Compositions and methods for antibiotic potentiation and drug discovery

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.

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 with this significant antibiotic discovery effort, only a few agents that represent new chemical classes of antibiotic agents have been approved by regulatory agencies in recent years. 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 are one of the most widely used classes of antimicrobial agents worldwide and serve to exemplify the promise and the problems associated with antibiotic development and use. Their therapeutic indications in humans have evolved from urinary tract infections to infections of almost all body compartments. Various members of the class are also widely administered for veterinary purposes.

Quinolones exert their antimicrobial effect by targeting bacterial type II topoisomerases, namely gyrase and topoisomerase IV, essential enzymes that catalyze breakage and rejoining of DNA strands during normal cell growth. 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. As a class, quinolones have a broad spectrum of activity against Gram positive and Gram negative species, including both aerobic and anaerobic microorganisms. Activity against Gram negative microorganisms often depends strongly upon activity against DNA gyrase, while topoisomerase IV is often the primary target of quinolones that display activity against Gram positive species. A number of therapeutically useful quinolones display activity towards both of these targets.

Quinolones, like most other antibiotics, can have adverse effects with the potential to affect virtually every major system in the body. The most frequent side effects associated with fluoroquinolone use involve the GI tract, skin, and/or central nervous system (CNS). Fluoroquinolones can also cause hypersensitivity reactions. A mild transient increase in liver function is seen in 2-3% of the patients who are treated with a fluoroquinolone, and severe liver toxicity is a rare side effect of quinolone use. Cardiac side effects such as prolongation of the QT interval, which may predispose to serious arrhythmias, is also a concern.

The adverse effects of many potent quinolones has hindered efforts to develop them as therapeutic agents. Gemifloxacin, for example, has an enhanced affinity for its target relative to many other quinolones and displays potent activity against most Gram positive cocci, particularly against Streptomyces pneumoniae. Gemifloxacin is 30 fold more active than ciprofloxacin against this pathogen. 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. The spread of resistance has motivated calls for the development of policies that would restrict unnecessary use of these agents. Resistance can develop in a variety of ways including mutations in the genes encoding either DNA gyrase or topoisomerase IV and overexpression of efflux pumps.

Quinolones are only one example of antibiotic agents that exhibit side effects and/or to which resistance has been developed. Thus, there is clearly a need in the art both for new agents to combat microbial infection and for new approaches to antibiotic drug discovery. There is also a need in the art for new approaches to improving the safety profile of certain antibiotics.

The present invention provides compositions and methods for potentiating the activity of antibiotic agents and for discovering new agents of use in treating bacterial infection.

In one aspect, the invention provides a method of inhibiting growth or survival of a microbial cell comprising: (a) contacting the microbial cell with a antibiotic (e.g., an quinolone); and (b) contacting the cell with an agent that potentiates activity of the antibiotic.

In another aspect the invention provides a method of treating a subject in need thereof comprising: administering an antibiotic to the subject in combination with an agent that potentiates activity of the antibiotic. In certain embodiments of either of the foregoing methods, the agent preferably inhibits or interferes with activity of an expression product of an antibiotic potentiator target gene. In either of the methods the agent and the antibiotic may be administered individually or as components of a single composition. The antibiotic may be used at a concentration below its MIC, or may be administered at a dose below that at which it would be effective as a single agent.

In another aspect, the invention provides a method of identifying an antibiotic potentiator target gene comprising steps of: (a) contacting a microbial cell with an antibiotic, wherein the microbial cell has a genetic alteration that significantly reduces or eliminates expression of a gene; (b) comparing growth or survival of the microbial cell with growth or survival of a microbial cell having higher expression of the gene than the cell having the genetic alteration; and (c) determining that the gene is an antibiotic potentiator target gene if the growth, survival, or both of the microbial cell having the genetic alteration is lower than the growth, survival, or both of the microbial cell having higher expression of the gene.

In another aspect the invention provides a set of target genes whose functional inactivation potentiates the activity of antibiotics. The set comprises dapF, fabH, fis, fliN, jw5303, priA, recA, recB, recC, recG, resA, ruvA, ruvB, ruvC, ruvC, uvrD, ycjS, ydfL, yhfT, yqgC, aceE, araJ, aspA, atpA, atpF, atph, brnQ, cydB, degP, dnaT, fepD, folP, guaB, Hfq, iscS, JW53600, lipA, lpdA, nuoB, nuoJ, nuoL, osmB, pdxH, pnuC, priA, priB, rbfA, recA, rfaD, rpmJ, rpoD, resA, ruvC, sdhC, seqA, setA, sucB, surA, tar, tatE, tauB, trmU, ubiH, ubiX, xerC, yaaU, ycfM, yejO, ygfZ, ygiH, yheL, yheM, yheN, yicG, yjdL, yjeT, yjiO, yjjY, agaAk, atpA, atpF, atpH, bglF, cysE, cysI, fepC, fepD, frvR, guaA, guaB, hofF, hsdS, iscS, JW4016, JW5075, JW5227, JW5257, JW5360, kdgK, lipA, IysA, malG, mbhA, mdoG, Nei, nmpC, nudH, pdxH, phnB, phnL, phnO, pnuC, potE, pshM, ptsA, rhaT, rpiA, resA, Sbp, speA, sucB, sugE, tdcE, tdcG, tolC, trxA, ubiE, ubiH, ubiX, Xni, ybbY, ycfM, ydeJ, yeeY, yfeT, ygaA, ygfZ, yhdX, yheL, yheM, yiaY, yidK, yihV, yjbN, yjcR, yjcZ, ynjD, yqeC, yqiH, and yrfA.

In another aspect the invention provides an assay system comprising: (a) a test agent; and (b) components suitable for performing an assay that detects expression of an antibiotic potentiator target gene or detects activity of an expression product of an antibiotic potentiator target gene, wherein the gene is from the set described above.

The invention further provides methods of identifying an agent that modulates expression and/or activity of an antibiotic potentiator target gene or its expression product.

One such method comprises (a) providing an assay system comprising a test compound, wherein the assay system is suitable for performing an assay to detect expression of an antibiotic potentiator target gene or to detect activity of an expression product of an antibiotic potentiator target gene; and (b) determining whether the test compound inhibits expression of the antibiotic potentiator target gene or inhibits or interferes with activity of an expression product of the gene; and (c) identifying the test compound as a potentiator of the antibiotic if the test agent inhibits expression of the potentiator target gene or inhibits or interferes with activity of an expression product of the potentiator target gene.

A variety of assays suitable for identifying agents that inhibit polypeptides encoded by specific target genes, e.g., RecA, are provided. Assays provided include binding assays, reporter-based assays, and enzymatic assays. Also provided are collections of bacterial strains suitable for performing the target gene identification methods of the invention and/or for identifying agents that modulate expression of a target gene or modulate activity of an expression product of a target gene.

In another aspect, the invention provides a method of identifying an agent comprising steps of: (a) computationally analyzing interaction of each of a plurality of test agents with a protein comprising a polypeptide encoded by an antibiotic potentiator target gene with each of a plurality of test agents; and (b) identifying at least one test agent having favorable interactions with the active site. The agent may be further tested, e.g., to determine whether it potentiates activity of an antibiotic against bacteria.