Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2001-12-07
2003-10-07
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S004000, C435S006120
Reexamination Certificate
active
06630321
ABSTRACT:
This invention provides methods for the identification of novel biological targets and/or inhibitors. Such methods provide useful and convenient tools for high throughput screening. In particular, such methods may be used to identify novel antimicrobial agents.
The emergence of resistance to current therapeutic agents, such as antibacterial, antifungal, and antimalarial agents, necessitates the development of novel agents. Whilst target-based biochemical screens can be successful, classic anti-microbial screening has clearly outperformed rational target-selecting procedures in terms of discovered novel antimicrobials. Target-based biochemical screens are frequently seen to invoke a lack of cell activity, despite a good inhibition of the target enzyme, perhaps due to low permeation into or export out of the cell. Also the presence of inhibiting structures for a hand-picked target within a limited set of available molecules is by no way guaranteed.
In contrast, cell-based antimicrobial screening frequently identifies antimicrobial action but then either reveals this inhibition to be caused by general toxicity, for example membrane perturbation or DNA-intercalation, or fails to identify any specific interaction of the drug with the cellular machinery. This complicates chemical optimisation of initial leads, providing no guide for a structure-activity relationship.
A particular approach to identify target-based mode of action has been that of hypersensitivity.
Historically, the way to generate hypersensitive versions of a protein of interest has been the generation of temperature-sensitive (ts) mutations in its encoding gene (Schmid et al. Genetics 123, pp625-633, (1989)). Temperature sensitive (Ts) or other conditional phenotypes can be generated by either targeted (for example using PCR) or random (for example using chemicals or radiation) mutagenesis. However, such conditional mutants are frequently hypersensitive to inhibitors even under permissive conditions.
We have now found that hypersensitivity to inhibitors can be readily achieved by shutting down a particular essential function of viable cells followed by analysis of the effects in the presence/absence of a potential inhibitor. By “shutting down” we mean that the particular essential function is not available to the cells at any expression level.
Therefore in a first aspect of the present invention we provide a method for identifying a compound which modulates the function of the gene product of an essential gene, which method comprises providing viable cells wherein the gene is expressed under the control of a heterogeneous, regulatable promoter, switching off gene expression via the promoter, contacting the cells with a test compound and determining any modulatory effect on the function of the gene product.
The method provides a step jump in the feasibility of a complete genome analysis for compound hypersensitivity. It provides a number of significant advantages not least that the unaltered “normal” protein is used in contrast to for example extrapolation from the interaction of an altered, mutagenized protein with an inhibitor onto the interaction of the same chemical. Also no effort is needed to fine tune promoter activity to a higher or lower level. Inhibition of one specific biochemical entity, for example a protein required for cellular growth or survival is conveniently indicated by a hypersensitive response of the strain with the regulatable gene compared to its unaltered parent.
By “heterogeneous, regulatable promoter” we mean a promoter other than the native promoter for the essential gene and which can be regulated by the addition and/or removal of specific materials or for example by other environmental changes.
By “hypersensitivity” we mean a larger reduction in cell growth in the presence of an identical concentration of drug, compared to a wild-type cell.
Furthermore, hypersensitivity caused by underexpression of one specific enzyme can extend to a whole biochemical pathway, such as for example sterol biosynthesis. This is exemplified by the results shown in
FIG. 5
of this invention, where the sensitivity to terbinafine is altered both by shutdown of the target enzyme (encoded by ERG1) as well as shutdown of another enzyme (encoded by ERG11) in the same pathway.
By “viable” we mean cells that have grown sufficiently so that when gene expression of an essential gene is turned off, meaningful measurements and kinetic analysis may be made. The cells are preferably allowed to grow into early stationary phase by which time a final optical density reading is taken. A reduced optical density compared to no drug or no switchoff controls is interpreted as growth inhibition.
By “essential gene” we mean a gene required by a cell for example for cell growth and/or cell viability. This does include genes required only under certain assayable conditions (ie conditional essential). In its simplest manifestation essentiality is defined as necessary for growth of the organism on rich media (For a comprehensive summary of such media as well as general yeast methodology see Sherman et al in Methods in Enzymology, Vol 194, Guthrie and Fink eds, Academic Press (1991). Convenient genes include fungal and bacterial genes.
By “switching off gene expression” we mean that all gene expression is turned from ON to OFF. There is no low level gene expression in the OFF position.
By “cells” we mean cells from any convenient source, these include human, animal or microbial cells. New genome-based techniques are developing which combine screening of compounds with simultaneous target identification. Whilst this invention is of particular use with genetically tractable model organisms such as Schizosaccharomyces pombe and Saccharomyces cerevisiae, it is universally applicable and is limited only by practical considerations. Using model organisms such as
S. cerevisiae, S. pombe, C. elegans,
or
D. melanogaster
it is possible to study conserved functions of eukaryotes. The application of genetics using human or animal cell lines directly, then extends possibilities further.
Any convenient test compound such as a peptide, nucleic acid and low molecular weight compound, may be used in the methods of the invention. Preferred test compounds are potential therapeutic agents or may be used in further studies to identify therapeutic agents. Particular test compounds are low molecular weight compounds of, for example, molecular weight of less than 1000, such as less than molecular weight 600.
The modulatory effect of a compound on the gene product of an essential gene is conveniently investigated taking endpoint optical density readings (the higher the absolute optical density of the culture, the less inhibition is thought to have occured. Such analysis is conveniently effected over period of typically 24 hours for yeast cells. Bacteria may take significantly shorter (eg 12 hours) whereas higher eukaryotic cells will require several days to reach early stationary growth phase. The analysis is conveniently photometric (optical density of the culture at 600 nm wavelength).
Importantly, the combination of very recent techniques for site specific integration of DNA (for example with a switchable promoter directly replacing the native one) with the observed fixed timepoint optical density analysis of the hypersensitive response (as further elaborated below) is novel and allows exploitation for large-scale drug screening, compound and target profiling. Moreover, such strains are also very easily obtainable in very high quantity (ie. thousands of genes in
S. cerevisiae
).
Some examples of switchable promoters for use in
S. cerevisiae
include MET3 (repressible by added methionine) and GAL1 (repressed by glucose induced by galactose); for use in
S. pombe:
NMT1 (repressed by thiamine); for use in
C. albicans
: MAL1 (repressed by glucose, induced by maltose, sucrose); for use in
E. coli
: araB (repressed by glucose, induced by arabinose); for use in Gram-positive bacteria such as Staphylococci, Enterococci, Streptococci and Bacilli: xylA/xylR (
Chavda Jini Suberna
Schnell Norbert Friedeman
AstraZeneca UK Limited
Ketter James
Sullivan Daniel
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