WO2003101405A2 - Antibiotiques a base d'amino-glycosides et procedes d'utilisation correspondants - Google Patents

Antibiotiques a base d'amino-glycosides et procedes d'utilisation correspondants Download PDF

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WO2003101405A2
WO2003101405A2 PCT/US2003/007930 US0307930W WO03101405A2 WO 2003101405 A2 WO2003101405 A2 WO 2003101405A2 US 0307930 W US0307930 W US 0307930W WO 03101405 A2 WO03101405 A2 WO 03101405A2
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aminoglycoside
neamine
rna
binding
compound
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PCT/US2003/007930
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WO2003101405A3 (fr
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Robert R. Rando
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President And Fellows Of Harvard College
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Priority to US10/941,623 priority patent/US7244712B2/en
Publication of WO2003101405A3 publication Critical patent/WO2003101405A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
    • C07H5/06—Aminosugars
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/12—Acyclic radicals, not substituted by cyclic structures attached to a nitrogen atom of the saccharide radical

Definitions

  • the present invention relates to a new class of compounds which are capable of inhibiting bacterial growth. More particularly, the invention provides a new class of L- aminoglycoside compounds which inhibit bacterial growth including the inhibition of growth of bacterial strains which are resistant to D-aminoglycoside compounds. The present invention also relates to methods of inhibiting bacterial growth and methods of administering compounds of the invention to patients suffering from or susceptible to a bacterial infection.
  • RNA molecules are targets for small molecule drugs.
  • several clinically useful drugs operate by interfering with RNA function.
  • the most useful RNA binding drugs are the aminoglycosides (Gale, E. F., Cundliffe, E., Reynolds, P. E., Richmond, M. H., & Waring M. J. (1981) The Molecular Basis of Antibiotic Action, 2 nd ed., John Wiley & Sons, London, Great Britain, pp 419-439; Cundliffe, E. (1989) Annu. Rev. Microbiol. 43, 207-233).
  • Aminoglycoside antibiotics function by binding to the A-site decoding region on bacterial 16S ribosomal (r)RNA (Scheme l/fig9) (Noller, H. F. (1991) Annu. Rev. Biochem. 60, 191- 227; Woodcock, J., Moazed, D., Cannon, M., Davies, J. and Noller, H.F. (1991) EMBO J. 10, 3099-3103).
  • This binding alters the interactions between the codon-anticodon helix and the A-site RNA, causing mis-translation, and premature termination during protein synthesis in bacteria. This leads to the bactericidal effects of this class of drugs (Cundliffe, E.
  • Aminoglycosides have been found to bind to many different types of RNA structures (Zapp, M. L., Stern, S., & Green, M. R. (1993) Cell 74, 969-978; Werstuck, G., Zapp., M. L., & Green, M. R. (1996) Chem. Biol. 3, 129-137; Mei, H. Y., Cui, M., Heldsinger, A., Lemrow, S. M., Loo, J. A., Sannes-Lowery, K. A., Sharmeen, L., and Czarnik, A. W. (1998) Biochemistry 37, 14204-14212; Tok, J. B., Cho, J., & Robert, R.
  • RNA molecules that bind to aminoglycosides typically possess non-duplex structural elements (Cho, J. and Rando, R. R. (1999) Biochemistry 38, 8548-8554). Often these RNA molecules contain asymmetric bulges or bubbles, which allow aminoglycoside access to the purine and pyrimidine bases.
  • RNA constructs measured affinities for D-aminoglycosides in the 1-2 ⁇ M range, save for neomycin B, which had a somewhat higher affinity. Similar observations were also made in studies on the binding of D-aminoglycosides to human decoding region A-site constructs. Binding studies have established that a wide variety of structurally dissimilar D-aminoglycosides have similar affinities for RNA substrates. See, for example, Ryu, D. H. and Rando, R. R. Bioorganic and Medicinal Chemistry, (In Press); and Wang, Y., Hamasaki, K., & Rando, R. R. (1997) Biochemistry 36, 768-77.
  • D-aminoglycosides A structurally diverse family of D-aminoglycosides are known to be effective antibiotics (Chambers, H. F., & Sande, M. A. (1996) Goodman & Gilman's The Pharmacological Basis of Therapeutics (Hardman, J. G., Limbird, L. E., Molinoff, P. B., Ruddon, R. W., & Gilman, A. G., Eds.) 9 th ed., McGraw-Hill, New York. Chap. 46, pp 1103-1121).
  • Bacteria, protazoa and other single cell organisms have a number of deactivation pathways available which can render an amindoglycoside inactive.
  • bacteria have a number of enzymes which are capable of metabolizing an aminoglycolide into non-cytotoxic species such as by phosphorylation, saccharide bio-degradation and the like.
  • the ability of bacterium and other single cell organisms to quickly develop resistance to aminoglycosides limits current antibiotic applications.
  • the present invention provides new aminoglycoside compounds that inhibit bacteria growth, preferably compounds that inhibit growth of bacteria resistant to current aminoglycoside antibiotics.
  • the invention further includes methods of inhibiting bacteria growth and methods of administering an aminoglycoside compound of the invention to a patient suffering from or susceptible to a bacterial infection.
  • the present invention provides a new class of aminoglycoside antibiotics comprising one or more non-natural saccharide residues.
  • Preferred aminoglycoside compounds of the invention comprise at least two non-natural saccharide residues such as an L-neamine structure preferrably having one or more additional natural or non-natural saccharide residues coupled to the L-neamine core.
  • Particularly preferred aminoglycosides of the invention are mirror images of known aminoglycosides antagonists, e.g., non-naturally occurring enantiomers of known D-aminoglycoside compounds having antibiotic activity.
  • the present invention also provides methods of inhibiting bacterial growth using an aminoglycoside compound comprising at least on L-saccharide residue and preferably using an aminoglycoside that is a mirror image of a known D-aminoglycoside antagonist.
  • the present invention further provides methods of administering a compound of the invention to a patient who is suffering from or susceptible to an infection, particularly an infection of bacteria, protozoa, yeast or the like.
  • HIV human Immunodeficiency virus
  • mRNA messenger RNA
  • CD circular dichroism
  • HEPES 4-(2-hydroxyethyl)piperazine-l-sulfonic acid
  • EDTA ethylenediamine tetraacetic acid
  • APH aminoglycoside phosphotransferase
  • NPT neomycin phosphotransferase
  • RNA Aptamer A single stranded RNA molecule that binds to specific molecular targets such as a protein or metabolite.
