Microorganism have the property of producing a range of secondary metabolites of diverse chemical types. The secondary metabolites are of great economic importance in the pharmaceutical industry as well as the agro chemical sector. The microbial metabolites which are useful against non infectious disease is made to undergo screening in order to detect various specific biological responses. Some examples include ligand-receptor interactions, enzyme inhibitors involving in metabolic disorders and cell-cell mediators.
The exploration of microorganism for the production of these metabolites began in 1940's and continued for about a decade until the discovery Penicillin which was a breakthrough in the field of biotechnology( Berdy J, 1980). During this period, the screening methods for the antimicrobial activity were used for the identification of various new antibiotics which included tetracyclines, cephalospocines and aminoglycoide. Other pharmacologically significant natural products consist of immuno suppressants cyclosporine A and FK 506. The no of microbial metabolites having non antibiotic biological activities that had been reported were on a steady increase since 1970's and by 1990 it had overtaken the number of antibiotics that had been reported.
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The recent trend developed was to screen for the products that are known to interfere with enzyme involved in control of cell division; some targets like protein kinase C(PKC) and tyrosine kinase. Theoretically, these products may be effective in antitumor drug. However , numerous protein kinase C enzymes are present and the inhibition of any one of them may lead to range of spurious or unexpected biological effects. For the detection of physiological mediators or antagonist of hormones, several tests have been designed. These test are usually based in the competition assays for humoral receptors. Some examples include peptide hormones that regulate gut motility and gastric and pancreatic secretion and antagonists if cholecystokinin.
The rapid advances in biotechnology and progress with the human genome project have provided a range of new molecular targets that have been implicated in numerous human disease state (Rosteck, 1994). this target identification has paved way to many new opportunities for exploration of the molecular diversity that has been generated from the natural product sources.
There are various assays which are being used to screen microbial metabolites with pharmacological activity. These assays have to meet a criteria for finding novel lead compounds which can be used as a drug successfully.
These assays must be designed in such a way that they are able to function with the sample having range of physical and chemical properties. A large no of preparation techniques of the sample are employed to generate screening procedures. This leads to the formation of test mixtures which are filtered to remove material having high molecular weight. In this case the sample may have various properties which include a high or low pH or ionic strength, may consist of medium components or may be highly colored. On the other hand some other test mixtures could also be extracted in a range of solvents. Therefore, microbial screens must have the ability to detect the active metabolites even in the presence of potential interference. These assay must be sensitive as the range of its detection is in 20 to 100nM. For better metabolite detection, the assay must be designed specifically for a molecular or cellular target. These conversion of an assay to a high throughput for microbial screen is of a major consideration. Throughput of the microbial screen is a vital criteria that should be considered before the start of the assay. Most of the assay are converted to high throughput screens, but some assays like Cell Based Assay are complex and consume more resources an time for their operation. Therefore, a lot of emphasis should be put on resource management for screening which can be done by the use of robotics and by miniaturisation of the assay format.
For screening of natural products various assay types have been exploited. Cell based assays are utilized for searching the molecular which inhibits a cellular function. It is used where a specific molecular target is not known. Thus the interaction of the active compound takes place with the cells at the variety of targets. The major requirement for the development of cell based assay is the cells utilized for the assay. To make accurate conclusion, cell based assay are dependent on the source of cells in which cells can be fresh or cyropreserved. A cell line can be used as a source of the cells in case like neutralizing antibody. For determining the mechanism of action, there is a need to subject the compounds identified to various selectivity and secondary assays due to the presence of various intervention sites. Cytotoxic assay are vital and operation of similar screen types produce significant data which helps in elimination of activities which are non specific. For screening of compounds with immunosuppressive properties, assay based on mixed lymphocyte reaction(MLR) have been used. ISP1(myriocin), mycestericins are the compounds isolated with these properties. Myriocin was found to be 10 more effective than cyclosporine A in inhibiting the MLR(Fujita et al, 1994). FR901459, isolated from Stachybotrys chartarum is a novel cyclosporine analogue, was isolated using MLR based assay. In this assay, cell proliferation was measured using a [3H]-thymidine incorporation when the responder cells and the stimulator cells were mixed in a 96 well plate. Cell adhesions have been found to play an important role in inflammatory response and tumor metastasis. IC101 which was isolated from streptomyces albulus was found to be an inhibitor of cell adhesion (Ueno et al, 1993). IC101 was found to inhibit the mixed lymphocyte reaction (LMR). On cytotoxicity testing of the compound was found to be toxic invivo and invitro. Also, cytostaton produced by streptomyces species was found to inhibit tumor metastasis's in vivo and exhibit antimetastatisis activity on B16 melanoma cells in mice. For discovery of a new potent pharmacologically active compound for CVD used platelet aggregation and new blood vessel formation assay. Inhibition of platelet aggregation can be a useful technique for preventing vascular disease. A novel compound, FK409 was found to be an inhibitor of platelet aggregation. It is a semi artificial bioproduct of streptomyces gruseosprous(Hino et al, 1989).testing FK409 in vivo showed relaxation activity of rat aorta and shows hyposensitive effect equal to nitroglycerin.
