Screening And Identification Of Inhibitory Activity Of Triterpene And Glucose Compounds From Chinese Herbs On The Formation Of HIV Gp41 Six-helix Bundle

The process of HIV fusing with target cells,mediated by the HIV envelope glycoprotein subunit gp 120 and transmembrane subunit gp41,is an important step for drug intervention.The membrane fusion events leading to HIV entry into the target cells are initiated by binding of gp120 to CD4 receptor and subsequently to a coreceptor,CXCR4 or CCR5.Consequently,gp41 undergoes conformational changes, resulting in the fusion between the viral and cellular membranes or between the mernbrances of HIV-infected and uninfected cells.Therefore,gp120 and gp41 on the virions,and CD4 and coreceptors on the target cells may serve as targets for development of a new class of anti-HIV drugs,HIV entry inhibtors.OBJECTIVE:Triterpene compounds isolated from Rhoiptelea chiliantha Diels et Hand.-Mazz and glucose compounds isolated from Balanophora japonica Makino, were used as a compound library to screen and identify small molecular lead compounds which can effetively inhibit the formation of HIV gp41 core structure,the six-helix bundle structure.METHODS:Triterpene compounds was isolated and purified from Rhoiptelea chiliantha Diels et Hand.-Mazz,while glucose compounds were isolated and purified from Balanophora japonica Makino by n-hexane and ethyl acetate extraction and column chromatography.The inhibitory activity of those compounds on HIV gp41 six-helix bundle formation was screened by enzyme linked immunosorbent assay(ELISA) and futher identified by native-polyacrylamide gel electrophoresis (N-PAGE) and size exclusion-high performance liquid chromatography(SE-HPLC). The inhibitory activity of those compounds on HIV entry into the target cells was detected by a non-infectious cell-cell fusion assay.Peptide C34 derived from HIV gp41 CHR region and N36 from NHR region,can form a similar helix bundle in vitro like the six-helix bundle formed by CHR and NHR of gp41.Therefore,the inhibitory activity of HIV gp41 six-helix bundle can be determined by inhibiting the interaction of C34 with N36.RESULTS:We isolated five triterpene compounds with high purity from Rhoiptelea chiliantha Diels et Hand.-Mazz,named as myriceric acid B(R-3), myriceric acid C(DR-22B),myriceric acid B methyl ester(R3-Me),chilianthin A (R-1),chilianthin D(DR-22A).Firstly,we screened the compounds by ELISA based on the principle of antigen-antibody binding.From the five compounds,R-3, DR-22A,DR-22B had an obvious inhibitory activity on the formation of HIV gp41 six-helix bundle,with the IC₅₀ of 7.22±0.25μg/ml,3.26±0.41μg/ml and 4.01±0.54μg/ml,respectively.The positive control compounds,such as ADS-J1 and 90% theaflavin derivatives(90%TF) had the IC₅₀ of 3.54±0.20μg/ml and 5.00±0.12μg/ml,respectively.We then confirmed the inhibitory activity of the three triterpene compounds on the formation of six-helix bundle by using two biophysical methods, Native-polyacrylamide gel electrophoresis(N-PAGE) and Size exclusion-high performance liquid chromatography(SE-HPLC).Peptides could be separated by N-PAGE,according to the molecular weight and their electrical properties.The three terpenoids R-3,DR-22A,22B-DR were able to inhibit the formation of HIV gp41 six-helix bundle significantly.Therefore,it was furtherly proved that the three compounds have the inhibitory activities on HIV gp41 six-helix bundle formation, corresponding to the results of ELISA.The SE-HPLC method was used to detect the inhibitory activity of active triterpene compounds on the formation of HIV gp41 six-helix bundle based on the difference of peak area of the peak of six-helix bundle. The three terpenoids R-3,DR-22A,22B-DR could reduce the peak area of six-helix bundle significantly,corresponding to the former results of ELISA and N-PAGE. Combining together of these results,it showed that the three triterpene compounds could target HIV gp41 and inhibit the formation of six-helix bundle,and might play a role in blocking HIV entry into target cells.We also extracted nine purified glucose compounds from Balanophora japonica Makino,named 1-O-caffeoyl-β-D-glucopyranose(BJ-821),Coniferin (BJ-83),1-O-coumaroyl-β-D-glucopyranose(BJ-842),1,2-Di-O-caffeoyl-β-D-glucopyranose(BJ-862B4), 1,2,6-Tri-O-caffeoyl-β-D-glucopyranose(BJ-862D), 1,3-Di-O-caffeoyl-β-D-glucopyranose(BJ-851),1,3-Di-O-caffeoyl-4-O-galloyl-β-D-glucopyranose(BJ-8525), 1-O-caffeoyl-3-O-galloyl-β-D-glucose(BAT-17) and 1,2,6-Tri-O-galloyl-β-D-glucopyranose(1,2,6-Tri).Similar to above experiments,we used ELISA,N-PAGE and SE-HPLC to detect the inhibitory activity of these glucose compounds on the formation of HIV gp41 six-helix bundle.ELISA test showed that BJ-862D,BJ-8525,1,2,6-Tri had potent inhibitory activity on the formation of HIV gp41 six-helix bundle,with IC₅₀ of 2.00±0.35μg/ml,0.83±0.16μg/ml and 1.37±0.19μg/ml,respectively.N-PAGE and SE-HPLC confirmed furtherly that these compounds have inhibitory activities on the formation of six-helix bundle, corresponding to the results of ELISA.We also examined the inhibitory activity of 1,2,6-Tri on HIV envelope glycoprotein-mediated cell-cell fusion,and the results showed 1,2,6-Tri can inhibit the membrane fusion of HIV and target cells.CONCLUSION:We had screened several triterpene and glucose compounds from natural resouses on the effects of inhibiting the formation of HIV gp41 six-helix bundle.Some of these compounds showed inhibitory activities.Those compounds are rich in benzene ring and phenolic hydroxyl groups.Therefore,we hypnosized that these compounds could dock into the surface cavity of gp41 through their special structure.The benzene ring may bind to the hydrophobic conservative residues in the cavity,while the phenol hydroxyl groups may bind to the conservative positive residues K574 or R579 through their negative properties,thereby led to the inhibition of HIV gp41 six-helix bundle formation.The study shows a light on how to find HIV fusion inhibitors from Chinese herbs and other natural resourses.