# Patent

## Title
In vivo assay for identification of antimicrobial agents

## Abstract

The present invention provides novel in vivo assay systems and methods of using these assays systems to identify compounds that affect microbial cell division. The present invention further provides pharmaceutical compositions that have anti-microbial activity and methods of treating microbial infections.

## Background
PRIORITY INFORMATION
This application is a continuation-in-part of U.S. National application Ser. No. 10/153,268, filed May 22, 2002, which claims benefit of and priority to U.S. provisional application Ser. No. 60/292,883, filed May 22, 2001. This application is related to the subject matter in and claims benefit of and priority to co-pending U.S. provisional application Ser. No. 60/300,931, filed Jun. 26, 2001. The contents of each of these applications is incorporated herein by reference.


GOVERNMENT SUPPORT
Development of the present invention was funded by a grant from the Department of Defense Advanced Research Projects Agency (Grant Number N65236-98-1-5408. Accordingly, the United States Government may have certain rights in the invention.


BACKGROUND OF THE INVENTION
Anti-microbial agents, such as antibiotics, have been effective tools in the treatment of infectious diseases during the last half-century. The systematic screening of natural product libraries from soil samples or marine environments has generated most of the classes of anti-bacterial agents used today (e.g., β-lactams, aminoglycosides, macrolides, and sulfonamides, to name a few). Additionally, these initial leads have, in many cases, been subsequently modified to produce second and third generation therapeutics with one or more of broadened anti-microbial activity, enhanced oral bioavailability, and improved toxicological and pharmacokinetic properties.
From the time that antibiotic therapy was first developed to the late 1980s, there was almost complete control over bacterial infections in developed countries. However, the emergence of resistant bacteria, especially during the late 1980s and early 1990s, is changing this situation (see, for example, Breithaupt, H., “The New Antibiotics: Can Novel Anti-bacterial Treatments Combat the Rising Tide of Drug-Resistant Infections?” Nature Biotechnology, (1997) 17: 1165). The increase in antibiotic resistant strains has been particularly common in major hospitals and care centers. The consequences of the increase in resistant strains include higher morbidity and mortality, longer patient hospitalization, and an increase in treatment costs. (B. Murray, New Engl. J. Med. 330: 1229–1230 (1994)).
One major factor that is contributing to the increase in the number of resistance strains is the over-use and/or inappropriate administration of anti-microbials in the treatment arena. Newly acquired resistance is generally due to the relatively rapid mutation rate in bacteria. Another contributing factor is the ability of many microorganisms to exchange genetic material that confers resistance, e.g., exchanging of resistance plasmids (R plasmids) or resistance transposons.
For example, following years of use to treat various infections and diseases, penicillin resistance has become increasingly widespread in the microbial populations that were previously susceptible to the action of this drug. Some microorganisms produce β-lactamase, an enzyme that destroys the anti-microbial itself, while other microorganisms have undergone genetic changes that result in alterations to the cell receptors known as the penicillin-binding proteins, such that penicillin no longer effectively binds to the receptors. Other organisms have evolved in a manner that prevents the lysis of cells to which the drug has bound. The drug therefore inhibits the growth of the cell, but does not kill the cell. This appears to contribute to the relapse of disease following premature discontinuation of treatment, as some of the cells remain viable and may begin growing once the anti-microbial is removed from their environment.
The first report of penicillin resistance occurred in Australia in 1967. Since this initial report, increasing numbers of penicillin resistant strains have been reported worldwide. In addition, strains having resistance to numerous other antibiotics have also been reported, including strains that are resistant to chloramphenicol, erythromycin, tetracycline, clindamycin, rifampin, and sulfamethoxazole-trimethoprim.
Microorganisms that are resistant to this wide range of drugs include opportunistic and virulent pathogens that were previously susceptible to antibiotic treatment. Resistant opportunistic pathogens are problematic for debilitated or immunocompromised patients, while the development of tolerance and resistance in virulent pathogens poses a significant threat to the ability to treat disease in all patients, compromised and non-compromised. Infections resulting from these naturally resistant opportunistic or virulent pathogens are becoming more difficult to treat with currently available antibiotics.
Clearly, in order to maintain the standard of public health we enjoy today, there is an urgent medical need for the identification of compounds having anti-microbial activity that can override existing mechanisms of resistance. Preferably, the anti-microbial compounds are active against a broad spectrum of microorganisms, while remaining non-toxic to human and other mammalian cells.
SUMMARY OF THE INVENTION
The invention provides in vivo assay systems and methods of using these assay systems for screening compounds for anti-microbial activity. In particular, the present invention provides in vivo assay systems that utilize conditional-lethal and other non-lethal conditional bacterial mutants in target gene products to screen compounds for anti-microbial activity. For example, the present invention provides a phenotypic screen for compounds that inhibit bacterial cell division.
The present invention further provides pharmaceutical compositions including anti-microbial agents and methods of using such pharmaceutical compositions to treat microbial infections and/or disorders related to microbial infections. The compounds can be used in combination with other agents for the prophylaxis and treatment of conditions associated with microbial infections and/or disorders related to microbial infections.
In certain preferred embodiments, microorganisms are not resistant to the identified anti-microbial agents, exhibit improved bioavailability, and/or have minimal side effects. In a particularly preferred embodiment of the invention the compounds are effective against certain microorganisms that are resistant to some or even all of the anti-bacterial agents that are currently approved or in clinical trials.
The pharmaceutical compositions can be used alone or in combination with other agents for the prophylaxis and treatment of conditions associated with microbial infections or disorders related to microbial infections. In general, the inventive compositions comprise an effective amount of an anti-microbial compound or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier, such as a diluent or excipient.
In still another aspect, the invention provides methods for prophylaxis and/or treatment of conditions associated with microbial infections and/or disorders related to microbial infections by administering an effective amount of an inventive compound. In particular, the invention provides a method for the treatment or prophylaxis of conditions associated with microbial infections and/or disorders related to microbial infections comprising administering to a host (such as a bird, fish, or cell) or patient, such as a primate, an effective amount of a compound of the present invention.
In certain preferred embodiments combination therapies are provided wherein an effective amount of a compound of the present invention, and an effective amount of one or more other compounds useful in the treatment of conditions associated with microbial infections and/or disorders related to microbial infections, are administered to a host or patient.
In yet another aspect, the present invention also provides pharmaceutical packs or kits comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention, and in certain embodiments, includes an additional approved therapeutic agent for use as a combination therapy. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use or sale for human administration. The invention further provides novel assays for the identification of agents having anti-microbial activity, e.g., anti-bacterial, e.g., any eubacteria or archaebacteria. In particular, these assays inhibit the ubiquitous prokaryotic cell division protein FtsZ. Such anti-microbial agents have the activity of inhibiting cell division by blocking the formation of the FtsZ ring that is crucial for septation. In other embodiments, the identified compounds and compositions may be inhibitory to plant cell division and be useful to kill weeds.



BRIEF DESCRIPTION OF THE DRAWINGS
The invention is described with reference to the several Figures of the drawing.
FIG. 1 is a drawing depicting FtsZ mediated cell division in different cell types.
FIG. 2 is a phylogenetic tree of FtsZ orthologs.
FIG. 3 shows a diagram of an FtsZ ring structure and photographs of an FtsZ ring structure by immunofluorescence in predivisional E. coli cells.
FIG. 4 is a drawing showing proteins involved in septal ring assembly in E. coli. 
FIG. 5 is an electron micrograph of FtsZ protofilaments that form in the presence e of GTP.
FIG. 6 is an illustration showing four possible arrangements of a linear protofilaments assembled from identical subunits.
FIG. 7 is an illustration that depicts an overview of a screening process for identifying FtsZ inhibitors.
FIG. 8 shows photographs of immunofluorescent stains and phase contrast microscopy of ftsZ84 mutant cells with thermolabile FtsZ84 rings.
FIG. 9 is an illustration of the malachite green assay.
FIG. 10 is a flow chart representing the results of a library screen using the inventive assay.
FIG. 11 illustrates the chemical structure of various inhibitors of FtsZ activity.
FIG. 12 shows images of E. coli cells that contain an AcrAB mutation and express an FtsZ-GFP fusion protein, which have been treated with the compound 58P-18.
FIG. 13 is an electron micrograph image of the effect of compounds 18M04 and 27D12 that destabilize FtsZ polymers in a dose dependent manner.
FIG. 14 is an electron micrograph of protofilaments in the presence of compound 27D-12.
FIG. 15 is an electron micrograph image of the effect of compounds 16L-09, 27F02, and 58P18 that cause mild bundling of FtsZ protofilaments.
FIG. 16 illustrates the chemical structure of an inhibitor of FtsZ ring assembly.
FIG. 17 shows images that illustrate the effect of compound 26E-10 on FtsZ ring assembly in E. coli. 
FIG. 18 shows images that illustrate the effect of compound 26E-10 on FtsZ ring assembly in E. coli. 
FIG. 19 is a graph that identified activation or FtsZ polymerization that shows fluorescence vs. time in the presence and absence of compound 26E-10.
FIG. 20 shows images that illustrate that compound 26E-10 causes synthetic lethality of ftsZ84-ts E. coli cells (acrAB-null)/pBR322 at 30° C.
FIG. 21 illustrates the inhibition of FtsZ-mediated cell division in M. tuberculosis of several compounds.
FIG. 22 is a table illustrating the minimum inhibitory concentrations against gram-positive and gram-negative bacteria of a number of compounds.
FIG. 23 shows a Cartesian pin transfer robot a pin array and a fluorescence plate reader that may be used to high-throughput screening with the assay of the invention.
FIG. 24 is a table showing minimum inhibitory concentrations of compounds on growth of E. coli (WT), E. coli (acrAB), E. coli ftsZ84acr AB, and Vibrio cholera. 
FIG. 25 is a photograph of DRC39 cells, which are wild-type E. coli cells that have a knockout of the multidrug efflux pump AcrAB, treated for two hours with 27F02.
FIG. 26 shows graphs illustrating the effect of an activator on FtsZ orthologs from E. coli and M tuberculosis. 
FIG. 27 is a flow chart that depicts the in vitro FtsZ screen that was carried out to identify five inhibitors of FtsZ activity.
FIG. 28 illustrates the in vitro enzyme-coupled assay for assembly dependent FtsZ GTPase activity.
FIG. 29 illustrates a TLC assay for FtsZ GTPase activity in the presence of compound I-20.
FIG. 30 illustrates the effect of a variety of compounds on FtsZ assembly in E coli determined by electron micrograph.
FIG. 31 is a table illustrating data for the effect of various compounds on cell division.
FIG. 32 is a table illustrating data for the effect of various compounds on cell division.
FIG. 33 is a table illustrating data for the effect of various compounds on cell division.
FIG. 34 is a table illustrating data for the effect of various compounds on cell division.



