Novel Screen Methodologies for Identification of New Microbial Metabolites
77
bioassay guided purification and structure elucidation of biologically active
compounds.
1.3 Overview of General Assay Categories
There are many assay types which have been exploited in natural products
screening programmes. For the purposes of this review, assays have been
broadly characterised into three types: cell-based assays, receptor binding assays
and enzyme assays.
A range of cell-based screens have been used in the detection of novel
microbial metabolites. These are often employed in the search for molecules
which inhibit a cellular function, but where a specific molecular target has not
been defined. This means that active compounds may interact with the cells at
a variety of targets from ligand-receptor binding, through intracelllar signal
transduction, to effector protein synthesis and secretion. As there is a complex
series of available intervention sites, compounds identified in cell-based screens
need to be subjected to a series of selectivity and secondary assays to determine
their mechanism of action. Cytotoxicity assays are often essential, and simultaneous operation of similar screen types can generate valuable data which helps
to eliminate non-specific activities.
Receptor binding assays have provided a major focus for drug discovery
programmes. Preparations from animal tissues have been used as sources of
membrane receptor in a wide variety of targets. In addition, the use of cell lines
expressing target receptors has given the opportunity for screening against
whole cells and membrane receptors which can be expressed in vitro. Binding
assays have most commonly measured the binding of radioactive ligand to
receptors in the presence of test samples. The bound ligand is then separated
from the free ligand, either by centrifugation or filtration, and a percentage
inhibition calculated in comparison with controls.
The use of enzymes to discover pharmacologically active enzyme inhibitors
by in vitro screening was pioneered by Umezawa in 1966 [10]. Advances in
molecular biology have provided a huge array of enzyme systems as targets for
selective cytotoxicity or pharmacological intervention. However, screening
against isolated enzymes can result in too many false positive inhibitors which
are active in vitro and not in vivo. This is because compounds detected may
affect enzymes or their environment non-specifically. In addition, whilst compounds may be very active against the target enzyme, they may not be able to
reach their target when tested in whole cells or in vivo. Despite these limitations,
enzymes remain a popular choice when selecting targets to begin a primary
screening programme, and continue to be a rich source of novel inhibitors from
natural sources.
77
bioassay guided purification and structure elucidation of biologically active
compounds.
1.3 Overview of General Assay Categories
There are many assay types which have been exploited in natural products
screening programmes. For the purposes of this review, assays have been
broadly characterised into three types: cell-based assays, receptor binding assays
and enzyme assays.
A range of cell-based screens have been used in the detection of novel
microbial metabolites. These are often employed in the search for molecules
which inhibit a cellular function, but where a specific molecular target has not
been defined. This means that active compounds may interact with the cells at
a variety of targets from ligand-receptor binding, through intracelllar signal
transduction, to effector protein synthesis and secretion. As there is a complex
series of available intervention sites, compounds identified in cell-based screens
need to be subjected to a series of selectivity and secondary assays to determine
their mechanism of action. Cytotoxicity assays are often essential, and simultaneous operation of similar screen types can generate valuable data which helps
to eliminate non-specific activities.
Receptor binding assays have provided a major focus for drug discovery
programmes. Preparations from animal tissues have been used as sources of
membrane receptor in a wide variety of targets. In addition, the use of cell lines
expressing target receptors has given the opportunity for screening against
whole cells and membrane receptors which can be expressed in vitro. Binding
assays have most commonly measured the binding of radioactive ligand to
receptors in the presence of test samples. The bound ligand is then separated
from the free ligand, either by centrifugation or filtration, and a percentage
inhibition calculated in comparison with controls.
The use of enzymes to discover pharmacologically active enzyme inhibitors
by in vitro screening was pioneered by Umezawa in 1966 [10]. Advances in
molecular biology have provided a huge array of enzyme systems as targets for
selective cytotoxicity or pharmacological intervention. However, screening
against isolated enzymes can result in too many false positive inhibitors which
are active in vitro and not in vivo. This is because compounds detected may
affect enzymes or their environment non-specifically. In addition, whilst compounds may be very active against the target enzyme, they may not be able to
reach their target when tested in whole cells or in vivo. Despite these limitations,
enzymes remain a popular choice when selecting targets to begin a primary
screening programme, and continue to be a rich source of novel inhibitors from
natural sources.