AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 11, 1992 Mary Ann Liebert, Inc., Publishers
HIV-1 Reverse TAKAHIRO
OGATA,1 AKIHISA
Transcriptase Inhibitor from Phyllanthus niruri HIROTAKA HIGUCHI,1 SHUNJI MOCHIDA,1 HIDEKI MATSUMOTO,1 KATO,2 TOHRU ENDO,1 AKIRA KAJI,3 and HIDEKO KAJI2
ABSTRACT niruri (Euphorbiaceae) inhibited human immunodeficiency virus type-1 The inhibitor against HIV-l-RT in this plant was purified by combination of transcriptase (HIV-l-RT). three column chromatographies, Sephadex LH-20, cellulose, and reverse-phase high-performance liquid chromatography. The inhibitor was then identified by nuclear magnetic resonance (NMR) spectra as repandusinic acid A monosodium salt (RA) which was originally isolated from Mallotus répandus. The 50% inhibitory doses (IDS0) of RA on HIV-l-RT and DNA polymerase a (from HeLa cells) were 0.05 pvM and 0.6 p,M, respectively, representing approximately a 10-fold more sensitivity of HIV-l-RT compared with DNA polymerase a. RA was shown to be a competitive inhibitor with respect to the template-primer while it was a noncompetitive inhibitor with respect to the substrate. RA as low as 10.1 |jlM inhibited HIV-1-induced cytopathogenicity in MT-4 cells. In addition, 4.5 p,M of RA inhibited HIV-1-induced giant cell formation of SUP-T1 approximately 50%. RA (2.5 pM) inhibited up to 90% of HIV-1 specific p24 antigen production in a Clone H9 cell system. An aqueous extract of
Phyllanthus
reverse
significant inhibitory effect on the production of HIV-1 capsid protein in an in vitro culture system.
INTRODUCTION
to exert a
NIRURI (P. niruri; Euphorbiaceae) has traditionbeen used as a diuretic and also for the treatment of jaundice.1 It has been reported by several laboratories that this plant contains compounds such as lignans, flavonoids, triterpenoids, alkaloids, and tannins.2~7 Venkateswaran et al. reported that an aqueous extract of P. niruri inhibited an endogenous DNA polymerase of the hepatitis B virus (HBV-pol) and interaction of the surface antigen of hepatitis B virus with the antibody against this virus.8 Yanagi et al. also reported that an aqueous extract of this plant inhibited the avian myeloblastosis virus reverse transcriptase (AMV-RT), HBV-po/ and, other DNA-dependent DNA polymerases.9 However, active components responsible for these activities have not yet been identified. We report that an aqueous extract of P. niruri inhibited the human immunodeficiency virus type-1 RT (HIV-l-RT). One of the active compounds in this extract was identified as repandusinic acid A monosodium salt (RA)10 which was found
Phyllanthus ally
MATERIALS
Phyllanthus niruri was collected in Taiwan in January 1989 and kept dry until used. The plasmid coding HIV-1 -RT gene was kindly provided by Dr. Hughes (National Cancer InstituteFrederick Cancer Research Facility). Rous-associated virus 2 reverse transcriptase (RAV-2-RT) was purchased from Takara Shuzo. DNA polymerase I (DNA-pol I) minimal nuclease (Escherichia coli CM5197), poly(rA)-pdT,2_18, activated calf thymus DNA, yeast RNA, unlabeled deoxynucleoside triphosphates, and Sephadex LH-20 were obtained from Pharmacia. [3H]thymidine triphosphate was obtained from Du Pont. Foscarnet (trisodium phosphonoformate) was kindly provided by Dr. Öberg (Astra Alab AB Research and Development Laboratories Antiviral Chemotherapy). 3'-Azido-3'-deoxythymidine (AZT) and dextran sulfate (Av. Mol. Wt. —8000, DXS) was purchased
'Research Institute for Molecular Genetics. Tsumura & Co., 3586 Yoshiwara, Ami-Machi, Inashiki-Gun, Ibaraki-Ken 300-11, Japan.
2Department of Pharmacology, Jefferson Medical College, Philadelphia, PA 19107. 'Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, PA 1937
19104-6076.
