltr. [currently known as Boesenbergia rotunda (L.) Mansf., Kulturpfl.]. Their structures were assigned on the basis of their spectroscopic data. (-)-Hydroxypanduratin A and (-)-panduratin A showed significant topical anti-inflammatory activity in the assay of TPA-induced ear edema in rats.     

Analysis of antioxidant activities contained in the Boesenbergia pandurata Schult. Rhizome

The rhizome of Boesenbergia pandurata Schult. was found to possess potent antioxidant activity in a rat brain homogenate model. Bioassay-guided isolation of the active compounds from a CH2Cl2-MeOH (1:1) extract led to the isolation of 5-hydroxy-7-methoxyflavanone, panduratin A, 5,7-dihydroxyflavanone, 2',6'-dihydroxy-4'-methoxychalcone, 2',4'-dihydroxy-6'-methoxychalcone, and 4-hydroxypanduratin A. Panduratin A, 4-hydroxypanduratin A, and 2',6'-dihydroxy-4'-methoxychalcone were also found to exert neuroprotective effects.     

Anti-HIV-1 protease triterpenoids from Stauntonia obovatifoliola Hayata subsp. intermedia

Three triterpenoids, 16beta-hydroxy-2,3-seco-lup-20(29)-ene-2,3-dioic acid (1), 3beta,21beta,24-trihydroxy-30-noroleana-12,20(29)-dien-28-oic acid (2) and 16beta-hydroxylupane-1,20(29)-dien-3-one (3), along with eleven known triterpenes were isolated from stems of Stauntonia obovatifoliola Hayata subsp. intermedia (Y.C. Wu) T. Chen. Their structures were determined by analysis of HR-EI/FAB-MS and 1D and 2D NMR spectroscopic data and comparison with those in the literature. Ten of the compounds showed inhibitory activity against HIV-1 protease.     

Antiviral potential of 4-hydroxypanduratin A,secondary metabolite of Fingerroot,Boesenbergia pandurata (Schult.), towards Japanese Encephalitis virus NS2B/NS3 protease

4-hydroxypanduratin A is a secondary metabolite of Boesenbergia pandurata Schult. (Fingerroot) plant with various pharmacological activities such as neuroprotective, potent antioxidant, antibacterial and antifungal. Flaviviral NS2B/NS3 protease activity is essential for polyprotein processing and viral replication for Japanese Encephalitis Virus (JEV), a major cause of Acute Encephaltis in Asia. Inhibition of formation of this complex by arresting the binding of NS2B with NS3 would reduce the enzyme''s activity to meager proportions and hence would prevent further viral proliferation. The automated 3D structure of NS2B protein of the JEV GP78 was predicted based on the sequence-to-structure-to-function paradigm using I-TASSER and the function of NS2B protein was inferred by matching to other known proteins. The stereochemical quality of predicted structure was checked by PROCHECK. The antiviral activity of 4-hydroxypanduratin A against NS2B protein as a potential drug has been elucidated in this paper. Docking simulation analysis showed 4-hydroxypanduratin A as potential inhibitor of NS2B protein/cofactor which is necessary for NS3 protease activity. 220 derivatives of 4-hydroxypanduratin A were virtually screened with rigid criteria of Lipinski''s rule of 5 using Autodock4.2. 4-hydroxypanduratin A was found interacting with target hydrophilic domain in NS2B protein by two Hbonds (Gly80 and Asp81) with active residues, several hydrophobic interactions, Log P value of 5.6, inhibition constant (Ki) of 51.07nM and lowest binding energy of -9.95Kcal/Mol. Hence, 4-hydroxypanduratin A targeted to Site 2 will have sufficient profound effect to inhibit protease activity to abrogate viral replication. It could be a promising potential drug candidate for JEV infections using NS2B Site 2 as a Drug target.     

