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WO1999037800A1 - Composes antimicrobiens - Google Patents

Composes antimicrobiens Download PDF

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Publication number
WO1999037800A1
WO1999037800A1 PCT/US1999/001288 US9901288W WO9937800A1 WO 1999037800 A1 WO1999037800 A1 WO 1999037800A1 US 9901288 W US9901288 W US 9901288W WO 9937800 A1 WO9937800 A1 WO 9937800A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
compound
triclosan
antimicrobial
mutant
Prior art date
Application number
PCT/US1999/001288
Other languages
English (en)
Inventor
Stuart B. Levy
Laura M. Mcmurry
Original Assignee
Trustees Of Tufts College
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Trustees Of Tufts College filed Critical Trustees Of Tufts College
Priority to AU23324/99A priority Critical patent/AU2332499A/en
Priority to EP99903262A priority patent/EP1049799A1/fr
Priority to CA002319115A priority patent/CA2319115A1/fr
Priority to JP2000528707A priority patent/JP2002510463A/ja
Priority to IL13744499A priority patent/IL137444A0/xx
Publication of WO1999037800A1 publication Critical patent/WO1999037800A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material

Definitions

  • Triclosan is a trichlorinated biphenyl broad spectrum antibacterial/fungal agent (Furia, T.E., et al. Soap & Chemical Specialties 44, 47-50, 116-122 (1968); Regos, J., et al. Dermatologica 158, 72-79 (1979)). Because of its general biocidal activity, triclosan has been used as a topical disinfectant in soaps, cosmetics, and lotions (Regos, J., et al. Dermatologica 158, 72-79 (1979)), and more recently has been added to toothpastes (Cummins, D. J. Clin. Periodont. 18, 455-461 (1991), to fabrics for use in bedding and clothing, and to plastics for use in toys, cutting boards, and flooring.
  • the present invention is based, at least in part, on the discovery that triclosan (an antimicrobial compound commonly used in consumer products, e.g., soaps and detergents), has a genomic target which is involved in its ability to impart antimicrobial activity.
  • the present invention further includes the identification of the genomic target for triclosan in Escherichia coli and in Mycobacterium smegmatis, as FabI and InhA, respectively, and provides for methods of identifying antimicrobial compounds based upon this identification (hereinafter screening assays will be used interchangeably for such methods for discussion purposes).
  • ER will be used to refer to these reductase enzymes, and it should be understood that the descriptions apply to the ER polypeptide as well as to the FabI and InhA polypeptide embodiments.
  • the present invention includes the development of screening assays using these mutant polypeptides and triclosan-resistant microbial cells for antimicrobial compounds which can be used against triclosan-resistant microbial cells, e.g., in lieu of triclosan or in addition to triclosan.
  • NSAM nonspecific antimicrobial agent
  • NSAMs for the purpose of this invention is intended to include the broad class of antimicrobial compounds, e.g., found in consumer products, that (prior to the present discovery) were not believed to be target specific on the genomic level by those of ordinary skill in the art.
  • NSAMs are not intended to include antibiotics or other antimicrobials which one of ordinary skill in the art would have expected to be target specific on a genomic level prior to the discovery of the present invention.
  • the present invention includes the identification of genomic targets involved in an NSAM's ability to impart antimicrobial activity and the development of screening assays for antimicrobial compounds based upon these genomic targets.
  • aspects of the invention include the reagents used in the aforementioned screening assays, antimicrobial compounds identified using the screening assays, and methods of using the identified compounds in combination products, e.g., consumer products and in therapeutic methods.
  • the present invention pertains to a method for identifying an antimicrobial compound which interacts with an ER polypeptide, e.g., a FabI or InhA polypeptide.
  • an ER polypeptide e.g., a FabI or InhA polypeptide.
  • the method involves contacting the ER polypeptide with a compound under conditions which allow interaction of the compound with the ER polypeptide to occur.
  • the method further includes detecting the interaction of the compound with the ER polypeptide as an indication of whether the compound is an antimicrobial compound.
  • antibacterial compound is art-recognized and is intended to include a compound which inhibits the proliferation or viability of a microbe which is undesirable and/or which disrupts a microbial cell.
  • the language further includes significant diminishment of a biological activity which is undesirable and associated with the microbe, such that a subject would not be detrimentally affected by the microbe. Examples include antibiotics, biocides, antibacterial compounds.
  • ER polypeptides having enoyl-acyl carrier protein reductase activity include the full length polypeptides and/or biologically active fragments thereof.
  • the preferred fragments contain the reducing agent binding cleft and/or the triclosan binding portion and/or the substrate binding site, and are of a size which allows for their use in the screening methods of the present invention.
  • ER polypeptides of the present invention are discussed in further detail below.
  • the term "compound” is art-recognized and includes compounds being tested for antimicrobial activity.
  • the compound can be designed to incorporate a moiety known to interact with a ER polypeptide or can be selected from a library of diverse compounds, e.g., based on a desired activity, e.g., random drug screening based on a desired activity.
  • a desired activity e.g., random drug screening based on a desired activity.
  • the compound of the present invention is a small molecule.
  • examples of compounds of the present invention include NSAMs and triclosan compounds.
  • NSAM for the purpose of this invention is as defined above.
  • An NSAM compound includes functional and structural analogs of a parent NSAM compound. The analogs can be selected or designed either using the genomic target involved in its ability to impart antimicrobial activity and/or based upon knowledge derived from studying the interaction between the NSAM and the genomic target.
  • triclosan compound includes functional and structural analogs of triclosan.
  • the analogs can be selected or designed either using the genomic target and/ or based upon knowledge derived from studying the interaction between triclosan and the genomic target.
  • the compound can be a single compound or can be a member of a test library.
  • test libraries that can be used include combinatorial libraries or libraries of natural products.
  • a "diversomer library” is created by the method of Hobbs DeWitt et al. (Proc. Natl. Acad. Sci. U.S.A. 90:6909 (1993)).
  • Other synthesis methods including the "tea-bag” technique of Houghten (see, e.g., Houghten et al., Nature 354:84-86 (1991)) can also be used to synthesize libraries of compounds according to the subject invention.
  • the language "interacts with an ER polypeptide” include interactions with the polypeptide which result in the identification of a compound having antimicrobial activity. Such interactions include binding of the compound to the polypeptide, e.g., direct or indirect binding, which allows for identification of a compound having antimicrobial activity. In one embodiment, the interaction occurs with the reducing agent binding cleft of the ER polypeptide. In another embodiment, the interaction occurs with the triclosan binding portion of the ER polypeptide. - 5 -
  • reducing agent binding cleft is intended to include that portion of the ER polypeptide which interacts with, e.g., binds with, a reducing agent.
  • An example of a reducing agent cleft is the NAD (or NADH+)/NADP (or NADPH+) binding cleft of the ER polypeptide.
  • the language “triclosan binding portion” is that portion of the ER polypeptide which binds, e.g. directly or indirectly, triclosan. In one embodiment the triclosan binding portion is within the reducing agent binding cleft.
  • detecting the interaction of the compound with the ER polypeptide includes means of detection which result in the identification of a compound having antimicrobial activity.
  • the interaction can be detected based on the presence or absence of enzyme activity, e.g., using art-recognized techniques.
  • the present invention further pertains to a method for identifying an antimicrobial compound by contacting an enoyl reductase molecule with a compound under conditions which allows enzyme activity to occur.
  • the presence or absence of enzyme activity is detected as an indication of whether the compound is an antimicrobial compound.
  • ER enoyl reductase molecule
  • the present invention further pertains to a method for identifying an antimicrobial compound by exposing or contacting a microorganism to a compound under conditions which allow fatty acid biosynthesis to occur.