1938
OGATA ET AL.
from Sigma. Cellulose (Avicel) was obtained from Asahi-kasei. 'H- and l3C-nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AM-500. Fast atom bombardment mass spectra (FAB-MS) were measured by a JEOL DX-300 mass
spectrometer.
MgCl2, 2 mM dithiothreitol (DTT), 0.25 pg of poly(rA) pdT,2_18, 10p.M [3H]dTTP(30GBq/mmol), and 10 p.1 aliquots of the crude HIV-l-RT (supernatant of bacterial lysates prepared as described below) or 2.5 p.1 of purified HIV-l-RT. Test compounds were dissolved in H20, and the 8 mM
•
The dried powder of P. niruri (40 g) was extracted with 400 ml of H20 at 60°C for 2 h followed by filtration with Whatman 3MM filter paper. The filtrate was evaporated under reduced pressure to yield 6.6 g of a brown powder. This powder was suspended in 100 ml of MeOH to obtain 3 g of a soluble portion and 2.5 g of an insoluble portion. Part (1.6 g) of the MeOH insoluble portion was dissolved in H20 and loaded on a Sephadex LH-20 column (33 x 260 mm). The column was eluted with 400 ml of H20 and each 40 ml fraction was collected (Fraction 1-10), followed by elution with 200 ml of MeOH (Fraction 11). Fractions 4-10 and Fraction 11 were combined (158 mg) and further chromatographed on a cellulose column with 2% acetic acid as the elution buffer. Each fraction (2 ml) was collected and pooled on the basis of thin layer chromatography (TLC) pattern of ultraviolet (UV)-absorbing material. Fractions 2-4 were combined (19.4 mg) and further subjected to a high-performance liquid chromatography (HPLC) on YMC AM-312 (C|8) with a linear gradient of solution A (10 mM sodium dihydrogen phosphate) and solution B (acetonitrile). The gradient condition was 3% B at 0 min and 43% B at 20 min. The eluates were desalted with Sephadex LH-20 and 3.6 mg of amorphous powder was obtained. This powder was treated with IN HC1 at 25°C for 1 min and subjected to Sephadex LH-20 column chromatography with MeOH. The resulting pure compound which gave single peak on a HPLC was subjected to 'H-NMR, l3C-NMR, and FAB-MS spectra.
solution was added to the reaction mixture before the addition of the enzyme. For a negative control, only H20 was added to the reaction mixture. The reaction mixture was incubated at 37°C for 30 min, and the reaction was stopped by cooling to 0CC, followed by the addition of 20 p.1 of 0.1 M EDTA (pH 8.0). After incubation, 20 pi of the reaction mixture was placed on DEAE ion exchange filter paper. The paper was treated three times with 5% disodium hydrogen phosphate solution and washed with H20 and 70% ethanol, followed by air drying. Radioactivity was counted with a Beckman scintillation counter. In some cases, a ß-plate counting system (Pharmacia) was used. The crude HIV-l-RT was prepared as described by Hizi et al. '4 Briefly, E. coli DH5 harboring the plasmid containing the HIV-1 -RT gene was grown at 37°C for 23 h. Bacterial cells were collected by centrifugation and disrupted with a lysis buffer, containing Triton X-100. The bacterial debris was removed by centrifugation and the supernatant was used as the crude HIVl-RT. HIV-l-RT was purified from this crude preparation by DEAE sephacel, phosphocellulose, and heparin sephalose chromatographies essentially according to the procedure reported by Weber and Grosse. The reaction mixture (50 pi) for RAV-2-RT assay contained 50 mM Tris HC1 (pH 8.3), 50 mM KC1, 10 mM MgCl2, 3 mM DTT, 0.5 pg of poly(rA) pdT12_18, 10 pJvl [3H]dTTP (30 GBq/mmol), and 0.1 units of RAV-2-RT. The reaction mixture was incubated at 37°C for 30 min and treated as described in the HIV-l-RT assay. DNA-poll activity was measured essentially according to the method reported by Hanajima et al.16 Briefly, the reaction mixture (50 p.1) for the DNA polymerase assay contained 40 mM glycine NaOH (pH 9.2), 5 mM MgCl2, 0.7 mM DTT, 1.25 p,g of activated calf thymus DNA, 5 pg of yeast RNA, 20 u,M dATP, dGTP, dCTP, 20 pJvl [3H]dTTP (30 GBq/mmol), and 0.625 units of E. coli DNA-poll. The reaction mixture was incubated at 37°C for 30 min and treated as described in the HIV-l-RT assay. DNA polymerase a (DNA-pol a) was partially purified from cultured human HeLa cells as previously described.17 Briefly, HeLa cells were maintained at 37°C in suspension culture in Eagle's minimal essential medium supplemented with 10% fetal calf serum. The harvested cells were sonicated and removed by centrifugation. The resulting supernatant was applied to a DE-52 (Whatman) and partially purified DNA-pol a was obtained. The reaction mixture (50 p.1) for the assay of this enzyme contained 50 mM Tris HC1 (pH 7.5), 4 mM MgCl2, 5 mM DTT, 15% (v/v) glycerol, 4 pg of activated calf thymus DNA, 10 p.M dATP, dGTP, dCTP, 10 pM [3H]dTTP (30 GBq/mmol), and 2 pi aliquots (0.3 units) of the partially purified DNA-pol a. The reaction mixture was incubated at 37°C for 30 min and treated as described in the HIV-l-RT assay.