DEFINITIONS
As discussed above, the present invention provides pharmaceutical compositions including compounds useful in the eradication or inactivation (i.e., affect their inability to replicate) of harmful microorganisms prior to infection and thus can be utilized as preventative and/or disinfectant agents.
It will be appreciated by one of ordinary skill in the art that numerous asymmetric centers may exist in the compounds of the present invention. Thus, inventive compounds and pharmaceutical compositions thereof may be in the form of an individual enantiomer, diastereomer or geometric isomer, or may be in the form of a mixture of stereoisomers.
Additionally, the present invention provides pharmaceutically acceptable derivatives of the foregoing compounds, and methods of treating animals using these compounds, pharmaceutical compositions thereof, or either of these in combination with one or more additional therapeutic agents. The phrase, “pharmaceutically acceptable derivative”, as used herein, denotes any pharmaceutically acceptable salt, ester, or salt of such ester, of such compound, or any other adduct or derivative which, upon administration to a patient, is capable of providing (directly or indirectly) a compound as otherwise described herein, or a metabolite or residue thereof. Pharmaceutically acceptable derivatives thus include among others pro-drugs. A pro-drug is a derivative of a compound, usually with significantly reduced pharmacological activity, which contains an additional moiety that is susceptible to removal in vivo yielding the parent molecule as the pharmacologically active species. An example of a pro-drug is an ester that is cleaved in vivo to yield a compound of interest. Pro-drugs of a variety of compounds, and materials and methods for derivatizing the parent compounds to create the pro-drugs, are known and may be adapted to the present invention. Certain exemplary pharmaceutical compositions and pharmaceutically acceptable derivatives will be discussed in more detail herein below.
Certain compounds of the present invention, and definitions of specific functional groups are also described in more detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, the entire contents of which are incorporated herein by reference.
It will be appreciated that the compounds, as described herein, may be substituted with any number of substituents or functional moieties. In general, the term “substituted” whether preceded by the term “optionally” or not, and substituents contained in formulas of this invention, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. Furthermore, this invention is not intended to be limited in any manner by the permissible substituents of organic compounds. Combinations of substituents and variables envisioned by this invention are preferably those that result in the formation of stable compounds useful in the treatment of and/or prevention of bacterial infections, protozoal infections, or for disorders related to microbial infections. The term “stable”, as used herein, preferably refers to compounds which possess stability sufficient to allow manufacture and which maintain the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein.
The term “aliphatic”, as used herein, includes both saturated and unsaturated, straight chain (i.e., unbranched), branched, cyclic, or polycyclic aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, “aliphatic” is intended herein to include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term “alkyl” includes both straight, branched and cyclic alkyl groups. An analogous convention applies to other generic terms such as “alkenyl”, “alkynyl” and the like. Furthermore, as used herein, the terms “alkyl”, “alkenyl”, “alkynyl” and the like encompass both substituted and unsubstituted groups.
Unless otherwise specified, alkyl and other aliphatic groups preferably contain 1–6, or 1–3, contiguous aliphatic carbon atoms. Illustrative aliphatic groups thus include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, —CH2-cyclopropyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, —CH2-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl, —CH2-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, —CH2-cyclohexyl moieties and the like, which again, may bear one or more substituents.
In certain embodiments of the present invention C1–C3 or C1–C6 alkyl moieties are employed. As used herein, the terms “C1–C3-alkyl” and “C1–C6-alkyl” refer to saturated, substituted or unsubstituted, straight- or branched-chain hydrocarbon radicals derived from a hydrocarbon moiety containing between one and three, and one and six carbon atoms, respectively, by removal of a single hydrogen atom. Examples of C1–C3-alkyl radicals include, but are not limited to, methyl, ethyl, propyl and isopropyl. Examples of C1–C6-alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl and n-hexyl.
In certain embodiments of the present invention, C2–C6 alkenyl moieties are employed. The term “C2–C6-alkenyl” denotes a monovalent group derived from a hydrocarbon moiety containing from two to six carbon atoms and having at least one carbon-carbon double bond by the removal of a single hydrogen atom. Additionally, the C2–C6 alkenyl moieties, as used herein, may be substituted or unsubstituted. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.
In certain embodiments of the present invention, C2–C6 alkynyl moieties are employed. The term “C1–C6-alkynyl” as used herein refers to a monovalent group derived from a hydrocarbon containing from two to six carbon atoms and having at least one carbon-carbon triple bond by the removal of a single hydrogen atom. Additionally, the C2–C6 alkenyl moieties, as used herein, may be substituted or unsubstituted. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl and the like.
The term “C1–C6-alkoxy” as used herein refers to a C1–C6-alkyl group, as previously defined, attached to the parent molecular moiety through an oxygen atom. Examples of C1–C6-alkoxy, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy and n-hexoxy.
The term “alkylamino” refers to a group having the structure —NHR′ wherein R′ is alkyl, as defined herein. Examples of alkylamino include, but are not limited to, methylamino, ethylamino, iso-propylamino and the like. In certain embodiments, C1–C3 alkylamino groups are utilized in the present invention. The term “C1–C3-alkylamino” as used herein refers to one or two C1–C3-alkyl groups, as previously defined, attached to the parent molecular moiety through a nitrogen atom. Examples of C1–C3-alkylamino include, but are not limited to methylamino, dimethylamino, ethylamino, diethylamino, and propylamino.
Some examples of substituents of the above-described aliphatic (and other) moieties of compounds of the invention include, but are not limited to: F, Cl, Br, I, OH, NO2, CN, C(O)—C1–C6-alkyl, C(O)-aryl, C(O)-heteroaryl, CO2-alkyl, CO2-aryl, CO2-heteroaryl, CONH2, CONH—C1–C6-alkyl, CONH-aryl, CONH-heteroaryl, OC(O)—C1–C6-alkyl, OC(O)-aryl, OC(O)-heteroaryl, OCO2-alkyl, OCO2-aryl, OCO2-heteroaryl, OCONH2, OCONH—C1–C6-alkyl, OCONH-aryl, OCONH-heteroaryl, NHC(O)—C1–C6-alkyl, NHC(O)-aryl, NHC(O)-heteroaryl, NHCO2-alkyl, NHCO2-aryl, NHCONH-heteroaryl, SO2—C1–C6-alkyl, SO2-aryl, C3–C6-cycloalkyl, CF3, CH2CF3, CHCl2, CH2OH, CH2CH2OH, CH2NH2, CH2SO2CH3, aryl, heteroaryl, benzyl, benzyloxy, aryloxy, heteroaryloxy, C1–C6-alkoxy, methoxymethoxy, methoxyethoxy, amino, benzylamino, arylamino, heteroarylamino, C1–C3-alkyl-amino, thio, aryl-thio, heteroarylthio, benzyl-thio, C1–C6-alkyl-thio, or methylthiomethyl. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein.
The term “aprotic solvent” as used herein refers to a solvent that is relatively inert to proton activity, i.e., not acting as a proton-donor. Examples include, but are not limited to, hydrocarbons, such as hexane and toluene, for example, halogenated hydrocarbons, such as, for example, methylene chloride, ethylene chloride, chloroform, and the like, heteroaryl compounds, such as, for example, tetrahydrofuran and N-methylpyrrolidinone, and ethers such as diethyl ether, bis-methoxymethyl ether. Such compounds are well known to those skilled in the art, and it will be obvious to those skilled in the art that individual solvents or mixtures thereof may be preferred for specific compounds and reaction conditions, depending upon such factors as the solubility of reagents, reactivity of reagents and preferred temperature ranges, for example. Further discussions of aprotic solvents may be found in organic chemistry textbooks or in specialized monographs, for example: Organic Solvents Physical Properties and Methods of Purification, 4th ed., edited by John A. Riddick et al., Vol. II, in the Techniques of Chemistry Series, John Wiley & Sons, NY, 1986.
In general, the terms “aryl” and “heteroaryl”, as used herein, refer to stable mono- or polycyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated moieties having preferably 3–14 carbon atoms, each of which may be substituted or unsubstituted. Substituents include, but are not limited to, any of the previously mentioned substituents, i.e., the substituents recited for aliphatic moieties, or for other moieties as disclosed herein, resulting in the formation of a stable compound. In certain embodiments of the present invention, “aryl” refers to a mono- or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl and the like. In certain embodiments of the present invention, the term “heteroaryl”, as used herein, refers to a cyclic aromatic radical having from five to ten ring atoms of which one ring atom is selected from S, O and N; zero, one or two ring atoms are additional heteroatoms independently selected from S, O and N; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.
It will be appreciated that aryl and heteroaryl groups (including bicyclic aryl groups) can be unsubstituted or substituted, wherein substitution includes replacement of one, two or three of the hydrogen atoms thereon independently with any one or more of the following moieties including, but not limited to: F, Cl, Br, I, OH, NO2, CN, C(O)—C1–C6-alkyl, C(O)-aryl, C(O)-heteroaryl, CO2-alkyl, CO2-aryl, CO2-heteroaryl, CONH2, CONH-C1–C6-alkyl, CONH-aryl, CONH-heteroaryl, OC(O)—C1–C6-alkyl, OC(O)-aryl, OC(O)-heteroaryl, OCO2-alkyl, OCO2-aryl, OCO2-heteroaryl, OCONH2, OCONH—C1–C6-alkyl, OCONH-aryl, OCONH-heteroaryl, NHC(O)—C1–C6-alkyl, NHC(O)-aryl, NHC(O)-heteroaryl, NHCO2-alkyl, NHCO2-aryl, NHCONH-heteroaryl, SO2—C1–C6-alkyl, SO2-aryl, C3–C6-cycloalkyl, CF3, CH2CF3, CHCl2, CH2OH, CH2CH2OH, CH2NH2, CH2SO2CH3, aryl, heteroaryl, benzyl, benzyloxy, aryloxy, heteroaryloxy, C1–C6-alkoxy, methoxymethoxy, methoxyethoxy, amino, benzylamino, arylamino, heteroarylamino, C1–C3-alkyl-amino, thio, aryl-thio, heteroarylthio, benzyl-thio, C1–C6-alkyl-thio, or methylthiomethyl. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein.
The term “cycloalkyl”, as used herein, refers specifically to groups having three to seven, preferably three to ten carbon atoms. Suitable cycloalkyls include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the case of other aliphatic, heteroaliphatic or hetercyclic moieties, may optionally be substituted. F, Cl, Br, I, OH, NO2, CN, C(O)—C1–C6-alkyl, C(O)-aryl, C(O)-heteroaryl, CO2-alkyl, CO2-aryl, CO2-heteroaryl, CONH2, CONH—C1–C6-alkyl, CONH-aryl, CONH-heteroaryl, OC(O)—C1–C6-alkyl, OC(O)-aryl, OC(O)-heteroaryl, OCO2-alkyl, OCO2-aryl, OCO2-heteroaryl, OCONH2, OCONH—C1–C6-alkyl, OCONH-aryl, OCONH-heteroaryl, NHC(O)—C1–C6-alkyl, NHC(O)-aryl, NHC(O)-heteroaryl, NHCO2-alkyl, NHCO2-aryl, NHCONH-heteroaryl, SO2—C1–C6-alkyl, SO2-aryl, C3–C6-cycloalkyl, CF3, CH2CF3, CHCl2, CH2OH, CH2CH2OH, CH2NH2, CH2SO2CH3, aryl, heteroaryl, benzyl, benzyloxy, aryloxy, heteroaryloxy, C1–C6-alkoxy, methoxymethoxy, methoxyethoxy, amino, benzylamino, arylamino, heteroarylamino, C1–C3-alkyl-amino, thio, aryl-thio, heteroarylthio, benzyl-thio, C1–C6-alkyl-thio, or methylthiomethyl. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein.
The term “heteroaliphatic”, as used herein, refers to aliphatic moieties which contain one or more oxygen, sulfur, nitrogen, phosphorous or silicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moieties may be branched, unbranched or cyclic and include saturated and unsaturated heterocycles such as morpholino, pyrrolidinyl, etc. In certain embodiments, heteroaliphatic moieties are substituted by independent replacement of one or more of the hydrogen atoms thereon with one or more moieties including, but not limited to: F, Cl, Br, I, OH, NO2, CN, C(O)—C1–C6-alkyl, C(O)-aryl, C(O)-heteroaryl, CO2-alkyl, CO2-aryl, CO2-heteroaryl, CONH2, CONH—C1–C6-alkyl, CONH-aryl, CONH-heteroaryl, OC(O)—C1–C6-alkyl, OC(O)-aryl, OC(O)-heteroaryl, OCO2-alkyl, OCO2-aryl, OCO2-heteroaryl, OCONH2, OCONH—C1–C6-alkyl, OCONH-aryl, OCONH-heteroaryl, NHC(O)—C1–C6-alkyl, NHC(O)-aryl, NHC(O)-heteroaryl, NHCO2-alkyl, NHCO2-aryl, NHCONH-heteroaryl, SO2—C1–C6-alkyl, SO2-aryl, C3–C6-cycloalkyl, CF3, CH2CF3, CHCl2, CH2OH, CH2CH2OH, CH2NH2, CH2SO2CH3, aryl heteroaryl, benzyl, benzyloxy, aryloxy, heteroaryloxy, C1–C6-alkoxy, methoxymethoxy, methoxyethoxy, amino, benzylamino, arylamino, heteroarylamino, C1–C3-alkyl-amino, thio, aryl-thio, heteroarylthio, benzyl-thio, C1–C6-alkyl-thio, or methylthiomethyl. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein.
The terms “halo” and “halogen” as used herein refer to an atom selected from fluorine, chlorine, bromine and iodine.
The term “haloalkyl” denotes an alkyl group, as defined above, having one, two, or three halogen atoms attached thereto and is exemplified by such groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.
The term “heterocycloalkyl”, as used herein, refers to a non-aromatic 5-, 6- or 7-membered ring or a bi- or tri-cyclic group comprising fused six-membered rings having between one and three heteroatoms independently selected from oxygen, sulfur and nitrogen, wherein (i) each 5-membered ring has 0 to 1 double bonds and each 6-membered ring has 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally be oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocyclic rings may be fused to a benzene ring. Representative heterocycles include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl. In certain embodiments, a “substituted heterocycloalkyl” group is utilized and as used herein, refers to a heterocycloalkyl group, as defined above, substituted by independent replacement of one, two or three of the hydrogen atoms thereon with but are not limited to: F, Cl, Br, I, OH, NO2, CN, C(O)—C1–C6-alkyl, C(O)-aryl, C(O)-heteroaryl, CO2-alkyl, CO2-aryl, CO2-heteroaryl, CONH2, CONH—C1–C6-alkyl, CONH-aryl, CONH-heteroaryl, OC(O)—C1–C6-alkyl, OC(O)-aryl, OC(O)-heteroaryl, OCO2-alkyl, OCO2-aryl, OCO2-heteroaryl, OCONH2, OCONH—C1–C6-alkyl, OCONH-aryl, OCONH-heteroaryl, NHC(O)—C1–C6-alkyl, NHC(O)-aryl, NHC(O)-heteroaryl, NHCO2-alkyl, NHCO2-aryl, NHCONH-heteroaryl, SO2—C1–C6-alkyl, SO2-aryl, C3–C6-cycloalkyl, CF3, CH2CF3, CHCl2, CH2OH, CH2CH2OH, CH2NH2, CH2SO2CH3, aryl, heteroaryl, benzyl, benzyloxy, aryloxy, heteroaryloxy, C1–C6-alkoxy, methoxymethoxy, methoxyethoxy, amino, benzylamino, arylamino, heteroarylamino, C1–C3-alkyl-amino, thio, aryl-thio, heteroarylthio, benzyl-thio, C1–C6-alkyl-thio, or methylthiomethyl. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein.