Anti-HIV-1 activity of low molecular weight sulfated chitooligosaccharides

Chitooligosaccharides are nontoxic and water-soluble compounds obtained by enzymatic degradation of chitosan, which is derived from chitin by a deacetylation process. Chitooligosaccharides possess broad range of activities such as antitumour, antifungal, antibacterial activities. Sulfated chitooligosaccharides (SCOSs) with different molecular weights were synthesized by a random sulfation reaction. In the present study, anti-HIV-1 properties of SCOSs and the impact of molecular weight on their inhibitory activity were investigated. SCOS III (MW 3-5 kDa) was found to be the most effective compound to inhibit HIV-1 replication. At nontoxic concentrations, SCOS III exhibited remarkable inhibitory activities on HIV-1-induced syncytia formation (EC₅₀ 2.19 μg/ml), lytic effect (EC₅₀ 1.43 μg/ml), and p24 antigen production (EC₅₀ 4.33 μg/ml and 7.76 μg/ml for HIV-1_RF and HIV-1_Ba-L, respectively). In contrast, unsulfated chitooligosaccharides showed no activity against HIV-1. Furthermore, it was found that SCOS III blocked viral entry and virus-cell fusion probably via disrupting the binding of HIV-1 gp120 to CD4 cell surface receptor. These results suggest that sulfated chitooligosaccharides represent novel candidates for the development of anti-HIV-1 agent.     

Anti-HIV-1 integrase diterpenoids from Dichrocephala benthamii

Five new clerodane diterpenoid dichrocephnoids A–E (1,2, 6–8) together with three known analogues were isolated from the whole herb of Dichrocephala benthamii C.B. Clarke. Their structures were elucidated on the basis of spectroscopic analyses (UV, IR, MS, 1D and 2D NMR). The relative configurations of the new compounds were established by NOESY correlations, and the absolute configuration of 1 was determined on the basis of CD methods. Dichrocephnoid A (1) possesses a very rare pentacyclic ring system with highly oxygenated functionalities. All of the isolated compounds exhibited inhibitory activity against HIV-1 integrase, however, dichrocephnoid C (6) showed the most promising inhibition of the enzyme with IC₅₀ value of 12.35 ± 1.27 μM.     

Anti-HIV-1 activity of poly(mandelic acid) derivatives

Homo- and heterochiral poly(mandelic acid)s (PMDAs) were synthesized under strongly acidic, mildly acidic, and nonacidic conditions. The water-soluble fractions of these polymers were evaluated with respect to their inhibitory activity against the human immunodeficiency virus (HIV-1). Polymers were prepared via a step-growth mechanism, yielding linear polyesters. The polymers were characterized by CHS elemental microanalysis, X-ray fluorescence (XRF), and FT-IR spectroscopy. Polymers prepared by the three methods have different structures. Both elemental microanalysis and XRF indicated the presence of S in those polymers prepared by treatment with concentrated H2SO4, which were the only ones exhibiting inhibitory and virucidal activity against HIV-1, mediated by their binding to cellular co-receptor binding sites on the virus envelope glycoprotein gp120. Additionally, FT-IR spectroscopy indicated the complete absence of C=O functionality in the H2SO4-prepared PMDA.     

Anti-HIV-1 efficacy of extracts from medicinal plants

The anti-HIV-1 activities of butanol, hexane, chloroform and water extracts from four widely used folk medicinal plants (Sophora flavescens, Tulipa edulis, Herba ephedra, and Pachyma hoelen Rumph) were evaluated in this study. The hexane extract of Pachyma hoelen Rumph, PH-4, showed effective inhibition against HIV-1. The 50% effective concentration (EC₅₀) of PH-4 was 37.3 μg/ml in the p24 antigen assay and 36.8% in the HIV-1 recombinant RT activity test (at 200 μg/ml). In addition, the PH-4 showed the protective effect on the infected MT-4 cells, with a 58.2% rate of protection. The 50% cytotoxic concentration (CC₅₀) of PH-4 was 100.6 μg/ml. These results suggest that PH-4 from Pachyma hoelen Rumph might be the candidate for the chemotherapy agent against HIV-1 infection with further study.     

