US20040242532A1 - Method for the treatment of microorganism infections by inhibiting energy storage and utilization - Google Patents

Method for the treatment of microorganism infections by inhibiting energy storage and utilization Download PDF

Info

Publication number
US20040242532A1
US20040242532A1 US10/867,959 US86795904A US2004242532A1 US 20040242532 A1 US20040242532 A1 US 20040242532A1 US 86795904 A US86795904 A US 86795904A US 2004242532 A1 US2004242532 A1 US 2004242532A1
Authority
US
United States
Prior art keywords
inhibitor
enzyme
adp
pathogenic bacteria
glucose
Prior art date
Legal status (The legal status 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 status listed.)
Abandoned
Application number
US10/867,959
Inventor
Christopher Meyer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
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 Individual filed Critical Individual
Priority to US10/867,959 priority Critical patent/US20040242532A1/en
Publication of US20040242532A1 publication Critical patent/US20040242532A1/en
Abandoned legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/66Phosphorus compounds
    • A61K31/675Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90Enzymes; Proenzymes
    • G01N2333/91Transferases (2.)
    • G01N2333/91091Glycosyltransferases (2.4)
    • G01N2333/91097Hexosyltransferases (general) (2.4.1)
    • G01N2333/91102Hexosyltransferases (general) (2.4.1) with definite EC number (2.4.1.-)
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90Enzymes; Proenzymes
    • G01N2333/91Transferases (2.)
    • G01N2333/912Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205Phosphotransferases in general
    • G01N2333/91245Nucleotidyltransferases (2.7.7)
    • G01N2333/9125Nucleotidyltransferases (2.7.7) with a definite EC number (2.7.7.-)
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is directed to the use of bacterial enzymes as targets for antibiotic therapy and the treatment of microorganism infections, particularly by inhibiting enzymes involved in energy storage and utilization.
  • Starch a complex polymer of glucose, is present in most green plants in practically every type of tissue and is the major intracellular reserve polysaccharide in photosynthetic organisms.
  • the glucan accumulates during development of storage or seed tissues and is catabolized to serve as a source of energy.
  • the primary reserve polysaccharide is glycogen.
  • Glycogen is a polysaccharide containing linear molecules with ⁇ -1,4 glucosyl linkages and is branched via ⁇ -1,6-glucosyl linkages.
  • glycogen is analogous to starch with regard to linkages, glycogen exhibits a different chain length and a different degree of polymerization.
  • step (1) ADP Glucose (ADPGlc) is synthesized from ATP and glucose-1-phosphate in the rate-limiting reaction which is catalyzed (in plants and bacteria) by ADP-glucose pyrophosphorylase (also referred to as ADPGlc PPase; or ADPG PPase, or glucose-1-P adenyltransferase, or as enzyme EC. 2.7.7.27).
  • the chain elongation step (2) is catalyzed by glycogen synthase (also referred to a GS, gly A, or as enzyme EC. 2.4.1.21).
  • U.S. Pat. No. 6,184,438 to Hannah describes mutant genes encoding plant ADP-glucose pyrophosphorylases and the use of those genes to produce transformed plants having enhance germination characteristics but without any diminishment in food quality or flavor.
  • U.S. Pat. No. 6,057,493 to Willmitzer et al. describes the use of anti-sense DNA sequences encoding ADP-glucose pyrophosphorylase from potato to produce transformed plants with a reduction in starch concentration and an increase in the concentration of at least sucrose.
  • DNA sequences have also been elucidated for certain bacterial ADP-glucose pyrophosphorylase enzymes.
  • U.S. Pat. No. 5,349,123 to Shewmaker et al. describes a nucleic acid construct which encodes an E. coli ADP-glucose pyrophosphorylase and the use of that construct to transform plant cells to modify biosynthesis of a glucan in the plant.
  • nucleoside diphosphate sugar substrate is UDP glucose
  • catalyzing enzyme is EC 2.4.1.11.
