Bioactive Agents from Natural Sources: Trends in Discovery and Application - Advances in Biochemical Engineering Biotechnology - page 135

glucocorticoid [336],the estrogen [337-340],the progesterone receptor [341-343]
and the vitamin D receptor [344,345].
Easy handling, short generation times, ready genetic manipulation, con-
tinuous heterologous gene expression, resistance to solvents and low cost of
growing make yeast an attractive option for cell-based HTS attempts.However,
screening assays in Saccharomyces cerevisiae can only serve as primary assays
that have to be followed up by advanced profiling of the “hit”in tissue culture
systems, organ systems and animal models, respectively, before lead structure
commitment.
With the cell-based screening approach, a recombinant cell or microorga-
nism is engineered to respond in a specific manner to an effector of the mole-
cular target of interest permitting the examination of complex multiprotein
interactions without having to reconstitute the entire system in vitro.If the
system is properly formatted,the probability of a lead compound derived from
a cell-based screening being active in pharmacological models is supposed to be
greater than if the compound was derived from a cell-free assay using purified
macromolecules.Furthermore,the need for the agent to cross the cell membra-
ne to demonstrate activity in a cell-based assay probably eliminates many non-
specific false positives obtained from screening attempts using isolated enzymes
and receptors [333].For many targets,cell penetration is required for pharma-
cological activity. On the other hand, potential lead compounds that inhibit
the target but cannot cross the membrane will not be discovered. These leads
may still be useful as subsequent chemical modification could introduce cell
penetration properties.Therefore,the use of cells with permeability mutations
provides a good compromise,eliminating many false positives while allowing a
variety of potential lead compounds to be detected.
A major problem of cell-based screening assays is that cytotoxicity derived
from mechanisms unrelated to the target of interest prevents further evaluation
of a compound or, more feasible, of an extract from natural sources. Further-
more,depending on the assay endpoint,false positives may be selected.There-
fore,secondary assays are required to eliminate cytotoxic false positives as well
as to prove the mode of interaction that gave positive endpoint detection in the
screening assay.
In the next few years,quantitative changes in both genetic and instrumenta-
tion engineering,including advances in nano-technology,will affect cell-based
HTS approaches.New reporter systems resulting in sensitive fluorescent read-
outs without cell disruption will probably contribute to assay miniaturization
and faster screening runs.The construction of assays that take advantage of the
ability ofcells to grow and to proliferate will cause a qualitative shift in how HTS
is conducted.Application ofsuch positive selections where cell growth depends
on the addition of the bioactive principle of interest should allow examination
of hundreds-of-thousands to millions of test samples per day, rather than the
tens-of-thousands now accessible using the best screening formats [330].
Furthermore,novel methods for single molecule detection will contribute to
improved drug discovery. For instance, fluorescence correlation spectroscopy
(FCS) is characterized by the ability to measure single molecules in sub-mi-
croliter sample volumes with a typical range of sensitivity between 10 -6 and