Enzyme assays
Anti-HIV-1 activities and
Cells and viruses
HumanCD4+ T-cell lines, MT-4cells," Sup-Tl cells,'2 and Clone H9(H9) cells'3 were used for the anti-HIV-1 assay. MT-4 cells were kindly provided by Dr. J.A. Levy (University of California, San Francisco, CA), Sup-Tl cells were kindly provided by Dr. F. Gonzalez and J.A. Hoxie (University of Pennsylvania, Philadelphia, PA), and H9 cells were kindly provided by Dr. H. Mitsuya of the National Cancer Institute. These cells were grown in 25 mM HEPES buffered RPMI-1640 (GIBCO) and supplemented with 10% fetal calf serum, 50 U/ml penicillin, and 50 pg/ml streptomycin. HIV-1 (strain HTLVIIIB) was prepared from a culture supernatant of H9/HTLV-IIIB cells (American Type Culture Collection).13 After removal of the cells by centrifugation (3000 xg,5 min), the virus-containing supernatant was stored at 70°C until used. The virus titerof the supernatant was 104 TCID50 (50% tissue culture infectious dose) on MT-4 cells. —
METHODS
•
Isolation and
identification of RA
performed according to the method The reaction mixture (50 pi) for HIVl-RT assay contained 25 mM Tris HC1 (pH 8.0), 75 mM KC1, HIV-l-RT assay
was
previously reported.14
•
•
cytotoxic activities of RA The procedure for measuring anti-HIV-1 activity in MT-4 cells has been described18 and was performed with slight modifications; MT-4 cells were suspended at 4 x 105/ml in
HIV-l-RT INHIBITOR FROM PHYLLANTHUS NIRURI
1939
RPMI-1640 media (without Phenol Red) and subsequently 100 pi portion of this cell suspension was seeded into each well of a 96-well microplate containing 50 pi of various concentrations of RA. Cells were cultured in the presence or absence of 50 p,l of solution containing HIV-1, adjusted at 20 TCID5O/50 pi in each well. Negative controls as color blank wells which contained various concentrations of RA without cells were also prepared. The microplate was incubated for 5 days at 37°C in a 5% C02 atmosphere. Viable cells were determined by the MTT [3-
the MeOH-insoluble portion. These active fractions were combined and rechromatographed with cellulose column. Each eluted fraction was combined to five fractions which exhibited similar TLC pattern of UV absorbing materials. The yields and inhibitory effects of these fractions are shown in Table 1. Fraction 3 gave several distinct peaks upon reverse phase HPLC as indicated in Figure 1. Major peaks (peaks 1 and 2) were isolated and these structures were examined. Peak 2 was not a very strong inhibitor (ID50:60 p,g/ml) and was identified as brevifolin carboxylic acid by comparing of 'H-NMR, 13CNMR, and FAB-MS data with that of the reported values of this compound. This compound has been reported to have inhibitory activity against rat lens aldose reductase.20 In contrast to peak 2, peak 1 exhibited a very strong HIV-l-RT inhibitory activity (ID5O:0.3 pg/ml). As shown in Table 1, altogether a 167-fold purification of the inhibitory substance was achieved by these procedures. The pure material was desalted with hydrochloric acid to obtain free acid form. This purified free acid was subjected to 'H-NMR and 13C-NMR analysis as shown in Figure 2. These data were in good agreement with the corresponding 'H-NMR and l3C-NMR values of repandusinic acid A.10 In addition, the non-desalting compound was subjected to positive and negative FAB-MS and the values [positive FAB-MS m/z: 993 (M + H) + negative FAB-MS m/z: 969 (M-Na)-, 991 (M-H)~J were obtained which correspond to the monosodium salt of repandusinic acid A.