Anti-HIV-1 property of trichosanthin correlates with its ribosome inactivating activity

Trichosanthin (TCS) is a type I ribosome inactivating (RI) protein possessing anti-tumor and antiviral activity, including human immunodeficiency virus (HIV). The mechanism of these actions is not entirely clear, but is generally attributed to its RI property. In order to study the relationship between the anti-HIV-1 activity of TCS and its RI activity, three TCS mutants with different RI activities were constructed by using site-directed mutagenesis. The anti-HIV-1 activities of the three mutants were tested in vitro. Results showed that two TCS mutants, namely TCS(M(120-123)), TCS(E160A/E189A), with the greatest decrease in RI activity, lost almost all of the anti-HIV activity and cytopathic effect. Another mutant TCS(R122G), which exhibited a 160-fold decrease in RI activity, retained some anti-HIV activity. The results from this study suggested that RI activity of TCS may have significant contribution to its anti-HIV-1 property.     

Anti-HIV-1 phorbol esters from the seeds of Croton tiglium

Five phorbol diesters, together with three known ones, were isolated from a MeOH extract of the seeds of Croton tiglium, and their structures were determined by spectroscopic methods and selective hydrolysis of acyl groups. These compounds were assessed for their abilities to inhibit an HIV-induced cytopathic effect (CPE) on MT-4 cells and to activate protein kinase C (PKC) associated with tumor-promoting action. 12-O-Acetylphorbol-13-decanoate and 12-O-decanoylphorbol-13-(2-methylbutyrate) effectively inhibited the cytopathic effect of HIV-1 [complete inhibitory concentration (IC100) values of 7.6 ng/ml and 7.81 microg/ml, and minimum cytotoxic concentration (CC0) value of 62.5 and 31.3 microg/ml, respectively]; however, 12-O-acetylphorbol-13-decanoate showed no activation of PKC at concentrations of 10 and 100 ng/ml. 12-O-Tetradecanoylphorbol-13-acetate (TPA) was found to be not only the most potent inhibitor of HIV-1-induced CPE (IC100 value of 0.48 ng/ml), but also the most potent activator of PKC (100% activation at 10 ng/ml).     

Effectors of HIV-1 protease peptidolytic activity

High concentrations of salts dramatically affect the interaction of small ligands with HIV-1 protease. For instance, the Km and kcat values for Abz-Thr-Ile-Nle-p-nitro-Phe-Gln-Arg-NH2 (S) increased 120-fold and 3-fold, respectively, as the NaCl concentration in the assay decreased from 4.0 to 0.5 M. The Kd value for the competitive inhibitor amprenavir increased 12-fold over this concentration range of NaCl. The bimolecular rate constant for association of enzyme with amprenavir was independent of NaCl concentration, whereas the dissociation rate constant decreased with increasing NaCl concentration. Polyanionic polymers such as heparin or poly A substituted for NaCl. For example, the value of kcat/Km for S was 0.18 microM(-1) x s(-1) when the enzyme (<10 nM) was assayed in the standard buffer supplemented with 5 mM NaCl. If 0.01% poly A were included, the value of kcat/Km increased to 8.6 microM(-1) x s(-1). A DNA oligomer (23-mer) with an hexachlorofluoresceinyl moiety linked to the 5' end was studied as a model polyanionic polymer. The enzyme bound HF23 (Kd < 1 nM) with concomitant quenching of the hexachlorofluoresceinyl fluorescence. The stoichiometry for binding was 3 mol of enzyme per mol of oligomer. The hydrolytic activity of the enzyme with this oligomer was similar to that observed with poly A or high salt concentration when the molar ratio of oligomer to enzyme was greater than one. The results presented herein demonstrate that polyanionic polymers substitute for salts as effectors of HIV protease.     

Anti-HIV-1 activity of a new scorpion venom peptide derivative Kn2-7

For over 30 years, HIV/AIDS has wreaked havoc in the world. In the absence of an effective vaccine for HIV, development of new anti-HIV agents is urgently needed. We previously identified the antiviral activities of the scorpion-venom-peptide-derived mucroporin-M1 for three RNA viruses (measles viruses, SARS-CoV, and H5N1). In this investigation, a panel of scorpion venom peptides and their derivatives were designed and chosen for assessment of their anti-HIV activities. A new scorpion venom peptide derivative Kn2-7 was identified as the most potent anti-HIV-1 peptide by screening assays with an EC(50) value of 2.76 μg/ml (1.65 μM) and showed low cytotoxicity to host cells with a selective index (SI) of 13.93. Kn2-7 could inhibit all members of a standard reference panel of HIV-1 subtype B pseudotyped virus (PV) with CCR5-tropic and CXCR4-tropic NL4-3 PV strain. Furthermore, it also inhibited a CXCR4-tropic replication-competent strain of HIV-1 subtype B virus. Binding assay of Kn2-7 to HIV-1 PV by Octet Red system suggested the anti-HIV-1 activity was correlated with a direct interaction between Kn2-7 and HIV-1 envelope. These results demonstrated that peptide Kn2-7 could inhibit HIV-1 by direct interaction with viral particle and may become a promising candidate compound for further development of microbicide against HIV-1.     