“Hydroxy-protecting group”, as used herein, refers to an easily removable group, which is known in the art to protect a hydroxyl group against undesirable reaction during synthetic procedures and to be selectively removable. The use of hydroxy-protecting groups is well known in the art for protecting groups against undesirable reactions during a synthetic procedure and many such protecting groups are known, cf., for example, T. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd edition, John Wiley & Sons, New York (1991). Examples of hydroxy-protecting groups include, but are not limited to, methylthiomethyl, tert-dimethylsilyl, tert-butyldiphenylsilyl, ethers such as methoxymethyl, and esters including acetyl benzoyl, and the like.
The term “oxo” denotes a group wherein two hydrogen atoms on a single carbon atom in an alkyl group as defined above are replaced with a single oxygen atom (i.e. a carbonyl group).
The term “protected-hydroxy” refers to a hydroxy group protected with a hydroxy protecting group, as defined above, including benzoyl, acetyl, trimethylsilyl, triethylsilyl, methoxymethyl groups, for example.
The term “protogenic organic solvent” as used herein refers to a solvent that tends to provide protons, such as an alcohol, for example, methanol, ethanol, propanol, isopropanol, butanol, t-butanol, and the like. Such solvents are well known to those skilled in the art, and it will be obvious to those skilled in the art that individual solvents or mixtures thereof may be preferred for specific compounds and reaction conditions, depending upon such factors as the solubility of reagents, reactivity of reagents and preferred temperature ranges, for example. Further discussions of protogenic solvents may be found in organic chemistry textbooks or in specialized monographs, for example: Organic Solvents Physical Properties and Methods of Purification, 4th ed., edited by John A. Riddick et al., Vol. II, in the Techniques of Chemistry Series, John Wiley & Sons, NY, 1986.
The term “treating”, as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. Them term “treatment”, as used herein, refers to the act of treating, as “treating” is defined immediately above.
DRC39 is the MC 1000 (ftsZ+) delta acrAB::kan strain of E. coli. 
DRC40 is the DRC13 (ftsZ84) delta acrAB::kan strain of E. coli. 
DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS
As discussed above, the invention relates to assay systems and the uses of these assay systems for screening compounds for anti-microbial activity, and more particularly, to using bacterial proteins in vivo to detect compounds that interfere with cell division. In one preferred embodiment, the present invention provides in vivo cellular assays that utilize mutant bacterial strains that have a defect in cell division to screen compounds for anti-microbial activity.
The present invention further relates to pharmaceutical compositions including compounds useful in the treatment and/or prevention of one or more microbial infections. Those skilled in the art will appreciate that this includes compounds that inhibit the growth of microbial cells, such as yeast, fungi, protozoa, bacteria, and the like.
Assay Systems and Methods of Use
Bacterial cells divide by first initiating DNA replication. At the end of the bacterial cell cycle, the chromosomes segregate and the cells divide by forming a septum that divides the cells in two, a process known as septation.
A large collection of mutants that block DNA replication and/or cell division have been identified in a wide range of microbial cells. In many cases, the gene(s) responsible for the mutant phenotypes and their wild-type counterparts have been cloned and characterized. The in vivo and in vitro activities of such wild-type and mutant proteins may be employed to identify inhibitors of DNA replication and/or cell division and thus identify inhibitors of microbial cell growth. Furthermore, a protein that is a key player in one type of microbial cell, for example, a bacterial cell, may be conserved in another type of microbial cell, e.g., a fungal cell. Thus, inhibitors that block the activity of these proteins to prevent cell division might also overlap between different microbial cell types.
Such anti-microbial agents may be used as broad-spectrum therapeutics, e.g., as anti-microbial agents. Alternatively, such anti-microbial agents may be used for decontamination, e.g., decontamination of water having a high microbial count. It may also be appreciated that molecules that activate the activity of a protein involved in the cell cycle may also be identified, which may spur further basic research.
One protein that participates in bacterial cell division is the FtsZ protein. FtsZ is essential for bacterial cell multiplication and is ubiquitous in the prokaryotic kingdom, being present in eubacteria (gram-positive/gram-negative), archaea, mycoplasmas, chloroplasts, and mitochondria of lower eukaryotes), while it is absent from the mitochondria of higher eukaryotes (yeast to humans). The constriction of organelles in cyanobacteria, a-proteobacteria, chloroplast, algae, and yeast or animal mitochondria is depicted in FIG. 1. It is also absent from the obligate intracellular bacterial pathogen, Clamydia trachomatis. Other potential targets that each harbor a single copy of the ftsZ gene, which is likely to be essential for cell division, include Vibrio cholerae, Hemophilus influenzae, Staphylococcus aureus, Clostridium perfringens, Mycobacterium tuberculosis, Bacillus anthracis, Francisella tularensis, Shigella flexneri, and Brucella abortus. A phylogenetic tree of FtsZ orthologs that might have FtsZ activity is shown in FIG. 2. Therefore, because inhibitors of FtsZ activity are expected to block cell division in a wide range of prokaryotic organisms, molecules that modulate FtsZ function may be developed as broad-spectrum anti-bacterial agents against known and unknown bacterial pathogens.
FtsZ is a tubulin-like GTPase that forms a membrane-associated cytokinetic contractile ring structure in vivo at the site of division in bacterial cells (see FIG. 3, which shows localization of FtsZ at the cytokinetic ring structure in predivisional E. coli cells). The tubulin signature sequence is GGGTGSG. The FtsZ signature sequence is GGGTGTG. During the process of cell division, FtsZ becomes concentrated at the inner membrane into a ring-like structure at the prospective division site immediately before the start of cell division. During septation, the diameter of the FtsZ ring (also referred to herein as the Z ring) becomes progressively smaller as it remains at the leading edge of the invaginating cell wall.
FtsZ is believed to interact with several different molecules that also play specific roles in one or more cell division processes (see FIG. 4). Genetic studies have suggested possible interactions between FtsZ and several other proteins. For example, FtsZ has been shown to interact with FtsA by yeast two-hybrid analysis and by the ability of the FtsZ ring to recruit FtsA. Indeed FtsA can be co-purified with FtsZ and vice-versa. FtsZ also is known to interact with ZipA, a protein essential for cell viability. Cells lacking sufficient ZipA activity die. Thus, those skilled in the art will appreciate that large screens for compounds that either inhibit or activate the ability of FtsZ to interact with FtsA or ZipA have great flexibility in their design and implementation.
In vitro, FtsZ polymerizes in a guanine nucleotide-dependent manner into structures (protofilaments or protofilament bundles or sheets (see FIG. 5 and FIG. 6)) that are similar to tubulin polymers (microtubules). These activities, or more particularly, the inhibition or activation of these activities, may also be used to identify test compounds, such as peptide and small molecule compounds that are inhibitors or activators of FtsZ-mediated cell division.
The likelihood of prokaryotic cells developing resistance to molecules that inhibit FtsZ is relatively low for several reasons. First, FtsZ orthologs have a high degree of sequence conservation, especially in domains involved in GTP binding and hydrolysis, in subunit interaction required for polymerization, and in the interaction with proteins such as FtsA and ZipA. Second, as demonstrated herein in Example 1, low, sub-stoichiometric levels of FtsZ inhibitors are likely to be required to affect FtsZ function. Third, and most importantly, FtsZ is an essential, non-redundant protein, required for cell division. Use of FtsZ inhibitors may further provide an advantage when used in combination with other drug treatments in that it may provide a valuable time window for other drug treatments to have an effect by slowing down the rate of multiplication of the infectious organism.
In preferred embodiments, the present invention provides methods of identifying compounds that are inhibitors or activators of FtsZ activity (the first anti-microbial compounds identified to target a bacterial cell division protein). In related embodiments, the present invention provides methods of identifying compounds that are inhibitors or activators of proteins that interact with FtsZ, such as FtsA and ZipA.
In related embodiments, the present invention provides assays for FtsZ activity that are based on cell morphology and FtsZ ring assembly in vivo in wild-type and ftsZ mutant cells. A visual assay may be used to determine the effect of a compound on polymerization, e.g., destabilizing or stabilizing polymerization (see FIG. 7, panel D). Other available assays include charcoal-based and thin-layer chromatographic assays for GTPase activity, negative-stain transmission electron microscopy to assess the activity of a compound on FtsZ polymers, and growth assays for assessing the anti-microbial activity of a compound. Such assays may include experiments that assess cell culture growth by, for example, culture turbidity in response to addition of compound.
For example, inhibition of FtsZ activity results in a block in the ability to form a cytokinetic ring structure, which results in abnormally long cells due to a decrease in septation without affecting cellular mass increase. An inhibition of FtsZ activity can be verified in vitro, e.g., by detecting a decrease in GTP-dependent polymerization of FtsZ and the concomitant GTPase activity see U.S. patent application Ser. No. 10/153,268. Alternatively, activation of FtsZ in vivo, or increased FtsZ abundance, results in hyper-formation of ring structures in the cell, which yields shortened cells due to polar septation. Similarly, an increase in in vitro polymerization-dependent GTPase activity may be observed in the presence of an FtsZ activator.
More particularly, the present invention provides an in vivo cellular assay that utilizes FtsZ wild-type and/or FtsZ-mutant microbial cells to screen compounds for anti-microbial activity. In certain preferred embodiments, a bacterial cell is provided that has, in addition to the mutation in FtsZ, a second mutation that effects the amount of drug that gets into the cell, e.g., a mutation that affects the multidrug efflux pump or cell permeability, and may further include an expression vector encoding the FtsZ protein. It will be appreciated that expression of proteins in bacteria is standard in the art, as demonstrated below (see also Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, New York, V. 1&2, 1996, each of which is incorporated by reference herein).
Without limitation, the present invention is based on the discovery that ftsZ mutants are, in some cases, significantly more sensitive to the adverse effect of the compound on its division ability at the permissive temperature relative to the isogenic wild-type strain. In addition, the enhanced sensitivity is reversible, i.e., is reversed to equal that of the wild-type strain if a normal wild-type copy of the ftsZ gene is introduced into the ftsZ mutant cell on a plasmid. This suggests that the compound is either affecting FtsZ directly in vivo or a critical FtsZ-related step in the assembly of the division apparatus. In preferred embodiments, the present invention provides high throughput differential phenotypic screens using the wild-type parent and the isogenic ftsZ-ts (ts) mutant of E. coli. The rationale for such a screen is that some compounds in combination with the ftsZ mutation may cause enhanced lethality for the mutant at the permissive temperature, while the wild-type or isogenic strain is used as a good control to examine the extent of filamentation and cytotoxicity induced by the compound.
In certain preferred embodiments differential phenotypic screens are carried out with wild-type and isogenic ftsZ84-ts E. coli strains. This mutant is conditional-lethal because it grows and divides at 30° C., but undergoes a cell division block at 42° C. that leads to lethal cell filamentation. The division block of the ftsZ84 mutant at 42° C. is due to a drastic destabilization of the FtsZ ring (Z-ring) at high temperature (within a few minutes after the temperature shift up). The Z-ring is highly dynamic and is continually remodeled within a halftime of 30 seconds. In contrast the Z-ring in the ftsZ84 mutants displayed a 9-fold slower turnover at the permissive temperature. According to the present invention, the mutant Z-ring structure in ftsZ84 would be less robust than the wild-type ring at the permissive temperature of 30° C. and the inherent weakness of the mutant ring may be exacerbated in the presence of small molecules and other compounds that target FtsZ or the septal ring in vivo.
Those skilled in the art will appreciate that the in vivo screen can easily be extended to other conditional mutant alleles of ftsZ to conduct allele-specific screening of chemical libraries. In certain preferred embodiments, a putative mutation in a second gene that may interact with the conditional mutant allele, e.g., the ftsZ84-ts mutation, is substituted with a small molecule to elicit a phenotypic response, thus establishing a chemical genetic approach to identify small molecule modulators/antagonists of bacterial cell division. The hits obtained against different mutant alleles vis-à-vis the wild-type parent are compared to facilitate identification of chemical structures that show high affinity for targeting FtsZ or an FtsZ related step in cell division. Those skilled in the art will appreciate that this would be helpful in establishing downstream chemistry for the synthesis of structural analogs to perform SAR studies.
