Академический Документы
Профессиональный Документы
Культура Документы
Syamsurizal1), Satoru Tamura2), Mohammad Mostaqul Huq2), Masafumi Kaneko2), Nobutoshi Murakami2) 1) Jurusan Pendidikan MIPA, FKIP Universitas Jambi, Jambi 2) Graduate School of Pharmaceutical Sciences, Osaka University,Osaka e-mail: syamsurizal@unja.ac.id Received 20 September 2006, accepted for publication 22 June 2007
Abstract Isoquercitrin (1), a flavonol monoglycoside showed a strong antimalarial activity. In order to identify the target protein in Plasmodium falciparum, a photoreactive isoquercitrin analog, 2-[2-[2-(biotinylaminoethoxy) ethoxy]-4[3-(trifluoromethyl)-3H-diazirin-3-yl] benzoic acid isoquercitrinyl ester (2) has been synthesized which closely mimics the action of 1. Photoaffinity labeling of 2 revealed the apparent molecular mass of ca 70 kDa on the uninfected erythrocyte membrane. Keywords: Isoquercitrin, Antimalarial, P. falciparum, Photoaffinity labeling, 70 kDa Protein Abstrak Isokuersitrin (1), suatu flavonoid monoglikosida, menunjukkan aktivitas kuat sebagai antimalaria. Untuk mengidentifikasi protein target pada Plasmodium falciparum, telah disintesis suatu turunan isokuersitrin yang fotoreaktif, 2-[2-[2-(biotinilaminoetoksi)etoksi]-4-[3-(trifluorometil)-3H-diazirin-3-il] benzoat isokuersitrinil ester (2) yang memiliki aktivitas menyerupai 1. Pelabelan fotoafinitas 2 berhasil mengidentifikasi protein dengan berat molekul ca 70 kDa pada membran sel darah merah yang tidak terinjeksi. Kata kunci: Isokuersitrin, Antimalaria, P. falciparum, Pelabelan fotoafinitas, Protein 70 kDa 1. Introduction parasite at a concentration of 1 M along with high selectivity index (Murakami et al., 2001). Nowadays, photoaffinity labeling has been used to elucidate target molecules and ligand binding regions. This method requires functional groups that can be activated photochemically to generate highly reactive intermediates, usually nitrenes and carbenes. Photoaffinity labels, such as those containing diazirine or azides, have been used to covalently modify molecules in a variety of biological experiments. Photolysis of phenyldiazirine affords a reactive carbene intermediate and that the insertion of the carbene establishes an irreversible, covalent cross-link to a variety of amino acid residues within the target protein. In contrast, recent evidence suggested that the photolysis of the phenyl azide category of probes generated a less reactive compound. It was expected, therefore, phenyldiazirines would result in more efficient cross-linking than their phenyl azide counterparts and would afford covalent adducts that would tolerate the subsequent digestion, purification, and sequencing process (Platz et al., 1991). To screen the target molecule or identify binding domains, radiochemical probes bearing 3H or 125 I were commonly used previously. However, detection techniques with fluorescence or chemiluminescence acquiring a similar sensitivity to radioligands 74
In the framework of screening for novel antimalarials from medicinal plants, a new flavonol oligoglycoside together with two known congeners (structure not shown) from Hydrangeae dulcis (Amacha in Japanese) were isolated. These compounds inhibit 60% of P. falciparum proliferation at low concentration of 0.5 g/mL without showing any toxicity against KB 3-1 cells, human epidermis carcinoma cells (Murakami et al., 2001). However, the flavonol oligoglycosides showed little dosedependency and were unable to kill the parasite completely (Murakami et al., 2001). Furthermore, several naturally occurring flavonol