Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-12-21
2004-11-30
Lacourciere, Karen A. (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007370, C435S170000
Reexamination Certificate
active
06824979
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns methods of screening for active agents useful as an antibacterial agent against
Pseudomonas,
as well as active agents and methods of use thereof in treating
Pseudomonas.
BACKGROUND OF THE INVENTION
Pseudomonas
infection is a leading cause of death in cystic fibrosis, and one of the top causes of serious hospital-acquired infections.
Pseudomonas
infection is particularly serious in burn victims and leukemia patients, and can cause blindness by infection in patients afflicted with trauma to the eye through surgery or contact lenses. Further,
Pseudomonas
has a high intrinsic resistance to current antibiotics. Hence, there is a need for the development of new antibiotics to treat
Pseudomonas
infections.
The use of antisense oligonucleotides in the treatment of bacterial infections is known. U.S. Pat. No. 5,294,533 to J. Lupski and L. Katz (assigned to Baylor College of Medicine and Abbott Laboratories, Inc.) describes a method of interrupting the expression of a macromolecular synthesis operon in bacteria comprising the step of binding an antisense oligonucleotide to a single stranded DNA or to a mRNA transcribed from the macromolecular synthesis operon. The antisense oligonucleotide can be either sequence specific to a unique intergenic sequence or a sequence specific to a bacterial homologous sequence. By interrupting the expression of the macromolecular synthesis, it is said that bacterial infections can be treated.
U.S. Pat. No. 6,060,241 to I. Corthesy-Theulaz (assigned to Kieta Holding SA) describes a poly-3-hydroxybutyrate metabolic pathway essential for
Helicobacter pylori
survival in a host. A
Helicobacter pylori
Coenzyme A transferase (Hp CoA-t), thiolase and PHB synthase, as well as methods for their preparation and use are provided. Pharmaceutical compositions containing Hp CoA-t protein fragments, antisense nucleic acids or other inhibitors of Hp CoA-t, thiolase and PHB synthase, as well as methods for their use in the treatment of some types of gastric disease are also described. This reference is not concerned with
Pseudomonas.
PCT Application WO98/03533A1 to A. Arrow et al. (assigned to Oligos Etc. and Oligos Therapeutics Inc.) describes the general therapeutic use of nuclease resistant oligonucleotides for treating animals having an infection caused by a pathogenic bacterium. The method is a general one and involves the integration of (1) methods for selecting the correct oligonucleotide, (2) synthesis and purification of nuclease resistant oligonucleotides, and (3) methods for in vitro analysis of potential antimicrobial oligonucleotides.
There remains a need for new ways to screen for antibiotics effective against
Pseudomonas,
along with compounds and methods of treating
Pseudomonas
infections.
SUMMARY OF THE INVENTION
The present invention provides a method of screening for compounds that inhibit the virulence of
Pseudomonas
bacteria. The method comprises the steps of: providing a culture medium comprising
Pseudomonas
bacteria; administering a test compound to said bacteria; and then detecting the presence or absence of inhibition of the catabolite repression control (Crc) protein in said bacteria, the inhibition of the Crc protein indicating said compound has antivirulence activity against
Pseudomonas
bacteria.
A second aspect of the present invention is an antivirulence compound (for example, an antisense oligonucleotide) that inhibits expression or activity of the Crc protein in a
Pseudomonas
bacteria. Such compounds are useful as antivirulence compounds. When such compounds are antisense oligonucleotides they are preferably from 8 to 25, 40 or 80 nucleotides in length, and preferably are nuclease resistant.
A third aspect of the present invention is an antivirulence compound as described above in a pharmaceutically acceptable carrier.
A fourth further aspect of the present invention is a method of inhibiting the virulence of
Pseudomonas
bacteria, comprising administering to
Pseudomonas
bacteria an antivirulence compound as described above in an effective antivirulence amount. The administering step may be carried out in vitro, for example in drug testing or screening studies, or may be carried out in vivo in the treatment of a subject.
A further aspect of the present invention is a method of treating
Pseudomonas
infection in a subject in need thereof, comprising administering to said subject an antivirulence compound as described above in an amount effective to treat said
Pseudomonas
infection.
A still further aspect of the present invention is the use of an antivirulence compound as described above for the preparation of a medicament for carrying out a method as described above.