  • FIG. 1A is a circular dichroism (CD) spectra of L-RNA construct B performed by mixing 50mL stock solution+450mL lOOmM sodium chloride, lOmM sodium hydrogen phosphate pH 7.0 at 4 ° C on an Aviv 202 spectropolarimeter;
  • CD circular dichroism
  • FIG. IB is a circular dichroism (CD) spectra of L-RNA construct H performed by mixing 50mL stock solution+450mL lOOmM sodium chloride, lOmM sodium hydrogen phosphate pH 7.0 at 4 ° C on an Aviv 202 spectropolarimeter;
  • CD circular dichroism
  • FIG. IC is a circular dichroism (CD) spectra of L-RNA construct J6fl performed by mixing 50mL stock solution+450mL lOOmM sodium chloride, lOmM sodium hydrogen phosphate pH 7.0 at 4 ° C on an Aviv 202 spectropolarimeter;
  • FIG. 2 A is a plot of fluorescence anisotropy of fluorescently labeled paromomycin (CRP) (20nM) as a function of L-RNA construct B concentration;
  • FIG. 2B is a plot of fluorescence anisotropy of fluorescently labeled paromomycin (CRP) (20nM) as a function of L-RNA construct H concentration;
  • FIG. 2C is a plot of fluorescence anisotropy of fluorescently labeled tobramycin (CRT) (20nM) as a function of L-RNA construct J6fl concentration;
  • FIG. 3 A is a plot of fluorescence anisotropy of fluorescently labeled paromomycin (CRP) (20nM) containing L-RNA construct B as a function of paromomycin concentration
  • FIG. 3B is a plot of fluorescence anisotropy of fluorescently labeled paromomycin (CRP) (20nM) containing L-RNA construct H as a function of paromomycin concenfration;
  • FIG. 3C is a plot of fluorescence anisotropy of fluorescently labeled tobramycin (CRT) (20nM) containing L-RNA construct J6fl as a function of tobramycin concenfration;
  • FIG. 4A is a plot of fluorescence anisotropy of fluorescently labeled paromomycin (CRP) (20nM) as a function of 70S ribosome concenfration;
  • FIG. 4B is a plot of fluorescence anisotropy of fluorescently labeled paromomycin (CRP) (20nM) as a function of 80S ribosome concenfration;
  • FIG. 5 A is a plot of fluorescence anisotropy of fluorescently labeled paromomycin (CRP) (20nM) containing 70S ribosomes, as a function of neomycin concenfration;
  • FIG. 5B is a plot of fluorescence anisotropy of fluorescently labeled paromomycin (CRP) (20nM) containing 70S ribosomes, as a function of paromomycin concentration;
  • FIG. 5C is a plot of fluorescence anisotropy of fluorescently labeled paromomycin (CRP) (20nM) containing 70S ribosomes, as a function of kanamycin concenfration;
  • FIG. 5D is a plot of fluorescence anisotropy of fluorescently labeled paromomycin (CRP) (20nM) containing 70S ribosomes, as a function of tobramycin concentration;
  • FIG. 6A is a plot of fluorescence anisotropy of fluorescently labeled paromomycin (CRP) (20nM) containing 70S ribosome as a function of D-neamine concentration;
  • FIG. 6B is a plot of fluorescence anisotropy of fluorescently labeled paromomycin (CRP) (20nM) containing 70S ribosome as a function of L-neamine concenfration;
  • FIG. 7A is a image of an antibiotic activity disk assay of E.coli (ATCC 25922) wild type where each numbered disc is impregnated with an aminoglycoside: 1, D-neamine (100 nmol), 2, D-neamine (200 nmol), 3, D-neamine (500 nmol), 4, L- neamine (200 nmol), 5, L- neamine (500 nmol) 6, L- neamine (1000 nmol); FIG.
  • 7B is a image of an antibiotic activity disk assay of E.coli (ATCC 25922) fransformed with the plasmid pMM, bearing the APH(3')IIa gene where each numbered disc is impregnated with an aminoglycoside: 1, D-neamine (100 nmol), 2, D-neamine (200 nmol), 3, D-neamine (500 nmol), 4, L- neamine (200 nmol), 5, L- neamine (500 nmol) 6, L- neamine (1000 nmol);
  • FIG. 7B is an image of an antibiotic activity disk assay of P.aeruginosa (ATCC 27853) where each numbered disc is impregnated with an aminoglycoside: 1, D-neamine (100 nmol), 2, D-neamine (200 nmol), 3, D-neamine (500 nmol), 4, L- neamine (200 nmol), 5, L- neamine (500 nmol) 6, L- neamine (1000 nmol);
  • FIG. 8 is a plot of bactericidal profiles of D- and L-neamine against E.coli (ATCC 25922) after a 3h incubation with antibiotics. Each point is the average of duplicate measurements;
  • FIG. 9 is an image of the Bacterial (B), Human (H) A-Site Decoding Region Constructs and the Tobramycin Binding Aptamer Construct (J6fl);
  • FIG. 10 A is a gel-electrophoresis for In vitro protein translation in the presence of neomycin B;
  • FIG. 10 B is a gel-electrophoresis for In vitro protein translation in the presence of D- neamine
  • FIG. 10 C is a gel-electrophoresis for In vitro protein translation in the presence of L- neamine
  • FIG. 11 is an image of the Bacterial A-Site Decoding Region L-RNA Construct with (D-A) 3 ;
  • FIG. 12 is an gel-electrophoresis of lead acetate footprinting studies of aminoglycoside binding to D- and L- A-site RNA constructs.
  • FIG. 13A is a gel-electrophoresis of footprinting studies of aminoglycoside binding to D- A-site RNA constructs. 0, untreated A-site RNA ( 32 P-labeled at 5' end); Ctrl, control experiment in the absence of binders; D- Nea, L-Nea, NeoB - RNA footprinting in the presence of D-neamine, L-neamine and neomycin B; Concenfrations of the aminoglycosides: 50 and 250 ⁇ M.
  • FIG. 13B is a gel-electrophoresis of footprinting studies of aminoglycoside binding to L- A-site RNA constructs.
  • FIG. 14 is a summary of D- and L- A-site RNA construct footprinting studies. Upper row- D-RNA construct, lower row- L-RNA construct. Circles- nucleotides, which found protected by lead acetate footprinting method. Arrows- nucleotides, which found enhanced by lead acetate footprinting method. Squares- nucleotides, which found protected by DMS footprinting method.
  • the present invention provides aminoglycoside compounds comprising at least one saccharide residue having at least one sugar hydroxyl residue is replaced with an amino residue.
  • Such amino functionalized sugars are interchangeably referred to as azasugars, aza- saccharides or aminoglycosides.
  • Preferred aminoglycosides of the invention include diastereomers or enantiomers of D-neamine, a common aminoglycoside structural unit, or a derivative thereof.
