Whole-Cell Screening

The selection of cell-targetingpeptides may involve targeting a known cell surface molecule or screening whole cells without a priori knowledge of the chemical nature of the cell surface. The latter approach can lead to identification of hitherto unknown cell surface receptors or target molecules. 

In a pioneering forward chemical genetic screen (whole-cell mitotic arrest assay detected by fluorescence microscopy), a cell-permeable small molecule, monastrol (Figure 1.9), was identified, as it caused inhibition of the normal mitotic spindle formation but did not affect normal tubulin formation. In subsequent studies, testing the inhibition of the formation of the mutant phenotype led to the identification of the primary molecular target in the signaling cascade, a molecular motor protein, kinesin, Eg5. Monastrol treatment showed a phenotype identical to the blocking of Eg5 function by microinjection of Eg5-specific antibodies (Kapoor et al., 2000). 

Direct quantitation of receptor concentrations and dmg—receptor interactions is possible by a variety of techniques, including fluorescence, nmr, and radioligand binding. The last is particularly versatile and has been appHed both to sophisticated receptor quantitation and to dmg screening and discovery protocols (50,51). The use of high specific activity, frequendy pH]- or p lj-labeled, dmgs bound to cmde or purified cellular materials, to whole cells, or to tissue shces, permits the determination not only of dmg—receptor saturation curves, but also of the receptor number, dmg affinity, and association and dissociation kinetics either direcdy or by competition. Complete theoretical and experimental details are available (50,51). 

Biocatalysts these are essential for life and play a vital role in most processes occurring within the body as well as in plants. In the laboratory biocatalysts are usually natural enzymes or enzymes produced in situ from whole cells. They offer the possibility of carrying out many difficult transformations under mild conditions and are especially valuable for producing enantiomerically pure materials. Their huge potential is currently largely untapped, partially due to the time and expense of isolating and screening enzymes. 

One of the main obstacles for whole-cell microbial transformation in an organic solvent is its biocompatibility, which has led to screening for organic-solvent-tolerant microorganisms. Numerous organic-solvent-tolerant microorganisms have been found and their tolerance mechanisms have been reviewed [14,33,34]. Two-phase biotransformation systems have been successfully implemented for the production of pharmaceutically relevant metabolites. 

One advantage of whole-cell biotransformation that has not been addressed adequately in this chapter is the ability to modify compounds with complex structure, such as natural products. Natural products are ideal substrates for biotransformation reactions since they are synthesized in a series of enzymatic reactions by the whole cells. The modification of natural products by biotransformation has been reviewed recently by Azerad [ 13] and a majority of the modifications were carried out by whole-cell biotransformations. Additional examples of modification of natural products by whole-cell biotransformations can also be found in the review article by Patel [2]. Natural products are an important source of new drugs and new drug leads [53]. The use of biotransformation, especially whole-cell biotransformation, in modification of natural products for lead optimization and generating libraries of derivatives for S AR and screening studies is important for the pharmaceutical industry. 

Primary assays are devised to incorporate physiological or enzymatic targets for screening biological activity of potential drug compounds, ilie biological assays are then reconfirmed in specific biochemical and whole cell assays to characterize the target-compound interaction. 

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