In preferred embodiments, the in vivo screen described herein is conducted with other conditional-lethal mutations in cell division genes such as ftsA, ftsI, ftsQ ftsK etc. or with mutations in new, as yet unidentified, division genes in eubacteria. There are multiple temperature-sensitive alleles available in a number of known fts genes for allele-specific screening as well. The present in vivo screens provide advantages over other cell division screens in their rapidity, simplicity, and cost effectiveness, as well as the specificity for the process being targeted, namely cell division in bacteria. In addition to E. coli as the organism being used for screening, the inventive in vivo screen can be carried out with conditional cell division mutants in other model or pathogenic bacteria such as Vibrio cholerae (Gram-negative), Staphylococcus aureus (Gram-positive), Shigella flexneri (Gram-negative), Bacillus subtilis (Gram-positive) etc.
The strains listed above may further provide for broad spectrum testing for other strains. For example, FtsZ is subject to temporal, spatial, and developmental regulation in B. subtilis. During exponential growth, Z-rings form exclusively at midcell as a prelude to binary fission in a manner similar to that in E. coli. At the onset of sporulation in E. coli, FtsZ shifts from a medial to a bipolar pattern of localization, forming a Z-ring near each pole of the cell. Subsequently, one Z-ring matures into the sporulation septum, while the other Z-ring is dissipated. A similar Z-ring switching mechanism is expected to be operative in B. anthracis during spore formation. Therefore, FtsZ inhibitors could also be very useful tools to prevent anthrax spore formation, because asymmetric Z-ring assembly near one pole is a critical prerequisite for successful sporulation in B. subilis. 
In yet other preferred embodiments, sensitized strains carrying mutations in regulatory genes that affect the level or the stability of key cell division proteins are used in the inventive in vivo assay. For example, such regulatory mutations may reside in transcription factors or in proteolytic enzymes. Small molecules can be identified that may either alleviate or worsen the sensitized state of cell division in such bacterial strains.
In one particularly preferred embodiment, both the wild-type and the conditional mutation, e.g., ftsZ84-ts mutant, carries a null mutation in the major drug efflux pump, AcrAB, in E. coli. This ensures that some of the putative hits are not pumped out of the cell. The AcrAB mutation renders the strain temperature-sensitive by destabilizing the division machinery at the non-permissive temperature of 42° C., yielding long filamentous cells. However, at the permissive temperature of 30° C., the temperature-sensitive cells grow like wild-type and are rod-shaped. Thus, the present invention provides assays that detect the phenotype of wild-type and mutant bacterial cells (e.g., the congenic thermosensitive ftsZ84 E. coli mutant DRC13 and their derivatives, which lack the major multidrug efflux pump AcrAB) in the presence and absence of compound (see Example 1).
In preferred embodiments, the present invention provides assays including the steps of 1) expressing the FtsZ protein in a wild-type cell, 2) contacting the cell with a compound, and 3) detecting a defect in cell division. For example, the defect in cell division may be an activation of cell division, e.g., caused by excessive intracellular polymerization of the FtsZ protein. This would result in a phenotype of excessively short cells without DNA, called minicells, resulting from division activity at the cell poles. In addition, under conditions of excessive intracellular polymerization of the FtsZ protein, the FtsZ rings would persist longer and more stably, thereby impeding ring constriction essential for septation. Alternatively, the defect in cell division may be an inhibition of cell division, e.g., caused by a blockage to intracellular polymerization of the FtsZ protein or hyperstabilization of the FtsZ polymers This block in FtsZ activity may result is long filamentous cells that divide infrequently or completely fail to divide.
In another embodiment, the compound is used in an assay that determines its ability to decrease or exacerbate an ftsZ phenotype. In certain preferred embodiments, the invention provides a method of identifying compounds that affect cell division, comprising steps of contacting a cell that is defective in cell division (e.g., ftsZ84 cells having a thermosensitive mutation in the gene encoding FtsZ and a signature sequence of SGGTGTG) with a compound of interest; and detecting an alteration in the phenotype of the cell. More particularly, the method involves the steps of 1) contacting an ftsZ cell with a compound of interest, and 2) detecting an alteration in the phenotype of the ftsZ cell (e.g., a destabilization in the Z ring structure). Typically such ftsZ cells are temperature sensitive ftsZ cells, e.g., ftsZ84 cell that grow and divide at 30° C. and undergo a division block at 42° C. due to a destabilization of the Z-rings at high temperature (see FIG. 8). ftsZ84 cells display about a 10-fold reduced GTP binding and GTPase activities compared to wild-type ftsZ cells. ftsZ84 cells lacking the multidrug efflux pump AcrAB would have a decreased ability to expel compound from the cell, thus allowing increased concentrations of a compound to accumulate in the cell potentially resulting in an exacerbated phenotype caused by the compound in that cell at the permissive temperature of 30° C. The phenotype in the presence of a compound that inhibits FtsZ activity would be an exacerbation of the failure to form Z rings in the ftsZ84 cells (a phenotype akin to the synthetic lethal interactions between two genetic mutations).
A third assay system provided by the present invention that may be utilized to identify compounds that affect cell division includes a bacterial cell that has a mutation affecting a multidrug efflux pump and further contains an expression construct encoding the ZipA protein. As noted above, as but one example of a cell that has a mutation affecting a multidrug efflux pump is the bacterial cell strain ftsZ84. In addition, as noted herein, the ZipA protein stabilizes intracellular assembly of the FtsZ ring. It has been shown that ftsZ84 cells expressing increased concentrations of ZipA, e.g., via introduction of a second copy of a ZipA gene into the cell, have decreased thermosensitivity at the restrictive temperature of 42° C.
In related embodiments, the present invention provides a method of utilizing the ftsZ84 strain in combination with a multidrug efflux pump mutation and a second copy ZipA, described above, to identify a compound that affects cell division, or alternatively a method of validating whether a compound affects cell division. The method involves observing the effect of the compound on the phenotype of ftsZ84 cells expressing increased concentrations of ZipA (see U.S. Pat. No. 5,948,889, incorporated by reference herein). Compounds that are inhibitors will diminish the suppression of the thermosensitivity of the ZipA expressing ftsZ84 cells at increased temperatures, resulting in a destabilization in the ring structure. Alternatively, a second copy of ZipA may increase the stability of the FtsZ ring in ftsZ84 cells and may thereby alleviate the lethality of FtsZ compounds.
A key step in bacterial cell division is the assembly of the septal ring organelle at the division site that guides the circumferential syntheses of cell wall and membrane for biogenesis of the division septum. The septal ring is assembled by the ordered recruitment of eight essential division proteins to the FtsZ ring scaffold in E. coli (see FIG. 4). It is therefore likely that the inventive in vivo screen can identify small molecules that target the protein-protein interaction interfaces between FtsZ and other division components in the septal ring and not the FtsZ protein per se. Those skilled in the art would appreciate that such molecules are highly desirable as lead compounds for antibacterial drugs. The probability of spontaneous resistance arising against such drugs would be low as they target the area of interaction between two proteins and key amino acid residues contributed by both proteins for such interaction may need to be mutated for resistance to develop.
Using defined cell division mutants in the inventive in vivo screens increases the specificity of the screening process and allows compounds to be identified rather easily, which may disrupt one or the other of multiple protein-protein interactions that occur in the septal ring complex. As described herein, the screening can be done with any bacterial organism carrying cell division mutations, thus providing facile method to screen against division genes that may be present in one organism and not the other. The in vivo screen described above differs other in vivo screens such as the yeast two-hybrid screen in that the bacterial cell itself, and not yeast, is being used to screen for molecules that target a complex process such as cell division in bacterial cells.
According to the present invention, the hits obtained from the in vivo screens can be validated by growth and phenotype assays against a wide range of bacteria and by a range of in vitro biochemical assays to pinpoint the target of their actions, whether for example the target is FtsZ or the interaction between FtsZ and another essential division protein (see, e.g., Example 2). One such assay system is the charcoal-based GTPase assay described by Lee et al. J. Biol. Chem. 267:1212–1218 (1992), incorporated herein by reference. Another assay is the malachite green-phosphomolybdate assay, shown below (Akiyama, Y., Kihara, A., Tokuda, H. and Ito, K. 1996, J. Biol. Chem. 271:31196–31201, incorporated herein by reference (see FIG. 9)). Yet another assay includes negative-strain transmission electron microscopy of FtsZ polymers. Another in vitro assay includes right-angle light scattering. More traditional anti-microbial screening assays are described by de Boer et al. in U.S. Pat. No. 5,948,889 (col. 8–9), incorporated herein by reference.
It will be appreciated that the in vivo assay of the present invention may be used to validate any compounds identified by in vitro assays. Other in vivo assays may also be use for validation. For example, the effect on FtsZ ring assembly in live cells carrying an ftsZ mutation may be assessed. Alternatively, the effect on FtsZ ring dynamics in live cells using fluorescence photobleaching recovery assays may be assessed. In addition, the effect on protein-protein interactions between FtsZ and other essential division proteins, such as FtsA or Zip A may be assessed by methods standard in the art, e.g., antibody pull down assays.
It will be appreciated that any compound may be tested on the inventive in vivo assay system described herein to detect activators or inhibitors of cell division. It will also be appreciated that such compounds may be generated by any art available means. For example, the compounds of the galanthamine library, described in U.S. patent application Ser. No. 09/863,141, incorporated herein by reference in its entirety. In addition, marine extracts (e.g., available from National Cancer Institute (NCI)) can also be screened for FtsZ inhibiting or activating activity in vivo. For example, the NCI extracts contain a mixture of about 10–12 compounds. If the extract scored positive in the in vivo assay of the invention, the extract would be separated into its constituent components and rescreened to identify the active component.
As described in Example I, an in vivo screen for molecules that inhibit bacterial cell growth was performed by perturbing the cell division process. A total of 18,320 compounds, including the 16,320 member Chembridge DiversetE Library and 2000 member NCI Diversity Set, were screened against two isogenic strains of E. coli that both lack the major drug-efflux pump AcrAB, yet differ by a point mutation in the ftsZ gene. Compounds that inhibit bacterial division in the sensitized ftsZ mutant strain (the terminal phenotype of filamentation that could eventually lead to cell death), but do not exhibit such pronounced effect in the wild-type strain, were expected to target either the mutant FtsZ or the protein-protein interaction interface between FtsZ and other components of the cell-division machinery. Compounds that inhibited greater than or equal to 45% of cell growth were separated from the library of compounds as positive hits. From the total library, 173 hits were identified on an ftsZ-ts mutant, ftsZ84. A filamentation defect was observed for 52 of these compounds and cell lysis or change is cell shape was observed for the remaining 121 (see FIG. 10). Additional compounds were identified on wild-type E. coli cells (see FIG. 10).
The analysis of the in vivo screening results revealed the following categories of compounds: 1) compounds that inhibited growth in both the wild-type and the isogenic ftsZ84-ts mutant strains; 2) compounds that inhibited growth only in the wild-type strain; and 3) compounds that inhibited growth only in the isogenic ftsZ84-ts mutant. Regarding the category 2 compounds, the lack of any visible adverse effect on the ftsZ84-ts mutant raises the possibility that one or a subset of these compounds may stabilize the mutant FtsZ84 ring. The increased stability of the wild-type FtsZ ring in the presence of such compounds might retard the constriction of the septal ring and this would result in a defect in septum synthesis with consequent filamentation in the wild-type strain. One skilled in the art could easily test whether the category 2 compounds actually suppress the temperature-sensitive growth of the ftsZ84-ts mutant at the non-permissive temperature of 42° C.
From preliminarily screening the two libraries, five inhibitors were identified and later verified in various in vivo and in vitro assays for bacterial growth and the formation of the FtsZ ring structure in the cell including, e.g., a TLC assay, a GTPase assay using activated charcoal, ta polymer sedimentation assay, negative-stain electron microscopy, or a polymerization assay using fluorescent tubulin, each of which are known in the art. The five of the inhibitors include 58P-18, 16L-09,18M-04, 27D-12, and 27F-02, which are depicted in FIG. 11, FIG. 12.
These five compounds can be divided into two classes. The first class includes the compounds 18M04 and 27D12 that have a dose dependent destabilizing effect on the polymers (see FIG. 13 and FIG. 14). The second class of compounds includes 16L-09, 27F02, and 58P18, which cause mild bundling of FtsZ protofilaments (mostly via pairing of protofilaments) that could alter the FtsZ ring dynamics in vivo (FIG. 15). One compound, 26E-10, was identified in a cell based assay and is depicted in FIG. 16 and FIG. 17 and FIG. 18. Additional compounds 26E-10 and 58P-18 were identified and results therefor are depicted in FIGS. 19, 20, 21, 22, 23, and 24.
The IC50 values for some of these compounds are shown herein (see also FIG. 25). The data provided demonstrate that some of the compounds identified by the inventive assay kill a wide range of bacteria at concentrations between 2 and 40 μM (see FIG. 26). IC50 values obtained using the malachite green-phosphomolybdate assay are shown (Akiyama, Y., Kihara, A., Tokuda, H. and Ito, K. 1996, J. Biol. Chem. 271:31196–31201, incorporated herein by reference).