glycosides, namely rutin, isorhamnetin 3--O-rutinoside, multinoside A, multinoside A acetate, quercitrin, and isoquercitrin (1) as well as flavonols, quercetin, have been assessed for their antimalarial potency in order to evaluate the structure activity relationship. These naturally occurring flavonol glycosides also displayed antimalarial behavior similar to the flavonol oligoglycosides at the low concentration between 0.05 and 5 g/mL (Huq, 2003). On the contrary, the monoglycoside 1 was found to inhibit the proliferation of P. falciparum in a dose-dependent manner. It exhibited nearly complete growth inhibition of the
Syamsurizal et al., Photoaffinity Labeling Study for Identification the Target Protein 75
have been developed to date because of requirements for safe handling the tedious radio-chemical process (Bayley et al., 1983). Here, we report a photoaffinity labeling producing nonradioactive biotinylation to recognize the target protein of 1 as antimalarial. If protein involved in 1 action could be identified and characterized, a functional approach to design novel isoquercitrin analogs would become a possibility. Recently, a new photoaffinity ligand 2 was devised by Hatanaka et al. (2001) and successfully used in identification of binding domain on -1,4-galactosyl transferase (GalT). This tridentate ligand bears a biotin moiety for identifying a labeled protein and the protein can be isolated by affinity chromatography based on strong interaction of biotin with either avidin or streptavidin, a phenyldiazirinyl moiety as a carbene source for photoaffinity labeling, and a carboxylic group for linkage to ligands. The hydrophilic spacer introduced between biotin and phenyldiazirine improves solubility of a hydrophobic diazirine moiety into aqueous media which results in stronger interaction between avidin/streptavidin and photoaffinity labeled biotinylated proteins (Figure 1).
carbene source to bind target protein
N F 3C N
gel (Fuji Sylisia BW-200). Thin-layer chromatography (TLC) analysis was performed on precoated Kieselgel 60F254 plates (0.25 mm, Merck). The spots were monitored under UV light (254 or 365 nm) and visualized by spraying agents phosphomolibdic acid in ethanolic solution. Culture of P. falciparum strain CDC1 was cultivated in an incubator (Model-9200, Wakenyaku) at 37 oC under a gas-controlled environment of 5% O2 and 5% CO2. Detection of parasitemia was determined over microscopy (OLYMPUS BX51) observation under oil immersion. Quinine (Nacalai) was utilized as a positive control for in vitro antimalarial assay. D-PBS (Aldrich) was used to dilute saponin and to wash the pellet cell. Composition of lysis Buffer is 50 mM HEPES (pH 7.4), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM PMSF, 50 mM NaF, 10 mM Na4P2O7, 2 mM Na3VO4, 1% protease inhibitor cocktail (sigma). Biotinylated diazirine probe was prepared according to Hatanaka et al., (1994). 2.2 Preparation of biotinylated isoquercitrin probe (2) Solution of 1 (9.2 mg, 0.019 mmol) and a prepared biotinylated diazirin probe (11.3 mg, 0.019 mmol) in pyridine was mixed with EDCI.HCl (11.5 mg) and DMAP (1.1 mg). The mixture was stirred at room temperature for 3 hours in the dark. The solvent was evaporated in vacuo to dryness. The residue was chromatographed over SiO2 (BW-200, 5.5 g, CHCl3 : MeOH:H2O = 15:3:1) to furnish 2 (3.2 mg) in 45% yield. Compound 2: a pale yellow powder, 1H NMR (300 MHz, DMSO-d6) : 12.56 (1H, s), 8.06 (1H, d, J = 8.0 Hz), 7.97 (1H, brs), 7.78 (1H, t, J = 6.6 Hz,), 7.69 (1H, d, J = 9.0 Hz), 7.15 (1H, d, J = 8.0 Hz), 7.07 (1H, d, J = 9.0 Hz), 6.90 (1H, s), 6.43 (1H, brs), 6.41 (1H, brs), 6.35 (1H, s), 6.19 (1H, s), 5.49 (1H, d, J = 7.5 Hz), 5.38 (1H, m), 5.08 (1H, m), 4.93 (1H, m,), 4.26 (3H, m), 4.10 (2H, m), 3.76 (2H, m), 3.57 (2H, m), 3.50-3.47 (2H. m), 3.44-3.22 (8H, m, overlapped with H2O peak), 3.16 (2H, m), 3.12 (1H, m), 2.82 (1H, dd, J = 12.0, 5.4 Hz), 2.56 (1H, d, J = 12.0 Hz), 2.06 (2H, t, J = 8.1 Hz), 1.2-1.6 (6H, m). 