  • the inhibition of fatty acid biosynthesis is detected as an indication of whether the compound is an antimicrobial compound.
  • the language and terms of this method are as defined above and/or below.
  • inhibition of fatty acid biosynthesis is art recognized and includes the inhibition of the synthesis of at least one fatty acid in the microorganism .
  • the inhibition of fatty acid biosynthesis can be measured as discussed below.
  • microorganism is art- recognized and for purposes of this invention is used interchangeably with “microbe or microbial cell”.
  • the present invention further pertains to a method for identifying an antimicrobial compound which interacts with a mutant ER polypeptide by contacting the mutant ER polypeptide with a compound under conditions which allow interaction of the compound with the mutant ER polypeptide to occur.
  • the presence or absence of interaction of the compound with the mutant ER polypeptide is detected as an - 6 -
  • mutant of an ER polypeptide is intended to include polypeptides which differ from the ER polypeptide in an alteration of at least one amino acid residue but retain their ability to be useful within the screening assays of the present invention.
  • the mutant ER polypeptides of the present invention include the full length mutant ER polypeptide and/ or biologically active fragments thereof.
  • the preferred fragments contain the reducing agent binding cleft and/ or the triclosan binding portion and are of a size which allows for their use in the screening methods of the present invention.
  • the protein product of the mutant gene is capable of conferring resistance to triclosan in a microbial cell.
  • the protein product of the mutant gene is capable of conferring resistance to an NSAM in a microbial cell.
  • the mutant has a gly93val substitution. (The convention used here to describe the substitution mutation lists the wild-type amino acid followed by the position of the residue in the protein followed by the substituted mutant amino acid.)
  • the mutant has a metl59thr or phe2031eu substitution.
  • the mutant has an alteration of at least one amino acid in the reducing agent, e.g., NAD/NADP binding cleft of the ER molecule or an alteration of at least one amino acid residue in the triclosan binding portion.
  • the mutant ER protein is a mutant FabI polypeptide having an amino acid sequence as shown in SEQ ID NO: 3 except that it comprises an amino acid substitution at a position selected from the group consisting of G 13, SI 6, SI 9, 120, A21, S91, 192, G93, F94, A95, L100, L144, S145, Y156, M159, K163, G190, P191, 1192, R193, T194, L195, A196, 1200, K201, D202, F203, R204 and K205.
  • One of ordinary skill in the art would understand that the numbering system is based on the E. coli FabI polypeptide.
  • an alignment of FabI can be made with other, related ER molecules.
  • FabI and InhA are shown in Figure 2.
  • the language “corresponds to” is meant to include an approximate correspondence when the sequence are aligned in a biologically meaningful manner by one of ordinary skill in the art.
  • the language “corresponds to” also includes residues which spatially correspond, e.g., are in the same functional position upon crystallography, but which may not correspond when aligned using an - 7 -
  • the present invention further pertains to a method for identifying an antimicrobial compound capable of inhibiting proliferation or viability of a triclosan- resistant microbial cell.
  • the method involves contacting a triclosan-resistant microbial cell with a compound under conditions which allow a triclosan-resistant microbial cell to proliferate or remain viable.
  • the method further includes determining whether the compound is capable of inhibiting proliferation or viability of the cell thereby identifying an antimicrobial compound capable of inhibiting proliferation or viability of a triclosan-resistant microbial cell.
  • the language and terms of this method are as defined above and/or below.
  • the presence of lysis of the triclosan-resistant microbial cell can be used to identify an antimicrobial compound capable of inhibiting proliferation or viability, and/or disrupting, a triclosan-resistant microbial cell.
  • the present invention further pertains to a method for identifying an antimicrobial compound capable of inhibiting proliferation or viability of a triclosan- resistant microbial cell by contacting a polypeptide capable of conferring resistance to - 8 -
  • polypeptide capable of conferring resistance to triclosan is intended to include a polypeptide which when present in the microbial cell under appropriate conditions confers resistance to triclosan to the microbial cell, e.g., the microbial cell can proliferate and remain viable in the presence of triclosan.
  • the invention further pertains to a method for identifying an antimicrobial compound capable of inhibiting proliferation or viability of a NSAM-resistant microbial cell.
  • the method involves contacting a polypeptide capable of conferring resistance to a NSAM with a compound under conditions which allow interaction of the compound with the polypeptide to occur.
  • the method further involves detecting the presence or absence of interaction with the polypeptide as an indication of whether the compound is an antimicrobial compound capable of inhibiting proliferation or viability of a NSAM- resistant microbial cell.
  • the language and terms of this method are as defined above and or below.
  • infectious disease is meant to include disorders caused by one or more species of bacteria, viruses, fungi, and protozoans, species of which that are disease-producing organisms collectively referred to as "pathogens.”
  • pathogens are exemplified, but not limited to, Gram-positive bacteria such as Actinomyces bovis, Enterococcus fecalis, Hemophilus pneumoniae, Listeria monocytogenes, Mycobacterium tuberculosis, M. leprae, M. smegmatis, Proprionibacterium acnes, Sarcina ventriculi, Staphylococcus aureus, S. epidermis, S.
  • Streptococcus hemolyticus S. pneumoniae
  • Gram-negative bacteria such as Campylobacter fetus, Erwinia carotovora, Flavobacterium meningosepticum, Helicobacter pylori, Hemophilus pneumoniae, H. influenzae, Klebsiella pneumonia, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Shigella dysenteria, Salmonella typhi, S.
  • viruses such as HIV- 1,-2, and -3, HSV-I and -II, non-A non-B non-C hepatitis virus, pox viruses, rabies viruses, and Newcastle disease virus; fungi such as Candida albicans, C. tropicalis, C. krusei, C. pseudotropicalis, C parapsilosis, C quillermondii, C. stellatoidea, Asperg ⁇ lius fumigatus, A. niger, A. nidulans, A. flavus, A.
  • the compound can be applied prior infection by the organism to prevent a subject from becoming infected.
  • the compounds can be used for cleaning surfaces, e.g., counter tops, instruments, or the skin of the subject, to inhibit the growth of the organism and reduce the possibility of the subject actually becoming infected with one of the organisms.
  • Treating or treatment of a state characterized by the presence of an unwanted cell is intended to include the alleviation of or diminishment of at least one symptom, for example, fever or inflammation, typically associated with the state.
  • the treatment also includes alleviation or diminishment of more than one symptom.
  • the treatment cures, e.g., substantially eliminates, the symptoms associated with the state.
  • therapeutically effective dose or “therapeutically effective amount” of a compound described herein, is that amount necessary or sufficient to perform its intended function, e.g., on a surface or on or within a subject, e.g., to eradicate or inhibit growth of an unwanted pathogen, e.g., microorganism.
  • the therapeutically effective amount can vary depending on such factors as the species or strain of the pathogen, the amount of the pathogen to be inhibited ant the manner in which the compound is to be used.
  • One of ordinary skill in the art would be able to study the aforementioned factors and make a determination regarding the effective amount of the compound required without undue experimentation.
  • an in vitro or in vivo assay can be here used to determine an "effective amount" of the compounds described herein to achieve inhibition of growth or proliferation of the cell by binding and inhibiting the specific target.
  • the present invention also pertains to antimicrobial soap or detergent preparations containing triclosan in amounts which are much lower than the amounts contained in the commercially available antimicrobial soap or detergent preparations.
  • the commercially available antimicrobial soap or detergent preparations contain triclosan in higher amounts and part of the present invention includes the realization that higher amounts, e.g., than 0.3 % triclosan found in Total® toothpaste, or 3 mg mH, are not necessary for the triclosan to interact with its genomic target.