  • ADP-glucose is critical to viability of bacteria, and since ADP-glucose pyrophosphorylase is not present in mammals, the enzyme provides an excellent target for inhibition of bacterial growth, thereby providing a means for inhibiting the growth of microorganisms and treating bacterial infections.
  • glycogen synthase in many pathogenic bacteria is different than the glycogen synthase in mammals, that enzyme also provides an excellent target for inhibition of bacterial growth and treating bacterial infections.
  • the present invention is directed to a method for treating a microorganism infection by administering an effective amount of a compound capable of inhibiting the production and/or utilization of ADP-glucose.
  • It is a further aspect of the invention to provide a method of identifying a compound capable of inhibiting the growth of pathogenic microorganisms which comprises identifying a compound which inhibits an enzyme important in the catabolism and metabolism of energy storage pathways, particularly a compound that inhibits the activity of ADP-glucose pyrophosphorylase and/or glycogen synthase.
  • FIGS. 1-5 are graphs plotting the results of experiments testing the inhibiting effect of ADP-glucose borano analogs on an ADP-glucose Ppase enzyme in in vitro enzyme tests.
  • the present inventor has discovered that while certain important pathogenic microorganisms require the activity of ADP-glucose pyrophosphorylase (EC 2.7.7.27) to produce ADP-glucose, that enzyme is not present in mammals, particularly not in humans.
  • ADP-glucose pyrophosphorylase EC 2.7.7.27
  • the present inventor has recognized that the glycogen synthase (EC 2.4.1.21) in many pathogenic bacteria as the ADPGlucose dependent glucan chain lengthening enzyme is not present in mammals, particularly not in humans.
  • ADP-glucose pyrophosphorylase EC 2.7.7.27
  • glycogen synthase EC 2.4.1.21
  • ADP-glucose pyrophosphorylase catalyzes the reaction of ⁇ -glucose-1-phosphate with ATP to produce ADP-glucose.
  • the “corresponding” reaction is catalyzed by UDP-glucose pyrophosphorylase by transferring a glucosyl residue from UDP-glucose, as shown below:
  • Step (1) in this pathway in mammals, including humans, is catalyzed by UDP-glucose pyrophosphorylase. Recognizing the difference between the critical use of UDPGlc PPase in mammals as compared to the use of ADPGlc PPase in certain pathogenic bacteria, the present inventor first recognized that such bacteria could be selectively killed by inhibiting the activity of ADPGlc PPase, without adversely affecting any mammal so infected with the bacteria. This selectivity provides a basis and target for a novel and important new class of antibiotics, antibiotics which are inhibitors of energy storage and utilization enzymes, namely ADPGlc PPase inhibitors.
  • Step (2) in the above is catalyzed in humans by glycogen synthase (EC 2.4.1.11), whereas in many pathogenic bacteria the corresponding enzyme is EC 2.4.1.21 because the sugar substrate is ADP-based, not UDP-based. Again, this provides a target for a novel and important new class of antibiotics which inhibit glycogen synthase (EC 2.4.1.21).
  • antibiotics are currently used to treat a wide range of bacterial infections, ranging from minor to life threatening infections.
  • Broad spectrum antibiotics treat a variety of gram-positive and gram-negative organisms, while mild spectrum antibiotics only cover limited types of bacterial organisms and are useful for curing infections with known bacterial strains.
  • pathogenic bacteria and fungi increasingly exhibit resistance to existing classes of antibiotics, such as penicillin, vancomycin and erythromycin. According to the Center for Disease Control, pathogenic resistance has significantly increased mortality rates, maling infectious disease the third largest cause of death in the United States. The rates of antibiotic resistant bacteria have particularly increased recently with respect to S. aureus, Enterococcus strains, S. pneumoniae and M. tuberculosis .
  • a first aspect of the invention relates to a method for identifying compounds capable of inhibiting the growth of pathogenic microorganisms which comprises:
  • an enzyme in an energy storage or utilization pathway which is important to continued growth and viability of a pathogenic microorganism but which is absent in humans provides a unique, specific target for compounds which can inhibit infections of such pathogenic microorganisms without causing undesirable side effects or toxicity to a mammalian patient.
