Current Drug Discovery Technologies (v.8, #1)
Editorial [Hot Topic: New Strategy for Drug Discovery Using Monoclonal Antibody (Guest Editor: Yukihiro Shoyama)] by Yukihiro Shoyama (1-2).
The authors in this thematic issue provide a comprehensive summary of most recent knowledge on the relation of natural products and monoclonal antibody (MAb) in order to expand the new technologies for drug discovery. Although MAbs are now indispensable in the biological field, they are rare phytochemical research areas except naturally occurring therapeutic medicines like morphine, taxol and vinblastine and so on because the preparation of MAb against naturally occurring bioactive compounds is not so easy depending on the complex structure and they cannot determine hapten molecule. Therefore, first we opened the determination methodology of hapten molecule number in the carrier protein conjugate by MALDI-tof mass. From this success we have been accumulating many MAbs that resulted to make it possible to create new methodologies such as [eastern blotting], [knockout extract] and [missile type molecular breeding]. In this thematic issue we will be able to cover the wide phytochemical fields from the quality control of traditional Chinese medicine (TCM) to the biotechnology using compact MAb gene for medicinal plant breeding regarding bioactive components. Quality control and standardization of the medicinal plants and/or the traditional Chinese medicine (TCM) are necessary factors affecting the difference of chemical constituent to the composition and concentration, many reasons such as genetic distinction, botanical sources, production area, harvest time, processing method, etc. are also considered. Quality control of the medicinal plants and/or TCM by using MAb is quick, without any pre-treatment, very small scale, reproducible and soft for circumstances as they do not need organic solvent compared to HPLC analysis. For example, ELISA kit for glycyrrhizin contained in licorice is now available in the market. The immunoblotting method is based on western blotting technique that utilizes antigen-antibody binding properties and has been developing a specific and sensitive detection of higher molecule analyte like peptides and proteins. Despite being impossible against small molecular compounds, a new immuno-staining method for natural glycosides has been developed and named as eastern blotting. This new methodology may open a new field of the quality control for natural products, therefore this methodology will be incorporated in this thematic issue. Eastern blotting can stain only hapten molecule on membrane. TLC plate developed will be covered by PVDF membrane, followed by treatment of the membrane with sodium periodide and further, the addition of carrier protein like BSA. The membrane will initially be treated by MAb and then the secondary antibody modified by the enzyme. Finally, a substrate will be added resulting in staining. In this case we succeeded to stain glycosides having sugar linkage in a molecule like ginseng saponins, solasodine glycosides, glycyrrhizin and so on. Moreover, the polyethersulphone (PES) membrane is also available. There is no need to develop TLC as it is possible to investigate the quantitative analysis by using NIH imaging soft. Glycosides are complicated and can not be elucidated automatically like amino acid analysis and the DAN sequencing results show that the structure elucidation of glycosides needs much efforts. Application of the double staining of eastern blotting for the ginseng saponins can suggest the structure of unknown ginseng saponins, depending on the staining color related to the aglycone and the Rf value related to the sugar number. Recently, without the affinity purification of proteins the promotion of almost all investigations might be impossible. But the succees of immunoaffinity purification against small molecule compounds is almost nothing until now. Knockout extract can be prepared by the elimination of only an antigen molecule from the crude extract by an immunoaffinity column. More over, an immunoaffinity concentration of glycoside can function for the exact determination of concentration for important glycosides even though at very lower concentration. This newly developed knockout extract evidently demonstrated the real bioactive compound contained in the extract of crude drug. Therefore, we incorporated three applications for ginseng, licorice and American ginseng in this thematic issue. Technology of compact MAb has been expanding and accumulating in the field of cancer therapy. However, the natural products having small molecule are rare therefore, a review has been planned to introduce the important natural products. In general, many attempts of increasing secondary metabolites by the stimulation of biosynthetic enzyme related to the natural products have been investigated, but no success has been reported so far. In this thematic issue one application of technology of compact MAb, single chain fragment-variable (scFv) antibody named as [missile type molecular breeding] is incorporated for the breeding of solasodine glycoside producing plant. In this case, if the MAb against a target molecule is available, the construction would be simple and quick. This way it resembles with the neutralizing antibody for cancer therapy. In this issue of Current Drug Discovery Technologies, seven reviews have been incorporated and highlighted in detail related to MAbs. It can be evaluated that the evidences reviewed in this issue may be applied widely in the field of drug discovery. Moreover, more than 260 references found at the end of the individual reviews, will make this thematic issue useful not only for phytochemical researchers but also for many scientists working in the numerous fields of natural products, emphasizing the high scientific standing of the issue.