  • the antimicrobial soap or detergent preparations contain triclosan at a concentration of less than about 500 ⁇ g per milliliter of soap or detergent preparation forming an antimicrobial soap or detergent preparation.
  • the antimicrobial soap or detergent preparations contain triclosan at a concentration of less than about, e.g., 500 ⁇ g ml ⁇ l (one ml being roughly equivalent to one gram of solid, which can be corrected by the density of the solid), less than about 100 ⁇ g mH , less than about 50 ⁇ g mH, e.g., less than about 10 ⁇ g ml ⁇ l, less than about 5 ⁇ g ml ⁇ l> less than about 1 ⁇ g mHand e.g., less than about O.S ⁇ g ml "1 .
  • a skin antiseptic a safe, nonirritating, antimicrobial-containing preparation that prevents overt skin infection
  • a patient preoperative skin preparation a safe, fast-acting, broad-spectrum, antimicrobial- containing preparation that significantly reduces the number of micro-organisms on intact skin
  • a surgical hand scrub a safe, nonirritating, antimicrobial-containing preparation that significantly reduces the number of microorganisms on the intact skin.
  • a skin wound cleanser a safe, nonirritating, liquid preparation (or product to be used with water) that assists in the removal of foreign material from small, superficial wounds and does not delay wound healing
  • a skin wound protectant a safe, nonirritating preparation applied to small cleansed wounds that provides a protective barrier (physical, chemical, or both) and neither delays healing nor - 13 -
  • an antimicrobial soap a soap containing an active ingredient with in vitro and in vivo activity against skin microorganisms.
  • the present invention also pertains to antimicrobial soap or detergent preparations containing triclosan compounds, e.g., structural analogs of triclosan, in a soap or detergent preparation.
  • triclosan compounds e.g., structural analogs of triclosan
  • the structural analog of triclosan is a compound capable of inhibiting the proliferation and viability of a triclosan-resistant microbial cell.
  • Antimicrobial compound is art-recognized and is intended to include a compound which inhibits the proliferation or viability of a microbe which is undesirable and/or which disrupts a microbial cell. The language further includes diminishment of an activity which is undesirable and associated with the microbe. Examples include antibiotics, biocides, antibacterial compounds.
  • antibiotics is art recognized and includes antimicrobial agents synthesized by an organism in nature and isolated from this natural source, and chemically synthesized antibiotics.
  • the term includes but is not limited to: polyether ionophore such as monensin and nigericin; macrolide antibiotics such as erythromycin and tylosin; aminoglycoside antibiotics such as streptomycin and kanamycin; ⁇ -lactam antibiotics such as penicillin and cephalosporin; and polypeptide antibiotics such as subtilisin and neosporin. Semi-synthetic derivatives of antibiotics, and antibiotics produced by chemical methods are also encompassed by this term.
  • Chemically-derived antimicrobial agents such as isoniazid, trimethoprim, quinolines, and sulfa drugs are considered antibacterial drugs, although the term antibiotic has been applied to these. These agents and antibiotics have specific cellular targets for which binding and inhibition by the agent or antibiotic can be measured. For example, erythromycin, streptomycin and kanamycin inhibit specific proteins involved in bacterial ribosomal activity; penicillin and cephalosporin inhibit enzymes of cell wall synthesis; and rifampicin inhibits the ⁇ subunit of bacterial RNA polymerase. It is within the scope of the screens of the present invention to include compounds derived from natural products and compounds that are chemically synthesized.
  • biocidal is art recognized and includes an agent that those ordinarily skilled in the art prior to the present invention believed would kill a cell "non- specifically," or a broad spectrum agent whose mechanism of action is unknown, e.g., prior to the present invention, one of ordinary skill in the art would not have expected the agent to be target-specific.
  • biocidal agents include paraben, chlorbutanol, phenol, alkylating agents such as ethylene oxide and formaldehyde, - 14 -
  • bactericidal refers to an agent that can kill a bacterium
  • bacteriostatic refers to an agent that inhibits the growth of a bacterium.
  • an antibiotic or an "anti-microbial drug approved for human use” is considered to have a specific molecular target in a microbial cell.
  • a microbial target of a therapeutic agent is sufficiently different from its physiological counterpart in a subject in need of treatment that the antibiotic or drug has minimal adverse effects on the subject.
  • a specific target for drug or antibiotic therapy can be ribosomal protein (SI 2 of the 30s ribosome); an RNA polymerase subunit ( ⁇ of bacterial RNA polymerase); a cell wall (a cross-linking enzyme of a bacterial cell wall); or a DNA polymerase-associated proteins (e.g., a gyrase).
  • SI 2 of the 30s ribosome
  • ⁇ of bacterial RNA polymerase RNA polymerase subunit
  • cell wall a cross-linking enzyme of a bacterial cell wall
  • a DNA polymerase-associated proteins e.g., a gyrase
  • an enzymatic component of fatty acid biosynthesis, enoyl-ACP reductase is determined to be a specific target of an effective dose of an agent which was previously classified as a non-specific biocidal agent when used at significantly higher concentrations than the effective dose.
  • the term "enzyme” includes polymorphic variants that are silent mutations naturally found within the microorganism population of a strain or species.
  • the enzymes in the preferred embodiment of the invention are fatty acid biosynthesis enzymes, preferably enoyl-ACP reductase (enoyl reductase) enzymes,.however, there is no intent to limit the invention to these enzymes.
  • fatty acid biosynthesis enzymes (and its equivalent term fatty acid biosynthetases) is intended to include those components of a proteins or polypeptides capable of synthesizing fatty acids via the three-carbon intermediate, malonyl CoA.
  • antibody as used herein is intended to include fragments thereof which are also specifically reactive with one of the components in the methods and kits of the invention. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab)2 fragments can be generated by treating an antibody with pepsin. The resulting F(ab)2 fragment can be treated to reduce disulfide bridges to produce F(ab) fragments.
  • antibody is further intended to include single chain, bispecific and chimeric molecules. The term “antibody” includes possible use both of monoclonal and polyclonal antibodies (Ab) directed against a target, according to the requirements of the application.
  • proteins that bind specific DNA sequences may be engineered (Ladner, R.C.,et. al., U.S. Patent 5,096,815), and proteins, polypeptides, or oligopeptides ("miniproteins") that bind - 16 -
  • Radioimmunoassays utilizing radioactively labeled ligands, for example, antigen directly labeled with ⁇ H, or ⁇ C, or 125j 5 measure presence of ER as antigenic material.
  • a fixed quantity of labeled mutant ER for example, competes with unlabeled antigen from the sample for a limited number of antibody binding sites.
  • the radioactivity in the bound fraction, or free fraction, or both is determined in an appropriate radiation counter.
  • the concentration of bound labeled antigen is inversely proportional to the concentration of unlabeled antigen present in the sample.
  • substantially pure or “isolated” with respect to a population of genetically modified cells means that the cells contain fewer than about 20%, more preferably fewer than about 10%, most preferably fewer than about 1%, non-modified cells.
  • genetically modified refers to mutation, including without limitation point mutation, substitution, transition, transversion, deletion, insertion, inversion and translocation mutation of nucleic acid. It includes manipulation of a recipient cell by introduction of recombinant or genetically engineered nucleic acid such as transformation and transfection.
  • the e > gene had been characterized as encoding a protein involved in biosynthesis of lipopolysaccharide (Hogenauer, G. et al, Nature 293: 662-664 (1981)), and this mutation was shown to reduce virulence in E. coli clinical isolates Ol 11 :B4 and Ol :K1.
  • the envM + gene was then shown to encode the FabI enoyl ACP reductase ((Turnowsky, F., et al. J. Bacteriol. 171, 6555-6565 (1989); Bergler, H., et al. J. Biol. Chem. 269, 5493-5496 (1994)).