  • Preferred aminoglycoside compounds include those aminoglycoside compounds wherein substantially all of the azasugar residues of the aminoglycoside compound are L-azasugar residues which are diastereomers or enantiomers of D-neamine or a derivative thereof.
  • Preferred embodiments of the invention provides L-aminoglycoside compounds which are enantiotopic, mirror images of naturally occurring or synthetic D-aminoglycoside compounds possessing anti-bacterial properties.
  • Other aminoglycoside compounds provided by the invention include diastereotopic aminoglycoside compounds which differ from preferred L-aminoglycoside compounds of the invention by the stereochemical identity of one or more stereogenic centers.
  • the present invention provides L-aminoglycoside compounds which are exact mirror images of naturally occurring or synthetic D- aminoglycosides and diastereomers of the mirror-image L-aminoglycosides (L- aminoglycoside diasteromers)of the invention which have opposite stereochemical identity at one or more stereocenters.
  • Preferred L-aminoglycoside diasteromers of the invention include those diasteromers which differ from the mirror image L-aminoglycoside by the stereochemistry at one, two or three stereogenic centers.
  • Preferred aminoglycoside compounds of the invention include, e.g., L-neamine, L- neamine diasteromers differing from L-neamine in the stereochemical identity of between 1 and 3 stereocenters, and aminoglycosides having a L-neamine or L-neamine diasteromer coupled to one or more D- or L-sugar or D- or L-azasugar residues.
  • Preferred aminoglycosides of the invention may be optionally substituted at one or more hydroxyl or amino functional groups.
  • L-aminoglycoside compounds include L-neamine or a L- neamine derivative selected from the group consisting of L-neomycin, L-paromomycin, L- kanamycin, and L-tobramycin.
  • Aminoglycoside compounds provided in the present invention including L-neamine, L-neamine derivatives and diasteromers of L-neamine and L-neamine derivatives are capable of inhibiting bacterial growth.
  • Particularly preferred aminoglycoside compounds of the invention include those L-aminoglycoside compounds which possess antibiotic activity against strains of bacteria or yeast which are resistant to the enantiotopic D-aminoglycoside compounds.
  • Certain preferred L-aminoglycoside compounds of the invention are not susceptible to enzymatic degradation processes.
  • Preferred compounds provided by the present invention include those compounds according to Formula I:
  • E is independently selected at each occurrence of E in the formula from the group consisting of O, NH, and N-C ⁇ alkyl, such that at least one occurrence of E is not oxygen;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are independently selected at each occurrence from the groups consisting of hydrogen, d- ⁇ alkanoyl, monosaccharides, disaccharides, mono-azasaccharides, and di-azasaccharides, where each saccharide or azasaccharide residue is either a D-saccharide or an L-saccharide.
  • Particularly preferred compounds of the invention include compounds according to Formula II:
  • R 1A , R 2A , R 4A , R 5A , R 6A , R 7A , and R 9A are independently selected at each occurrence from the groups consisting of hydrogen, alkyl, alkanoyl, monosaccharides, disaccharides, mono- azasaccharides, and di-azasaccharides, where each saccharide or azasaccharide residue is either a D-saccharide or an L-saccharide; and
  • R 3A and R 8A are independently selected at each occurrence from the groups consisting of hydrogen, hydroxy, amino, d- ⁇ alkoxy, amino, mono and diCj- 6 alkylamino, carboxamide, monosaccharides, disaccharides, mono-azasaccharides, and di-azasaccharides, where each saccharide or azasaccharide residue is either a D-saccharide or an L-saccharide.
  • compositions which comprise an aminoglycoside compound of the invention, preferably an L- aminoglycoside according to either Formula I or II, and a pharmaceutically acceptable carrier.
  • the present invention also provides methods of inhibiting growth of a bacteria or yeast comprising the steps of providing an aminoglycoside compound of Formula I or II; contacting the L-aminoglycoside compound with the bacteria or yeast under conditions conducive to the inhibition of growth.
  • Preferred methods of inhibiting growth of a bacterial or yeast include the use of an aminoglycoside selected from those aminoglycosides having at least one azasugar residue which is a diasteroemer or enantiomer of D-neamine or a derivative thereof.
  • Preferred L-aminoglycoside compounds of the invention are capable of binding to one or more RNA sequences.
  • binding affinity is measured by a dissociation constant which quantifies the binding efficiency with which a compound binds to one or more RNA sequences.
  • tightly associated RNA-compound complex have small dissociation constants.
  • Particularly preferred L-aminoglycoside compounds have a RNA dissociation constant of less than about 100 ⁇ M, typically the dissociation constant is between about 0.01 ⁇ M and about 100 ⁇ M. More preferably, the dissociation constant is less than about 100 ⁇ M, 75 ⁇ M, 50 ⁇ M, 25 ⁇ M, 10 ⁇ M, 5 ⁇ M, or 1 ⁇ M.
  • Particularly preferred aminoglycoside compound comprising at least one azasugar residue, e.g., a sugar residue in which one or more hydroxyl groups are replaced with amino groups, which is a diasteroemer or enantiomer of D-neamine or a derivative thereof have a RNA dissociation constant of between about 0.1 ⁇ M and about 20 ⁇ M.
  • Preferred L-aminoglycoside compounds of the invention are capable of inhibiting bacterial growth.
  • antibiotic activity is measured using a standard disk assay using sterile paper disks soaked with various concenfrations of solutions of compounds of the invention.
  • minimal inhibitory concentrations (MIC) were measured using the technique published by Greenberg et al. in J. Am. Chem. Soc. 1999, 121:6527-6541.
  • Particularly preferred L-aminoglycoside compounds have a MIC of less than about 5000 ⁇ M, typically the MIC is less than about 4000 ⁇ M, 3000 ⁇ M, 2500 ⁇ M, 2000 ⁇ M, 1500 ⁇ M, or 1000 ⁇ M.
  • Particularly preferred aminoglycoside compound comprising at least one azasugar residue, e.g., a sugar residue in which one or more hydroxyl groups are replaced with amino groups, which is a diasteroemer or enantiomer of D-neamine or a derivative thereof have a minimal inhibitory concentration of less than about 2000 ⁇ M.
  • the present invention also provides methods for treating a mammal suffering or susceptible to a bacterial or yeast infection or disorder, comprising administering to the mammal an effective amount of an aminoglycoside compound comprising at least one azasugar residue which is a diasteroemer or enantiomer of D-neamine or a derivative thereof.
  • aminoglycoside compounds are administered to a mammal already suffering from a bacterial or yeast infection.
  • Preferred mammals include humans and domesticated animals such as dogs, cats, pigs, bovine, sheep and the like. Humans are a particularly preferred mammal for administration of an aminoglycoside compound of the present invention.