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method, as previously reported.1819 Experiments with Sup-Tl cells and H9 cells were performed in an identical fashion to the experiments with MT-4 cells. The anti-HIV-1 activity with Sup-Tl cells was determined by counting multinuclear-giant cells on the fourth day after viral infection. On the H9 cells, the viral antigen (p24) was determined in the culture supernatant on the fifth day using the enzyme immunoassay specific for HIV-1 (Coulter Co., Hialeah, FL).
RESULTS
Identification of the inhibitory substance
as
RA
,
An aqueous extract of P. niruri and its fractions were evaluated for the inhibitory effects on HIV-l-RT. These results are summarized in Table 1. The inhibitory activity was purified as follows. An aqueous extract (ID5O:50 pg/ml) was partitioned into MeOH-soluble and -insoluble portions. The MeOH-soluble portion did not inhibit HIV-l-RT at concentrations up to 100 pg/ml. The activity was found in the MeOH-insoluble portion (ID5O:20 pg/ml) with 95% recovery. The MeOH-insoluble portion was subjected to Sephadex LH-20 and the components were separated on the basis of molecular weights. Of the MeOH-insoluble material, 85% was recovered in Fraction 1-3 (1322 mg) corresponding to 1 bed volume. In contrast, most of the inhibitory activity was found in Fractions 4-10 (ID5O:3.0 p,g/ml) and Fraction 11 (ID50:3.6 pg/ml). The specific activities of Fractions 4-10 and 11 were about 6-fold higher than that of
The
MeOH insoluble Sephadex LH-20 Fr. 4-1 Ie Cellulose Fr. 1 Fr. 2 Fr. 3 Fr. 4 Fr. 5 RA a
Yield
(mg) 6600 2500 247
189 24 18 9 14 5.9
on
HIV-l-RT
The structure of the free acid of RA is shown in Figure 3. RA possesses three ester groups (galloyl, hexahydroxydiphenoyl and dehydrochebloyl) linked to the glucose moiety. The components of RA, ellagic acid, and gallic acid, were therefore evaluated for inhibitory effects on HIV-l-RT. As shown in Table 2, gallic acid did not inhibit HIV-l-RT at concentrations up to 588 pM, while ellagic acid inhibited HIV-1 -RT (ID50:11.6 pM). The inhibitory activity of ellagic acid was about 1/77 of
Table 1. Purification
Purification procedure
comparison of inhibitory effects of RA, gallic
acid, and ellagic acid
of
RA
from
P.
niruri
activity
IDso"
Specific activity
50 20 3.0-3.6
4 10 56-67
2640 2500 1616
7.8 5.0 2.4 3.4 1.8 0.3
26 40 83 58 111 668
484 96 150 52 156 394
(\iglml)
(xlO2 IU/mg)
Total
(X103IU)b
ID50 indicates the effectiveness of inhibitors expressed as concentrations which cause 50% inhibition of HIV-l-RT. Crude HIV-l-RT was used in this experiment. b IU are arbitrary inhibitory activity units obtained by dividing the total weight of the fraction at each step by the weight of each fraction required to achieve 50% inhibition of [3H]dTTP into the polymer in the HIV-l-RT assay. incorporation c Fractions 4-10 and Fraction 11 were combined because both fractions had the inhibitory activity.
OGATA ET AL.