Immunochemical Approach Using Monoclonal Antibody against Δ 9-Tetrahydrocannabinolic Acid (THCA) to Discern Cannabis Plants and to Investigate New Drug Candidates by Hiroyuki Tanaka (3-15).
A monoclonal antibody (MAb-4A4) against and#x394;9-tetrahydrocannabinolic acid (THCA) showing extensive crossreactivity against various cannabinoids was prepared. Using this antibody, a competitive enzyme-linked immunoassay (ELISA) was developed to detect and#x394;9-THCA in the range of 1 to 100 and#956;g/ml. Various cannabinoids including and#x394;9-THC (and#x394;9- tetrahydrocannabinolic acid), and#x394;8-THCA (and#x394;8-tetrahydrocannabinolic acid), and#x394;8-THC (and#x394;8-tetrahydrocannabinol), CBD (cannabidiol), and CBN (cannabinol) were recognized by MAb-4A4, and their cross-reactivities were 55-1600and#x25; compared with and#x394;9-THCA (100and#x25;). This novel characteristic of this MAb enabled detection of marijuana residues in biological samples by detection of residual cannabinoids. The ELISA using MAb-4A4 was found to be applicable even for withered samples which contained only trace amounts of and#x394;9-THCA and and#x394;9-THC. In addition, this method using MAb-4A4 could be useful in forensic analysis since the MAb-4A4 also shows cross-reactivities against cannabinoid metabolites in body fluids. As well as forensic applications using this MAb, an investigation of new drug candidates focusing on cannabinoid metabolites arising from biotransformation in plant tissue was performed using immunochemical screening. The resulting new drug candidates were cannabinoid glycosides biotransformed by Pinellia ternata whose bioactivity is as yet unidentified. Our results indicate the utility of the application of ELISA using MAb-4A4 for further experiments involving marijuana and cannabinoids not only in the forensic field but also in the context of drug discovery.
Preparation of Knockout Extract for Determination of Really Active Compound Using MAb by Takuhiro Uto, Indree Tuvshintogtokh, Yukihiro Shoyama (16-23).
The crude-rhizome extract of P. japonicus was loaded on the immunoaffinity column conjugated with antiginsenoside- Rb1 monoclonal antibody (MAb) and washed with the washing solvent, followed by elution solvent, to give fraction 2 containing higher concentration of compound 1. Compound 1 clearly indicated a dammarane saponin having protopanaxadiol as a framework and three sugars in a molecule suggesting that compound 1 is chikusetsusaponin III. Compound 2 was also determined as chikusetsusaponin VI compared to the staining color, its Rf value and the comparison with ginsenoside Rb1. We succeeded in one step purification of ginsenoside-Rb1 by immunoaffinity column conjugated with anti- ginsenoside-Rb1 MAb leading to the knock-out extract which will be useful for pharmacological investigation. The antibody was stable when exposed to the eluent, and the immunoaffinity column showed almost no decrease in capacity after repeated use more than 10 times under the same conditions. From the crude extract of licorice we isolated glycyrrhizin by one-step purification by the immunoaffinity column using anti-glycyrrhizin MAb. Washing fraction contained all components except for only glycyrrhizin and was named as the knockout extract. We confirmed the synergic effect of glycyrrhizin with some other components for the inhibition of nitric oxide (NO) production by blocking inducible nitric oxide synthase (iNOS) expression by using its knockout extract.
Technology of Compact MAb and its Application for Medicinal Plant Breeding Named as Missile Type Molecular Breeding by Waraporn Putalun (24-31).
Single-chain variable fragment (scFv) enhanced solasodine glycoside accumulation in Solanum khasianum hairy root cultures transformed by the scFv against solamargine (As-scFv) gene. The scFv protein was expressed at a high level in inclusion bodies of E. coli. After being renatured, the scFv protein was purified in a one-step manner by metal chelate affinity chromatography. The yield of refolded and purified scFv was 12.5 mg per 100 ml of cell culture. The characteristics of the As-scFv expressed in E. coli and transgenic hairy roots were similar to those of monoclonal antibody (MAb). The expression of scFv protein provides a low cost and a high yield of functional scFv antibody against solamargine. The full linear range of the ELISA assay using scFv was extended from 1.5-10 and#956;g/ml. The expressed antisolamargine scFv protein could be useful for determination of total solasodine glycoside content in plant samples by ELISA. Solasodine glycoside levels in the transgenic hairy root were 2.3-fold higher than that in the wild-type hairy root based on the soluble protein level and binding activities. The As-scFv expressed in S. khasianum hairy roots enhanced solasodine glycosides accumulation and provide a novel medicinal plant breeding methodology which can produce a high yield of secondary metabolites.