2.3 Photolysis of (2) Solution of 2 (0.59 mol: 0.62 mg) in 1 mL methanol was placed in a glass petri dish (d, 5 cm) and irradiated for 20 minutes until all diazirine consumed with a 100 W longwave mercury spot lamp (Model B-100 A) at a distance of 5 cm from the surface of light source, afforded the OCH3 insertion product 3. Compound 3: 1H-NMR (500 MHz, DMSO-d6) : 7.82 (1H, brd, J = ca. 8.0 Hz, 2), 7.80 (1H, t like, J = 6.0 Hz,14b), 7.76 (1H, brs, 6), 7.51 (1H, d like, J = ca.8.0, Hz, 6a), 7.43 (1H, brd, J = 7.3 Hz, 5a), 7.21 (1H, brs, 3a), 7.14 (1H, d, J = 8.5 Hz, 3), 6.78 (1H, brs, 8),
spacer
*)
HN S O
NH
O O HO O O Glc OH O O OH
H N
for detection *) to gain better binding between streptaavidin and photobiotinylated product
isoquercitrin(ligand)
Figure 1. Structure of isoquercitrin probe (2). In this study, we have synthesized a photoreactive analog of isoquercitrin coupled with unit bearing phenyldiazirine and biotin, which was separately prepared according to the method described by Hatanaka et al. (1994) and used it to identify the target protein (Figure 1). 2. Methods
2.1 General The 1H-NMR spectra were measured with a JEOL GX-500 (1H: 500 MHz) using Me4Si (0 ppm) as internal reference. Chemical Shifts were reported in parts per million (ppm). FAB mass spectra was recorded on a JEOL SX-102, while ESI mass spectra with Waters 2695. All reactions were carried out under an argon atmosphere unless otherwise indicated. Column chromatography was conducted using silica
6.68 (1H, s, 6), 6.41 (1H, brs, 6b or 4b), 6.35 (1H, brs, 6b or 4b), ca. 5.40 (1H, d like, J = ca. 7.0 Hz, 1), 4.43 (1H, q like, J = 7.0 Hz, 7a), 4.30 (1H, brs, 7b), 4.28 (2H, t like, J = 4.3 Hz, 22b), ca.4.10 (1H, brs, 3b), 3.75 (2H, t, J = 4.0 Hz, 21b), 3.60 (2H, t like, J = 4.9 Hz, 18b or 19b), 3.49 (3H, s, 7a-OCH3), ca. 3.01 3.50 (6H, obscured, 2, 3, 4, 5, 6), ca.3.10 (1H, m, 8b), 2.80 (1H, dd, J = 12.8, 6.0 Hz, 2b), 2.57 (1H, d, J = 12.2 Hz, 2b), 2.04 (2H, t, J = 7.3 Hz, 12b), 1.20 1.60 (6H, m, 9b, 10b, 11b). 2.4 Photoaffinity labeling experiment Probe, 2 (6 L) was added from a stock in dimethyl sulfoxide (DMSO) to 600 L of uninfected red blood cell (RBC) or infected RBC with mature stage parasites and incubated for 3 hours in reduced light. The samples were then irradiated for 20 minutes with a 100 W long wave mercury spot lamp to induce photoactivation. The mixture was centrifugated at 3000 rpm for 5 minutes at 4 oC and washed with D-PBS. The pellet was suspended in three folds volume of D-PBS and four folds volume of 0.15% saponin and incubated in a shaking water bath for 10 min to allow lysis of the RBC membrane and release of the intact parasites. To isolate the proteins of P. falciparum, the parasite was lysed in lysis buffer at 0 oC by stirring vigorously and sonicated for 3 minutes. The lysate was centrifuged at 5000 rpm for 10 minutes at 4 oC. The supernatant was immobilized with streptavidin beads for 12 hours at 4 oC. The lysate samples were then subjected to sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) and photoaffinity labeled proteins were visualized by enhancer chemiluminescent (ECL) detection (Syamsurizal, 2006). For competition experiments, isoquercitrin (1) was added from a concentrated stock in DMSO and incubated with uninfected RBC for 1 h prior to addition of probe 2 in the range of concentration of 0, 6, 30, 150, 600 M. Afterwards 2, with final concentration of 30 M, was incorporated into each culture of RBC, and continued with incubation for 2 hours. Following photoactivation, samples were treated in the same manner as mentioned above and finally the band was visualized by ECL (Syamsurizal, 2006). 