Screening Methods Lecture – 4 Tahir

Early screening strategies tends to be empirical, labour intensive and low success rates. As the no of commercially important compounds isolated increased, the success rates of such screens decreased further. Thus new screening methods have been developed which are more precisely targeted to identify the desired activity. Eg antibiotics

Antibiotics initially detected by growing on ager plates to check the antimicrobial action. Some antibiotics shows high activity and some shows low. Advances made in the understanding of cell wall biosynthesis and the mode of action of antibiotics allowed the development of mode of action screens in the 1970s which resulted in a very significant increase in the discovereis of new B-lactam antibiotics. Nagarajan et al 1971 – discovery of the cephamycines was based on the detection of compounds which induced morphological changes in the susceptible bacteria. Action of Penicillins was the inhibition of transpeptidase enzmes crosslinking mcopeptide molecules led to the development of enzymes inhibitor assay. Fleming et al 1982 described the development of an automated screen for the detection of carboxypeptidase inhibitors which led to the detection of several novel cephamycin and carbapenem compunds.

Increasing frequency of penicillin and cephalosporin resistance amongst clinical bacteria led to the development of mechanism based screens for the isolation of more effective antibacterials. Brown et al 1976 search for compounds which inhibit B-lactamase and could incorporate with ampicillin as a combination therapeutic agent. Samples were tested for their ability to increase the inhibitory effects of ampicillin on a B-lactamase producing Klebsiella aerogenes & the strategy resulted in the discovery of clavulanic acid.

The concept of using the inhibition of enzymes as a screening mechanism was pioneered by Umezawa Alberts et al 1980 isolated mevinolin which is inhibitor of hydroxy methyl glutaryl reductase, the rate limiting step in the biosynthesis of sterols, it is now marketed as an agent to lower the cholesterol level. Bull 1992 microbial enzymes have been shown to inhibit key enzymes: hypercholesterolemia, hypertension, gastric inflammation, muscular dystrophy, benign prostate hyperplasia and systemic lupus erythemosus. Hashimoto et al 1990 – activity of carbapenem antibiotic is lost in therapy due to renal dehydropeptidase activity. They isolated microbial products capable of inhibiting the enzymes which could then be administered along with the antibiotic to maintian its clinical activity.

The detection of pharmacological agents by receptor ligand binding assay has been developed rapidly by pharmaceutical companies (Bull 1992). These are very effective even at low concentration The gastrointestinal hormone cholecystokinin (CCK) controls a range of digestive activities such as pancreatic secretion & gall bladder contraction. Receptor screening identified a fungal metabolite, aperlicin (Aspergillus allianceus) that has a very high affinity for CCK receptors. The progress in molecular biology, genetics and immunological has also contributed extensively to the development of innovative screens, by enabling the construction of specific detector strains, increasing the availability of enzymes and receptors and constructing extremely sensitive assays.

Bull 1992 contributions Super sensitive strains. Cloning of gene coding for enzymes or receptors Development of receptors gene assays. Molecular probes for particular gene sequences may enable the detection of organisms capable of producing certain product. Development of immunologically based assays such as ELISA