  • FabI wild type and mutant proteins were expressed on plasmids in E. coli cells (Bergler, H., et al. Eur. J. Biochem. 242, 689-694 (1996)), and the proteins were overproduced, facilitating purification and assay.
  • FabI has been engineered as an N- terminal insertion of six histidine residues, enabling purification using a Ni ++ -agarose column (Qiagen, Hilden, Germany) for use in reconstitution of purified fatty acid biosynthesis components for synthesis and assay in vitro (Heath, R. et al, J. Biol. Chem. 270: 26538-26542 (1995)).
  • ⁇ noyl reductase activity can be measured using crude cell extracts or substantially purified enzymes by following NADH oxidation at 340 ran with a Uvikon 93310 spectrophotometer (Kontron Instruments), with 2-tr ⁇ , -octenoyl-ACP as a substrate (Dessen, A., et al Science 267, 1638-1641 (1995)).
  • This reaction can be carried out in small volumes in 96-well or 384- well multi-well plastic dishes, and can be automated for use in large-scale screens of antimicrobial agents using FabI or InhA as the specific target.
  • ⁇ noyl reductase activity can also be measured in whole cells by growth with
  • Potential drug candidates can be assayed by ability to bind to an ⁇ R protein which has been immobilized on a bead or on a plastic surface, for example, the plastic of multi-well plastic dishes.
  • a plastic surface for example, the plastic of multi-well plastic dishes.
  • Candidate agents can be incubated with immobilized ⁇ R protein under appropriate conditions, for example, in the presence of NAD or NADP, and under conditions of different temperature and pH, using the known inhibitors and mutants of the invention to optimize the assay.
  • the immobilized ⁇ R is separated from unbound compounds, washed to remove non-specifically bound materials, and then bound materials are eluted, for example, with solutions of decreased pH, or increased - 20 -
  • Agents that are found to bind immobilized ER in this primary screen can be tested for ability to inhibit the enoyl reductase activity, and for antimicrobial activity using whole cells.
  • Viability assays, and assays of cell lysis can also be performed in multi-well plastic dishes, in which viability is measured by cfu content following incubation in the presence and absence of drug, of dilutions of the contents of each well. Lysis can be measured by loss of optical density at, e.g., 540 nm, using an automated plate reader.
  • Genes, Nucleic Acids, Hybridization to Clone Homologs of ER, and Vectors Homologs of ⁇ R proteins can be generated by mutagenesis, such as by at least one of a discrete point mutation which can give rise to a substitution, or by at least one of deletion or insertion.
  • the present invention also is intended to encompass homologs of the ⁇ R polypeptide and mutant ⁇ R polypeptides described above. These fragments and homologs, which are biologically active in a manner which is the same or similar to the parent ⁇ R polypeptide.
  • a polypeptide or protein has ⁇ R biological activity if it can bind and reduce the double bond of an enoyl such as an octenoyl which is linked to ACP.
  • nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
  • gene or “recombinant gene” refers to a nucleic acid comprising an open reading frame encoding a ⁇ R of the present invention.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • expression vector includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a promoter).
  • plasmid and “vector” are used interchangeably, as a plasmid is a commonly used form of vector.
  • the invention is intended to include other vectors which serve equivalent functions. - 21 -
  • gene product includes an RNA molecule transcribed from a gene, or a protein translated from the RNA transcribed from the gene.
  • Expression vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors".
  • Transcriptional regulatory sequence is a generic term to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked.
  • transcription of a recombinant ER gene, a marRAB sequence or acrAB sequence is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell- type in which expression is intended.
  • the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally- occurring form of the ⁇ R protein.
  • telomere sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • any of a wide variety of expression control sequences, that control the expression of a DNA sequence when operatively linked to it, may be used in these vectors to express DNA sequences encoding the ER proteins of this invention.
  • "Homology” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the - 22 -
  • Cells are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Recipient cells are "sensitive" for the drug that is used to select for the particular drug-resistant trait of interest encoded by the transducing or transforming nucleic acid, e.g., in the invention, the cell can be sensitive to one or more of ampicillin, kanamycin, or triclosan.
  • the invention includes a nucleic acid which encodes a peptide having enoyl reductase enzyme activity, e.g., FabI or InhA.
  • the nucleic acid is a PCR product molecule comprising at least a portion of the nucleotide sequence represented in SEQ ID NO: 1 or SEQ ID NO: 2 from nucleotide (nt) 404 to 1189, or a homolog or variant thereof.
  • Preferred nucleic acids encode a bacterial FabI protein comprising an amino acid sequence at least 50% homologous, more preferably 75% homologous and most preferably 80%, 90%), or 95% homologous with an amino acid sequence shown in one of SEQ ID NO: 3.
  • Nucleic acids which encode polypeptides having an activity of a FabI protein and having at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology with a sequence shown in SEQ ID NO: 2 are within the scope of the invention.
  • Preferred nucleic acids encode a bacterial InhA protein comprising an amino acid sequence at least 50% homologous, more preferably 75% homologous and most preferably 80%, 90%, or 95% homologous with an amino acid sequence shown in one of SEQ ID NO: 12.
  • Nucleic acids which encode polypeptides having an activity of a InhA protein and having at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology with a sequence shown in SEQ ID NO: 11 are within the scope of the invention.
  • nucleic acid which hybridizes under high stringency conditions to a "probe", which is a nucleic acid molecule which binds specifically to a nucleic acid molecule encoding an ER enzyme.
  • a suitable probe is at least 12 nucleotides in length, is single-stranded, and is labeled, for example, radiolabeled or fluorescently labeled.
  • suitable stringency conditions include selecting the salt concentration in the wash step from a low stringency of about 2.0 x SSC at 50°C, and then using a wash of a high stringency condition, of about 0.2 x SSC at 50°C.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C.
  • Exemplary probes for DNA sequencing and for PCR analysis of FabI are shown in SEQ ID NOs: 4-10.
  • Tm melting temperature
  • the Tm the temperature in °C at which half the molecules of a given sequence are melted or single-stranded.
  • the Tm can be estimated in degrees C as 2(number of A+T residues) + 4(number of C+G residues).
  • Hybridization or annealing of the probe to the nucleic acid being probed should be conducted at a temperature lower than the Tm, e.g., 15°C, 20°C, 25°C or 30°C lower than the Tm.
  • nucleic acids encoding ER proteins are within the scope of the invention.
  • a fragment of the nucleic acid encoding a portion of a ER protein refers to a nucleic acid molecule having fewer nucleotides than the nucleotide sequence encoding the entire amino acid sequence of ER protein but which nevertheless encodes a peptide having the biological activity, e.g., enoyl-ACP reductase activity.
  • Nucleic acid fragments within the scope of the present invention include those capable of hybridizing under high stringency conditions with nucleic acids from other species for use in screening protocols to detect ER homologs and naturally occurring polymorphic alleles.
  • Useful expression control sequences include, for example, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda , the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast -mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • a useful translational enhancer sequence is described in U.S.P.N. 4,820,639. - 24 -
  • the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed.
  • the expression vector includes a recombinant gene encoding a peptide having an activity of a ER protein.
  • Such expression vectors can be used to transfect cells and thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.
  • a "derivative" or “analog” of an antimicrobial compound refers to a form of that compound in which one or more reaction groups on the compound have been derivatized with a substituent group (e.g. , alkylated or acylated peptides).
  • an “analog” of a compound refers to a compound that retains chemical structures necessary for functional activity yet that also contains certain chemical structures that differ.
  • An example of an analog of a naturally-occurring peptide is a peptide that includes one or more non-naturally-occurring amino acids.