The foregoing and other objects and aspects of the present invention are explained in greater detail in the specification set forth below.
REFERENCES:
patent: 5989912 (1999-11-01), Arrow et al.
patent: 6015886 (2000-01-01), Dale et al.
patent: 2002/0077272 (2002-06-01), Mahan et al.
patent: WO 98/03533 (1997-07-01), None
patent: WO 98/03533 (1998-01-01), None
Ch MacGregor et al., Journal of Bacteriology, “The Nucleotide Sequence of thePseudomonas aeruginosapyrE-crc-rph Region and the Purification of the crc Gene Product,” Oct. 1996, vol. 178, No. 19, pp. 5627-5635.*
Ga O'Toole et al., Journal of Bacteriology, “The Global Carbon Metabolism Regulator Crc is a Component of a Signal Transduction Pathway Required for Biofilm Development byPseudomonas aeruginosa,” Jan. 2000, vol. 182, No. 2, pp. 425-431.*
Bd Bright et al., 96thASM General Meeting, “crc Mutants ofP.aeruginosaHave Aletations in the Production of Diverse Virulence Factors,” May 1996, p. B-377.*
Bd Bright et al., 1995 Cystic Fibrosis Conference, “Involvement of the crc Locus in the Regulation of the Expression ofPseudomonas aeruginosaVirulence Factors,”Sep. 1995, p. 244.*
Wolf et al. Isolation and Characterization of Catabolite Repression Control Mutants ofPseudomonas aeruginosaPAO. Journa of Bacteriology, 1991 vol. 173:4700-4706.*
Symth et al. Catabolite Repression ofPseudomonas aeruginosaamidase: Isolation of Promoter Mutants.*
International Search Report for International application Ser. No. PCT/US01/51047 dated Aug. 9, 2002.
Bright et al.,Involvement of the crc Locus in the Regulation of the Expression of Pseudomonas aeruginosa Virulence Factors, 231,1995 Cystic Fibrosis Conference, pp. 244 (1995).
Phibbs et al.,crc Mutants of P.aeruginosa Have Alterations in the Production of Diverse Virulence Factors, B-377, 96th ASM General Meeting, pp. 220 (May 1996).
Collier, et al.,Isolation and Characterization of Pseudomonas aeruginosa Mutants Containing Suppressors of Defective crc Alleles, K-94,Microbial Physiology and Metabolism, 97thASM General Meeting, pp. 357 (May 1997).
Hester, et al.,Crc is Involved in Catabolite Repression Control of the bkd Operons of Pseudomonas putida and Pseudomonas aeruginosa, vol. 182, No. 4,Journal of Bacteriology, pp. 1144-1149 (Feb. 2000).
MacGregor, et al.,The Nucleotide Sequence of the Pseudomonas aeruginosa pyrE-crc-rph Region and the Purification of the crc Gene Product, vol. 178, No. 19, pp. 5627-5635 (Oct. 1996).
Collier, et al.,Isolation and Characterization of Pseudomonas aeruginosa Mutants Containing Suppressors of Defective crc Alleles, FEMS Microbiology Letters, vol. 196, pp. 87-92 (2001).
Breithaupt, Holger,The new antibiotics—Can novel antibacterial treatments combat the rising tide of drug-resistant infections?, Nature Biotechnology, vol. 17, pp. 1165-1169 (Dec. 1999).
Larsen, H. Jakob, et al.,Antisense properties of peptide nucleic acid, Biochimica et Biophysica Acta, col. 1489, pp. 159-166 (1999).
White, D.G., et al.,Inhibition of the Multiple Antibiotic Resistance(mar)Operon in Escherichia coli by Antisense DNA Analogs, Antimicrobial Agents and Chemotherapy, vol. 41, No. 12, pp. 2699-2704 (Dec. 1997).
Moellering, Robert C.,Antibiotic Resistance: Lessons for the Future, Clinical Infectious Diseases, vol. 27 (Suppl 1), pp. S135-40 (1998).
MacGregor, C.H., et al.,Cloning of a C
Collier David N.
Hager Paul W.
Phibbs Paul V.
East Carolina University
Gibbs Terra C.
Lacourciere Karen A.
Myers Bigel & Sibley & Sajovec
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