TABLE 1







IC50 values against M. tuberculosis for FtsZ.











IC50 (μM) against



Compound
M. tub. FtsZ







58-P18
~30–35



27-F02
~60



16-L09
~50



27-D12
~55










Subsets of the inhibitors kill bacterial cells at surprisingly low concentrations, ranging from 2–25 μg/ml. Two compounds, 26E-10 and 58-P18, appeared to affect cell division by targeting FtsZ ring formation in vivo. The compound 26E-10 has a strong in vivo phenotype, as demonstrated below. Moreover, 58P-18 shows both in vitro inhibition and an in vivo phenotype.
Upon screening a number of compounds using the inventive in vivo assay, it was determined that many of the compounds showing in vivo effects do not effect GTPase and/or filament assembly in vitro. Several possible explanations for this lack of in vitro activity include 1) these compounds may target protein-protein interactions between FtsZ and other essential septal ring components, rather than FtsZ per se (molecules that disrupt protein-protein interactions are highly desirable as lead compounds because the probability of resistance developing against them is low); 2) these compounds may cause cell filamentation by activating a checkpoint, such as the SOS-inducible division inhibitor SulA, that blocks Z-ring assembly; 3) these compounds induce the MinC division inhibitor to be activated to block cell division, though we consider this scenario less likely.
Mutant Strains
In preferred embodiments, strains other than acrAB are generated to contain ftsZ mutations for screening for FtsZ interactive compounds in vivo. In certain preferred embodiments, the inventive in vivo assay may be conducted with strains carrying a mutation in the tolC gene that encodes a channel-tunnel protein linking the inner and the outer membrane in E. coli. The tolC mutation is known to enhance the permeability of cells to a variety of molecules. In other preferred embodiments, both the acrAB and tolC mutations can be combined as this is known to confer maximum permeability to E. coli. After identifying compounds that affect cell division, the molecules can be tested for their cell permeability in an E. coli strain that is wild-type for acrAB and tolC genes. If some of the hits prove impermeable, downstream chemistry can be employed to enhance their cell permeability.
In certain preferred embodiments, the screen in carried out in the context of using ftsI-ts mutants, which code for the penicillin-binding protein 3 (PBP 3) in E. coli and is essential for the synthesis of septal cell wall in eubacteria. PBP 3 is the only one out of eight PBPs characterized in E. coli dedicated to septum synthesis and cell division. The rest of the PBPs are involved in cell wall synthesis along the long axis of the cell and presumably do not participate in cell division. As is well known, the PBPs are the lethal targets of penicillin and its derivatives, as well as cephalosporins (the beta-lactam class of antibiotics). The beta-lactams covalently acylate the active site serine residue of PBPs, which are generally bifunctional enzymes carrying out both transglycosylation and transpeptidation activities essential for cell wall synthesis. PBP 3, however, is believed to be a monofunctional transpeptidase. To date, no allosteric inhibitor outside the active site of PBPs has been described.
In vivo screens using ftsI-ts strains have the potential to identify molecules that may either allosterically affect PBP 3 function or disrupt protein-protein interactions between PBP 3 and other cell division proteins. An allosteric PBP 3 inhibitor could be a useful starting point to design or screen for such allosteric inhibitors against other PBPs that are involved in the essential and delicate process of cell wall elongation and in the maintenance of the osmotic integrity of the eubacterial cell.
Other embodiments of the invention provide a novel thermosensitive ftsZ mutation, designated ftsZ26, for use in the in vivo screen that consists of a 6-codon insertion near the 5′-end of the gene. This mutation, like the ftsZ84 mutation, causes morphological changes in E. coli even at the permissive temperature. The changes include cells with blunt poles and carrying protrusions to one side, Y-shaped cells, and minicells. The altered polar morphology is a result of cell division and is due to the aberrant geometry of FtsZ rings that assembled non-perpendicular to the long axis of the cell. Upon shift to the non-permissive temperature, ftsZ26 cells form filaments that are morphologically similar to the filaments formed by the ftsZ84 mutant, except for the poles that form at 30° C. The thermosensitivity and the altered geometry of ftsZ26 rings argue that FtsZ-specific small molecules may likely exacerbate the assembly defect of ftsZ26 under permissive conditions, thus rendering this mutant amenable for use in the inventive high throughput whole-cell screens. In certain preferred embodiments, the acrAB deletion is introduced into both ftsZ26 and its isogenic parent for screening purpose.
In other preferred embodiments, a strain in which expression of ftsZ is kept at a low level sufficient for viability and subject to modulation by an inducer is provided. The chromosomal ftsZ copy is rendered non-functional in the presence of a plasmid that express the essential ftsZ gene from of an inducible, ectopic promoter. A frame-shifted allele of ftsZ, e.g., obtained by cutting and filling-in the unique EcoRI site in ftsZ, is available in the art. This allele is designated ftsZo. This mutation is introduced into the strain background DRC39 (MC1000 ΔacrAB::Kan) by P1 transduction with the closely linked leu::Tn10 marker. Before transducing the null ftsZo allele, a plasmid is introduced into DRC39 that contains the wild-type ftsZ under the control of arabinose(Ara)-inducible pBAD promoter. The transductants are plated on LB plates containing Ara (a range of Ara concentrations are tested) to maintain viability) and the transductants are screened on glucose plates to look for cell death in the absence of ftsZ expression from pBAD. This would confirm the presence of the ftsZo allele in the chromosome.
After obtaining the desired low expression ftsZ strains, growth and viability assays are carried out at different Ara concentrations to identify the minimum inducer concentration sufficient for maintaining cell viability. Screening is performed at this low inducer concentration to make the strain hypersusceptible to FtsZ specific small molecule hits.
As mentioned above, many strains of microorganisms have conserve FtsZ and thus may be used in the in vivo screen of the invention. One of these strains is B. subtilis. The Gram-positive bacterium B. subtilis may be used as a surrogate for the closely related pathogen B. anthracis, the causative agent of anthrax, because cell division is an essential, conserved cellular process. A temperature-sensitive allele of ftsZ has been described in B. subtilis in which the only comply of ftsZ is fused to wild-type green fluorescent protein (gfp) (strain designated PL642). Since wild-type gfp is prone to misfolding at higher temperatures (37° C. and above), the defect in PL642 is likely due to misfolding of the entire fusion at the high temperature such that cells are viable at 30° C. but unable to form colonies between 42–45° C. Thus, according to the present invention, PL642 can be easily adopted for the in vivo screens of the invention. However, for screening against E. coli, standing growth of 40-μl cultures in 384-well plates is allowed at 30° C. In contrast, B. subtilis is an aerobic organism and requires vigorous aeration for vegetative growth to avoid sporulation.
As demonstrated herein, the in vitro FtsZ hits as well as the in vivo hit 26E-10 efficiently killed B. subtilis. This demonstrates first that E. coli screens can identify broad-spectrum compounds that would be lethal for B. anthracis and other biothreat bacterial agents. Furthermore, using E. coli as a model organism in whole-cell screens allows us to exploit the well-characterized division mutants and to circumvent the need for BL3 facility required for handling virulent pathogens. However, according to the invention, in some cases compounds may be screened directly in B. subtilis. 
Pharmaceutical Compositions
As described above, the present invention provides compounds useful for the treatment of microbial infections and/or disorders relating to a microbial infection. It will be appreciated that the compounds of the present invention can exist in free form for treatment, or where appropriate, as a pharmaceutically acceptable derivative thereof. Additionally, it will be appreciated that one or more of the inventive compounds can be formulated with a pharmaceutically acceptable carrier or excipient to provide a pharmaceutical composition.
The composition may be prepared in various forms for administration, including tablets, caplets, pills or dragees, or can be filled in suitable containers, such as capsules, or, in the case of suspensions, filled into bottles. As used herein, “pharmaceutically acceptable carrier medium” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences, Fifteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1975) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the anti-microbial compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. In the pharmaceutical compositions of the invention, the active agent may be present in an amount of at least 0.1% and not more than 50% by weight based on the total weight of the composition, including carrier medium and/or auxiliary agent(s). Preferably, the proportion of active agent varies between 0.1 to 5% by weight of the composition. Pharmaceutical organic or inorganic solid or liquid carrier media suitable for enteral or parenteral administration can be used to make up the composition. Gelatine, lactose, starch, magnesium, stearate, talc, vegetable and animal fats and oils, gum, polyalkylene glycol, or other known carriers for medicaments may all be suitable as carrier media.
The compounds of the invention may be administered using any amount and any route of administration effective for attenuating infectivity of the microorganism. Thus, the expression “amount effective to attenuate infectivity of a microorganism”, as used herein, refers to a nontoxic but sufficient amount of the anti-microbial agent to provide the desired treatment of microbial infection. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular anti-microbial agent, its mode of administration, and the like. The anti-microbial compounds of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression “dosage unit form” as used herein refers to a physically discrete unit of anti-microbial agent appropriate for the patient to be treated.
Each dosage should contain the quantity of active material calculated to produce the desired therapeutic effect either as such, or in association with the selected pharmaceutical carrier medium. Typically, the anti-microbial compounds of the invention will be administered in dosage units containing from about 5 mg to about 500 mg of the anti-microbial agent with a range of about 0.1 mg to about 50 mg being preferred.
The compounds of the invention may be administered orally, parenterally, such as by intramuscular injection, intraperitoneal injection, aerosol, intravenous infusion or the like, depending on the severity of the infection being treated. The compounds of the invention may be administered orally or parenterally at dosage levels of about 0.1 mg/kg to about 50 mg/kg and preferably from about 2 mg/kg to about 25 mg/kg, of patient body weight per day, one or more times a day, to obtain the desired therapeutic effect.
According to the present invention, a pharmaceutically acceptable derivative includes, but is not limited to, pharmaceutically acceptable salts, esters, salts of such esters, or any other adduct or derivative which upon administration to a patient in need is capable of providing, directly or indirectly, a compound as otherwise described herein, or a metabolite or residue thereof, e.g., a prodrug.
As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1–19 (1977), incorporated herein by reference. The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.
Additionally, as used herein, the term “pharmaceutically acceptable ester” refers to esters that hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms. Examples of particular esters include formates, acetates, propionates, butyrates, acrylates and ethylsuccinates.
Furthermore, the term “pharmaceutically acceptable prodrugs” as used herein refers to those prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention. The term “prodrug” refers to compounds that are rapidly transformed in vivo to yield the parent compound of the above formula, for example by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference.
As described above, the pharmaceutical compositions of the present invention additionally comprise a pharmaceutically acceptable carrier, which, as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The pharmaceutical compositions of this invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), bucally, or as an oral or nasal spray.
Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.
The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissues.
Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar—agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.
Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.
The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, and eye drops are also contemplated as being within the scope of this invention.
The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to the compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.
Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.
Uses of Compounds and Pharmaceutical Compositions
According to the methods of treatment of the present invention, microbial infections are treated or prevented in a patient or organism such as a human, lower mammal, fish, bird, or other organism, by administering to the patient a therapeutically effective amount of a compound or pharmaceutical composition of the invention, in such amounts and for such time as is necessary to achieve the desired result. In certain preferred embodiments, the compounds of the present invention are capable of acting as broad spectrum antibiotics and are effective against Gram-negative bacteria. By a “therapeutically effective amount” of a compound of the invention is meant a sufficient amount of the compound to treat microbial, e.g., bacterial infections, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
As discussed above and as exemplified in greater detail below, the compounds of the present invention are useful as anti-microbial agents, and thus may be useful in the treatment or prevention of microbial infections. As used herein, unless otherwise indicated, the terms or phrases “microbial infection” and “disorder relate to a microbial infection” include, but are not limited to, infection by the following, bacterial, fungi, yeast, or protozoa.
It will also be appreciated that the compounds and pharmaceutical compositions of the present invention can be employed in combination therapies, that is, the compounds and pharmaceutical compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, an inventive compound may be administered concurrently with another antibiotic), or they may achieve different effects (for example, surgery for removal of a tumor, administered concurrently with an inventive antibiotic).
In but one example of the usefulness of combination therapy, it has been shown that treatment with an antibiotic appears to have protective effects against atherosclerosis complications. Specifically, it has been shown that infection with Chlamydia pneumoniae is a contributing factor in the pathogenesis of atherosclerosis (Movahed, M. R. J.S.C. Med. Assoc. 1999, 95, 303). C. pneumoniae and its constituents, such as specific antigens and even DNA, have been detected in atherosclerotic plaques and also in endothelium, smooth muscle cells, and macrophages of arterial walls with atherosclerosis, but have not been found in normal arteries. Thus, treatment with an antibiotic may be used in combination with other therapies, such as surgery or other medication, to more effectively mitigate the symptoms of this disorder.
In yet another aspect, the present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention, and in certain embodiments, includes an additional approved therapeutic agent for use as a combination therapy. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
Equivalents
The representative examples that follow are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples which follow and the references to the scientific and patent literature cited herein. It should further be appreciated that the contents of those cited references are incorporated herein by reference to help illustrate the state of the art. The following examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and the equivalents thereof.
Examples
EXAMPLES
Example 1
In Vivo Assay for FtsZ Inhibitors or Activators
Materials and Methods
First Screen Variation: A differential in vivo screen was carried out at 30° C. by looking for greater than or equal to 45% growth inhibition and phenotypic aberrations (e.g., changes in filamentation or cell shape (e.g., rod to sphere) or minicell formation) of the DRC39 strain, which is deleted for the multidrug efflux pump AcrAB, and its isogenic fts84 variant DRC 40. Libraries were screened with two different concentrations of compound using 5- and 100 nl pin transfer devices.
Bacterial cultures were grown overnight at 30° C. in 0.5% NaCl Luria Broth (LB) with 20 μg/ml kanamycin and diluted 1:1000 (OD600 of 0.005) in fresh 0.5% NaCl LB. The Labsystems 384-well liquid dispenser was used to dispense 40 μl of the diluted culture to the first 23 columns of a clear NUNC 384-well plate, leaving one column empty for manual addition of the blank samples. 100 nl or 5 nl of compounds were transferred from a 384-well library plate to the assay plate using a Cartesian pin-transfer robot (see FIG. 27). After compound addition, 40 μl of LB-Amp (50 μg/ml) was added in quadruplicate to the empty column of each plate, serving as blanks. For the DMSO-only control, 1 μl of 10% DMSO was added in quadruplicate to wells lacking compounds. 5 μM 26-E10, a compound that has been shown to cause filamentation in E. coli cells, was added as a positive control for inhibition of bacterial cell division. After the controls were set up, the plates were incubated at 30° C. in a humidified chamber. At 24 hours and 48 hours, the Wallac Victor Multiwell Plate Reader measured the absorbance (650 nm) of each well. The data for each plate was processed in Microsoft Excel in order to yield a list of each well ranked in order of increasing turbidity. We then inspected microscopically samples from wells, showing 45–50% or greater growth inhibition, for filamentation or minicells, phenotypes one would expect upon perturbation of the cell division process. Cells were visualized with DIC microscopy at 100× magnification using the Zeiss AxiophotiTM Microscope.
Second Screen Variation: For setting up whole-cell screens, DRC39 and DRC40 are grown overnight at 30° C. in 0.5% NaCl-containing Luria-Bertani (LB) medium in the presence of 20 μg/ml kanamycin to ensure the presence of the disrupted acrAB::kan allele. The overnight cultures are diluted 1:1000 (OD650=005) in fresh LB. The Labsystems 384-well liquid dispenser is used to dispense 40-μl diluted cultures to the first 23 columns of a transparent NUNC 384-well plate, leaving one column empty for manual addition of control samples. Appropriate amounts (5–100 nl) of library compounds are transferred from a 384-well library plate to the assay plate using a Cartesian pin-transfer robot. 40-μl of diluted cultures supplemented with ampicillin (50 μg/ml) were manually added in duplicate to empty wells in the 24th row to serve as blanks. 5 μM 26E-10, a compound that was previously shown to cause filamentation of E. coli, is added in duplicate to culture wells (24th row) to serve as positive controls for hits and for cell filamentation. The assay plates are incubated in a humidified chamber at 30° C. After 24 and 48 hours, the optical density (OD 650 nm) in each well is read using a Wallac Victor Multiwell Plate Reader. The data is processed in Microsoft Excel to rank each well in order of increasing turbidity. Wells that show greater than or equal to 45% growth inhibition are visually inspected for phenotypes of the survivors using DIC microscopy at 100× magnification under a Zeiss AxiophotTM Microscope.
Results
First Screen: In an initial screen, a total of 18,320 compounds were screened against the wild-type and temperature-sensitive E. coli strains. A compound that caused greater than or equal to 45% decrease in turbidity of the culture in a well was considered a “hit.” There were 98 compounds that caused 45% or greater growth inhibition in the wild-type strain and 173 hits against the temperature-sensitive strain. Exactly 81 of the hits caused virtually complete growth inhibition/lysis in both strains, indicating that they are highly potent and may affect other targets required for cell viability in addition to, or besides the cell division process (see FIG. 10).
We also observed that 92 compounds only exhibited a marked effect in the temperature sensitive strain, of which 27 compounds caused cells to form long filaments. These molecules are of particular interest as they support the original hypothesis that the mutant will be more sensitive to the effects of compounds that target the cell division machinery, than the wild-type strain. Interestingly, there were 17 compounds that affected the wild-type strain yet did not show any significant effect on the mutant strain. Out of these 17 hits, there were 2 compounds that caused cell filamentation. These compounds could be potential stabilizers of the FtsZ-ring, acting as chemical suppressors of the more labile structure in the temperature-sensitive strain, while effectively hyperstabilizing the FtsZ-ring in the wild-type strain, causing filamentation. This is an exciting possibility. Further in vivo and in vitro validation of the hits generated from the in vivo screen may be carried out as described, e.g., in Example 2.
Second Screen: In a second screen of the two libraries using 100 nl of library compound, a total of 190 hits were identified. The library was rescreened using 5 nl of compound identifying at total of 39 hits. Of the 39 hits, 18 inhibited growth greater than or equal to 45% in both the DRC39 and DRC40 strains; 16 inhibited growth greater than or equal to 45% in DRC40 (ftsZ84), but not in DRC39 (FtsZ+); and 5 inhibited growth greater than or equal to 45% in DRC39, but not in DRC40.
The 16 compounds that inhibited growth in only DRC40 may preferentially destabilize Z-rings in ftsZ84 cells, while the 5 compounds that inhibited growth in only DRC39 may have low affinity fro the FtsZ84 mutant protein vis-a-vis FtsZ. Alternatively, some of the compounds that inhibited growth in only DRC39 may bind and stabilize the mutant Z-ring in DRC39 cells, whereas such stabilization may hinder the constriction of the wild-type Z-rings in DRC39 cells, thus blocking cell growth and division. This scenario would be similar to the differential effects of stabilization of Z-rings in wild-type (constriction blocked) and in ftsZ84 mutant cells (suppression of thermosensitivity) by higher dosage of the essential division protein ZipA in







