Novel Screen Methodologies for Identification of New Microbial Metabolites
109
Again, in the latter two assay types, opportunities for automation of these
techniques are currently limited.
5.3 Developments in Assay Technology for New Drug Discovery
Due both to the anticipated explosion in targets available for screening and the
need to test a large number of samples quickly, a wealth of new assay formats are
becoming available.
Cell-based assays which are now commonly used in screening programmes
include those for inhibitors of extracellular protein secretion, adhesion assays,
and reporter gene assays [163] which are shown in Fig. 9.
One recent innovation in cell-based systems is melanophore technology,
which uses cells from the frog Xenopus laevis. The movement of pigment in this
system is sensitive to intracellular concentrations of cAMP and diacylglycerol. If
receptor stimulation activates adenyl cyclase and phospholipase C, darkening of
cells is induced. Conversely, stimulation of a receptor which inhibits adenyl
cyclase induces cell lightening. Recombinant receptors can be stably transfected
into this system and pigment hue measured following receptor stimulation. Such
changes are appreciable within minutes, making this technology a useful tool
for high throughput screening [164], although success is dependent on the
genuine coupling of the recombinant receptor with the secondary signalling
pathways.
In addition, scintillation proximity assay (SPA) for cell-based assays has
recently been developed. SPA is a homogeneous radioisotopic technique which
relies on the limited path length of a 13 particle travelling through aqueous
media. If the electron collides with a scintillant particle, energy is transferred and
light is emitted. In SPA, scintillant is coated onto the surface of microtitre plate
(MTP) wells, or incorporated into beads or the MTP itself, making it possible to
attach reagents to the scintillating surface and to monitor the proximity of
radiolabelled substances. In the case of cellular assays the scintillant is incorporated into the base of the MTP. The walls of the well are opaque to exclude
optical cross talk, and the base is transparent to facilitate visualisation of cells
during culture. Cells are grown in monolayers in the wells and radioisotope in
solution is too distant from the scintillating base plate to generate a signal.
Using this type of assay it is possible to monitor in situ receptor binding as well
as cellular metabolism and motility [165].
For receptor binding assays wider availability of cloned receptors has led to
changes in technology, including the development of "capture" assays, the use of
SPA [166, 167] and introduction of fluorescence polarization as a screening
tool, and examples of these are shown in Fig. 10.
In fluorescence polarization assays, the ligand is labelled with a fluorescent
tag and used in a technique based on molecular rotation which measures the
decrease in depolarization of fluorescence exhibited by small molecules when
their rotation is slowed by binding to large molecules [168]. The quantification
109
Again, in the latter two assay types, opportunities for automation of these
techniques are currently limited.
5.3 Developments in Assay Technology for New Drug Discovery
Due both to the anticipated explosion in targets available for screening and the
need to test a large number of samples quickly, a wealth of new assay formats are
becoming available.
Cell-based assays which are now commonly used in screening programmes
include those for inhibitors of extracellular protein secretion, adhesion assays,
and reporter gene assays [163] which are shown in Fig. 9.
One recent innovation in cell-based systems is melanophore technology,
which uses cells from the frog Xenopus laevis. The movement of pigment in this
system is sensitive to intracellular concentrations of cAMP and diacylglycerol. If
receptor stimulation activates adenyl cyclase and phospholipase C, darkening of
cells is induced. Conversely, stimulation of a receptor which inhibits adenyl
cyclase induces cell lightening. Recombinant receptors can be stably transfected
into this system and pigment hue measured following receptor stimulation. Such
changes are appreciable within minutes, making this technology a useful tool
for high throughput screening [164], although success is dependent on the
genuine coupling of the recombinant receptor with the secondary signalling
pathways.
In addition, scintillation proximity assay (SPA) for cell-based assays has
recently been developed. SPA is a homogeneous radioisotopic technique which
relies on the limited path length of a 13 particle travelling through aqueous
media. If the electron collides with a scintillant particle, energy is transferred and
light is emitted. In SPA, scintillant is coated onto the surface of microtitre plate
(MTP) wells, or incorporated into beads or the MTP itself, making it possible to
attach reagents to the scintillating surface and to monitor the proximity of
radiolabelled substances. In the case of cellular assays the scintillant is incorporated into the base of the MTP. The walls of the well are opaque to exclude
optical cross talk, and the base is transparent to facilitate visualisation of cells
during culture. Cells are grown in monolayers in the wells and radioisotope in
solution is too distant from the scintillating base plate to generate a signal.
Using this type of assay it is possible to monitor in situ receptor binding as well
as cellular metabolism and motility [165].
For receptor binding assays wider availability of cloned receptors has led to
changes in technology, including the development of "capture" assays, the use of
SPA [166, 167] and introduction of fluorescence polarization as a screening
tool, and examples of these are shown in Fig. 10.
In fluorescence polarization assays, the ligand is labelled with a fluorescent
tag and used in a technique based on molecular rotation which measures the
decrease in depolarization of fluorescence exhibited by small molecules when
their rotation is slowed by binding to large molecules [168]. The quantification