Bioactivity of American Ginseng by Knockout Extract Preparation Using Monoclonal Antibody by Chun-Su Yuan, Hiroyuki Tanaka (32-41).
We produced anti-ginsenoside Re (GRe)-4G10 monoclonal antibody that specifically recognizes GRe and used this to prepare an immunoaffinity column to remove GRe from American ginseng berry extract (AGBE) (thus producing GRe knock-out AGBE (GRe-KO-AGBE)). We compared the anti-diabetic and anti-obesity effects of AGBE and GRe- KO-AGBE in adult diabetic C57BL/6J ob/ob mice. Fasting blood glucose levels and body weight were measured in mice receiving daily intraperitoneal injections of 150 mg/kg of either extract for 27 consecutive days. On Day 6, the group taking AGBE had significantly lower fasting blood glucose levels compared to GRe-KO-AGBE-treated mice (70.5 mg/dl vs. 140 mg/dl). On Day 13, the glucose levels in both groups were 201 mg/dl for AGBE-treated group and 169 mg/dl for GRe-KO-AGBE, respectively. GRe (20 mg/kg) had dose-related effects on fasting blood glucose levels (reduced to 188 and 180 mg/dl on Day 5 and Day 12, respectively, compared to 229 and 235 mg/dl, respectively, in the vehicle group). This hypoglycemic effect persisted even after treatment discontinued (blood glucose levels at 3 days post-treatment: 198 and 253 mg/dl with ginsenoside and vehicle treatment, respectively). The monoclonal antibody technique can be used to prepare botanical knockout extracts for bioactivity studies.
Development of New Staining Technology “Eastern Blotting” Using Monoclonal Antibody by Osamu Morinaga, Yukihiro Shoyama (42-50).
Ginsenosides contained in Panax species were separated by silica gel TLC blotted to a polyvinylidene difluoride (PVDF) membrane which was dipped in a sodium periodide (NaIO4) solution and reacted with protein, preparing a ginsenoside-protein conjugate for binding a ginsenoside on a PVDF membrane. The blotted spots were stained by anti-ginsenoside-Rb1 monoclonal antibody (MAb) and anti-ginsenoside-Rg1MAb, respectively. The newly established immunostaining method, eastern blotting was applied for the determination of ginsenosides possessing protopanaxadiol and/or protopanaxatriol. Double staining of eastern blotting for ginsenosides using anti-ginsenoside-Rb1 MAb and antiginsenoside- Rg1 MAb promoted complete identification of ginsenosides in Panax species. This technique has been devised for the chromatographic separation and identification of ginsenosides using polyethersulfone (PES) membrane. It caused an acceptable separation of ginsenoside-Rb1, -Rc and -Rd in various ginseng extracts. Newly developed technique is quite simple and applies for immunoassay system. Ginsenosides separated using a PES membrane were directly treated with a NaIO4 solution and then reacted with bovine serum albumin (BSA) for making a ginsenoside-protein conjugate. After the blocking, anti-ginsenoside-Rb1 MAb recognized a ginsenoside on a PES membrane and then a second antibody labeled with enzyme reacted to the first antibody. Finally a substrate was oxidized with the enzyme and developed the staining of ginsenosides. The staining spots of ginsenosides on membrane were quantitatively evaluated by NIH Image indicating at least 62.5 ng of each ginsenoside-Rb1, -Rc and -Rd were detected with clarity. The determination range of three ginsenosides was from 0.125 to 2.0 and#956;g of direct amount on PES membrane.
Immunofluorescence and Immunoelectron Microscopic Localization of Medicinal Substance, Rb1, in Several Plant Parts of Panax ginseng by Sadaki Yokota, Yuko Onohara, Yukihiro Shoyama (51-59).
