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Compositions and methods for antibiotic potentiation and drug discovery

a technology of antibiotic potentiation and composition, applied in the field of compositions and methods for antibiotic potentiation and drug discovery, can solve the problems of few antibiotics that are effective against, the emergence of resistance to antibacterial agents is a growing problem, and the potentiating antibiotic agent is too toxic for clinical us

Inactive Publication Date: 2009-10-22
TRUSTEES OF BOSTON UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides methods for potentiating the activity of antibiotics and identifying new antibiotics. The invention involves contacting a microbial cell with an antibiotic and an agent that potentiates the antibiotic's activity. The potentiator may inhibit or interfere with the expression of a gene that is involved in the antibiotic's mechanism of action. The invention also provides an assay system for detecting the expression or activity of a potentiator target gene. The invention can help to treat bacterial infections more effectively by using existing antibiotics in a new way.

Problems solved by technology

The emergence of resistance to antibacterial agents is a growing problem for human and animal health, and new drugs to treat infections due to microorganisms that display resistance to currently used antibiotics are urgently needed.
In addition, few antibiotics that are effective against bacterial that have developed resistance to currently used antibiotics are in clinical development.
Furthermore, a number of potent antibiotic agents are too toxic for clinical use or have significant side effects that limit their therapeutic utility.
Quinolones form a ternary complex between DNA and either gyrase or topoisomerase IV, thereby blocking DNA replication and leading to events such as double-stranded DNA breaks that are rapidly lethal to the cell.
Quinolones, like most other antibiotics, can have adverse effects with the potential to affect virtually every major system in the body.
Fluoroquinolones can also cause hypersensitivity reactions.
The adverse effects of many potent quinolones has hindered efforts to develop them as therapeutic agents.
Unfortunately, the side effects of the compound were significant enough that the further development by the pharmaceutical company was terminated.
In addition, the rapid development of resistance to a number of quinolones is a cause for alarm and has led to a number of treatment failures.

Method used

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  • Compositions and methods for antibiotic potentiation and drug discovery
  • Compositions and methods for antibiotic potentiation and drug discovery
  • Compositions and methods for antibiotic potentiation and drug discovery

Examples

Experimental program
Comparison scheme
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example 1

Identification of Mutations that Potentiate Quinolone Activity Using a Growth Assay

[0342]In order to discover genetic targets for quinolone potentiating agents the present Applications have established a screen to identify E. coli mutants that would be largely unable to grow in the presence of a concentration of quinolone that would not allow the growth of wild type E. coli. They decided to initially focus on two important members of this compound class, i.e., norfloxacin (Nor) and ciprofloxacin (Cipro). The growth of wild type E. coli was first shown to be fully inhibited by 0.1 μg / mL of Nor (a lethal concentration) but not by 0.05 μg / mL (a sublethal concentration). While the latter dose was shown to activate the SOS response using microarray analysis (data not shown), it allowed bacterial growth to 80% of wild type levels. The wild type E. coli was also shown to be able to grow in the presence of 0.1 μg / mL of Cipro (a sublethal concentration).

[0343]0.05 μg / mL of Nor and Cipro at 0...

example 2

Identification of Mutations that Potentiate Quinolone Activity Using a Survival Assay

[0350]188 E. coli genes were identified for which a homolog existed in S. aureus (MRSA 252) by performing searches of publicly available sequence databases. Homologs were identified using the NCBI Genplot pairwise genome comparison of protein homologues. A 96 well plate format was then used to perform a survival assay on a set of E. coli deletion strains, each of which had a deletion in one of these highly conserved genes. The survival assay measured the number of cells which form colonies (colony forming units, or CFU) after growth in the presence of a compound. This assay was applied to identify deletion strains which cannot survive following a limited time period of exposure to a quinolone treatment at a lethal concentration as compared to survival of the parent strain. The lethal concentration one that inhibits growth and would eventually be lethal if exposure was continued indefinitely. 2 μg / mL...

example 3

recA Mutant Response to Nor and Cipro

[0354]As described in Examples 1 and 2, deletion of recA in E. coli was shown to result in an inability to grow in the presence of sublethal concentrations of either Nor or Cipro (Example 1) and a reduced survival following exposure to lethal concentrations of Nor or Cipro (Example 2). The present Applicants decided to focus on recA as an exemplary target gene. Loss of recA function was first confirmed not to alter growth of E. coli (data not shown). The ability of Nor-sensitive and Nor-resistant wild type E. coli and recA deletion mutants to survive in the presence of Nor was then tested using the survival assay described in Example 2.

[0355]Nor-resistant E. coli strains were then constructed by introducing mutations into the gyrA locus using a standard molecular genetics approach (Datsenko and Wanner, 2000) and selecting for cells able to form colonies on plates containing Nor. The transformants were plated on LB plus Nor at 0.1 μg / mL. The indiv...

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Abstract

The present invention provides methods for identifying target genes whose partial or complete functional inactivation potentiates the activity of an antibiotic agent, e.g., a quinolone antibiotic. The invention further provides methods for identifying agents that modulate expression of target genes or that modulate activity of expression products of target genes. Agents identified according to various methods of the invention potentiate the activity of antibiotics such as quinolones, aminoglycosides, peptide antibiotics and β-lactams. Also provided are agents that suppress and / or retard resistance to antibiotics. The inventive methods provide potentiating agents and compositions comprising potentiating agents and antibiotics. Such agents and compositions can be used for inhibiting growth or survival of a microbial cell or of treating a subject suffering from or susceptible to a microbial infection.

Description

RELATED APPLICATIONS[0001]The present application claims priority from U.S. Provisional Application No. 60 / 772,648 filed on Feb. 13, 2006 and entitled “Compositions and Methods for Antibiotic Potentiation and Drug Discovery”, and from U.S. Provisional Application No. 60 / 835,596 filed on Aug. 4, 2006 and entitled “Rec A Inhibition”. Each of the provisional applications is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION[0002]The emergence of resistance to antibacterial agents is a growing problem for human and animal health, and new drugs to treat infections due to microorganisms that display resistance to currently used antibiotics are urgently needed. Efforts to overcome the growing problem of resistance have included modification of known antibiotics, classical screening of new compound libraries and natural product libraries, and genomic efforts to identify novel targets to which no cross resistance with existing antibiotics would be anticipated. Even ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/00C12Q1/68C12N5/02A61K31/70A61K31/435A61K31/352
CPCA61K38/12A61K45/06C12Q1/18G01N33/5023A61K2300/00A61P31/04
Inventor COTTAREL, GUILLAUMEWIERZBOWSKI, JAMEYPAL, KOLLOLKOHANSKI, MICHAELDWYER, DANIELCOLLINS, JAMESALMSTETTER, MICHAELTHORMANN, MICHAELTREML, ANDREAS
Owner TRUSTEES OF BOSTON UNIV
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