Cell Permeability

An advantage of cell-free systems is that all active compounds are identified and then secondary cellular systems can be used to differentiate those hits that are cell-permeable and those that are not. Although cloning new targets of interest into microbial systems is easily achieved, microbial cells have evolved an impermeable cell wall and membrane that allow them to survive in the environment. Therefore the target may not be easily accessible to compounds in high-throughput screens that use microbial cells. Mammalian cells, in general, are more permeable than microbial cells, and therefore microbial cell-based screens are likely to miss those compounds that do not penetrate across the cell wall and membrane barrier. In addition, many microbes have very effective efflux systems that pump out compounds. These efflux systems are similar to the multidrug resistance (MDR) transporters found in tumor cells. One of the large classes of efflux systems, or transporters, is called the ATP-binding cassette transporters or ABC transporters. The ABC transporters are conserved from bacteria to man [5].

With such drawbacks, can microbial-based screening be effectively used in screening? Genetic and molecular technology has made it possible to remove some of these barriers and make screen development and screening in microbial systems a viable, inexpensive, and productive alternative to other screening systems. Among microbes, Saccharomyces cereviseae or yeast and Escherichia coli have been the most popular because of the genetic manipulations possible with these organisms. Since the yeast S. cerevisiae is an eukaryote, it is often considered to be a more realistic system for screen development against mammalian targets. However, yeast is slower to grow than E. coli, taking 48 hours to grow to measurable densities, while E. coli can be used within 6 to 8 hours of growth. Also, genetic manipulations in E. coli are considerably less difficult than with yeast, and E. coli are more permeable than yeast.

A. Development of Permeable S. cerevisiae

Wild-type S. cerevisiae are quite impermeable owing to their cell wall and membrane. The cell wall is considered to be latticelike and allows most small molecules to permeate through. However, the cell membrane is considered to be quite impermeable. More recently it has been noted that yeast cells are actually permeable, and the lack of drug effect is the result of the activity of multiple efflux systems, belonging to the family of ATP-binding cassette transporters (MDR), called PDR, that rapidly pump out compounds. Transcription factors, pdr1p and pdr3p, down-regulate the expression of hexose transporters, HXT11 and HXT9, which in turn up-regulate the expression of PDR. Thus deleting the hexose transporters, HXT11 or HXT9, confers pleiotropic drug resistance on yeast while overexpression of these transporters results in increased sensitivity to drugs (Fig. 3). Furthermore, deletion of the regulators of the promoter for ATP-binding transporters, PDR1 and PDR3, in HXT11 and HXT9 over-expressing strains, results in supersensitive yeast [6]. These mutant strains are ideal organisms for use as host strains for the development of screens. Improved cell permeability was also

Figure 3 Design of permeable S. cerevisiae. The transcription factors pdr1p and pdr3p decrease the expression of the hexose transporters. The hexose transporters increase the expression of the ATP transporters such as PDR5. Deletion of PDR1 and PDR5 increases expression of HXT11, and HXT9 down-regulates the expression of ATP transporters and enhances the susceptibility of S. cerevisiae to many compounds.

Figure 3 Design of permeable S. cerevisiae. The transcription factors pdr1p and pdr3p decrease the expression of the hexose transporters. The hexose transporters increase the expression of the ATP transporters such as PDR5. Deletion of PDR1 and PDR5 increases expression of HXT11, and HXT9 down-regulates the expression of ATP transporters and enhances the susceptibility of S. cerevisiae to many compounds.

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