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Receptor binding assay have provided a large amount of information for drug discovery programs. In a wide range of targets, animal tissue preparations were used a sources of the membrane receptor. The screening against membrane receptors and whole cells which may be expressed in vitro can be done by the use of cell lines that express target receptors. The binding assays measure the binding of ligands which are radioactive to the receptors in presence of a range of test samples. The ligand that is bound may then be separated by filtration or centrifugation and this helps in the calculation of percentage inhibition which is compared with the controls.
The early discovery of asperlicin from aspergillus alliacers which was found to be an potent and selective antagonist of cholecystokinin(chang et al, 1985). The designing of receptor binding assay often results in compromise because of the use of non physiological enzyme substrate because of economical reasons. Generally, these type of assay results in high false positive rate due to the presence of compounds with non specific effects on enzyme or receptor or their environment. Inflammatory response are found due to the receptor ligand interaction and these have been targeted for screening of new pharmacologically active compounds. Leukotriene B4(LTB4) was found in the low concentration at the sire of inflammation. Due to the low concentration, the function of polymorphonuclear leucocytes(PMNL) were altered suggesting that LTB4 may serve as a mediator in inflammatory response. [3H]-LTB4 and a PMNL membrane suspension were incubated with the test sample. Unbound [3H]-LTB4 was separated from the free ligand using a fibre glass filter after incubation and the cells were harvested. This lead to the discovery of WF11605 which is a novel tetracycline triterpene glucoside produced by an unidentified strain of fungus(Tsujii et al, 1992).
Umezawa in 1966 discovered pharmacologically active enzyme inhibitors by using enzymes in the invitro screening. Enormous progress of the enzyme systems as targets for pharmacological intervention or selective cytotoxicity was provided by the advancements that took place in molecular biology. Screening against enzymes that are isolated may lead to a large no of false positive inhibitors that are active invitro and not invivo. This is due to the reason that the compounds that are detected may have a effect on the enzymes or their environment non specifically. Additionally, even though some compounds are known to be active against the target enzyme , they may not reach their targets when tested invivo or in whole cells. However, even after all these limitations enzymes are a vital choice in selecting the targets to initiate a primary screening program and are also advantageous as they comprise of a rich source of novel inhibitors from natural sources.
Human leukocyte elastase(HLE) hydrolyses several connective tissues is found to be one of the most destructive enzymes. PMNL release HLE by inflammatory stimuli and was found to play a role in destructive process in chronic inflammatory diseases. By using this assay, FR901277 a peptidic metabolite of streptomyces restitomycificus and FR901451 a tricyclic depsipeptide produced by flexibacter sp was discovered. FR901277 was found to inhibit the function of HLE, porcine pancreatic elastase and chymotrypsin. But it was a weak inhibitor of trypsin. FR901451 was also found to be competitive inhibitor of HLE.
Recently, a number of microbial genomes are fully sequenced. The discovery of new drugs and targets in increasing at a fast pace due to the advancement in technologies in genomics and proteomics. This is further assisted b bioinformatics and high throughput screening. The main challenge is in understanding the structure function relationships of proteins and receptors, the increase throughput target screening and identifying cations uses the functional genomics methods which are sequence based. The functional domain that exist within the protein(e.g. ATP binding sites) may be identified by sequence similarity with the known gene. The 'gene chip' technology is also a high throughput genomics screen which helps in comparison of gene expression patterns between normal and diseased tissues for example. Another process to identify putative ligand is the 'ligand fishing' where orphan receptors may be used to screen against a host of compounds or cell extracts. Surface Plasmon resonance(SPR) based biosensor technology in association with MALDI-TOF technology helps to detect and quantify the specific binding between the orphan receptors and various other complex material like the cell extracts or bacterial lysates. Non destructive SPR detection can also aid in providing the real time information on biomarker interaction. With the help of mass spectroscopy, molecular weight of the bound ligand may be determined accurately. Also, the proteolytic digestion of the bound ligand provides further characterization and identification of the sequence.
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