  • a "mimetic" of a compound refers to a compound in which chemical structures necessary for functional activity have been replaced with other chemical structures that mimic the conformation.
  • peptidomimetics include peptidic compounds in which the peptide backbone is substituted with one or more benzodiazapine molecules (see e.g., James, G.L. et ⁇ l, (1993) Science 260:1937-1942) and "retro-inverso" peptides (see U.S. Patent No. 4,522,752 by Sisto), described further below.
  • a “residue” refers to an amino acid in a position in a peptide, or an amino acid mimetic incorporated in the peptide compound by an amide bond or amide bond mimetic.
  • Approaches to designing peptide derivatives, analogs and mimetics are known in the art. For example, see Farmer, P.S. in Drug Design (E.J. Ariens, ed.) Academic Press, New York, 1980, vol. 10, pp. 119-143; Ball. J.B. and Alewood, P.F. (1990) J. Mol. Recognition 3:55; Morgan, B.A. and Gainor, J.A. (1989) Ann. Rep. Med. Chem. 24:243; and Freidinger, R.M. (1989) Trends Pharmacol. Sci. H):270.
  • amino acid mimetic refers to a moiety, other than a naturally occurring amino acid, that conformationally and functionally serves as a substitute for a particular amino acid in a peptide-like compound without adversely interfering to a significant extent with the function of the compound (e.g., inhibition of ER). In some circumstances, substitution with an amino acid mimetic may actually enhance properties of the inhibitor (e.g., interaction of the inhibitor with ER). Examples of amino acid mimetics include D-amino acids. Peptides substituted with one or more D-amino acids may be made using well known peptide synthesis procedures. The effect of amino acid substitutions with D-amino acids and other peptidomimetics can be tested using assays as described herein. - 25 -
  • the peptide analogs or mimetics of the invention include isosteres.
  • isostere refers to a sequence of two or more residues that can be substituted for a second sequence because the steric conformation of the first sequence fits a binding site specific for the second sequence.
  • the term specifically includes peptide backbone modifications (i.e. , amide bond mimetics) well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the ⁇ - carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks.
  • ⁇ indicates the absence of an amide bond.
  • the structure that replaces the amide group is specified within the brackets.
  • isosteres include peptides substituted with one or more benzodiazapine molecules (see e.g., James, G.L. et al. (1993) Science 260:1937-1942)
  • a parent peptide is Thr-Ala-Tyr
  • the retro modified form is Tyr-Ala-Thr
  • the inverso form is thr-ala-tyr
  • the retro-inverso form is tyr-ala-thr using lower case letters to refer to D-amino acids.
  • a retro-inverso peptide has a reversed backbone while retaining substantially the original spatial conformation of the side chains, resulting in a retro-inverso isomer with a topology that closely resembles the parent peptide and is able to bind the selected cysteine protease. See Goodman et al. "Perspectives in Peptide Chemistry" pp. 283-294 (1981). See also U.S. Patent No. 4,522,752 by Sisto for further description of "retro- inverso" peptides.
  • compositions which include a therapeutically-effective amount or dose of an antimicrobial compound, e.g., triclosan, and one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • a composition can also include a second antimicrobial agent, e.g., an inhibitor of an efflux pump.
  • the pharmaceutical compositions can be formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or - 26 -
  • suspensions for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream, foam, or suppository; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.
  • parenteral administration for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension
  • topical application for example, as a cream, ointment or spray applied to the skin
  • intravaginally or intrarectally for example, as a pessary, cream, foam, or suppository
  • aerosol for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the antimicrobial agents or compounds of the invention from one organ, or portion of the body, to another organ, or portion of the body without affecting its biological effect.
  • Each carrier should be “acceptable” in the sense of being compatible with the other ingredients of the composition and not injurious to the subject.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydrox
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. - 27 -
  • the absorption of the drug in order to prolong the effect of a drug, it is 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 having 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.
  • compositions of the present invention may be administered to epithelial surfaces of the body orally, parenterally, topically, rectally, nasally, intravaginally, intracisternally. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, etc., administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal or vaginal suppositories.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • systemic administration means the administration of a sucrose octasulfate and/or an antibacterial or a contraceptive agent, drug or other material other than directly into the central nervous system, such that it enters the subject's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • compositions of the invention can be topically administered to any epithelial surface.
  • An "epithelial surface” is defined as an area of tissue that covers external surfaces of a body, or which and lines hollow structures including, but not limited to, cutaneous and mucosal surfaces.
  • Such epithelial surfaces include oral, pharyngeal, esophageal, pulmonary, ocular, aural, nasal, buccal, lingual, vaginal, cervical, genitourinary, alimentary, and anorectal surfaces.
  • compositions can be formulated in a variety of conventional forms employed for topical administration. These include, for example, semi-solid and liquid dosage forms, such as liquid solutions or suspensions, suppositories, douches, enemas, gels, creams, emulsions, lotions, slurries, powders, sprays, lipsticks, foams, pastes, toothpastes, ointments, salves, balms, douches, drops, troches, chewing gums, lozenges, mouthwashes, rinses. - 28 -
  • Conventionally used carriers for topical applications include pectin, gelatin and derivatives thereof, polylactic acid or polyglycolic acid polymers or copolymers thereof, cellulose derivatives such as methyl cellulose, carboxymethyl cellulose, or oxidized cellulose, guar gum, acacia gum, karaya gum, tragacanth gum, bentonite, agar, carbomer, bladderwrack, ceratonia, dextran and derivatives thereof, ghatti gum, hectorite, ispaghula husk, polyvinypyrrolidone, silica and derivatives thereof, xanthan gum, kaolin, talc, starch and derivatives thereof, paraffin, water, vegetable and animal oils, polyethylene, polyethylene oxide, polyethylene glycol, polypropylene glycol, glycerol, ethanol, propanol, propylene glycol (glycols, alcohols), fixed oils, sodium, potassium, aluminum, magnesium or calcium salts (such as chloride,
  • compositions can be particularly useful, for example, for treatment or prevention of an unwanted cell, e.g., vaginal Neisseria gonorrhea, or infections of the oral cavity, including cold sores, infections of eye, the skin, or the lower intestinal tract.
  • Standard composition strategies for topical agents can be applied to the antimicrobial compounds, e.g., triclosan or a pharmaceutically acceptable salt thereof in order to enhance the persistence and residence time of the drug, and to improve the prophylactic efficacy achieved.
  • a rectal suppository for topical application to be used in the lower intestinal tract or vaginally, a rectal suppository, a suitable enema, a gel, an ointment, a solution, a suspension or an insert can be used.
  • Topical transdermal patches may also be used.
  • Transdermal patches have the added advantage of providing controlled delivery of the compositions of the invention to the body.
  • dosage forms can be made by dissolving or dispersing the agent in the proper medium.
  • Compositions of the invention can be administered in the form of suppositories for rectal or vaginal administration. These can be prepared by mixing the agent with a suitable non-irritating carrier which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum or vagina to release the drug.
  • compositions which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams, films, or spray compositions containing such carriers as are known in the art to be appropriate.
  • the carrier employed in the sucrose octasulfate /contraceptive agent should be compatible with vaginal administration and/or coating of contraceptive devices. Combinations can be in solid, semi-solid and liquid dosage forms, such as diaphragm, jelly, douches, foams, films, - 29 -
  • ointments creams, balms, gels, salves, pastes, slurries, vaginal suppositories, sexual lubricants, and coatings for devices, such as condoms, contraceptive sponges, cervical caps and diaphragms.
  • the pharmaceutical compositions can be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride.
  • the compositions can be formulated in an ointment such as petrolatum.
