Screening methods for the identification of novel antibiotics

Details Screening methods for the identification of novel antibiotics Screening methods for the identification of novel antibiotics

1998-05-29

2000-03-28

Leary, Louise N.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving transferase

435 32, 435 23, 435 24, 435 31, 435 72, 435 721, 435 4, 435 692, 435176, 435177, 435968, C12Q 150

Patent

active

060430451

DESCRIPTION:

BRIEF SUMMARY
TECHNICAL FIELD

The present invention relates to screening methods for the identification of compounds and compositions useful as novel antibiotics, antibacterial agents, and inhibitors of prokaryotic enzyme activity. The invention also discloses a high-throughput assay system useful in the large-scale screening of protein kinase inhibitors and related methods.


BACKGROUND OF THE INVENTION

Antimicrobial therapy has focussed on antibiotics that affect processes unique to bacteria, such as compositions that affect enzymes and components of the cell wall (e.g., penicillin), and prokaryotic ribosome inhibitors (e.g., streptomycin). To a lesser extent, antibiotic therapy has also exploited structural and catalytic differences between enzymes common to prokaryotes and eukaryotes.
Antimicrobial or antibiotic therapy is dependent upon the discovery of biochemical systems that are unique to bacteria and which can be safely inhibited--i.e., systems which can be inhibited without producing detrimental effects or undesired side effects in or upon the individual receiving such therapy. Further, it has been observed that antibiotic resistance increases in pathogen populations due to recruitment of resistance enzymes from the microbial gene pool, partially as a result of antibiotic overuse or misuse. As resistance develops, it has become increasingly difficult to identify unique biochemical pathways which may be inhibited in bacteria, which are not also represented in the cells of higher organisms, including man.
Therefore, one goal of the present disclosure is the revelation of a new, apparently ubiquitous biochemical and regulatory system unique to bacteria, which may be exploited for antimicrobial therapy. Unique regulatory proteins are described herein which are absent from eukaryotic cells and could provide novel targets for antimicrobial therapy.
One exemplary "target" regulatory system involves bacterial protein kinases. Histidine protein kinase plays an important role in bacterial signal transduction. Typically, histidine protein kinase activity is assayed via a two-step procedure including a phosphorylation reaction in the presence of [.gamma.-.sup.32 P]ATP followed by SDS-PAGE and autoradiography analysis. Application of this method in large-scale screening for histidine protein kinase inhibitor is limited, however, largely because of the need to use SDS-PAGE.
The regulation of biological activities of proteins by reversible phosphorylation plays an important role in control of cellular response to extracellular stimuli in both prokaryotic and eukaryotic organisms. Phosphorylation cascades mediated by bacterial two-component systems provide a conserved mechanism for coordinate regulation in response to signal input. In bacteria, diverse processes such as chemotaxis (Hess, et al., PNAS USA 84: 7609-7613 (1987); Wylie, et al., Biochem. Biophys. Res. Commun. 151: 891-896 (1988); Hess, et al., Cell 53: 79-87 (1988)), nitrogen starvation (Ninfa and Magasanik, PNAS USA 83: 5909-5913 (1986); Keener and Kotsu, PNAS USA 85 4976-4980 (1988); Weiss and Magasanik, PNAS USA 85: 8919-8923 (1988)), osmotic regulation (Aiba, et al., J. Biol. Chem. 264: 8563-8567 (1989); Forst, et al., PNAS USA 86: 6052-6056 (1989); Igo, et al., Genes & Dev. 3: 589-605 (1989)), sporulation (Perego, et al., J. Bacteriol. 171: 6187-6196 (1989)), and certain types of antibiotic resistance (Christopher, Science 261: 308-309 (1993); Guenzi, et al., Mol. Microbiol. 12: 505-515 (1994)) are regulated by a two-component system.
In general, two-component systems comprise a sensor protein (usually a histidine protein kinase) and a response regulator protein. The histidine protein kinase undergoes ATP-dependent autophosphorylation on a histidine residue in response to a stimulus. The phosphorylated sensor protein then transfers the phosphor group to an aspartyl residue of the response regulator protein, which protein either acts as a transcriptional regulator or interacts with another protein.
Inhibition of either the autophosphorylation or the subseque

REFERENCES:
patent: 5747276 (1998-05-01), Hoch et al.
Burbulys et al; Cell, vol. 64; pp. 545-552, 1991.
Perego et al; J. Bacteriol.; vol. 171, pp. 6187-6196 (1989).
Satola et al; Proc. Natl. Acad. Sci; USA, vol. 88, pp. 4533-4537, (1991).
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Trach et al; Res. Microbiol.; vol. 142, pp. 815-823, (1991).
Trach et al; Molec. Microbiol; vol. 8, pp. 69-79, (1993).

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