  • aminoglycoside compounds of the present invention are suitable for treatment of any bacterial or yeast infection.
  • bacterial infections and yeast infections which are suitable for treatment by administration of an aminoglycoside compound of the invention, include various bacteria and yeast strains associated with inducing illnesses and diseases.
  • the compounds of the invention are administered in effective amounts and in appropriate dosage form ultimately at the discretion of the medical or veterinary practitioner.
  • the amount of compounds of the invention required to be pharmaceutically effective will vary with a number of factors such as the mammal's weight, age and general health, the efficacy of the particular compound and formulation, route of administration, nature and extent of the condition being treated, and the effect desired.
  • the total daily dose may be given as a single dose, multiple doses, or intravenously for a selected period. Efficacy and suitable dosage of a particular compound can be determined by known methods.
  • a typical effective dose of the compound of the invention will be in the range of 0.1 to 100 milligrams per kilogram body weight of recipient per day, preferably in the range of 1 to 10 milligrams per kilogram body weight of recipient per day.
  • the desired dose is suitably administered once daily, or as several sub-doses, e.g. 2 to 4 sub-doses administered at appropriate intervals through the day, or other appropriate schedule.
  • Such sub-doses may be administered as unit dosage forms, e.g., containing from 0.2 to 200 milligrams of compound(s) of the invention per unit dosage, preferably from 2 to 20 milligrams per unit dosage.
  • formulation and typical dosage of a compound of the invention will vary depending on the mode of administration, e.g., oral or topical administration may require a higher or lower dosage than administration by injection.
  • the compounds of the present invention may be suitably administered to a subject as a pharmaceutically acceptable salt.
  • Such salts can be prepared in a number of ways.
  • salts can be formed from an organic or inorganic acid, e.g. hydrochloride, sulfate, hemisulfate, phosphate, nitrate, acetate, oxalate, citrate, maleate, etc.
  • the therapeutic compound(s) may be administered alone, or as part of a pharmaceutical composition, comprising at least one compound of the invention together with one or more acceptable carriers thereof and optionally other therapeutic ingredients.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and infradermal) administration.
  • the formulations may conveniently be presented in unit dosage form, e.g., tablets and sustained release capsules, and in liposomes, and may be prepared by any methods well known in the art of pharmacy.
  • compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, liposomes or finely divided solid carriers or both, and then if necessary shaping the product.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion, or packed in liposomes and as a bolus, etc.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.
  • compositions suitable for oral administration include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the ingredient to be administered in a suitable liquid carrier.
  • compositions suitable for topical administration to the skin may be presented as ointments, creams, gels and pastes comprising one or more compounds of the present invention and a pharmaceutically acceptable carrier.
  • a suitable topical delivery system is a transdermal patch containing the ingredient to be administered.
  • compositions suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
  • compositions suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size, for example, in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • Suitable formulations wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.
  • compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
  • compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include flavoring agents.
  • aminoglycoside compounds composing a neamine residue are provided such as L-neamine, ent-l, positional isomers of D-neamine, 2 and 3, and positional isomers of L-neamine, ent-2 and ent-3 (Table
  • L-neamine and L- enantiomers of the D-aminoglycoside derivatives of D-neamine which possess antibiotic activity such as D-neomycin, D-paromomycin, D-kanamycin B, and D-tobramycin.
  • Aminoglycoside antibiotics function by binding to the A-site decoding region of bacterial rRNA causing mistranslation and/or premature message termination. Given the broad range of aminoglycoside structural types that can act as anti-bacterial drugs it seems unlikely that aminoglycosides bind in a unique way to their functional target. One way to gauge specificity of drug-target interactions is to probe the stereospecificity of the interactions.
  • A-site decoding region rRNA constructs bind a series of aminoglycosides in a non-stereospecific manner with dissociation constants in the 1-5 ⁇ M range. Synthetic D and L-neamine were prepared and found also to bind to both bacterial and yeast ribosomes with similar affinities.
  • L-neamine like D-neamine, inhibits the growth of E.coli and P.aerugenosa. Moreover, L-neamine also inhibits the growth of aminoglycoside resistant E.coli which expresses a kinase that detoxifies aminoglycosides of the D-series, suggesting that mirror image aminoglycosides may avoid certain forms of enzyme-mediated resistance.
  • NMR investigations on the binding of aminoglycosides to the truncated A-site decoding region construct B suggests elements of specificity in the binding (Fourmy, D., Faculty, M. L, Blanchard, S. C, & Puglisi, J. D. (1996) Science 274, 1367-1371; and Lynch, S. R. & Puglisi, J. D. (2001) J. Mol. Biol. 306, 1037-1058.).
  • recent structural studies on ribosomal subunits demonstrate specificity in A-site interactions with mRNA and tRNA codon-anticodon complexes as well as with aminoglycosides.
  • tRNAs See, for example, Wimberly, B. T., Brodersen, D. E., Clemons, W. M., Jr., Morgan- Warren, R. J., Carter, A. P., Vonrhein, C, Hartsch, T., & Ramakrishnan, V.
  • aminoglycosides are arguably the most clinically important group of drugs known to target RNA molecules, it is important to establish the order of selectivity of this class of drugs.
  • Applicants have surprisingly discovered that a diverse structural family of aminoglycosides bind to similar common RNA decoding target region in a non-stereospecific manner.
  • structurally diverse aminoglycosides bind to a broad collection of RNA molecules with similar affinities. See, for example, the structurally diverse eukaryotic and prokaryotic decoding region constructs presented in FIG. 9A-C bind with several D- aminoglycosides with about the same affinity.
  • aminoglycoside-target interactions are non-stereospecific. It is generally accepted that aminoglycosides can bind to a myriad of RNA structures that contain non-duplex regions. An important question to address is how specifically aminoglycosides bind to their pharmacological targets, namely the A-site rRNA decoding regions of susceptible bacteria.
  • D-aminoglycosides bind to both natural RNA substrates and unnatural RNA substrates.
  • the D-enantiomer and the L-enantiomer of prokaryotic A-site RNA constructs (denoted hereinafter and in the figures as "B"), have been prepared and the ability of D-aminoglycosides to bind to each RNA enantiomer determined.
  • the D- aminoglycloside compounds bind to the naturally occurring RNA sequences and mirror image enantiomer thereof establishing that non-stereospecific binding behavior for aminoglycosides for RNA.
  • D-aminoglycoside compounds bind to both D-RNA and L-RNA constructs.
  • L-aminoglycoside compounds also bind to both D-RNA and L-RNA constructs.
  • binding of D-aminoglycosides to D-series RNA sequences occurs with greater affinity than binding to the 'unnatural' L-RNA sequence, e.g., K ⁇ j (natural) ⁇ ICj (unnatural).