1940 0.64
galloyl-H
H-NMR
-,
hexahydroxydiphenoyl-H
peak 2
peak 1 O oo M M
(0
o C CO c o (0
1
3'
4,
3 6 5
UUUL
0.32 7.0
8.0
6.0
ä
5.0
4.0
PPM
HIV-1 Reverse TAKAHIRO
OGATA,1 AKIHISA
Transcriptase Inhibitor from Phyllanthus niruri HIROTAKA HIGUCHI,1 SHUNJI MOCHIDA,1 HIDEKI MATSUMOTO,1 KATO,2 TOHRU ENDO,1 AKIRA KAJI,3 and HIDEKO KAJI2
ABSTRACT niruri (Euphorbiaceae) inhibited human immunodeficiency virus type-1 The inhibitor against HIV-l-RT in this plant was purified by combination of transcriptase (HIV-l-RT). three column chromatographies, Sephadex LH-20, cellulose, and reverse-phase high-performance liquid chromatography. The inhibitor was then identified by nuclear magnetic resonance (NMR) spectra as repandusinic acid A monosodium salt (RA) which was originally isolated from Mallotus répandus. The 50% inhibitory doses (IDS0) of RA on HIV-l-RT and DNA polymerase a (from HeLa cells) were 0.05 pvM and 0.6 p,M, respectively, representing approximately a 10-fold more sensitivity of HIV-l-RT compared with DNA polymerase a. RA was shown to be a competitive inhibitor with respect to the template-primer while it was a noncompetitive inhibitor with respect to the substrate. RA as low as 10.1 |jlM inhibited HIV-1-induced cytopathogenicity in MT-4 cells. In addition, 4.5 p,M of RA inhibited HIV-1-induced giant cell formation of SUP-T1 approximately 50%. RA (2.5 pM) inhibited up to 90% of HIV-1 specific p24 antigen production in a Clone H9 cell system. An aqueous extract of
Phyllanthus
reverse
significant inhibitory effect on the production of HIV-1 capsid protein in an in vitro culture system.
INTRODUCTION
to exert a
NIRURI (P. niruri; Euphorbiaceae) has traditionbeen used as a diuretic and also for the treatment of jaundice.1 It has been reported by several laboratories that this plant contains compounds such as lignans, flavonoids, triterpenoids, alkaloids, and tannins.2~7 Venkateswaran et al. reported that an aqueous extract of P. niruri inhibited an endogenous DNA polymerase of the hepatitis B virus (HBV-pol) and interaction of the surface antigen of hepatitis B virus with the antibody against this virus.8 Yanagi et al. also reported that an aqueous extract of this plant inhibited the avian myeloblastosis virus reverse transcriptase (AMV-RT), HBV-po/ and, other DNA-dependent DNA polymerases.9 However, active components responsible for these activities have not yet been identified. We report that an aqueous extract of P. niruri inhibited the human immunodeficiency virus type-1 RT (HIV-l-RT). One of the active compounds in this extract was identified as repandusinic acid A monosodium salt (RA)10 which was found
Phyllanthus ally
MATERIALS
Phyllanthus niruri was collected in Taiwan in January 1989 and kept dry until used. The plasmid coding HIV-1 -RT gene was kindly provided by Dr. Hughes (National Cancer InstituteFrederick Cancer Research Facility). Rous-associated virus 2 reverse transcriptase (RAV-2-RT) was purchased from Takara Shuzo. DNA polymerase I (DNA-pol I) minimal nuclease (Escherichia coli CM5197), poly(rA)-pdT,2_18, activated calf thymus DNA, yeast RNA, unlabeled deoxynucleoside triphosphates, and Sephadex LH-20 were obtained from Pharmacia. [3H]thymidine triphosphate was obtained from Du Pont. Foscarnet (trisodium phosphonoformate) was kindly provided by Dr. Öberg (Astra Alab AB Research and Development Laboratories Antiviral Chemotherapy). 3'-Azido-3'-deoxythymidine (AZT) and dextran sulfate (Av. Mol. Wt. —8000, DXS) was purchased
'Research Institute for Molecular Genetics. Tsumura & Co., 3586 Yoshiwara, Ami-Machi, Inashiki-Gun, Ibaraki-Ken 300-11, Japan.
2Department of Pharmacology, Jefferson Medical College, Philadelphia, PA 19107. 'Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, PA 1937
19104-6076.