  • Various biosynthetic pathways have been identified in the literature for various microorganisms and for mammals, and those pathways, which include an important enzyme present in pathogenic microorganisms but absent in mammals, provide a unique target for screening for compounds useful for inhibiting pathogenic microorganism infections.
  • the present inventor has specifically identified ADP-glucose pyrophosphorylase (EC 2.7.7.27) and glycogen synthase (EC 2.4.1.21) as enzymes present in a biosynthetic pathway important for energy storage and utilization in pathogenic microorganisms, but absent in mammals, specifically absent in humans. Since the biosynthetic pathway is important for energy storage or utilization in pathogenic microorganisms, inhibition of this pathway significantly decreases the viability of pathogenic microorganisms, leading ultimately to death of the microorganism, either by action of the inhibitor alone, or in combination with the patient's own immunological systems for resisting infections, or in combination with other antibiotics.
  • ADP-glucose pyrophosphorylase EC 2.7.7.27
  • glycogen synthase EC 2.4.1.21
  • inhibition of the biosynthetic pathways and/or inhibition of ADP-glucose pyrophosphorylase (EC 2.7.7.27) or glycogen synthase (EC 2.4.1.21) may not per se kill the bacteria, but will render the microorganisms non-infective or non-pathogenic. It has been known for quite some time that complex carbohydrates can act as virulence factors in bacteria responsible for invasive infections (Glazer, A. N. and Nikaido, H. Microbial Biotechnology. Fundamentals of Applied Microbiology (1995) W. H. Freeman and Co., pgs. 266-272). Specific to glycogen biosynthesis, Tamilo, A. D., and Ugalde, R. A.
  • the present inventor has specifically identified a number of important pathogenic microorganisms which require ADP-glucose pyrophosphorylase (EC 2.7.7.27) and glycogen synthase (EC 2.4.1.21), including Chlamydia pneumoniae, Chlamydia trachomatis, Esherichia coli O157, Haemophilus influenzae, Mycobacterium leprae, Mycobacterium tuberculosis, Salmonella typhimurium and Vibrio cholerae, Streptococcus pneumoniae, Yersinia pestis, Bacillus subtilus and Bacillus anthracis.
  • ADP-glucose pyrophosphorylase EC 2.7.7.27
  • glycogen synthase EC 2.4.1.21
  • Chlamydia pneumoniae Chlamydia trachomatis
  • Esherichia coli O157 Haemophilus influenzae
  • Mycobacterium leprae Mycobacterium tuberculos
  • the above listed bacteria comprise some of the most important pathogenic microorganisms which account for significant numbers of disease patients in the United States and around the world.
  • the following table summarizes the prevalence and current treatments available for these pathogenic microorganisms.
  • TABLE 1 Prevalence and Current Treatments Incidence Prevalence (estimated (estimated number of number of new people currently Microorganism Disease(s) cases/yr) infected) Treatment Chlamydia
  • Acute and chronic pneumoniae respiratory diseases including: pneumonia, pharyngitis, bronchitis, sinusitis, otitis media, COPD, asthma, Reiter syndrome, and sarciodosis.
  • Mycobacterium Leprosy (Hansen's 250 new 12 million world-wide. Dapsone, refampin, leprae disease) cases/yr in the It is a public health ethionamide U.S., 600,000 problem in 72 countries, new cases/yr 19 of which account for world-wide. 90% of all the cases in the world.
  • Salmonella Salmonellosis >50,000/yr Ampicillin, typhimurium abdominal cramps, non- world-wide chloramphenicol, bloody diarrhea.
  • one aspect of the present invention is a method for the identification of a compound capable of inhibiting the growth of pathogenic microorganisms by interfering with the activity of glycogen synthase and/or ADP-glucose pyrophosphorylase.
  • Compounds can be identified by growing bacteria on defined media in the presence or absence of a test compound, and assessing the effect on glycogen synthesis by iodine staining of colonies (Govons, S. et al (1969) J. Bacteriol. 97, 970-972).