  • Exemplary ophthalmic compositions include eye ointments, powders, solutions and the like.
  • Powders and sprays can contain, in addition to sucrose octasulfate and/or antibiotic or contraceptive agent(s), carriers 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 and volatile unsubstituted hydrocarbons, such as butane and propane.
  • an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers.
  • the carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (T weens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols.
  • Aerosols generally are prepared from isotonic solutions.
  • compositions of the invention can also be orally administered in any orally- acceptable dosage form including, but not limited to, capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of sucrose octasulfate and/or antibiotic or contraceptive agent(s) as an active ingredient.
  • capsules, cachets, pills, tablets, lozenges using a flavored basis, usually sucrose and acacia or tragacanth
  • powders granules
  • a compound may also be administered as a bolus, electuary or paste.
  • carriers which are commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried corn starch.
  • aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. - 30 -
  • Tablets, and other solid dosage forms may be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • opacifying agents include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • 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, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the antimicrobial agent(s) may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar and tragacanth, and mixtures thereof.
  • Sterile injectable forms of the compositions of this invention can be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. - 31 -
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • a nontoxic parenterally-acceptable diluent or solvent for example as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono-or di-glycerides.
  • Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • oils such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as Ph. Helv or similar alcohol.
  • the antimicrobial agent or a pharmaceutically acceptable salt thereof will represent some percentage of the total dose in other dosage forms in a material forming a combination product, including liquid solutions or suspensions, suppositories, douches, enemas, gels, creams, emulsions, lotions slurries, soaps, shampoos, detergents, powders, sprays, lipsticks, foams, pastes, toothpastes, ointments, salves, balms, douches, drops, troches, lozenges, mouthwashes, rinses and others.
  • Creams and gels for example, are typically limited by the physical chemical properties of the delivery medium to concentrations less than 20% (e.g., 200 mg/gm).
  • the pharmaceutical composition of the invention can comprise sucrose octasulfate in an amount of 0.001-99%, typically 0.01-75%>, more typically 0.1-20%>, especially 1-10% by weight of the total preparation.
  • a preferred concentration thereof in the preparation is 0.5-50%, especially 0.5-25%, such as 1-10%. It can be suitably applied 1-10 times a day, depending on the type and severity of the condition to be treated or prevented.
  • the minimum amount present in the materials forming combinations of this invention that is effective in treating or preventing bacterial disease due to direct interaction with the organism should produce be less than 0.1 ⁇ g ml ' l, less than 0.5 ⁇ g ml ⁇ l, preferably less than 1 ⁇ g ml" , even more preferably less than less than 5 ⁇ g ml" 1 , and most preferably less than 10 ⁇ g ml" 1.
  • the pharmaceutical composition of the invention can be applied prior to physical contact.
  • the timing of application prior to physical contact can be optimized to maximize the prophylactic effectiveness of the compound.
  • the timing of application will vary depending on the mode of administration, the epithelial surface to which it is applied, the surface area, doses, the stability and effectiveness of composition under the pH of the epithelial surface, the frequency of application, e.g., single application or multiple applications.
  • the timing of application can be determined such that a single application of composition is sufficient.
  • One skilled in the art will be able to determine the most appropriate time interval required to maximize prophylactive effectiveness of the compound.
  • a suitable daily dose of a composition of the invention will be that amount of the composition which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. It is preferred that administration be intravenous, intracoronary, intramuscular, intraperitoneal, or subcutaneous.
  • MIC minimal inhibitory concentration
  • Inhibition of growth rate was determined in broth culture by adding triclosan at various concentrations to log phase cells which had reached an absorbance (A530) of 0.1 and determining the effect on the rate of change of absorbance 1 h later; lysis was identified by a loss of absorbance (about 50%) accompanied by a 4-5 log loss in viable cfu per A53Q unit.
  • a chromosomal library was prepared from mutant AGT11 by cloning l-7kb Sau3al partial digestion fragments into the BamHI site of the tet gene in pBR322, transforming into strain DH5 ⁇ (Gibco/BRL, Bethesda MD), and selecting on ampicillin (Sigma, St. Louis, MO). Approximately 16,000 transformants were pooled to form the library, and the clones encoding triclosan resistance were found by plating about 80,000 cfu from the library on 0.3 ⁇ g mH triclosan.
  • Chromosomal DNA was prepared using a Puregene kit (Gentra Systems, Minneapolis, MN). PCR products of AGT23 and AGT25 were generated for sequencing using Taq DNA polymerase (Gibco) and oligonucleotide pairs LM011, SEQ ID No: 4, and LM010, SEQ ID No. 5 (respectively, nt 160-179 and 1168-1149). The numbering - 34 -
  • junctional DNA in pLYT6 and pLYT8 was sequenced using oligonucleotide BR346, SEQ ID No: 9 (nt 346-357 in pBR322, in which the BamHI site is at nt 375 (see the catalog of New England Biolabs, Beverly, MA).
  • the fabl gene in pLYT8 was sequenced using LM010, LM019, and LM011.
  • pLYT27 was sequenced using LM015 (nt 875-856; see SEQ ID No: 8) and LM021, SEQ ID No: 10 (in pBR322, nt 4068- 4086).
  • Strain JZM120 (AacrAB::kan; Ma, D., et al. Molec. Microbiol. 16, 45-55 (1995)) (from H. Nikaido) served as the donor strain for bacteriophage Pl -mediated transduction (Maritime, D.L. & Curtiss, R.I. in Methods for General and Molecular Bacteriology eds. Gerhardt, P., Murray, R.G.E., Wood, W.A. & Kreig, N.R. 317-347 American Society for Microbiology, Washington, D. C, (1994); Miller, J. Experiments in Molecular Genetics (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
  • the acrAB operon in Escherichia coli encodes a multidrug efflux pump which provides intrinsic resistance to many diverse compounds including antibiotics and disinfectants (Nikaido, H. J. Bacteriol. 178, 5853-5859 (1996)).
  • This operon can be up- regulated by MarA (Ma, D., et al. Molec. Microbiol. 16, 45-55 (1995), a transcriptional activator encoded by the marRAB operon involved in multiple antibiotic . resistance (Alekshun, M.N., et al. Antimicrob. Agents Chemother. 41, 2067-2075 (1997)).
  • MICs Minimal inhibitory concentrations
  • Inactivation of acrAB increased the susceptibility of all strains (including that of the triclosan susceptible parent AG100) approximately 7 - 24 fold (Table 1, MIC column 3). Increased triclosan resistance of fabl acrAB mutants was observed compared to acrAB inactivated in the fabl + AG100.
  • the AcrAB multidrug pump was an effective exporter of triclosan but was not the basis of the enhanced resistance in the fabl mutants.
  • Loss of the AcrAB multidrug efflux pump presumably permits a greater concentration of triclosan within the cytoplasm of the cell, where Fabl is located (Cronan, J.E., Jr. & Rock, CO. in Escherichia coli and Salmonella: Cellular and Molecular Biology (ed. Neidhardt, F.C.) 612-636 (ASM Press, Washington, DC, 1996)), resulting in the observed increase in susceptibility of cells to the drug.
  • the triclosan resistance phenotype of mutant AGT1 1 could be transduced to recipient strain AG100A using Pl phage (Maritime, D.L., et al. Methods for General and Molecular Bacteriology, eds. Gerhardt, P., et al. pp. 317-347, American Society for Microbiology, Washington, D. C, 1994); Miller, J. Experiments in Molecular Genetics (Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1972)), indicating that the mutation conferring the resistance phenotype might lie in one clonable locus.
  • a genomic Sau3AI library from strain AGT 11 was prepared in plasmid pBR322, and transformed into strain DH5 ⁇ (see Methods).