1938
OGATA ET AL.
from Sigma. Cellulose (Avicel) was obtained from Asahi-kasei. 'H- and l3C-nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AM-500. Fast atom bombardment mass spectra (FAB-MS) were measured by a JEOL DX-300 mass
spectrometer.
MgCl2, 2 mM dithiothreitol (DTT), 0.25 pg of poly(rA) pdT,2_18, 10p.M [3H]dTTP(30GBq/mmol), and 10 p.1 aliquots of the crude HIV-l-RT (supernatant of bacterial lysates prepared as described below) or 2.5 p.1 of purified HIV-l-RT. Test compounds were dissolved in H20, and the 8 mM
•
The dried powder of P. niruri (40 g) was extracted with 400 ml of H20 at 60°C for 2 h followed by filtration with Whatman 3MM filter paper. The filtrate was evaporated under reduced pressure to yield 6.6 g of a brown powder. This powder was suspended in 100 ml of MeOH to obtain 3 g of a soluble portion and 2.5 g of an insoluble portion. Part (1.6 g) of the MeOH insoluble portion was dissolved in H20 and loaded on a Sephadex LH-20 column (33 x 260 mm). The column was eluted with 400 ml of H20 and each 40 ml fraction was collected (Fraction 1-10), followed by elution with 200 ml of MeOH (Fraction 11). Fractions 4-10 and Fraction 11 were combined (158 mg) and further chromatographed on a cellulose column with 2% acetic acid as the elution buffer. Each fraction (2 ml) was collected and pooled on the basis of thin layer chromatography (TLC) pattern of ultraviolet (UV)-absorbing material. Fractions 2-4 were combined (19.4 mg) and further subjected to a high-performance liquid chromatography (HPLC) on YMC AM-312 (C|8) with a linear gradient of solution A (10 mM sodium dihydrogen phosphate) and solution B (acetonitrile). The gradient condition was 3% B at 0 min and 43% B at 20 min. The eluates were desalted with Sephadex LH-20 and 3.6 mg of amorphous powder was obtained. This powder was treated with IN HC1 at 25°C for 1 min and subjected to Sephadex LH-20 column chromatography with MeOH. The resulting pure compound which gave single peak on a HPLC was subjected to 'H-NMR, l3C-NMR, and FAB-MS spectra.
solution was added to the reaction mixture before the addition of the enzyme. For a negative control, only H20 was added to the reaction mixture. The reaction mixture was incubated at 37°C for 30 min, and the reaction was stopped by cooling to 0CC, followed by the addition of 20 p.1 of 0.1 M EDTA (pH 8.0). After incubation, 20 pi of the reaction mixture was placed on DEAE ion exchange filter paper. The paper was treated three times with 5% disodium hydrogen phosphate solution and washed with H20 and 70% ethanol, followed by air drying. Radioactivity was counted with a Beckman scintillation counter. In some cases, a ß-plate counting system (Pharmacia) was used. The crude HIV-l-RT was prepared as described by Hizi et al. '4 Briefly, E. coli DH5 harboring the plasmid containing the HIV-1 -RT gene was grown at 37°C for 23 h. Bacterial cells were collected by centrifugation and disrupted with a lysis buffer, containing Triton X-100. The bacterial debris was removed by centrifugation and the supernatant was used as the crude HIVl-RT. HIV-l-RT was purified from this crude preparation by DEAE sephacel, phosphocellulose, and heparin sephalose chromatographies essentially according to the procedure reported by Weber and Grosse. The reaction mixture (50 pi) for RAV-2-RT assay contained 50 mM Tris HC1 (pH 8.3), 50 mM KC1, 10 mM MgCl2, 3 mM DTT, 0.5 pg of poly(rA) pdT12_18, 10 pJvl [3H]dTTP (30 GBq/mmol), and 0.1 units of RAV-2-RT. The reaction mixture was incubated at 37°C for 30 min and treated as described in the HIV-l-RT assay. DNA-poll activity was measured essentially according to the method reported by Hanajima et al.16 Briefly, the reaction mixture (50 p.1) for the DNA polymerase assay contained 40 mM glycine NaOH (pH 9.2), 5 mM MgCl2, 0.7 mM DTT, 1.25 p,g of activated calf thymus DNA, 5 pg of yeast RNA, 20 u,M dATP, dGTP, dCTP, 20 pJvl [3H]dTTP (30 GBq/mmol), and 0.625 units of E. coli DNA-poll. The reaction mixture was incubated at 37°C for 30 min and treated as described in the HIV-l-RT assay. DNA polymerase a (DNA-pol a) was partially purified from cultured human HeLa cells as previously described.17 Briefly, HeLa cells were maintained at 37°C in suspension culture in Eagle's minimal essential medium supplemented with 10% fetal calf serum. The harvested cells were sonicated and removed by centrifugation. The resulting supernatant was applied to a DE-52 (Whatman) and partially purified DNA-pol a was obtained. The reaction mixture (50 p.1) for the assay of this enzyme contained 50 mM Tris HC1 (pH 7.5), 4 mM MgCl2, 5 mM DTT, 15% (v/v) glycerol, 4 pg of activated calf thymus DNA, 10 p.M dATP, dGTP, dCTP, 10 pM [3H]dTTP (30 GBq/mmol), and 2 pi aliquots (0.3 units) of the partially purified DNA-pol a. The reaction mixture was incubated at 37°C for 30 min and treated as described in the HIV-l-RT assay.