  • the amount of glucan accumulated in the absence or presence of test compounds can be assessed by collection of the glycogen from the culture and quantitatively converting it to glucose with glucoamylase and ⁇ -amylase (Preiss, J. et al (1975) J. Biol. Chem. 250: 7631-7638)
  • Compounds capable of inhibiting glycogen synthase and/or ADP-glucose pyrophosphorylase can also be identified by means of in vitro experiments by exposing a substrate comprising glycogen synthase and/or ADP-glucose pyrophosphorylase to a plurality of test compounds and identifying those compounds which inhibit the tested enzyme according to known catalytic measurement techniques.
  • ADPGlc PPase One particular in vitro method for assessing the activity of an inhibitor to purified ADPGlc PPase is the following:
  • Assaying for ADPGlc synthesis activity involves measuring the amount of 14-C labeled Glc-1-P converted to ADPGlucose (Preiss, J., Shen, L., Greenberg, E., and Gentner, N. (1966) Biochemistry 5, 1833-1845). Briefly, unreacted Glc-1-P is separated from product by the following steps: 1) digestion with alkaline phosphatase (thus removing the negatively charged phosphate); 2) spotting an aliquot of the reaction mixture on to positively charged DE-81 filters (Whatman); and 3) washing the filters with water (thus removing the now neutral C-14 glucose).
  • Initial screening of a putative inhibitor typically includes testing at two concentrations ( ⁇ 25 ⁇ M and 1 mM) ⁇ major activator with appropriate controls and blanks for a total of 9 assays/inhibitor/enzyme (2 control assays—appropriate enzyme concentrations in the absence of inhibitor; 4 experimental assays; 1 blank in the absence of inhibitors; 2 blanks in the presence of inhibitor (no activator required).
  • a quantitative assay to measure the activity of an inhibitor to bacterial (ADPG dependent) Glycogen Synthase involves the measurement of incorporated 14-C-labeled ADPGlc into a glucan molecule (a specific glycogen, or amylopectin) that serves as a primer wherein the labeled glucan can easily be separated from unincorporated ADPGlc by a precipitation step (methanol insoluble polysaccaride).
  • glucan molecule a specific glycogen, or amylopectin
  • methanol insoluble polysaccaride methanol insoluble polysaccaride
  • Useful inhibitors can also be identified, and potential inhibitors assessed, by in vitro treatment of bacteria in, for example, culture tubes or petri dish samples. Such assessments can be performed, for example, by spreading a measured aliquot of a diluted bacterial culture into nutrient agar plates, both treated and control, and counting the number of visible cells. Detailed procedures are well known to those in the art, as shown for example in Miller, Experiments in Molecular Genetics , Cold Spring Harbor Laboratory, 1972.
  • Compounds which inhibit ADPGlc PPase or glycogen synthase can also be assessed in an in vivo animal model test. Such tests can be conducted in an animal which is susceptible to infection by the pathogenic microorganism of interest. For example, the effect of the test compound on the virulence of H. influenzae is assessed by comparing the survival rates of animals which have been administered the test compound with the survival rate of animals which have not been administered the test compound, wherein a higher survival rate of animals administered the test compound is an indication that the test compound has an effect on the virulence of H. influenzae.
  • test compound is administered to the animals either prior to, at the time of, or after inoculation of the animals with H. influenzae .
  • the test compound may be administered directly into the nasopharynx, or may be administered by any other route including any one of the traditional modes (e.g., orally, parenterally, transdermally or transmucosally), in a sustained release formulation using a biodegradable biopolymer, or by on-site delivery using micelles, gels and liposomes, or rectally (e.g., by suppository or enema).
  • Precise formulations and dosages will depend on the nature of the test compound and may be determined using standard techniques, by a pharmacologist of ordinary skill in the art.
  • an amount of S. pneumoniae is inoculated into the left anterior naris of the animal. Colony counts are performed to ensure that the inocula are of the desired density and phenotype.