  • Clones mediating triclosan resistance were obtained in the library at a frequency of about 1 in 2500 transformants.
  • the plasmids isolated from these clones bore inserts of various sizes. Digestion of plasmids with Hindlll and Sphl revealed that all plasmid clones had a fragment of approximately 1530 bp. All clones gave the same MIC (about 4 ⁇ g mH, measured in hypersensitive strain AG100A), compared to 0.005 - 0.02 ⁇ g mH for the vector alone in the same host.
  • the level of resistance by the mutation present as a single copy on the chromosome was 2 to 4 ⁇ g ml" .
  • the fabl gene encodes enoyl ACP reductase, an enzyme involved in the synthesis of fatty acids (Cronan, J.E., Jr., et al. in Escherichia coli and Salmonella: Cellular and Molecular Biology, ed. Neidhardt, F.C., 612-636 ASM Press, Washington, DC, (1996)) which reduces a double bond using NADH or NADPH (Bergler, H., et al. Eur. J. Biochem. 242, 689-694 (1996)).
  • the mutation-bearing 606 bp Sspl-Hindlll fragment of pLYT12 was replaced with the wild type Sspl-Hindlll counterpart from a PCR product of chromosomal DNA from parental strain AG100.
  • the 606 bp region of the resulting plasmid, pLYT27 was sequenced to confirm that the DNA derived from AG100 in fact carried the wild type sequence identical to that in the database.
  • the triclosan MIC measured for pLYT27 in host AG100A was 20-30 fold greater than that for vector pBR322 itself, showing a clear multicopy effect.
  • Diazaborine resistance results from a gly93ser mutation (Bergler, H., et al. J. Gen. Microbiol. 138, 2093-2100 (1992); Turnowsky, F., et al. J. Bacteriol. 171, 6555-6565 (1989)), similar to the gly93val mutation shown here to cause a high level of triclosan resistance.
  • mutant AGTl 1 the growth rate in broth of mutant AGTl 1 was about 40% less, and that of mutant AGT23 about 15% less, than that of the wild type parent.
  • the sequence of the PCR product of strain AGT23 revealed a single point mutation (atg became acg), leading to replacement of methionine 159 by threonine.
  • Strain AGT25 had a single point mutation (ttc became etc), leading to replacement of phenylalanine 203 by - 39 -
  • Triclosan, diazaborine and isoniazid can interact in a related manner with enoyl- ACP reductases as indicated by the following facts.
  • InhA the gene encoding enoyl-ACP reductase of Mycobacterium tuberculosis, has 40% sequence identity with E. coli Fabl (Banerjee, A., et al. Science 263, 227-230 (1994)).
  • a mutation of serine 94 to alanine is associated with isoniazid resistance in both M. smegmatis and M. tuberculosis (Banerjee, A., et al. Science 263, 227-230 (1994)).
  • triclosan-resistant E. coli strain AGTl 1 had several times the isoniazid resistance of the isogenic parent AG100 (determined in the presence of 250 ⁇ M hydrogen peroxide to reduce the inherently high resistance of E. coli to isoniazid).
  • M. smegmatis is susceptible to triclosan, M. tuberculosis is not sensitive (Vischer, W.A., et al. Zbl. Bakt. Hyg., I. Abt. Orig. A 226, 376-389 (1974)).
  • Linkage of the triclosan resistance locus in three unsequenced mutants AGT7, AGT8, and AGT9 was used to map this gene to min 28.5, the location of fabl. This was done using Pl transduction of zci-3118::TnI0kan at approximately min 28.5 (Singer, M., et ⁇ l Microbiol. Rev. 53, 1-24 (1989)), from a wild type donor strain into each of the mutants. Of 10 kanamycin resistant transductants analysed for each mutant, 3 to 6 had - 40 -
  • Triclosan here was found to cause a loss of absorbance in broth cultures of growing susceptible E. coli, accompanied by a decrease in recoverable viable cells due to cell lysis, at concentrations of triclosan higher than those which affected the growth rate.
  • To inhibit the growth rate 50% about 0.15 ⁇ g ml ⁇ l triclosan was required for wild type strain AG100, about 0.02 ⁇ g mH triclosan for AG100A (deleted of acrAB), and about 1 ⁇ g ml"l for triclosan-resistant mutant derivative AGTl IK (gly93val, otherwise isogenic to AG100A).
  • the amount of triclosan required to give lysis was 2-8 ⁇ g mH for these strains.
  • strain AGTl 1 fabl gly93val otherwise isogenic to AG100, did not display lysis even when triclosan up to a level of 256 ⁇ g ml"l was added.
  • Mycobacterium smegmatis mutants were selected for resistance to triclosan and were found to have different mutations in InhA, an enoyl reductase involved in fatty acid synthesis. Isoniazid resistance accompanied triclosan resistance for the Metl ⁇ l Val mutation and to a lesser extent for Alal24Val, but not for - 41 -
  • smegmatis strain mc2l55 was grown in LB broth or 7H9 medium (see legend to Table 1) to stationary phase and approximately 10 ⁇ colony-forming units were plated onto LB agar (without Tween 80 or glycerol) containing 0.8-1.6 ⁇ gmH triclosan (a trichlorinated diphenyl ether, from Ciba-Geigy Corp., Greensboro, NC). After a 3 day incubation, the largest of the 20-200 colonies of various sizes which appeared per plate were selected. Three independent mutants, MTl, MT9, and MTl 7, were chosen for study. Each was 4-6 times more resistant to triclosan than was the parental strain (Table 2).
  • coli host STBL-2 [Gibco/BRL]) was digested with Pad and extracted with phenol/chloroform.
  • Cells in logarithmic phase in LB broth/0.2% Tween 80 were chilled on ice for 1.5hr and pelleted at 4°C. The pellets were resupended gently in 0.2 vol of cold 10% glycerol/ 0.1 % Tween 80, and then 10% glycerol was added up to 1 vol. Cells were pelleted and the resuspension and washing process repeated once, with final resuspension in 0.01 vol of glycerol/Tween 80. Electroporation was performed using 0.1 ml cell suspension with 0.2 ⁇ gDNA in 0.2 cm chilled cuvettes - 42 -
  • the inhA gene in each of the three triclosan-selected mutants was sequenced. Chromosomal DNA was prepared as described (Ausubel, F.M. et al. 1996. Current Protocols in Molecular Biology, vol 1 John Wiley Sons, p. 2.4.1.) using a 2 fir preliminary incubation at 37°C of cells with 4 mg mH lysozyme. Polymerase chain reaction (PCR) of the entire inhA gene was performed for each mutant using Taq DNA polymerase (Gibco/BRL) at 2 mM Mg ++ in EasyStart reaction tubes (Molecular Bio- Products).
  • PCR Polymerase chain reaction
  • Primers LM026 forward: 5'-AAAGCCCGGACACACAAGA-3') (SEQ ID NO: 13) and LM027 (reverse): 5'-CGAACGACAGCAGTAGCAAG-3' (SEQ ID NO: 14) were chosen from sequences bracketing inhA (see GenBank accession number (173544) using the PRIME program of GCG and were annealed at 52°C. Both strands of the resulting 890 bp PCR product were sequenced (Tufts Core Facility) using the same two primers.
  • the inhA structural gene of each mutant differed by a single nucleotide from the wild type sequence (GenBank accession number U02530). Together with the other results, this finding proved that a mutated inhA gene was responsible for the triclosan resistance in each mutant. Mutant MTl had replacement of methionine 161 (ATG) by valine (GTG), mutant MT9 had replacement of methionine 103 (ATG) by threonine (ACG), and mutant MTl 7 had replacement of alanine 124 (GCG) by valine (GTG).