Enzyme assays
Anti-HIV-1 activities and
Cells and viruses
HumanCD4+ T-cell lines, MT-4cells," Sup-Tl cells,'2 and Clone H9(H9) cells'3 were used for the anti-HIV-1 assay. MT-4 cells were kindly provided by Dr. J.A. Levy (University of California, San Francisco, CA), Sup-Tl cells were kindly provided by Dr. F. Gonzalez and J.A. Hoxie (University of Pennsylvania, Philadelphia, PA), and H9 cells were kindly provided by Dr. H. Mitsuya of the National Cancer Institute. These cells were grown in 25 mM HEPES buffered RPMI-1640 (GIBCO) and supplemented with 10% fetal calf serum, 50 U/ml penicillin, and 50 pg/ml streptomycin. HIV-1 (strain HTLVIIIB) was prepared from a culture supernatant of H9/HTLV-IIIB cells (American Type Culture Collection).13 After removal of the cells by centrifugation (3000 xg,5 min), the virus-containing supernatant was stored at 70°C until used. The virus titerof the supernatant was 104 TCID50 (50% tissue culture infectious dose) on MT-4 cells. —
METHODS
•
Isolation and
identification of RA
performed according to the method The reaction mixture (50 pi) for HIVl-RT assay contained 25 mM Tris HC1 (pH 8.0), 75 mM KC1, HIV-l-RT assay
was
previously reported.14
•
•
cytotoxic activities of RA The procedure for measuring anti-HIV-1 activity in MT-4 cells has been described18 and was performed with slight modifications; MT-4 cells were suspended at 4 x 105/ml in
HIV-l-RT INHIBITOR FROM PHYLLANTHUS NIRURI
1939
RPMI-1640 media (without Phenol Red) and subsequently 100 pi portion of this cell suspension was seeded into each well of a 96-well microplate containing 50 pi of various concentrations of RA. Cells were cultured in the presence or absence of 50 p,l of solution containing HIV-1, adjusted at 20 TCID5O/50 pi in each well. Negative controls as color blank wells which contained various concentrations of RA without cells were also prepared. The microplate was incubated for 5 days at 37°C in a 5% C02 atmosphere. Viable cells were determined by the MTT [3-
the MeOH-insoluble portion. These active fractions were combined and rechromatographed with cellulose column. Each eluted fraction was combined to five fractions which exhibited similar TLC pattern of UV absorbing materials. The yields and inhibitory effects of these fractions are shown in Table 1. Fraction 3 gave several distinct peaks upon reverse phase HPLC as indicated in Figure 1. Major peaks (peaks 1 and 2) were isolated and these structures were examined. Peak 2 was not a very strong inhibitor (ID50:60 p,g/ml) and was identified as brevifolin carboxylic acid by comparing of 'H-NMR, 13CNMR, and FAB-MS data with that of the reported values of this compound. This compound has been reported to have inhibitory activity against rat lens aldose reductase.20 In contrast to peak 2, peak 1 exhibited a very strong HIV-l-RT inhibitory activity (ID5O:0.3 pg/ml). As shown in Table 1, altogether a 167-fold purification of the inhibitory substance was achieved by these procedures. The pure material was desalted with hydrochloric acid to obtain free acid form. This purified free acid was subjected to 'H-NMR and 13C-NMR analysis as shown in Figure 2. These data were in good agreement with the corresponding 'H-NMR and l3C-NMR values of repandusinic acid A.10 In addition, the non-desalting compound was subjected to positive and negative FAB-MS and the values [positive FAB-MS m/z: 993 (M + H) + negative FAB-MS m/z: 969 (M-Na)-, 991 (M-H)~J were obtained which correspond to the monosodium salt of repandusinic acid A.