  • the nasopharynx is cultured for the presence of viable S. pneumoniae by the slow instillation of 20 to 40 ⁇ l of sterile PBS into the left naris and withdrawal of the initial 10 ⁇ l from the right naris. This procedure ensures that the fluid has passed through the nasopharynx. The quantity of organisms recovered is then assessed in a well known culture assay.
  • the effect of the test compound on colonization of the nasopharynx by S. pneumoniae is evaluated by comparing the degree of colonization of the nasopharynx in animals which have been administered the test compound with the degree of colonization in animals which have not been administered the test compound, wherein a lower degree of colonization in animals administered the test compound is an indication that the test compound inhibits colonization of the nasopharynx by S. pneumoniae.
  • the invasive capability of S. pneumoniae may also be measured in the same animal model. Essentially, bacteria which have entered the blood stream following inoculation of the nasopharynx are detected by culturing the same in a sample of blood obtained from the animal. Again, the effect of the test compound on the invasive capacity of S. pneumoniae is assessed by comparing the number of organisms found in the blood stream in animals which have been administered the test compound with the number of organisms in the blood stream in animals which have not been administered the test compound, wherein a lower number of organisms in the blood stream of animals administered the test compound is an indication that the test compound has an effect on the invasive capacity of S. pneumoniae.
  • the effect of the test compound on the virulence of S. pneumoniae is assessed by comparing the survival rates of animals which have been administered the test compound with the survival rate of animals which have not been administered the test compound, wherein a higher survival rate of animals administered the test compound is an indication that the test compound has an effect on the virulence of S. pneumoniae.
  • test compound is administered to the animals either prior to, at the time of, or after inoculation of the animals with S. pneumoniae .
  • the test compound may be administered directly into the nasopharynx, or may be administered by any other route including any one of the traditional modes (e.g., orally, parenterally, transdermally or transmucosally), in a sustained release formulation using a biodegradable biopolymer, or by on-site delivery using micelles, gels and liposomes, or rectally (e.g., by suppository or enema).
  • Precise formulations and dosages will depend on the nature of the test compound and may be determined using standard techniques, by a pharmacologist of ordinary skill in the art.
  • the present invention further provides a method for treating pathogenic microorganism infections in a patient by administering to the patient an effective amount of an inhibitor against glycogen synthase and/or ADP-glucose pyrophosphorylase, wherein an effective amount of the inhibitor will inhibit the activity of the enzyme so as to decrease viability of and/or kill the microorganism.
  • the inhibitor utilized in the treatment may be one identified by one of the methods described above, or inhibitors may be identified by any other method.
  • nucleoside ⁇ -P-boranodiphosphoglucose particularly its triethylammonium salt
  • adenosine ⁇ -P-boranodiphosphoglucose is a boran analog of ADP-glucose generating two sterioisomers and has the following structures:
  • A is adenosine
  • Adenosine boranophosphate exists as two stereoisomers, the preparation of which is described below:
  • the compound of formula (I) is a useful inhibitor of ADPGlc PPase according to the present invention.
  • ADPGlc PPase Other known regulators and inhibitors of ADPGlc PPase are the following: TABLE 2 Regulatory properties of ADP-glucose pyrophosphorylase in different organisms ADP-glucose pyrophosphorylase Allosteric regulators Organism Activator Inhibitor Prokaryotes Enterobacteria Escherichia coli Fructose-1,6-bisP AMP Salmonella typhimurium Enterobacter aerogenes Aeromonas formicans Fructose-1,6-bisP AMP Micrococcus luteus Fructose-6-P ADP Mycobacterium smegmatis Serratia mercescens None AMP Enterobacter hafniae Clostridium pasteurianum Agrobacterium tumefaciens Pyruvate AMP Arthrobacter viscosus Fructose-6-P ADP Chromatium vinosum Rhodobacter capsulata
  • Rhodobacter sphaeroides The APDGlc Ppase from Rhodobacter sphaeroides has been used as a model enzyme for initial testing of inhibitors as it has been cloned, sequenced, and expressed in the inventor's laboratory (Meyer et al (1999) Arch. Biochem. Biophys. 372, 179-188;
  • PREMIX A: 100 mM HEPES (pH 7), 0.5 mg/mL BSA, 0.5 mM Glc-1-P, 0.25 mM ATP subsaturating (near S0.5 value), 5 mM MgC12, (0.2 U Pyrophosphatase))
  • PREMIX B A+1 mM F6P
  • RBS ADPG PPASE WT ENZYME CONC. 17 mg/mL (use 1:8000 dilution in Dilution Buffer)
  • Analog I successfully inhibited the Rb.s . ADPG Ppase. While the response may be biphasic, the results showed about 75% inhibition by 500 ⁇ M in the absence of the activator F6P. The inhibition was less in the presence of F6P ( ⁇ 50% at 500 ⁇ M). Analog II also successfully inhibited the Rb.s . enzyme with about 70% inhibition at 500 ⁇ M in both the presence and absence of F6P. TABLE 3 Inhibition Study with ADPGlucose Borano compounds Analog I and II Analog I Analog II Blank 60.33 56.33 Blank + 50 Um 76.33 51.00 Blank + 500 uM 53.67 53.33 825 Cpm/nmol Volume dil.
