Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-09-30
2002-10-29
Smith, Lynette R. F. (Department: 1645)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S004000, C435S007200, C435S007340, C435S036000, C435S069100, C435S069200, C435S069700, C435S071300, C436S501000, C436S518000, C424S404000, C424S116000, C536S023100, C536S023600, C536S023700
Reexamination Certificate
active
06472377
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the use of yneS, an essential bacterial gene, in 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 yneS gene of the gram positive bacterium
Streptococcus pneumoniae
, termed “S-yneS,” and of
Bacillus subtilus
, termed “B-yneS,” are essential for survival. The terms “yneS gene” and “yneS polypeptide” refer to the S-yneS and B-yneS genes, as well as their homologs and orthologs, collectively. While “homologs” are structurally similar genes contained within a species, “orthologs” are functionally equivalent genes from other species (within or outside of a given genus). The yneS gene is considered an “essential” gene, and the yneS polypeptide is considered an “essential” polypeptide. A yneS gene and polypeptide can be used in methods for identifying similar genes in pathogenic and non-pathogenic microorganisms. A yneS polypeptide can be used to identify compounds that are inhibitors of the pathogens in which the yneS polypeptide is expressed. Such inhibitors attenuate bacterial growth by inhibiting the activity of a yneS polypeptide, or by inhibiting transcription of a yneS gene or translation of the mRNA transcribed from the yneS gene.
The amino acid and nucleic acid sequences of S-yneS are set forth in
FIG. 1
as SEQ ID NOs:1 and 2, respectively. The amino acid and nucleic acid sequences of B-yneS are set forth in
FIG. 2
as SEQ ID NOs:3 and 4, respectively.
Now that the yneS gene has been identified and shown to be essential for survival, the yneS gene and polypeptide (including homologs and orthologs of the sequences disclosed herein) can be used to identify antibacterial agents. Such antibacterial agents can readily be identified with high throughput assays to detect inhibition of the metabolic pathway in which the yneS polypeptide participates. This inhibition can be caused by small molecules interacting with (e.g., binding directly or indirectly to) the yneS 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 essential yneS polypeptide 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 yneS protein, the gene encoding the yneS protein is deleted from the genome and replaced with a version of the gene in which the sequence encoding the yneS protein is operably linked to a regulatable promoter. The cells containing this regulatable genetic construct are kept alive by the essential yneS polypeptide produced from the genetic construct containing the regulatable promoter. However, the regulatable promoter allows the expression of the yneS 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 a promoter that drives expression of the identified RNA species. Such promoters are up- or down-regulated by depletion of the yneS polypeptide.
Having identified a promoter(s) that is up- or down-regulated by depletion of the yneS 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 yneS polypeptide (or other polypeptides in the essential pathway in which the yneS polypeptide participates) will cause a functional depletion of the yneS 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 yneS 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 essential yneS 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 yneS polypeptide by measuring the ability of the small molecule to stabilize the yneS polypeptide in its folded, rather than unfolded, state. More specifically, one can measure the degree of protection against unfolding that is afforded by the test compound. Test compounds that bind the yneS polypeptide with high affinity cause, for example, a large shift in the temperature at which the polypeptide is denatured. Test compounds that stabilize the yneS 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 essential yneS polypeptide is used to isolate peptide or nucleic acid ligands that specifically bind the yneS polypeptide. These peptide or nucleic acid ligands are then used in a displacement screen to identify small molecules that interact with the yneS polypeptide. Such assays can be carried out essentially as described above.
Another suitable method for identifying inhibitors of the essential yneS 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.
The various yneS 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 interact with the yneS polypeptides are candidate antibacterial agents, in contrast to compounds that do not interact with the yneS polypeptides. As described herein, any of a variety of art-known methods can be used to assay for the interaction of test compounds with the yneS polypeptides.
The invention also includes a method for identifying an antibacterial agent where the method entails: (a) contacting an essential yneS polypeptide with a test compound; (b) detecting binding of the test compound to the polypeptide; and, optionally, (c) determining whether a test compound that binds to the yneS polypeptide inhibits growth of bacteria, relative to growth of bacteria cultured in the absence of the test compound that binds to the yneS polyepeptide, as an indication that the test compound is an antibacterial agent.
In still another method, interaction of a test compound with a yneS polypeptide (e.g., binding) can be detected in a conventional two-hybrid system for detecting protein/protein interactions (e.g., in yeast or mammalian cells). A test compound found to interact with the yneS polypeptide can be further tested for antibacterial activity in a conventional susceptibility assay. Generally, in such two-hybrid methods, (a) the yneS polypeptide is provided as a fusion protein that includes the polypeptide fused to (i) a transcription
Fritz Christian
Guzman Luz-Maria
Youngman Philip
Baskar Padma
Fish & Richardson P.C.
Smith Lynette R. F.
LandOfFree
Use of YNES, essential bacterial genes and polypeptides does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Use of YNES, essential bacterial genes and polypeptides, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Use of YNES, essential bacterial genes and polypeptides will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2927666