  • Non-sterospecific binding is also observed in the human (denoted hereinafter and in the figures as "H") decoding region constructs such that the difference in binding affinity of D-aminoglycosides for binding to the natural and non-natural enantiomers of the decoding region is negligible.
  • the naturally occurring aminoglycosides exhibited a modest 2-3 fold increase in selectivity for the D-series RNA construct.
  • binding to the unnatural L-RNA was favored by a factor of approximately 1.5 over the D-decoding region construct.
  • the general drift towards enhanced binding of the cognate RNA construct was also observed in the case of D and L-neamine. Both enantiomers were preferentially bound to their stereochemical cognate by a factor of approximately two-fold relative to the enantiomers.
  • RNA constructs shown in FIG. 9 were designed to probe the stereospecificity of aminoglycoside binding to the RNA constructs shown in FIG. 9 because these small rRNA constructs mimic ribosomal decoding region RNA function.
  • the three constructs shown include a bacterial 16S A-site rRNA construct (B) (27), its human counterpart (H), and an RNA aptamer (J6fl) selected to bind to the aminoglycoside tobramycin (12,15,16).
  • RNA construct, J6fl, (FIG 9C) is of interest because of the high-level of binding specificity this construct exhibits for D-aminoglycoside compounds such that RNA construct, J6fl, (FIG 9C) is a useful as a control for studying binding of aminoglycosides with RNA decoding region constructs.
  • L-aminoglycoside binding to D- RNA sequences are energetically equivalent to binding the enantiotopic D-aminoglycoside to an L-RNA sequence.
  • Initial binding experiments were carried out using enantiomerically pure L-RNA constructs, B, H, and J6fl (FIG 9A-C) and D-aminoglycoside compounds.
  • Circular dichroism spectra of the purified constructs confirm the enantiomeric relationship of the corresponding D-RNA and L-RNA constructs (FIG.l A-C).
  • the constructs were tested for aminoglycoside binding using the fluorescence anisotropy method described in Example 4. In these measurements, the fluorescent aminoglycoside CRP bearing a rhodamine chromaphore is used to monitor binding to the decoding region constructs (B and H) and fluorescent aminoglycoside CRT bearing a rhodamine chromaphore was used to monitor binding to RNA construct J6fl (FIG 9C). See, for example, Wang, Y. and Rando, R. R. Chem. Biol.
  • the binding data, shown in Figure 2 A-C, for the L-RNA constructs demonstrates non-stereospecificity of aminoglycoside binding to RNA. Competition experiments provide further evidence for non-stereospecific binding.
  • competition experiments using paromomycin for the decoding regions B and H (FIG. 9A and 9B) and tobramycin for decoding region RNA construct, D-J6fl, (FIG 9C) are shown in Figure 3 A- C.
  • Table 2 provides binding data for the various D-aminoglycosides for complexation with the D-RNA and L-RNA enantiomers of decoding region constructs B and H and complexation with the D- and L- enantiomers of J6fl.
  • aminoglycoside binding to the prokaryotic decoding region A-site construct is weakly stereospecific.
  • aminoglycoside binding to the aptamer J6fl is strongly stereospecific.
  • the measured affinities for the D-decoding region construct is consistent with results of binding measurements on prokaryotic ribosomes using radioactive tobramycin (Le Goffic, F., Capmau, M. -L., Tangy, F. and Baillarge, M. (1979) Eur. J. Biochem. 102,73-81). Binding affinities in ⁇ M range are to be expected in the naturally occurring series.
  • the enantiomeric aminoglycoside L-neamine (Example 1) was synthesized and tested as a competitive inhibitor for CRP binding to the A-site constructs described above (Table 2).
  • the binding of L-neamine to both the D- and L- A-site constructs was compared to the binding of the naturally occurring enantiomer D-neamine.
  • Straightforward competitive binding of L-neamine to the RNA constructs was observed.
  • D-neamine binds with an approximately two-fold higher affinity to the D-construct than does L-neamine (Table 2).
  • L-neamine binds with a twofold higher affinity than D-neamine does to the L- A-site RNA.
  • neomycin B binds to the D- A-site RNA construct with an approximately two-fold higher affinity than to the L- RNA construct (Table 2). Overall these results indicate a very modest degree of stereospecificity in aminoglycoside- decoding region rRNA binding.
  • the present invention has established that non-stereospecificity in the binding of aminoglycoside compounds to RNA occurs for short RNA constructs as shown in FIG 9A-C and also for native rRNA, which in certain situations may be complexed with ribosomal proteins.
  • the present invention observed non-stereospecific binding of a D-aminoglycoside and a L-aminoglycoside, e.g., D-neamine and L-neamine, to ribosomes and observed the effect of this binding on bacterial cell growth (FIG. 7 A-C).
  • RNA footprinting techniques (lead acetate and DMS footprinting) were used to reveal the binding sites for the D-, L-neamine and neomycin B on the D- and L- A-site of 16S RNA constructs (FIG. 12, 13). Both D- and L- RNA (with D-A 3 tail) were then 32 P- labeled at the 5' end.
  • the L- A-site construct with a tail of three D-adenosines at 5' end was prepared (FIG. 11).
  • the L-RNA oligonucleotide containing a single D-adenosine at 5' end was radiolabled using T4 polynucleotide kinase with only 1-3% efficiency, while the three-adenosine tail provided over 25% labeling efficiency.
  • FIG. 14 A summary of the footprinting results are presented in FIG. 14. Both footprinting methods used in this study indicate that both L- and D- neamine appear to bind to the same site on the D- A-site RNA construct.
  • the binding site is located in the A-rich bulge and part of the stem below it.
  • the putative binding site is formed by nucleotides C6-G22, A7 » A21 and A20 (FIG. 14). This site is similar to neomycin B binding site; which is larger and spans C6-G22, and A7 » A21, as well as involving G4, U5 and A20.
  • Binding of neomycin B to the D-A-site construct induces a significant conformational change in the RNA structure, protecting tefraloop nucleotides U13 and C14. These results are similar to previously reported specific binding site for aminoglycosides on A-site RNA construct.
  • the site for neomycin B on L-RNA is formed by A7-A21, C8-G19, A9, C10-G17, CI 1-G16 and A20, and thus spans the upper-stem and A-rich bulge region.
  • the tefraloop nucleotide C14 is also involved in the interactions with neomycin B.
  • the non-stereospecific binding observed for aminoglycosides with the decoding region RNA constructs B and H is a general characteristic of conformationally flexible aminoglycosides with flexible RNA constructs when the binding affinity is one a low micromolar range, e.g., about 0.1 ⁇ M to about 100 ⁇ M range.