  • Inhibitors useful for the treatment of pathogenic bacteria and microorganisms can be administered by a variety of means and dosage forms well known to those skilled in the art.
  • the present compounds are administered, for example, orally in the form of a tablet, capsule, powder, syrup, etc., or parenterally such as intravenous injection, intramuscular injection, or intrarectal administration.
  • the suitable administration forms as mentioned above may be prepared by mixing an active ingredient with a conventional pharmaceutically acceptable carrier, excipient, binder, stabilizer, etc.
  • a pharmaceutically acceptable buffering agent, solubilizer, isotonic agent, etc. may be added thereto.
  • the active compound may be administered per se, or in the form of a pharmaceutically acceptable salt thereof, or in the form of a pro-drug, such as an ester.
  • the dosage of the compound varies according to the conditions, ages, weights of the patient, the administration form, the frequency of the administration, etc., but it is usually in the range of 100 to 3000 mg per day for an adult, which is administered once or divided into several dosage units.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Zoology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

A method and pharmaceutical composition for inhibiting infections of pathogenic microorganisms by inhibiting the production of ADP-glucose, particularly by inhibiting the activity of ADP-glucose pyrophosphorylase or glycogen synthase.

Description

    FIELD OF THE INVENTION
  • The present invention is directed to the use of bacterial enzymes as targets for antibiotic therapy and the treatment of microorganism infections, particularly by inhibiting enzymes involved in energy storage and utilization. [0001]
  • BACKGROUND OF THE INVENTION
  • Starch, a complex polymer of glucose, is present in most green plants in practically every type of tissue and is the major intracellular reserve polysaccharide in photosynthetic organisms. The glucan accumulates during development of storage or seed tissues and is catabolized to serve as a source of energy. In the animal kingdom, as well as in fungi, yeast and bacteria, the primary reserve polysaccharide is glycogen. Glycogen is a polysaccharide containing linear molecules with α-1,4 glucosyl linkages and is branched via α-1,6-glucosyl linkages. Although glycogen is analogous to starch with regard to linkages, glycogen exhibits a different chain length and a different degree of polymerization. [0002]
  • The following common reactions are shared by the biosynthetic pathways of bacterial glycogen and of starch in algae and higher plants: [0003]
  • ATP+α-Glc-1-P
    Figure US20040242532A1-20041202-P00900
    ADPGlc+PPi  (1)
  • ADPGlc+α-1,4-glucan→ADP+α-1,4-glucosyl-α-1,4-glucan  (2)
  • Elongated αa-1,4-glucan chain→Branched α-1,6-α-1,4-glucan  (3)
  • In step (1), ADP Glucose (ADPGlc) is synthesized from ATP and glucose-1-phosphate in the rate-limiting reaction which is catalyzed (in plants and bacteria) by ADP-glucose pyrophosphorylase (also referred to as ADPGlc PPase; or ADPG PPase, or glucose-1-P adenyltransferase, or as enzyme EC. 2.7.7.27). The chain elongation step (2) is catalyzed by glycogen synthase (also referred to a GS, gly A, or as enzyme EC. 2.4.1.21). [0004]
  • 1. ADP-glucose phyrophosphorylase [0005]
  • The reaction scheme catalyzed by ADPGlc PPase is shown below: [0006]
    Figure US20040242532A1-20041202-C00001