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method, as previously reported.1819 Experiments with Sup-Tl cells and H9 cells were performed in an identical fashion to the experiments with MT-4 cells. The anti-HIV-1 activity with Sup-Tl cells was determined by counting multinuclear-giant cells on the fourth day after viral infection. On the H9 cells, the viral antigen (p24) was determined in the culture supernatant on the fifth day using the enzyme immunoassay specific for HIV-1 (Coulter Co., Hialeah, FL).
RESULTS
Identification of the inhibitory substance
as
RA
,
An aqueous extract of P. niruri and its fractions were evaluated for the inhibitory effects on HIV-l-RT. These results are summarized in Table 1. The inhibitory activity was purified as follows. An aqueous extract (ID5O:50 pg/ml) was partitioned into MeOH-soluble and -insoluble portions. The MeOH-soluble portion did not inhibit HIV-l-RT at concentrations up to 100 pg/ml. The activity was found in the MeOH-insoluble portion (ID5O:20 pg/ml) with 95% recovery. The MeOH-insoluble portion was subjected to Sephadex LH-20 and the components were separated on the basis of molecular weights. Of the MeOH-insoluble material, 85% was recovered in Fraction 1-3 (1322 mg) corresponding to 1 bed volume. In contrast, most of the inhibitory activity was found in Fractions 4-10 (ID5O:3.0 p,g/ml) and Fraction 11 (ID50:3.6 pg/ml). The specific activities of Fractions 4-10 and 11 were about 6-fold higher than that of
The
MeOH insoluble Sephadex LH-20 Fr. 4-1 Ie Cellulose Fr. 1 Fr. 2 Fr. 3 Fr. 4 Fr. 5 RA a
Yield
(mg) 6600 2500 247
189 24 18 9 14 5.9
on
HIV-l-RT
The structure of the free acid of RA is shown in Figure 3. RA possesses three ester groups (galloyl, hexahydroxydiphenoyl and dehydrochebloyl) linked to the glucose moiety. The components of RA, ellagic acid, and gallic acid, were therefore evaluated for inhibitory effects on HIV-l-RT. As shown in Table 2, gallic acid did not inhibit HIV-l-RT at concentrations up to 588 pM, while ellagic acid inhibited HIV-1 -RT (ID50:11.6 pM). The inhibitory activity of ellagic acid was about 1/77 of
Table 1. Purification
Purification procedure
comparison of inhibitory effects of RA, gallic
acid, and ellagic acid
of
RA
from
P.
niruri
activity
IDso"
Specific activity
50 20 3.0-3.6
4 10 56-67
2640 2500 1616
7.8 5.0 2.4 3.4 1.8 0.3
26 40 83 58 111 668
484 96 150 52 156 394
(\iglml)
(xlO2 IU/mg)
Total
(X103IU)b
ID50 indicates the effectiveness of inhibitors expressed as concentrations which cause 50% inhibition of HIV-l-RT. Crude HIV-l-RT was used in this experiment. b IU are arbitrary inhibitory activity units obtained by dividing the total weight of the fraction at each step by the weight of each fraction required to achieve 50% inhibition of [3H]dTTP into the polymer in the HIV-l-RT assay. incorporation c Fractions 4-10 and Fraction 11 were combined because both fractions had the inhibitory activity.
OGATA ET AL.
1940 0.64
galloyl-H
H-NMR
-,
hexahydroxydiphenoyl-H
peak 2
peak 1 O oo M M
(0
o C CO c o (0
1
3'
4,
3 6 5
UUUL
0.32 7.0
8.0
6.0
ä
5.0
4.0
PPM