TABLE 1







Major division-related phenotypes associated with the in vivo hits.











Phenotypic Data
WT (100 nl)
TS (100 nl)
WT (5 nl)
TS (5 nl)





f
9
16
3
10 


mf
8
11
8
2


F
1
25
1
5


mc
40 
 3
1
0





f = mixed population of rods with short filaments;


mf = medium filaments (4X–16X);


F = Long filaments (>16X); and


mc = minicells.


WT = DRC39, TS = DRC40.






From the combined screens, 54 compounds were selected for follow-up experiments. These compounds fell into 2 categories: 23 molecules reproducibly caused filamentation or minicell formation (in one or both strains), while the rest 31 caused greater than 45% growth inhibition of both strains regardless of the phenotype. A few of the second category molecules induced morphological transitions such as round or sausage-shaped cells.
We have tested all 54 compounds for their effects on FtsZ GTPase using the malachite green-phosphomolybdate assay. Surprisingly, only 6 compounds showed significant inhibition of FtsZ GTPase, with IC50 values ranging between 25–80 μM. We have also examined by electron microscopy the effects of 13 in vivo hits on FtsZ assembly. These 13 molecules caused cell filamentation with no effect on FtsZ GTPase. Only 3 out of 13 molecules showed effects on FtsZ assembly: one induced protofilament pairing, the second reduced protofilament assembly, while the third led to superstructure formation consisting of 5 protofilament-wide bundles. These 3 hits affect FtsZ assembly without any noticeable effect on its GTPase activity.
Third Screen: A collection of  ̃50,000 “drug-like” compounds from various sources was recently acquired and  ̃21,000 compounds are currently available for screening. These 21,000 compounds were screen using 5- and 40-nl volumes of the compounds. A total of 35 hits were obtained that caused E. coil to filament significantly in either the wild-type or in the ftsZ84 strain background and/or showed a significant difference in lethality between the strains. The effects of these 35 hits were examined on FtsZ GTPase in vitro and surprisingly none of the hits caused any significant inhibition of FtsZ GTPase activity. It is possible that these hits affect the ability of FtsZ to assemble into protein filaments. Besides the 35 hits, 15 molecules were also identified that caused significant growth inhibition and loss of cell shape, the survivors in the wells assuming a round shape from being normal rods (see FIG. 28). It is known that mutations in certain morphogenetic proteins, such as PBP2 (penicillin-binding protein 2), RodA, or the bacterial actin homolog MreB, cause E. coil to undergo a dramatic shape transition (rod to sphere). Those skilled in the art will appreciate that the hits causing shape transition can be investigated to determine whether they target any of these proteins or some unknown protein(s). PBP2 is essential for rod-shaped bacteria as it is one of the major enzymes involved in the synthesis of the longitudinal cell wall peptidoglycan. Loss of the actin homolog MreB causes E. coli to become sick and Bacillus subtilis to die. PBP2 is one target for investigation.
Example 2
Verification FtsZ Inhibitor Activity Using Vivo Assays
This example describes the use of an in vivo assay for the identification of small molecule inhibitors that effect bacterial cell division through the protein FtsZ.
Microscopy
A small molecule inhibitor of FtsZ activity, 26E-10, 27F02, 58P-18, was identified using the in vitro NADH assay (see U.S. patent application Ser. No. 10/153,268. Additional compounds were preliminarily identified from the MDS1 galanthamine library using the NADH assay. For example, FIG. 29 in U.S. patent application Ser. No. 10/153,268 depicts the percent inhibition measured by the NADH assay, described below. In some cases positive identification of a compound was validated using the GTPase assay with charcoal, described below. These studies confirmed that low, levels of FtsZ inhibitors affect FtsZ function. Those skilled in the art will appreciate that any of a variety of assays, some of which are described herein, may be used to verify activity initially detected in any in vitro or in vivo assay for compounds that affect FtsZ activity. It is also possible to use the inventive in vivo assay to verify hits identified in vitro.
The effect of the identified small molecule inhibitor 26E-10 on FtsZ ring assembly was tested in vivo by employing a single-copy ftsZ-gfp fusion construct that was integrated at the phage lambda attachment site on the E. coli chromosome. The wild-type, untagged ftsZ gene is also present on the chromosome at its normal locus. The expression of the fusion gene was placed under the control of a mutationally weakened, IPTG inducible tac promoter. Since the level of FtsZ protein expression in the cell is critical for proper cell division, the fusion gene was expressed from a single-copy and at the lowest possible inducer concentration to generate a low level of fluorescently tagged FtsZ-GFP, which did not cause any noticeable cell division aberrations. However, this low level expression was sufficient for imaging the in vivo assembly of the FtsZ-GFP fusion protein by fluorescence microscopy.
We found that the compound-induced phenotype is more pronounced in an acrAB deletion background, suggesting the likely involvement of the drug pump in reducing the intracellular concentration of compounds such as 26E-10. Therefore, in order to test the effect of 26E-10, a culture of the E. coli K-12 strain MC1000 containing acrAB deletion (henceforth designated as strain DRC 39) was grown.
To an early log-phase culture of DRC 39 ( ̃1–2×108/ml), 26E-10 was added at its MIC (minimum inhibitory concentration) of 10 μM. Aliquots were withdrawn at 15, 30, 60, and 90 min intervals after the addition of the compound and the cells in the aliquots were fixed immediately with a mixture of glutaraldehyde and para-formaldehyde. This fixation step was carried out to ensure that the handling of cells before microscopy did not cause any artifactual destabilization of the FtsZ ring structure. The fixed cells were washed with PBS, stained with DAPI to visualize the nucleoids, and observed under a fluorescence microscope. In some experiments, the cells were embedded on a thin layer of agarose before microscopy for ease of visualization. After examining the cells for GFP (FITC filter set) and DAPI (DAPI filter set) fluorescence, the DIC digital images of cell morphology were recorded using Nomarski optics (differential interference contrast, DIC, microscopy). Images were taken using an Olympus fluorescence microscope equipped with a CCD camera. Images were finally imported into Adobe Photoshop for processing and presentation.
Those skilled in the art will appreciate that MIC determinations for the compounds identified are made for a wide range of bacteria, including BW pathogens such as Bacillus anthracis, to determine which compounds have a broad-spectrum effect. Bacillus anthracis, Francisella tularensis, Shigella flexneri, and Brucella abortus each harbor a single copy of the ftsZ gene, which is likely to be essential for cell multiplication in these organisms.
As shown in FIG. 30, the control culture (no 26E-10 added) displayed a distinct equatorial FtsZ-GFP ring structure (Z-ring) at the center of the cells. DAPI staining revealed that the cells carrying Z-rings had segregated nucleoids present on either side of the ring. However, upon treatment with 26E-10, DRC 39 cells started filamenting ( ̃4× long cells seen within 60 min of treatment), which is indicative of a defect in cell division. Concurrently, the Z-rings appeared markedly reduced in number and diminished in intensity in these elongated cells, with appreciable GFP fluorescence distributed along the length of the cell body. This suggests that 26E-10 is inhibiting de novo Z-ring assembly, perhaps by destabilizing the ring structures.
It is important to note that the fluorescence intensity in the treated cells was significantly higher compared to the control culture and the exposure time for imaging the treated cells was 5–10 fold lower than that for the control cells. It is known that aberrant polymerization of FtsZ-GFP fusion in the cell cytoplasm causes the emitted GFP fluorescence to be intense. Even though we could not discern any such aberration under the microscope, it is possible that 26E-10 may induce inappropriate polymers to form.
We would also point out that DAPI staining showed that the filamenting cells contained mostly regularly spaced nucleoids, suggesting that 26E-10 does not affect DNA replication or chromosome segregation. However, DAPI staining of the elongated cells was not uniform because the cells were fixed but not permeabilized. To avoid this problem, blue Hoechst 33342 dye, which efficiently stains nucleoids in unpermeabilized E. coli cells, was used.
FIG. 31 shows two fields of the phenotype of a 26E-10 treated culture. Images were captured as described above. After 90 min treatment: there was a mixture of 1× to 8× long cells and most of the cells were devoid of distinct Z-rings irrespective of their age as evident from the cell length distribution. This indicates that 26E-10 is targeting Z-ring assembly and the effect increases in severity with the time of treatment. The fact that there were 1× cells in the culture indicates that cell division was continuing at a low level presumably because Z-rings were stochastically forming in some cells in the presence of 26E-10 and some of these rings could complete the septation process.
Cell Growth Assay
The in vivo effects of the compounds were also tested in a number of other microbial cells, including E. coli ΔacrB::kan, Hemophilus influenzae, Staphylococcus aureus, and Vibrio cholerae, as shown in FIG. 32. Specifically, a cell culture was grown up and diluted 1:5000 (105 to 5×105 cells/ml) as a starter inoculum. Thereafter, one of the identified test compounds was added at concentrations ranging from 1.25 μM to 40–80 μM in DMSO. The samples were incubated at 37° C. and aerated on a rotary wheel for 16 hours and the level of growth assessed by the turbidity of the culture visually. The data in FIG. 32 represents the minimum inhibitory concentration (MIC) of compound that was required to completely inhibit growth of the bacterial culture.
