Transcript Screening Methods - WELCOME aboard UG3!

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 1972.
• 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
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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