3. Results and Discussion Isoquercitrin (1) has been shown to be active against P. falciparum, and its antimalarial activity was reported for the first time by Murakami et al. (2001). This compound was also proven to arrest the proliferation of malaria parasite in the trophozoite stage and the parasite was unable to develop to the next stage even after 72 hours (Huq, 2006). Therefore, to identify the target protein, a photoreactive isoquercitrin analog, 2-[2-[2-(biotinylaminoethoxy)ethoxy]-4-[3- (trifluoro-
methyl)-3H-diazirin-3-yl] benzoic acid isoquercitrinyl ester (2) has been synthesized as a photoaffinity probe for isoquercitrin-interacting proteins both in uninfected and malaria-infected erythrocytes. Prior to perform the photoaffinity labeling by using 2, photolysis of 2 was undertaken to examine the photoreactivities of diazirine, whether it could be used as a probe to identify the target protein (Fuji et al., 2003). Photolysis of 2 was carried out for 20 min with a 100 W UV lamp until all diazirine was consumed, to furnish a single product dominantly. In the 1H-NMR spectrum of 2, the signals due to an arylmethyne proton [ 4.43 (q, J=7.0 Hz)] and a methoxyl group [3.49 (3H, s)] were newly observed. Furthermore, in the electrospray ionization mass spectrometry (ESI-MS) the peak at m/z 1053 [(M-H)-] was given which corresponds to [M-H]- ion designated for the OCH3 insertion product, 3. Based on these physicochemical properties, the chemical structure of 2 could be determined as shown in Figure 2. This result indicated that probe 2 could bind with the target proteins by irradiation of UV light for 20 minutes. The ability of a carbene to insert into aliphatic C-H bonds is indicative of an extremely reactive species and is commonly regarded as a prerequisite for photolabeling reagents (Brunner et al., 1997).
O N F3C N S O O O OH HO O O OH O Glc CH3 OH h, 365 nm HO
6 8 2'
O NH F3C
5a 7a
HN
OCH3 H
3a
HN
7b
NH
3b
S H N 3
H N 3
O O O
OH
6'
O O Glc
OH O
Figure 2. Photolysis of the isoquercitrin probe 2. The ability of 2 to arrest the growth of P. falciparum, was examined and compared to that of 1 in order to determine whether 2 mimicked the pharmacological action of 1. Apparently, compound 2 inhibited parasite growth in a dose-dependent manner with an IC50 of 30.4 M, only three fold lower than that for 1. This data suggest that 2 inhibited the proliferation of malaria parasite in an analogous manner to 1 and was a suitable reagent for use in photoaffinity labeling studies. Photoaffinity labeling experiments were performed in an attempt to identify the isoquercitrin-binding proteins both in uninfected and malaria infected erythrocyte. The isoquercitrin probe, 2 was photoactivated in the presence of uninfected or malaria infected erythrocyte. After successive lysis of erythrocytes and malaria parasites, fractions containing proteins were respectively immobilized with streptavidine beads. SDS-PAGE and Western blot analysis revealed preferential labeling of protein with
Syamsurizal et al., Photoaffinity Labeling Study for Identification the Target Protein 77
B.