Genetic Evidence of FtsZ Inhibition In Vivo
Results presented in FIGS. 29, 31, and 32 provide the cell biology perspective on the effect of 26E-10 in E. coli cells. In order to understand whether 26E-10 indeed specifically targets FtsZ in vivo, it was tested on the thermosensitive ftsZ84 mutant of E. coli. This mutant is conditional-lethal because it grows and divides at 30° C., but undergoes a cell division block at 42° C. that leads to lethal cell filamentation. The division block of the ftsZ84 mutant at 42° C. is due to a drastic destabilization of the Z-rings at high temperature (within a minute after temperature shift-up). Based on the theory that 26E-10 was inhibiting the in vivo assembly of FtsZ, we reasoned that in the presence of 26E-10, the mutant Z-ring may not be as robust as the wild-type ring at the permissive temperature of 30° C. and this inherent weakness of the mutant ring may be exacerbated.
We assessed the sensitivity of the ftsZ84 mutant strain DRC40 (harboring the plasmid vector pBR322) to 26E-10 and 27F02 at 30° C. in comparison to the congenic parent DRC39. Both DRC39 and DRC40 lack the major multidrug efflux pump AcrAB.
DRC39 cells were treated for 2 hours with inhibitor 27F02. It was found initially that 27F02 kills cells, but no filamentation phenotype was observed (see FIG. 33). The high fluorescent background of 27F02 itself was precluding FtsZ-GFP ring imaging or FtsZ ring imaging using immunofluorescence. Viewing the effect of 27F02 on FtsZ ring structure in vivo in DRC39 cells (using the RITC filter set for GFP imaging), the majority of cells have distinct bipolar fluorescent foci and infrequently a central focus that did not span the entire circumference of the cell. (see FIG. 33).
One way to interpret this result is that 27F02 is interacting in vivo with division ring components such as FtsZ. The polar foci may be remnants of old division rings that did not disassemble completely. Alternatively, nascent ring machinery may be assembling inappropriately at the poles. Most strikingly, the majority of cells lack a distinct central ring, suggesting inhibition of medial FtsZ ring assembly. Moreover, the partial ring-like foci at midcell are also indicative of aberrant assembly or destabilization of division rings in the presence of 27F02.
Whereas the MIC of 26E-10 for DRC39 is 10 μM, it was between 2.5 to 5 μM for DRC40. The DRC40 cell density at 2.5 μM compound was very low compared to the untreated control, with predominantly long filaments (16×), filamentous ghosts, and few short cells present (FIG. 34). DRC40 showed absolutely no growth at 5 μM, whereas DRC39 had a mixture of filaments of varying lengths and short cells present at a low density at this concentration of 26E-10. The cell density of DRC39 at 5 μM was appreciably higher than that of DRC40 at 2.5 μM. These results indicate that the ftsZ84 mutant (DRC40) has higher sensitivity toward 26E-10 compared to its wild-type parent (DRC39), likely because the presence of 26E-10 augments the inherent weakness of the ftsZ84 ring structure, akin to a synthetic lethal genetic interaction.
Whether the higher sensitivity of ftsZ84 to 26E-10 could be reversed in the presence of the recombinant pBR322 plasmid carrying the wild-type ftsZ gene was also tested. Indeed, when wild-type ftsZ was provided in trans, the MIC of 26E-10 for the mutant DRC40 was 10 μM, identical to that seen with the DRC39 parental cells. As shown in FIG. 34 panel B, DRC40/pBR-ftsZ+ cells underwent robust cell division at 2.5 μM 26E-10 unlike the situation with DRC40/pBR322. Moreover, at 5 μM 26E-10, there was higher cell density and less pronounced filamentation with DRC40/pBR-ftsZ+ in contrast to DRC40/pBR322 (compare FIGS. 34 panels A and B). These results provide compelling genetic evidence that 26E-10 targets FtsZ rings in vivo.
Phenotypic Screen
In addition, the phenotype of ftsZ84 cells was examined in the presence of 26E-10 over time to determine the thermolability of the ftsZ84 rings. Specifically, the phenotype of the thermosensitive ftsZ84 was assessed at 30° C., and also at 42° C. at 10 and 120 minutes. At 42° C. the mutant FtsZ rings were rapidly destabilized, within 10 minutes. A similar phenotype is expected with small molecules that inhibit or activate polymerization-dependent FtsZ GTPase activity. (See FIG. 35).
ZipA interacts with FtsZ both in vitro and in vivo and it has been shown that a second copy of zipA can suppress the thermosensitivity of the ftsZ84 mutant at the restrictive temperature of 42° C. This is because ZipA is a stabilizer of FtsZ ring assembly and doubling the number of ZipA molecules in the cell leads to the stabilization of the thermolabile FtsZ84 ring in vivo.
A new result is that a second copy of the essential division gene zipA can also decrease or ameliorate the toxicity of 26E-10. The fact that a second copy of zipA can reverse the toxicity of 26E-10 to a significant degree suggests that 26E-10 may be destabilizing the FtsZ ring structure in vivo.
Effect of an In Vitro Inhibitor 58P-18 on FtsZ GTPase In Vivo
The effect of 58P-18 on Z-ring assembly and cell division was tested in DRC39 cells in a similar manner as that described above for 26E-10. FIG. 36 shows that cells start elongating within 60 min of treatment with 40 μM 58P-18 and, more strikingly, none of the treated cells appear to contain a medial FtsZ ring. Even though DAPI staining shows that the nucleoids have replicated and segregated in the presence of the compound, none of the short cells in the field has been able to assemble a Z-ring between segregated nucleoids. Instead, the GFP fluorescence is delocalized all over the cell body (fluorescence is much more intense than in the control cells, similar to that seen with 26E-10), suggesting the possibility of aberrant FtsZ-GFP polymerization. Interestingly, in contrast to 26E-10, treatment with 58P-18 seems to generate a pattern of FtsZ-GFP distribution in cells that is very similar to that seen with DAPI stained nucleoids. Thus, without limiting the mechanism of the invention, 58P-18 may promote inappropriate association of FtsZ with the chromosome.
Charcoal Based Assay
A high throughput enzyme-coupled FtsZ assay is used to preliminarily screen the combinatorial libraries, synthesized in the candidate inhibitors of FtsZ GTPase activity identified in the FtsZ assay and are then tested in the downstream charcoal-based GTPase assay, also described herein. The inhibitory activities of the compounds are tested at two concentrations (e.g., 3.5 μM or 17.5 μM) relative to the DMSO-only control in the downstream charcoal-based assay for GTPase activity described herein. FIG. 37 depicts the effect of an activator on FtsZ orthologs E. coli and M. tuberculosis. 
Initial Library Screening: Inhibition of FtsZ Activity In Vivo
This assay was miniaturized for high throughput screening of the Chembridge 16,320-member small molecule library and the  ̃2000 member NCI mini diversity library against untagged, assembly-competent FtsZ purified from Escherichia coli. [The MDS1 library was also screened, but no downstream validation assay could be carried out due to lack of compound, so none of the inhibitors identified are from the MDS1 library.] From this screen, the in vitro assay identified 172 compounds. From this assay 23 compounds were identified that inhibited FtsZ activity. In a secondary screen, five inhibitors were verified (see FIG. 38).
As depicted in FIGS. 11 and 16, five inhibitors (58-PIS (NCI library), 16-L09 (Chembridge library), 18-M04, 27-D12 (Chembridge library), and 27-F02 (Chembridge library)) and of FtsZ were identified in these initial screens. A phenotypic screen using AcrAB efflux pump knockout strains identified 26E-10.
NADH Assay
Purified FtsZ protein is combined in a reaction vessel with the enzymes, pyruvate kinase, and lactate dehydrogenase, and the substrates GTP, PEP, and NADH. As shown in FIG. 39, upon reaction with GTP, FtsZ yields the products GDP and phosphate, providing a substrate, GDP, for pyruvate kinase in combination with PEP to generate pyruvate. Pyruvate in turn becomes a substrate for lactate dehydrogenase with NADH to yield NAD+ and lactate. Test molecules may be added to the reaction mixture to assess their effect on FtsZ activity. Activation of FtsZ activity can be determined by measuring a decrease in the rate of NADH fluorescence compared to the absence of the test molecule (excitation: 355 nm, emission: 460). This assay was miniaturized by testing compounds in a multi-well plate and assessing fluorescence using a Wallac plate reader.
TLC Assay
As noted above, several downstream assays were developed to validate the primary hits, including the NADH assay, described above. Another such assay is the radioactive thin-layer chromatographic (TLC) analysis that measures the conversion of [α-32P]GTP to [(α-32P]GDP catalyzed by FtsZ. In this assay, the two nucleotides (GTP and GDP) are present in reaction aliquots and are separated on a polyethyleneimine-cellulose thin-layer plate. This affords rapid and direct estimation of the GTPase activity of FtsZ under reaction conditions that are known to promote FtsZ assembly. The reaction mixture does not contain other enzymes or substrates unlike the situation with the enzyme-coupled assay used for primary screening. Therefore, the TLC assay result is a reliable indicator of the in vitro efficacy of the candidate molecules identified as hits using the coupled assay.
TLC analysis of FtsZ GTPase activity in the presence or absence of test compounds was also conducted. 5 μM FtsZ was incubated with 1 mM [α-32P]GTP (1.5 μCi) at 30° C. and 2 μl aliquots were withdrawn at 5, 15, and 30 min intervals into an equal volume of 1% SDS-20 mM EDTA to quench the reaction. The aliquots were incubated at 70° C. for 2 minutes prior to spotting 0.5 μl samples on a PEI-cellulose plate. The TLC plate was developed in 0.75 M potassium phosphate buffer (pH 3.4), air-dried, and exposed to a film or a phosphorimager.
The compound I-20 inhibited FtsZ GTPase activity in a dose-dependent manner, see FIG. 40.
Example 3
Analysis of Compound 26E-10
Below, is shown the MIC values of 26E-10, which causes filamentation in both DRC39 and DRC40 but has no effect on FtsZ in vitro.