  • the decoding region RNA constructs prepared from either L- or D- ribonucleosides described herein bind aminoglycosides in the ⁇ M range, an affinity typical of many aminoglycoside RNA interactions.
  • CRP was used as the binding probe.
  • the fluorescence assay was found to be well adapted to measuring aminoglycoside binding to 70S bacterial (E.coli) and 80S ribosomes (yeast). Save for neomycin B where the dissociation constant is approximately 0.1 ⁇ M, the typical dissociation constants for aminoglycoside-RNA aptamer complexes are in the 1-2 ⁇ M range.
  • binding data reported herein for aminoglycoside compounds with eukaryotic yeast ribosomes are similar to binding data for said aminoglycoside compounds with ribosoimes in bacteria and with eukaryotic A-site rRNA constructs.
  • Table 3 Dissociation constants for complexes of aminoglycosides with 70S and 80S ribosomes ( M .
  • Binding affinities of D, L-neamine 1, ent-l and their isomers 2, 3, ent-2, ent-3 (Table 1) to ribosomes were determined by the competitive binding assay using the fluorescence anisotropy method recently developed in our laboratory. The binding results are summarized in Table 4. All of D, L-neamine and their isomers bind to ribosomes with dissociation constants in the 0.9 ⁇ 33 ⁇ M range. 5-Isomers of neamines, 2 and ent-2 shows better binding affinities to bacterial ribosome than other positional isomers. 4-Isomer of L-neamine ent-3 shows the highest preference for the bacterial ribosome, fivefold over eukaryotic, yeast ribosome.
  • L-neamine like other aminoglycosides, is bactericidal and exhibits very weak activity against Gram-(+) bacteria-another hallmark of aminoglycosides. For example, when L-neamine was tested against S. aureus and E. faecalis little to no antibacterial activity was found. Antibacterial activity of the neamine enantiomers against aminoglycoside resistant bacteria was also determined.
  • FIG. 10 shows the results of typical in vitro franslation reactions in the presence of neomycin B (A), D-neamine (B) and L-neamine (C).
  • IC 50 50% inhibitory concentration, was defined as concenfrations of aminoglycoside producing 50% inhibition of translation.
  • Quantitation of the gel bands provided the following IC 50 values: neomycin B - 15 ⁇ M; D-neamine - 38 ⁇ M and L-neamine - 195 ⁇ M. These functional assays indicate a clear stereoselective advantage of D-neamine versus its unnatural L-neamine enantiomer.
  • Protein franslation was performed from chloramphenicol acetyl transferase (CAT) mRNA.
  • CAT mRNA was transcribed from a plasmid, bearing the CAT gene under the confrol of T7 promotor (PROTEINscript TM -PRO Linked TranscriptiomTranslation kit. Ambion ® instruction manual; and Pratt J. M. (1984) Coupled transcription-translation in prokariotic cell-free system. In Transcription and Translation (Hames, B. C, and Higgins, S. J.), pp 179-209, URL Press, Oxford). A commercial E. coli ribosomal suspension was supplemented with an E.coli extract containing the required elements for franslation.
  • franslation factors include franslation factors, tRNAs, and aminoacyl tRNA synthases. Plasmid, T7 RNA polymerase and amino acids were added to the reaction mixture, and both transcription and franslation steps were performed in the same vial. The effects of the aminoglycosides on the franslation process were evaluated by measuring the incorporation of 35 S methionine in the franslated chloramphenicol acetyl fransferase protein. Both D- and L-neamine are capable of the inhibiting protein synthesis in vitro. However, D-neamine appeared over 5-fold more potent as a translation inhibitor than L-neamine.
  • L-aminoglycosides such as L- neamine maintains antibiotic activity against bacterial sfrains resistant to D-aminoglycosides.
  • L-aminoglycosides such as L- neamine maintains antibiotic activity against bacterial sfrains resistant to D-aminoglycosides.
  • plasmid pMM bearing the gene for the aminoglycoside kinase APH(3')IIa, which confers kanamycin resistance
  • the transformed sfrain had selective resistance towards D-neamine, but L-neamine maintained antibiotic activity.
  • Aminoglycoside metabolizing enzymes including APH(3')Iia, acetylases, the adenylylases, and the kinases, are stereospecific for D-aminoglycoside substrates.
  • L- aminoglycosides enantiomers amy avoid common cellular resistance mechanisms which degrade or otherwise deactivate their corresponding D-enantiomer, including enzymatic degredation which targets D-aminoglycoside substrates, while maintaining effective binding to their target bacterial rRNA molecules.
  • aminoglycoside analogs which escape these modification reactions could be of substantial use as antibiotics and other applications where resistance to enzymatic degradation would be advantageous.
  • L-aminoglycoside compounds of the invention were determined suing two Gram-(-) sfrains were investigated, E.coli (ATCC 25922) and P.aerugenosa (ATCC 27853). These two strains were chosen because: (1) both sfrains are Gram-(-) and aminoglycosides preferentially inhibit the growth of Gram-(-) bacteria; and (2) both sfrains are potentially resistant to aminoglycosides by mechanisms involving the enzymatic modification of these drugs.
  • An attractive property of L-aminoglycoside compounds of the invention is that they might avoid resistance mechanisms that depend on stereospecific interactions between modifying enzymes and their aminoglycoside substrates.
  • L-aminoglycosides such as L-neamine possess antibacterial activity.
  • the antibacterial activitity of L-neamine and D-neamine were studied using antibiotic disk assays.
  • L-neamine displayed a somewhat lower activity with WT E.coli (ATCC 25922) than D-neamine (Fig 7 A), but L-neamine is still clearly active.
  • a disk assay using an E.coli bacterial stain that is resistant to kanamycin/neomycin were studied, the activity of D- neamine was essentially abolished, while L-neamine maintaines substantially the same level of activity as observed for disc assays with WT E.coli (Fig 7B).
  • Aminoglycoside resistance of E.coli was induced by transformation with pMM plasmid.
  • This plasmid contains the neomycin phosphofransferase gene, NTP ⁇ , derived from the Tn5 fransposon, a generic antibiotic marker for kanamycin/neomycin resistance.
  • the protein product of the NTP II gene is the APH(3') Ila phosphofransferase. This gene is derived from the Tn5 fransposon, and comprises a generic antibiotic marker for kanamycin and neomycin resistance (Wright, G. D. and Thompson, P. R. (1999) Front. Biosci. 4, D9- 21).
  • P.aerugenosa contains gene for APH(3') lib phosphotransferase, homologous to APH(3') Ila which provides the inherent kanamycin resistance of P.aeruginosa. It is possible that the enhanced antibiotic activity relative (with respect to WT E.coli) activity of L-neamine observed here is related to the selective deactivation of D-neamine. See, for example, Wright, G. D. & Thompson, P. R. (1999) Front. Biosci. 4, D9-21.