Figure 3. Photoaffinity labeling of proteins by isoquercitrin probe 2. apparent molecular mass of 70 kDa (Figure 3 A, lane 3 and 4). The band observed in lane 3 was lysate of uninfected erythrocyte treated with saponin and similar apparent band was seen in lane 4 as apart of uninfected erythrocyte when unsolved in 0.15% saponin (diluted in D-PBS) where it was treated with lysis buffer. Judging from intensity of both bands, the band in lane 4 was derived from the saponin treatment due to incomplete washing with D-PBS. Saponin treatment was intended to disrupt the erythrocyte membrane to release the intact malaria parasite and then lysis buffer was added to extract the proteins of malaria parasite. The ca 70 kDa proteins may be present within erythrocyte membrane where the target of 2 was presumed to be located. Mefloquine, an antimalarial drug, was also reported to bind with high affinity to membrane of uninfected erythrocytes, although the specific interacting components have not been identified (Dayton et al., 1975). In order to examine the ability of 1 to inhibit the insertion of 2 into the ca 70 kDa protein in uninfected erythrocyte, photoaffinity labeling was conducted by pre-incubation with increasing levels of 1. Western blot analysis revealed that at the same concentration with 2, compound 1 inhibited labeling of the apparent band at ca 70 kDa. This result suggests that 1 was competing with 2 for the binding site on the 70 kDa protein as shown in Figure 3B. Thus interaction of 2 with the 70 kDa protein was specific in nature, as insertion of the photolabel into this polypeptide was competitively inhibited in the presence of 1. In the future, it might be possible to study the role of 70 kDa protein in invasion of malaria parasites into erythrocytes through proteomic approach. 4. Conclusion Isoquercitrin-binding protein has been identified at the apparent molecular weight of ca 70 kDa from erythrocyte membrane which may be involved in the mechanism of action of 1 as antimalarial. Affinity binding of 1 in a protein of uninfected erythrocyte membrane may protect erythrocyte from malaria parasite invasion. This effect is of particular interest in connection to the prophylactic potential of compound 1. Acknowledgements This work was supported in part by Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture, Japan. I am profoundly indebted to Professor Toshihiro Horii, Research Institute for Microbial Diseases, Department of Molecular Protozoology, Osaka University, for his kind and enthusiastic collaborative support regarding the culture of malaria parasites. References Bayley, H., T. S. Work, and R. H. Burdon, 1983, Laboratory Techniques in Biochemistry and Molecular Biology: Photogenerated Reagents in Biochemistry and Molecular Biology, Elsevier, Amsterdam. Brunner, J., H. Senn, and F.M. Richards, 1997, 3-Trifluoromethyl-3-phenyl-diazirine; a New Carbene Generating Group for Photolabeling Reagents, J. Biol. Chem., 255:8, 3313. Dayton, P. G., Z. H. Israili, and J. Y. Mu, 1975, Studies of the Disposition and Metabolism of Mefloquine HCl (WR142490). A Quinolinemethanol Antimalarial in the Rat, Drug. Metab. Dispos., 3., 110-198. Fuji, T., T. Sugimoto, S. Yamamura, and M. Ueda, 2003, Synthesis of a Novel Bioactive Photoaffinity Probe Based on a LeafMovement Factor with Potential High Binding Affinity to its Receptor Molecule, Tetrahedron Lett., 44, 2497. Hatanaka, Y., M. Hashimoto, and Y. Kanaoka, 1994, A Novel Biotinylated Heterobifunctional Cross-Linking Reagent Bearing an Aromatic Diazirine, Bioorg. Med. Chem., 2, 1367. Hatanaka, Y., M. Ishiguro, M. Hashimoto, L. N Gastineld, and K. Nakagomia, 2001, A Model of Photoprobe Docking with 1,4-Galactosyltransferase Identifies a Possible Carboxylate Involved in Glycosylation Steps, Bioorg. Med.
Chem. Lett., 11, 411-413. Huq, M. M., 2003, Studies on Antimalarial Flavonoid from Medicinal Plants. Ph.D. Thesis, Osaka University, Osaka. Murakami, N., M. M. Huq, S. Tamura, S. Itagaki, T. Horii, and M. Kobayashi, 2001, New Antimalarial Flavonol Glycoside from Hydrangea Dulcis folium, Bioorg. Med. Chem. Lett., 11, 2445-2447.
Plazt., M., A. S. Admasu, S. Kwiatkowski, P. Cracker, J. N. Imay, and D. S. Watt, 1991, Photolysis of 3-Aryl-3-(trifluoromethyl)diazirines: a Caveat Regarding Their Use in Photoaffinity Probes, Bioconjugate Chem., 2, 337-341. Syamsurizal, 2006, Antimalarial Principal from African Medicinal Plants, Ph.D, Thesis, Osaka University, Osaka.