Recombinant Der p 1 and Der f 1 exhibit cysteine protease activity but no serine protease activity

Although mite major group 1 allergens, Der p 1 and Der f 1, were first isolated as cysteine proteases, some studies reported that natural Der p 1 exhibits mixed cysteine and serine protease activity. Clarifying whether the serine protease activity originates from Der p 1 or is due to contamination is important for distinguishing between the pathogenic proteolytic activities of group 1 allergens and mite-derived serine proteases. Recombinant mite group 1 allergens would be useful tool for addressing this issue, because they are completely free from contamination by mite serine proteases. Recombinant Der p 1 and Der f 1, and highly purified natural forms exhibited only cysteine protease activity. However, commercially available natural forms exhibited both activities, but the two activities were eluted into different fractions in size-exclusion column chromatography. The substrate specificity associated with the serine protease activity was similar to that of Der f 3. These results indicate that the serine protease activity does not originate from group 1 allergens.     

Anti-HIV-1 activity of an antisense phosphorothioate oligonucleotide bearing imidazole and primary amine groups

We have previously shown that RNA cleaving reagents with imidazole and primary amine groups on the 5'-end of antisense oligodeoxyribonucleotides could site-specifically cleave CpA as the target sequence of the substrate tRNA in vitro. In this study, a RNA cleaving reagent, composed of imidazole and primary amine groups on an antisense phosphorothioate oligonucleotide (Im-anti-s-ODN), was synthesized and evaluated for anti-HIV-1 activity in MT-4 cells. The sequence of the Im-anti-s-ODN was designed to be complementary to the HIV-1 gag-mRNA and to bind adjacent to the CpA cleavage site position. Im-anti-s-ODN encapsulated with the transfection reagent, DMRIE-C, had higher anti-HIV-1 activity than the unmodified antisense phosphorothioate oligonucleotide (anti-s-ODN) at a 2 microM concentration. Furthermore, the Im-anti-ODN encapsulated with DMRIE-C conferred sequence-specific inhibition.     

Anti-HIV-1 activity and mode of action of mirror image oligodeoxynucleotide analogue of Zintevir

Zintevir is an oligonucleotide analogue, which has the phosphorothioate modification at both termini, that forms a K(+)-induced quadruplex structure and shows potent anti-human immunodeficiency virus (HIV)-1 activity. We synthesized the non-modified analogue (D-17mer) of Zintevir and its enantiomer (L-17mer), and compared their anti-HIV-1 activity and molecular mechanism of action. Although L-17mer forms the exact mirror image quadruplex structure of D-17mer, which has a very similar structure with Zintevir, L-17mer showed comparable anti-HIV-1 activity with Zintevir. The results obtained by the time-of-addition experiments and the immunofluorescence binding assay strongly suggest that the primary molecular target of L-17mer is the viral gp120 envelope protein as well as Zintevir, regardless of their reciprocal chirality.     

Inhibitory activity of cyclohexenyl chalcone derivatives and flavonoids of fingerroot, Boesenbergia rotunda (L.), towards dengue-2 virus NS3 protease

Boesenbergia rotunda (L.) cyclohexenyl chalcone derivatives, 4-hydroxypanduratin A and panduratin A, showed good competitive inhibitory activities towards dengue 2 virus NS3 protease with the Ki values of 21 and 25 microM, respectively, whilst those of pinostrobin and cardamonin were observed to be non-competitive. NMR and GCMS spectroscopic data formed the basis of assignment of structures of the six compounds isolated.