  • Triclosan might, like isoniazid (Rozwarski, DA. 1998. Science 279:98-102) and diazaborine (Baldock, C. et al. 1996. Science 274:2107-2110), bind covalently to NADH. Resistance then might be explained, as for isoniazid (Basso, L. A. et al. 1998. J. Infect. Dis. 178:769-775) (Dessen, A. et al. 1995.
  • M. smegmatis InhA which influence triclosan resitance, S94, Ml 03, A 124, and Ml 61, are conserved in M. tuberculosis. They would not, therefore, identify any residues unique to M. tuberculosis InhA which might account for the intrinsic resistance. On the other hand, that resistance may be due to mechanisms unrelated to InhA, such as the activity of endogenous efflux pump(s) analogous to those which operate on triclosan in other organisms (McMurry, L.M. et al. FEMS Microbiol. Lett.; Schweizer, H.P. 1998. Antimicrob. Agents Chemother. 42:394-398). 44
  • MICs Minimal inhibitory concerntrations
  • All MICs wre determined on agar plates by 2-fold serial dilutions using logarithmic phase cells as described (McMurry, L.M. et al. 1998. Nature 394:531-532). Cells were grown with 0.05% Tween 80 either in LB broth or in 7H9 medium supplemented with ADC plus 0.2% glycerol and were tested on the corresponding solid media without Tween 80. All plates with triclosan also contained 0.1%) ethanol.
  • acrAB locus Deletion of the acrAB locus increased the susceptibility to triclosan approximately 10-fold.
  • Four of five clinical E. coli strains which overexpressed mar A or soxS also showed enhanced triclosan resistance.
  • the acrAB locus was involved in the effects of triclosan upon both cell growth rate and cell lysis.
  • Triclosan inhibits the synthesis of lipids in Escherichia coli, presumably by action upon Fabl, an enoyl reductase required for the synthesis of fatty acids (McMurry et al. (1998) Triclosan targets lipid synthesis. Nature 394, 531-532). At higher concentrations, triclosan also causes cell lysis (McMurry et al.
  • GC4488 Greenberg et al (1991) Wild-type 1 0 Activation of oxidative stress genes by mutation at the soxQl/cfxBl/marA locus of Escherichia coli J Bacteriol 173, 4433-4439)
  • a MIC of strain divided by MIC of corresponding wild-type strain MIC for AG100 was 0 17 ⁇ g ml "1 , for HH180, 0 07 ⁇ g ml "1 , and for GC4488, 0 08 ⁇ g ml "1 MIC values are means from two to five determinations "Has a 39 kb chromosomal deletion encompassing the mar locus - 48 -
  • Strains AG102 and AP5 are chromosomal Mar mutants and overexpress mar A. a MIC of strain divided by MIC of AG100 control strain (with no deletion). The MIC for AG100 was 0.17 ⁇ g mH.
  • the concentration of triclosan required to slow the growth rate by 50% (or 90%) an hour after addition was determined using OD 530 to monitor growth 'Lysis' was defined in such cultures by a 30-50% loss of OD 530 within 2 h of triclosan addition accompanied by a 4-6 log loss in viability (as indicated by colony-forming units) a Deter ⁇ mned using agar dilution plates

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Abstract

Procédés et mutants servant à identifier un composé antimicrobien exerçant une interaction avec un polypeptide ER. Ce procédé consiste, en particulier, à identifier par criblage un composé antimicrobien au moyen de cellules mutantes ou de polypeptides FabI ou InhA.
PCT/US1999/001288 1998-01-23 1999-01-22 Composes antimicrobiens WO1999037800A1 (fr)

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CA002319115A CA2319115A1 (fr) 1998-01-23 1999-01-22 Methodes pour identifier les peptides antimicrobiens interagissant avec l'enoyl acp reductase
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US6346391B1 (en) 1999-07-22 2002-02-12 Trustees Of Tufts College Methods of reducing microbial resistance to drugs
US6531291B1 (en) * 1999-11-10 2003-03-11 The Trustees Of Columbia University In The City Of New York Antimicrobial activity of gemfibrozil and related compounds and derivatives and metabolites thereof
US6713043B2 (en) 1996-02-29 2004-03-30 The Trustees Of Columbia University In The City Of New York Antimicrobial activity of gemfibrozil
US6951729B1 (en) * 1999-10-27 2005-10-04 Affinium Pharmaceuticals, Inc. High throughput screening method for biological agents affecting fatty acid biosynthesis
FR3000104A1 (fr) * 2012-12-21 2014-06-27 Oxoid Ltd Derives de triclosane et leurs utilisations
US10568960B2 (en) * 2015-05-22 2020-02-25 Consejo Superior De Investigaciones Científicas Molecular adjuvant and vaccine

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CA2641988A1 (fr) * 2006-02-10 2007-08-16 Tokai University Procede ou agent pour inhiber la fonction de pompe a ejection de pseudomonas aeruginosa

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US5837480A (en) * 1994-04-28 1998-11-17 Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University Method and compounds for inhibiting lipid biosynthesis of bacteria and plants
WO1998002139A1 (fr) * 1996-07-12 1998-01-22 Indústria e Comércio de Cosméticos Natura Ltda. Savon liquide antiseptique dermatologique
EP0826774A2 (fr) * 1996-08-28 1998-03-04 Smithkline Beecham Corporation Fab I enoyl-ACP réductase des Staphylocoques

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H. BERGLER ET AL.: "The enoyl-(acyl-carrier-protein) reductase (FabI) of Escherichia coli, which catalyzes a key regulatory step in fatty acid biosynthesis, accepts NADH and NADPH as cofactors and is inhibited by palmitoyl-CoA", EURPEAN JOURNAL OF BIOCHEMISTRY, vol. 242, no. 3, 1996, Berlin FRG, pages 689 - 694 *
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6713043B2 (en) 1996-02-29 2004-03-30 The Trustees Of Columbia University In The City Of New York Antimicrobial activity of gemfibrozil
US7132096B2 (en) 1996-02-29 2006-11-07 The Trustees Of Columbia University In The City Of New York Antimicrobial activity of gemfibrozil
US6346391B1 (en) 1999-07-22 2002-02-12 Trustees Of Tufts College Methods of reducing microbial resistance to drugs
US6677133B2 (en) 1999-07-22 2004-01-13 Trustees Of Tufts College Methods of reducing microbial resistance to drugs
US7026136B2 (en) 1999-07-22 2006-04-11 Trustees Of Tufts College Methods of reducing microbial resistance to drugs
US6951729B1 (en) * 1999-10-27 2005-10-04 Affinium Pharmaceuticals, Inc. High throughput screening method for biological agents affecting fatty acid biosynthesis
US6531291B1 (en) * 1999-11-10 2003-03-11 The Trustees Of Columbia University In The City Of New York Antimicrobial activity of gemfibrozil and related compounds and derivatives and metabolites thereof
US6881553B2 (en) 1999-11-10 2005-04-19 The Trustees Of Columbia University In The City Of New York Antimicrobial activity of gemfibrozil and related compounds and derivatives and metabolites thereof
FR3000104A1 (fr) * 2012-12-21 2014-06-27 Oxoid Ltd Derives de triclosane et leurs utilisations
US9908912B2 (en) 2012-12-21 2018-03-06 Oxoid Limited Triclosan derivatives and uses thereof
US10568960B2 (en) * 2015-05-22 2020-02-25 Consejo Superior De Investigaciones Científicas Molecular adjuvant and vaccine
US11179462B2 (en) 2015-05-22 2021-11-23 Consejo Superior De Investigaciones Científicas Molecular adjuvant and vaccine

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