TABLE 2







MIC values of 26E-10 against different bacteria.










Organism
MIC (μM)















E. coli MC1000 (wild-type)
>40




E. coli DRC39 (acrAB::kan)
10
(filaments)



E. coli DRC40 (DRC39 ftsZ84)
2.5*
(filaments)



E. coli DRC42 (DRC39 recA::cat)
10




Vibrio cholerae
40




Bacillus subtilis
40




Staphylococcus aureus
20




Clostridium perfringens
>80
(filaments)










As seen before with the in vitro inhibitors, 26E-10 permeates E. coli cells significantly better in the absence of the AcrAB pump. Consistent with the inventive in vivo assay, the ftsZ84 mutant is significantly more sensitive to 26E-10 compared to the isogenic parent strain.
To rule out if SOS-mediated SulA induction might be responsible for cell division inhibition with 26E-10, we introduced the null recA::cat allele into DRC39 by P1 transduction. To confirm the absence of the recA gene product in the transductants, we screened them for UV-sensitivity and chose one chloramphenicol-resistant, UV-sensitive transductant, designated DRC42, for testing. RecA is essential for the induction of SOS regulons in E. coli and in its absence, the division inhibitor SulA is not induced. DRC42 was as sensitive to 26E-10 as its parent DRC39 (Table 2), indicating that 26E-10 is unlikely to cause division inhibition via SulA induction. 26E-10 is also active against other wild-type organisms at somewhat higher concentrations (Table 2;  ̃10–20 μg/ml corresponding to 20–40 μM, the molecular weight of 26E-10 being 500). Interestingly, C. perfringens showed filamentation phenotype in the presence of 80 μM 26E-10 without any overt effect on cell growth under anaerobic conditions. Any of the in vivo hits may be tested on DRC42 to rule out indirect inhibition of cell division through SOS induction as well as testing the compounds against a plethora of organisms in broth cultures to check the broad-spectrum nature of the hits.
Example 4
Screening for FtsZ Inhibitors Using Small Molecule Microarrays
Small molecule libraries are printed on glass slides to create small molecule microarrays to provide an opportunity to explore the feasibility of using such microarrays to identify FtsZ antagonists. The microarray is created by using a high-precision robot to pick up a small volume of dissolved compounds from the original 384 well plates and repetitively deliver 1 nL of solution to defined locations on a chemically derivatized glass microscope slide (see FIG. 27). Each compound is immobilized on the glass slide via a covalent linkage between a common functional group on the small molecule and the maleimide-derivatized glass slides. Interactions between FtsZ and small molecules are determined by incubating the microarray slide with purified FtsZ-GFP fusion protein and then visualizing the location of the bound protein by the ArrayWoRx fluorescent slide scanner. This experiment is performed in the absence of GTP to identify compounds that bind FtsZ monomers and in the presence of GTP to identify compounds that bind FtsZ polymers. Data obtained from screening the microarray library may validate the initials hits identified in the enzyme-coupled biochemical screen and provide evidence for the utility of small molecule microarrays as a fast and efficient method for screening future chemical libraries.
Alternatively, compound may be delivered to the well containing cells. An overnight culture of the wild-type E. coli lacking the major drug efflux pump AcrAB (MC1000 ΔacrAB=DRC 39) is diluted to 1:5000 in fresh medium and 40 μl is inoculated into each well of a clear bottom NUNC 384-well plate. 100 nL of compounds is pin transferred to each well using the Cartesian robot, resulting in a final screening concentration of 17 μM. The 384-well plates are incubated at 37° C. in a humid chamber and the culture turbidity is measured at 650 nm using the Wallac Plate Reader after 5 h and after 24 h.
The reduction in cell density in the presence of a compound after 24 h or 48 h is expressed as the standard deviation from the average final density of the other wells on the plate. The effect of the compounds is also expressed as percent growth inhibition compared to the DMSO only control. A compound was characterized as a “hit” if it caused the cell density in a well to decrease by two standard deviations from the mean density of all wells in the plate. Samples from the growth-inhibited wells are then visualized by DIC microcopy and inspected for filaments or mini cells, phenotypic markers for cell division aberrations.
Screening a chemical library in a microarray format improves the speed of the screening method and also increases the reliability of the assay by comparing the hits identified by microarray analysis with those obtained from other in vitro and cell-based screening assays. In addition, the combination of the microarray assay with other assay methods will also assist in the validation of the targets identified, e.g., by comparing the targets identified in one assay to the targets identified in the other assay. Validation of inhibiting and activating structures is important for molecular modeling and generation of more potent derivatives against a given target.
A number of interesting compounds have been identified as shown in FIGS. 42–46.
Other embodiments of the invention will be apparent to those skilled in the art from a consideration of the specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

## Claims

1. A method for identifying compounds that affect cell division comprising steps of: providing a bacterial strain expressing a mutant ftsZ protein under permissive conditions, wherein the bacterial strain is more sensitive to an inhibitor of cell division under permissive conditions than an isogenic wild type strain; contacting cells of the bacterial strain with a compound; and detecting a defect in cell division.

2. A method for identifying compounds that affect cell division, comprising steps of: providing a strain of bacteria that expresses a reduced amount of FtsZ compared to a wild-type strain; contacting the bacteria with a compound; and detecting a defect in cell division.

3. The method of claim 2, wherein the bacteria have a non-functional chromosomal copy of ftsZ and express a wild-type copy of the ftsZ gene from a plasmid containing an inducible promoter.

4. The method of claim 1 or 2, wherein the step of detecting comprises detecting an alteration in the phenotype of the bacteria.

5. The method of claim 4, wherein the alteration comprises a destabilization in Z-ring structure.

6. The method of claim 4, wherein the alteration comprises stabilization in Z-ring structure.

7. The method of claim 1 or 2, wherein the step of detecting comprises detecting a decrease in cell division.

8. The method of claim 1 or 2, wherein the step of detecting comprises detecting a greater than or equal to 45% decrease in cell growth.

9. The method of claim 1 or 2, wherein the step of detecting comprises detecting an increase in cell division.

10. The method of claim 1 or 2, wherein the step of detecting comprises detecting an activator of cell division by detecting bacteria that are shorter than wild-type cells.

11. The method of claim 1 or 2, wherein the step of detecting comprises detecting an inhibitor of cell division by detecting bacteria that are longer and more filamentous than wild-type cells.

12. The method of claim 1 or 2, wherein the bacteria further comprise a mutation in the multidrug efflux pump.

13. The method of claim 12, wherein the mutation is an acrAB mutation.

14. The method of claim 1 or 2, wherein the bacteria further comprise a tolC mutation.

15. The method of claim 1 or 2, wherein the bacteria further comprise ZipA.

16. The method of claim 1 or 2, wherein the bacteria comprise a conditional-lethal mutant allele of ftsZ.

17. The method of claim 16, wherein the bacteria comprising a conditional-lethal mutant allele of ftsZ are selected from the group consisting of V. cholerae, S. aureus, S. flexneri, and B. subtilis.

18. The method of claim 1 or 2, wherein the bacteria are E. coli.

19. The method of claim 18, wherein the E. coli contain an ftsZ84 mutation.

20. The method of claim 19, wherein the ftsZ84 mutation generates an enhanced sensitivity to the adverse effects of a compound on cell division at under permissive conditions compared to the isogenic wild-type strain.

21. The method of claim 18, wherein the E. coli contain an ftsZ26 mutation.

22. A method of identifying compounds that affect bacterial cell division, comprising steps of: contacting a cell that is defective in cell division under non-permissive conditions with a compound of interest; and detecting an alteration in the phenotype of the cell.

23. The method of claim 22, wherein the bacterial cell contains a conditional-lethal mutant allele of ftsZ.

24. The method of claim 22, wherein the bacterial cell is an E. coli cell containing a conditional-lethal mutant allele of ftsZ.

25. The method of claim 24, wherein the conditional-lethal mutant allele of ftsZ is ftsZ84.

26. The method of claim 24, wherein the conditional-lethal mutant allele of ftsZ is ftsZ26.

27. The method of claim 23, wherein the bacterial cell comprising a conditional-lethal mutant allele of ftsZ is selected from the group consisting of V. cholerae, S. aureus, S. flexneri, and B. subtilis.

28. The method of claim 22, wherein the alteration in phenotype is a destabilization in the Z ring structure.

29. The method of claim 22, wherein the alteration in phenotype is an exacerbation of the failure to form Z rings.

30. An assay system for identifying compounds that effect cell division, the assay system comprising a bacterial cell comprising a mutation affecting a multidrug efflux pump, wherein the bacterial cell further comprises a mutant FtsZ protein.

31. The assay system of claim 30, wherein the bacterial cell is an E. coli ftsZ84 bacterial cell.

32. The assay system of claim 30, wherein the bacterial cell is an E. coli ftsZ26 bacterial cell.

33. A method for identifying compounds that affect cell division, comprising steps of: providing a bacterial strain expressing a mutant ftsZ protein under permissive conditions; contacting cells of the bacterial strain with a compound; and detecting a defect in cell division, wherein the step of detecting comprises detecting an inhibitor of cell division by detecting a decrease in cell division or detecting bacteria that are longer and more filamentous than wild-type cells.

34. The method of claim 33, wherein the bacteria selected from the group consisting of: E. coli, V. cholerae, S. aureus, S. flexneri, and B. subtilis.

35. A method for identifying compounds that affect cell division, comprising steps of: providing a bacterial strain expressing mutant ftsZ protein under permissive conditions; contacting cells of the bacterial strain with a compound; and detecting a defect in cell division, wherein the bacterial strain is of a bacterial species selected from the group consisting of E. coli, V. cholerae, aureus, and S. flexneri.

36. The method of claim 35, wherein the detecting step comprises detecting a decrease in cell division.

37. A method of screening for compounds that affect cell division comprising steps of: providing a bacterial strain expressing a mutant ftsZ protein under permissive conditions; contacting cells of the bacterial strain with a compound not previously known to be an inhibitor of cell division; and detecting a defect in cell division.

38. A method for identifying compounds that affect cell division comprising steps of: providing a temperature-sensitive bacterial strain expressing a mutant ftsZ protein under permissive conditions; contacting cells of the bacterial strain with a compound; and detecting a defect in cell division.

39. A method for identifying compounds that affect cell division comprising steps of: providing a bacterial strain expressing a mutant ftsZ protein under permissive conditions; contacting cells of the bacterial strain with members of a compound library; and detecting a defect in cell division in cells contacted with a member of the library.