Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-09-30
2003-02-04
Yucel, Remy (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C435S252300, C435S320100, C435S254200
Reexamination Certificate
active
06515119
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the use of S-ydcB and B-ydcB, which are essential bacterial genes useful for identifying antibacterial agents.
BACKGROUND OF THE INVENTION
Bacterial infections may be cutaneous, subcutaneous, or systemic. Opportunistic bacterial infections proliferate, especially in patients afflicted with AIDS or other diseases that compromise the immune system. Most bacteria that are pathogenic to humans are gram positive bacteria. The bacterium
Streptococcus pneumoniae
, for example, typically infects the respiratory tract and can cause lobar pneumonia, as well as meningitis, sinusitis, and other infections.
SUMMARY OF THE INVENTION
The invention is based on the discovery that the S-ydcB gene in the gram positive bacterium
Streptococcus pneumoniae
and the B-ydcB gene in
Bacillus subtilis
are essential for survival. These genes are considered “essential” genes, and the S-ydcB and B-ydcB polypeptides are considered “essential” polypeptides. The invention features methods for using these genes and polypeptides to identify antibacterial compounds that inhibit a wide variety of bacteria (e.g., gram-positive and gram-negative bacteria, such as Streptococcus, Bacillus, and
E. coli
). Such inhibitors attenuate bacterial growth by inhibiting the activity of an S-ydcB or B-ydcB polypeptide (or homolog or ortholog thereof), or by inhibiting transcription of an S-ydcB or B-ydcB gene (or homolog or ortholog), or by inhibiting translation of the mRNA transcribed from an S-ydcB or B-ydcB gene (or homolog or ortholog). The S-ydcB and B-ydcB genes and polypeptides also are included within the invention and can be used in methods for identifying homologous genes and polypeptides in other bacterial strains.
The amino acid and nucleic acid sequences of S-ydcB are set forth in
FIG. 1
as SEQ ID NOs:2 and 1, respectively. The amino acid and nucleic acid sequences of B-ydcB are set forth in
FIG. 2
as SEQ ID NOs:4 and 3 respectively.
Now that the S-ydcB and B-ydcB genes described herein have been identified and shown to be essential for survival, these genes and polypeptides and their homologs can be used to identify antibacterial agents. “Homologs” are structurally similar genes contained within a species, while “orthologs” are functionally equivalent genes in other species. The identified antibacterial agents can readily be identified with high throughput assays to detect inhibition of the S-ydcB or B-ydcB polypeptide, or essential polypeptides with which S-ydcB and B-ydcB associate (e.g., in a pathway). This inhibition can be caused by small molecules interacting with (e.g., binding directly or indirectly to) the S-ydcB or B-ydcB polypeptide or other essential polypeptides in that pathway.
In an exemplary assay, but not the only assay, a promoter that responds to depletion of the B-ydcB or S-ydcB polypeptide (or homolog thereof) by upregulation or downregulation is linked to. a reporter gene. To identify a promoter that is up- or down-regulated by the depletion of a B-ydcB or S-ydcB protein, the gene encoding the B-ydcB or S-ydcB protein is deleted from the genome and replaced with a version of the gene in which the sequence encoding the B-ydcB or S-ydcB protein is operably linked to a regulatable promoter. The cells containing this regulatable genetic construct are kept alive by the B-ydcB or S-ydcB polypeptide produced from the genetic construct containing the regulatable promoter. However, the regulatable promoter allows expression of the B-ydcB or S-ydcB polypeptide to be reduced to a level that causes growth inhibition. Total RNA prepared from bacteria under such growth-limiting conditions is compared with RNA from wild-type cells. Standard methods of transcriptional profiling can be used to identify mRNA species that are either more or less abundant (i.e., up- or down-regulated) when expressed under the limiting conditions. Genomic sequence information, e.g., from GenBank, can be used to identify the promoter that drives expression of the identified RNA species. Such promoters are up- or down-regulated by depletion of the B-ydcB or S-ydcB polypeptide.
Having identified a promoter(s) that is up- or down-regulated by depletion of a B-ydcB or S-ydcB polypeptide, the promoter(s) is operably linked to a reporter gene (e.g., &bgr;-galactosidase, gus, or green fluorescent protein (GFP)). A bacterial strain containing this reporter gene construct is then exposed to test compounds. Compounds that inhibit the B-ydcB or S-ydcB polypeptide (or other polypeptides in an essential pathway in which the B-ydcB or S-ydcB polypeptide participates) will cause a functional depletion of the B-ydcB or S-ydcB polypeptide and therefore lead to an upregulation or downregulation of expression of the reporter gene. Because the polypeptides described herein are essential for the survival of bacteria, compounds that inhibit the B-ydcB or S-ydcB polypeptides in such an assay are expected to be antibacterial and can be further tested, if desired, in standard susceptibility assays.
Another suitable method for identifying antibacterial compounds involves screening for small molecules that specifically interact with (i.e., bind directly or indirectly to) the B-ydcB or S-ydcB polypeptide. A variety of suitable interaction and binding assays are known in the art as described, for example, in U.S. Pat. Nos. 5,585,277 and 5,679,582, incorporated herein by reference. For example, in various conventional assays, test compounds can be assayed for their ability to interact with a B-ydcB or S-ydcB polypeptide by measuring the ability of the small molecule to stabilize the B-ydcB or S-ydcB polypeptide in its folded, rather than unfolded, state. More specifically, one can measure the degree of protection from unfolding that is afforded by the test compound. Test compounds that bind the polypeptide with high affinity cause, for example, a large shift in the temperature at which the polypeptide is denatured. Test compounds that stabilize the B-ydcB or S-ydcB polypeptide in a folded state can be further tested for antibacterial activity in a standard susceptibility assay.
In a related method for identifying antibacterial compounds, the B-ydcB or S-ydcB polypeptide is used to isolate peptide or nucleic acid ligands that specifically bind the B-ydcB or S-ydcB polypeptide. These peptide or nucleic acid ligands are then used in a displacement screen to identify small molecules that interact with the B-ydcB or S-ydcB polypeptide. Such assays can be carried out essentially as described above.
Another suitable method for identifying inhibitors of the B-ydcB or S-ydcB polypeptide involves identifying a biochemical activity of the polypeptide and then screening for small molecule inhibitors of the activity using, for example, a high throughput screening method. S-ydcB and B-ydcB catalyze a reaction of CoenzymeA plus apo-Acyl Carrier Protein to produce holo-Acyl Carrier Protein and 3′,5′-ADP (PAP). Based on this activity, various biochemical assays can be set up as high throughput screens to detect compounds that inhibit the enzymatic activity of ydcB. For example, incorporation of a labelled :version of CoenzymeA into the holoACP protein can readily :be detected. The label can be fluorescent, radioactive, or any easily detectable moiety, such as biotin. The holo-ACP protein can be the Acyl Carrier Protein derived from; any of a wide variety of bacteria, or it can be a peptide fragment thereof or a fusion portein containing ACP sequences. In an alternative assay, the production of PAP from the catalytic reaction described above is detected. PAP can be detected in a calorimetric assay in which sulfotransferase uses PAP as a cofactor (Lin et al., Analytical Biochemistry).
The various B-ydcB and S-ydcB polypeptides can be used, separately or together, in assays to identify test compounds that interact with these polypeptides. Test compounds that interact with these polypeptides then can readily be tested, in conventional assays, for their ability to inhibit bacterial growth. Test compounds that in
Fritz Christian
Guzman Luz-Maria
Youngman Philip
Fish & Richardson P.C.
Katcheves Konstantina
Millennium Pharmaceuticals Inc.
Yucel Remy
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