  • Minimal inhibitory concenfrations (MIC) of D- and L-neamine were measured in WT bacterial sfrains, in kanamycin/neomycin resistant E.coli, and in P.aeruginosa. The results are summarized in Table 5.
  • the bactericidal profiles for D- and L-neamine are presented in FIG. 8. As can be seen here, both compounds are bactericidal towards E.coli (ATCC 25922). However the activity of L-neamine is significantly lower then the activity of natural D- neamine under the conditions of the experiments.
  • D-neamine begins to kill bacteria at 75 ⁇ M, while the bactericidal effects of L-neamine are manifest 600 ⁇ M and, thus, L-neamine is approximately 8 times less potent than its D-enantiomer. This ratio is confirmed by MIC measurements as L-neamine is observed to be approximately 8.7 times less active than D- neamine. Although the MIC values for L-neamine are close for the resistant and susceptible bacterial strains, there is a sharp drop off for D-neamine in the resistant strains. This is especially true for the resistant E. coli sfrain (Table 5)
  • Neomycin sulfate, paromomycin sulfate, kanamycin B sulfate. tobramycin sulfate, streptomycin sulfate were purchased from Sigma Inc. and used without further purification.
  • 5-Carboxytetramethylrhodamine-labeled paromomycin (CRP) and tobramycin (CRT) were prepared as previously reported (19).
  • L-ribonucleoside phosphoramidites and L- ribonucleoside CPG supports were from Chemgenes Inc. Nick Sephadex G-50 columns were purchased from Pharmacia Inc. D-Neamine hydrochloride was synthesized from Neomycin sulfate as described previously (20).
  • Example 1 The synthesis of L-neamine hydrochloride.
  • D-thioglycoside donor 13 was synthesized from protected Dglucosamine derivative 8 (Aiper, P. B., Hung, S. -C. Wong, C. -H. tetrahedron lett. 1996, 34, 6029-6032) in five steps.
  • Acetate deprotection of 9 affoded friol 10 which was monotosylated to give 11 in 94% yield.
  • the tosylate 11 was displaced with sodium azide in DMF providing 12 in quantitative yield.
  • the oligomers were synthesized on an Applied Biosystems INC (ABI) 381 A DNA/RNA synthesizer using a modified 1 ⁇ mol RNA cycle, L(D)-ribonucleoside CPG supports, L(D)-ribonucleoside phosphoramidites and a coupling time of 600 seconds. After deblocking with ethanolic ammonium hydroxide and triethylamine trihydrofluoride, the oligoribonucleotides were precipitated from n-butanol at -20 ° C (21) and purified by Nick Sephadex G-50 column. Circular dichroism (CD) spectra were recorded in 0.1M NaCl and lOmM sodium phosphate, pH 7.0 at 4 ° C on an Aviv 202 spectropolarimeter.
  • CD Circular dichroism
  • the oligomers were synthesized on an ExpediteTM oligonucleotide synthesizer using a modified 1 ⁇ mol RNA cycle, L(D)-ribonucleoside CPG supports, DMT-L(D)-ribonucleoside phosphoramidites and a coupling time of 600 seconds. After deblocking with ethanolic ammonium hydroxide and triethylamine trihydrofluoride, and the oligoribonucleotides were precipitated with n-butanol at -20 °C (23) and desalted on a Nick Sephadex G-50 column.
  • RNA oligonucleotides were purified by gel-electrophoresis on a 12% polyacrylamide/7M urea denaturing gel. All stock solutions were prepared in nuclease-free water, and were diluted with the appropriate buffers prior to use. The concenfrations of RNA oligonucleotides were determined specfrophotometrically, by absorption at 260 nm. The RNA molecules were annealed by heating to 95 °C followed by cooling to room temperature. For the comparison, D- A-site RNA construct was also purchased from Dharmacon and was deprotected using the buffer provided and according to the company's instructions.
  • RNA constructs were renatured by incubating in binding buffer ⁇ 150 mM NaCl, 5mM KC1, ImM CaCl 2 , ImM MgCl 2 , and 20 mM HEPES (pH 7.5) ⁇ for 3 minutes at 90 ° C followed by slow cooling to 25 ° C.
  • Equation 1 was used for the determination of the dissociation constant (Kd ) for the interactions between RNA or ribosome and CRP.
  • RNA and CRP are the initial concenfrations of RNA and CRP, respectively.
  • Equation 2 is used for the calculation of the K D values in the competition binding assay.
  • K D is the dissociation constant between the RNA and the aminoglycosides
  • (aminoglycoside)o is the initial concentration of the aminoglycosides
  • A. is anisofropy of completely bound tracer. Both K d and K D were determined by non-linear curve fitting using the equations described above, and are presented as mean values of three independent measurements.
  • E.coli (MRE600) 70S ribosomes The purification of E.coli (MRE600) 70S ribosomes was carried out according to the published procedure (Lazaro, E., van den Broek, L. A. G. M., Felix, A. S., Ottenheijm, H. C. J., Ballesta, J. P. G. (1991) Biochemistry 30, 9642-9648).
  • E.coli MRE600 were grown for 12 h in 6L of LB medium (10 g trypton, 5g yeast extract, 5g NaCl per 1 L, pH 7.0) at 37 ° C. Crude ribosomes were precipitated from clarified supernatant by 4 h centrifugation at 25,000 rpm in Beckman SW 28 rotor at 4 ° C.
  • the ribosome pellets were collected in 5 mL of buffer BR1, containing 10 mM HEPES, pH 7.5, 500 mM ammonium acetate, 100 mM magnesium chloride and 2.5 mM DTT.
  • the ribosome suspension was centrifuged for 20 min at 15000 rpm to be clarified.
  • the clarified sample was loaded over two layers of 20% and 40% sucrose in buffer BR1 and centrifuged for 18 hr at 19000 rpm in Beckman SW28 rotor at 4 ° C.
  • the concenfrations of ribosomes were determined by absorbance at 260 nm, assuming 23 pmol/ 1 OD 260 , ribosomal suspension was frozen in liquid nitrogen and stored at -80 ° C.
  • yeast 80S ribosomes from S.cerevisiae sfrain YSB 758 was carried out according to the published procedure (Verschoor, A., Warner, J. R., Srivastava, S., Grassucci, R. A., & Frank, J. (1997) Nucl. Acid Res. 26, 655-661).
  • S.cerevisiea YSB 758 were grown for 12 hrs at 30 ° C in 2L of YPD medium, containing: 20g bacto peptone, 20 g glucose, 10 g yeast extract and 0.15 g tryptophan per 1 L, pH 7.0.