It is important to know the localization of medicinal substance, Rb1, of Ginseng, Panax ginseng, in this plant in order to achieve efficient extraction of Rb1 or to culture producing cells. In this report, we describe the localization of Rb1 in various parts of the plant as determined by immunofluorescence (IF) and immunoelectron microscopies (IEM). Using IF, we show that Rb1 is localized to chloroplasts, peroxisomes and cytoplasm but not to vacuoles of leaf parenchymal cells. In the leaf stem, Rb1 is localized to the vascular bundles as well as vacuoles. In the root, vacuoles of parenchymal cells are stained at various intensities. Using IEM, gold particles showing Rb1 antigenic sites are present in the compartments stained by IF technique. In addition, Rb1 is localized in the sieve elements of the phloem and degrading primary cell wall of xylem, and in the root parenchymal cells Rb1 is associated with electron dense polymorphic materials but not in starch granules. Translocation and storage of Rb1 and effective utilization of leaves are discussed.
Quality Control of Traditional Chinese Medicine by Monoclonal Antibody Method by Ming-Ying Shang, Min Tian, Hiroyuki Tanaka, Xiao-Wei Li, Shao-Qing Cai, Yukihiro Shoyama (60-65).
In a previous study, we reported the preparation, characterization, variation, specificity, and sensitivity of an anti-aristolochic acid-II (AA-II) monoclonal antibody. The preparation procedure was as follows. AA-II conjugated with bovine serum albumin was used as an antigen for immunizing BALB/c mice. Splenocytes isolated from the immunized mice were fused with an aminopterin-sensitive mouse myeloma cell line to produce hybridoma cells secreting a monoclonal antibody (MAb) against AA-II. The selected MAb was subsequently cloned. Hapten number, isotype, and an estimated dissociation constant (KD) of the secreted MAb were determined. This MAb was used to establish an ELISA method. The linear range was 0.19-13 and#956;g/ml. Anti-AA-II MAb showed extremely high specificity for AA-II, low crossreactivity (CR) against other AAs or aristololactam-I, and negligible CR ( andlt; 0.5and#x25;) toward other natural compounds with different chemical structures. This study describes the successful application of the ELISA method using anti-AA-II MAb to determine AA-II concentration in several crude drugs derived from Aristolochia species. The highest AA-II concentration (2.82 and#956;g/mg) was observed in the stem of A. manshuriensis, followed by that in the fruit of A. contorta (0.81 and#956;g/mg). In case of A. indica, AA-II concentration in the root was higher than that in the aerial parts. These data indicated that the established ELISA method can be used for the quality control of crude drugs derived from Aristolochia plants.
Asparaginyl-tRNA Synthetase Pre-Transfer Editing Assay by Franck Danel, Patrick Caspers, Claude Nuoffer, Michael Hartlein, Michael A. Kron, Malcolm G.P. Page (66-75).
Aminoacyl-tRNA synthetases (AARSs) are a structurally heterogeneous family of enzymes present in prokaryotes, archaea and eukaryotes. They catalyze the attachment of tRNA to its corresponding amino acid via an aminoacyl adenylate intermediate. Errors in protein synthesis will occur if an incorrect amino acid is attached to the tRNA. To prevent such errors, AARSs have evolved editing mechanisms that eliminate incorrect aminoacyl adenylates (pre-transfer editing) or misacylated tRNAs (post-transfer editing). Various AARSs are the targets of natural antibiotics and are considered validated targets for chemotherapy. We have developed a high-throughput screening (HTS) assay measuring the pre-transfer editing activity of pathogen-derived asparaginyl-tRNA synthetase (AsnRS). This was achieved by monitoring the formation of pyrophosphate via cleavage to phosphate, which was quantified by reaction with Malachite Green. L-Aspartate-and#946;-hydroxamate, an asparagine analogue, was most effective in promoting the editing activity of AsnRS from Brugia malayi (BmAsnRS) and Staphylococcus epidermidis (SeAsnRS) with KM values close to 100 and#956;M. The assay sensitivity was enhanced by the thiol agents, DTT and L-cysteine, which significantly increased the turn-over of aminoacyl adenylate by BmAsnRS, but not SeAsnRS. The HTS assay was used to screen a library of 37,120 natural-product extracts for inhibitors of BmAsnRS. A small number of extracts that inhibited the pre-transfer editing by BmAsnRS was identified for future isolation of the active component(s). The principle of this assay can be applied to all enzymes having a pre- or post-editing activity.