  • the cells were lysed by 5- 10 cycles of 1 min vigorous shaking with glass beads with a vortex, followed by cooling on ice.
  • the ribosomes were collected by centrifugation in Beckman TL100.3 rotor at 75000 rpm for 3 h.
  • Ribosome concenfrations were determined by absorbance at 260 nm, assuming 18 pmol of 80S ribosome particles per 1 OD 260 . Ribosomes were frozen in liquid nitrogen and stored at -80 ° C.
  • Example 8 In vitro protein franslation.
  • In vitro translation was performed by using PROTEINscriptTM-PRO (Ambion) kit.
  • This kit is designed for coupled in vitro transcription and translation using a highly active E. coli S30 extract, containing 70S ribosomes. Reaction mixtures contained appropriate concenfrations of antibiotic (neomycin B, D- and L-neamine).
  • the protein was franslated from CAT mRNA, transcribed from confrol DNA template containing CAT gene under confrol of the T7 promotor.
  • the product, -25 kDa chloramphenicol acetyl fransferase was labeled during franslation with 1 ⁇ Ci 35 S methionine (New England Nuclear) per reaction (no cold methionine was added).
  • E.coli ATCC 25922
  • P.aeruginosa ATCC 27853
  • BD Bioscience P.aeruginosa
  • Kanamycin/neomycin resistant E.coli was prepared by transformation (elecfroporation) of wild type (WT) E.coli (ATCC 25922) with pMM plasmid, bearing the gene of aminoglycoside phosphofransferase II (APH(3')IIa), also known as neomycin phosphofransferase II (NPT II, IUBMB Enzyme nomenclature: EC 2.7.1.95) (Wright, G. D. & Thompson, P. R. (1999) Front. Biosci. 4, D9-21). Kanamycin/ neomycin resistant E.coli was propagated in the same medium 2, supplemented with 30 mg/mL kanamycin.
  • each bacterial inoculum was mixed with 10 mL of melted agar (1.5 % medium 2 agar, BD Bioscience) at 50-55 ° C and poured onto 100 mm Petri dishes. Sterile Whatmann 3MM 5 mm paper disks were soaked with antibiotic solutions and allowed to dry. The disks were positioned evenly on the surface of bacterial- inoculated solid agar and the plates were incubated at 37 ° C for 1-2 days. MIC and bactericide index measurements.
  • MIC Minimal inhibitory concenfrations
  • the inoculi were diluted to OD 600 -0.1 in medium 2 and 1 mL of the culture was placed in 13 mL test tubes. The desired concentrations of antibiotic were added from stock solutions. The samples were incubated at 37 ° C for 3-5 h when the control culture had an OD 600 of 1.2-1.5. The absorbance at 600 nm of each sample was read and MIC was taken as the lowest antibiotic concentration inhibiting bacterial growth by greater than 90%.
  • the bactericidal activity of each antibiotic was investigated at concenfrations of 0.5- 4xMIC in medium E.coli (ATCC 25922) inoculum was added to 0.5 mL medium 2 to OD 600 -0.1 together with different antibiotic concentrations. The samples were incubated at 37 ° C for 3 hrs, and the bacteria were collected by centrifugation. The bacterial pellets were washed with 0.5 mL of medium 2 and were plated on 1.5 % medium 2 agar in 100 mm Petri dishes. The plates were incubated for 1-2 days at 37 ° C and mean log 10 change in viable count was calculated and plotted vs drug concenfration.
  • the L- A-site RNA construct was synthesized with a 3-D-adenosine (A 3 ) tail at 5' end (FIG. 11).
  • the RNA was purified by gel-electrophoresis on a 12% polyacrylamide/7M urea gel as described above.
  • the purified D and L- RNA were radioactively labeled at 5' end with 1 ⁇ L of P ⁇ -ATP (6000 Ci/mmol, New England Nuclear) per 1 nmol of RNA using T4 polynucleotide kinase (Ambion) in a buffer containing 70 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , and 5 mM DTT.
  • RNA samples were annealed by heating them to 95°C for 3 min, followed by cooling to room temperature in a 10 mM HEPES buffer (pH 7.0).
  • RNA was precipitated by the addition of 1 ⁇ L 3M sodium acetate and 32 ⁇ L of ice-cold ethanol (75% final ethanol concenfration) followed by incubation for 1-2 hrs at -20 °C.
  • the pellets were washed with 75% ethanol, dried and dissolved in 10 ⁇ L of 1M Tris HC1, pH 7.3. 10 ⁇ L of freshly prepared 0.2 M sodium borohydride (Sigma) solution was added to the samples, and further incubation was allowed for 30 minutes on ice in the dark.
  • the samples were precipitated from 75% ethanol as described above. After the pellets were washed and dried, they were dissolved in 20 ⁇ L of fresh aniline acetate solution, prepared by addition to 210 ⁇ L of glacial acetic acid, 90 ⁇ L of water and 30 ⁇ L of freshly distilled aniline. The RNA samples were incubated at 60 °C for 20 min, and the reaction was stopped by freezing on dry ice. The samples were lyophilized, the pellets were washed twice with ethanol, dissolved in 20 ⁇ L water and lyophilized again. The samples then were dissolved in 10 ⁇ L of formamide/bromphenol blue/xylene cyanol loading buffer and gel-electrophoretic analysis was performed as described above.

Abstract

La présente invention concerne des composés à base d'aminoglycosides possédant une activité antibiotique. En outre, la présente invention concerne des composés à base de L-aminoglycosides et de leurs diastéréoimères qui ont une activité antibiotique et ne sont pas vulnérables au développement de souches bactériennes. La présente invention concerne aussi des procédés de traitement et des compositions pharmaceutiques qui utilisent ou comprennent un ou plusieurs composés à base d'aminoglycosides décrits dans l'invention.
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WO2005116041A3 (fr) * 2004-05-28 2006-08-24 Univ Leeds Nouveaux composes aminoglycoside et derives
WO2005116041A2 (fr) * 2004-05-28 2005-12-08 University Of Leeds Nouveaux composes aminoglycoside et derives
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US8114856B2 (en) 2005-12-02 2012-02-14 Isis Pharmaceuticals, Inc. Antibacterial 4,5-substituted aminoglycoside analogs having multiple substituents
US8569264B2 (en) 2005-12-02 2013-10-29 Isis Pharmaceuticals, Inc. Antibacterial 4,5-substituted aminoglycoside analogs having multiple substituents
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US8742078B2 (en) 2008-09-10 2014-06-03 Isis Pharmaceuticals, Inc. Antibacterial 4,6-substituted 6′, 6″ and 1 modified aminoglycoside analogs
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US8372813B2 (en) 2008-10-09 2013-02-12 Achaogen, Inc. Antibacterial aminoglycoside analogs
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