  • Significant research has led to cloning and sequencing genes which code for ADP-glucose pyrophosphorylase in plants for the purpose of modulating sucrose and starch content in plants. For example, U.S. Pat. Nos. 5,498,831 and 5,773,693 to Burgess et al. described the sequence of Pea ADP-glucose pyrophosphorylase subunit genes and the use of those genes to transform plant cells in order to provide plants that have increased sucrose content. [0007]
  • U.S. Pat. No. 6,184,438 to Hannah describes mutant genes encoding plant ADP-glucose pyrophosphorylases and the use of those genes to produce transformed plants having enhance germination characteristics but without any diminishment in food quality or flavor. [0008]
  • U.S. Pat. No. 6,057,493 to Willmitzer et al. describes the use of anti-sense DNA sequences encoding ADP-glucose pyrophosphorylase from potato to produce transformed plants with a reduction in starch concentration and an increase in the concentration of at least sucrose. [0009]
  • The genes which code for several bacterial ADPGlc PPases have also been cloned and recombinant enzymes have been prepared. See for example, Preiss, J., and M. N. Sivak, M. N. (1998) [0010] Genet. Eng. (N.Y.) 20, 177-223; Preiss, J. (1996) In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (Neidhart, F. C., Ed.), 2nd ed., Vol 1 pgs. 10115-1024, ASM Press; Preiss, J., and Romeo,-T. (1989) Advances in Microbial Physiology 30, 183-238. DNA sequences have also been elucidated for certain bacterial ADP-glucose pyrophosphorylase enzymes. For example, U.S. Pat. No. 5,349,123 to Shewmaker et al. describes a nucleic acid construct which encodes an E. coli ADP-glucose pyrophosphorylase and the use of that construct to transform plant cells to modify biosynthesis of a glucan in the plant.
  • 2. Glycogen Synthase [0011]
  • The reaction scheme in bacteria catalyzed by glycogen synthase is shown below: [0012]
    Figure US20040242532A1-20041202-C00002
  • The “corresponding” reaction in mammals is similar except that the nucleoside diphosphate sugar substrate is UDP glucose, and the catalyzing enzyme is EC 2.4.1.11. [0013]
  • Genes encoding mammalian glycogen synthases have been cloned and sequenced (See Browner et al., Proc. Nat. Acad. Sci. (1989) 86:1443-1447; Bai et al., J. Biol. Chem. (1990) 265:7843-7848), and the genes encoding bacterial glycogen synthases have also been cloned and sequenced. (See for example U.S. Pat. No. 5,969,214.) [0014]
  • SUMMARY OF THE INVENTION
  • While previous research has focused on modifying the activity of glycogen synthase or ADP-glucose pyrophosphorylase to modify starch content in plants, the present inventor has determined that, since the catabolism and metabolism of energy storage pathways are critical to viability of bacteria, the inhibition of such pathways in bacteria provides a novel class of antibiotics for the treatment of bacterial infections. Further, numerous studies have indicated that glycogen plays an important role in the survival of the bacterial cell (Strange, R. E. (1968) [0015] Nature 220, 606; Strange et al (1961) J. Gen. Microbiol. 25, 61; Van Houte, J., and Jansen, H. M. (1970) J. of Bacteriol. 101, 1083). Whereas current antibiotics are characterized by inhibition of protein synthesis, DNA synthesis and cell wall synthesis, this novel class of antibiotics is characterized by inhibition of energy storage and utilization pathways.
  • The inventor has particularly noted that production of ADP-glucose is critical to viability of bacteria, and since ADP-glucose pyrophosphorylase is not present in mammals, the enzyme provides an excellent target for inhibition of bacterial growth, thereby providing a means for inhibiting the growth of microorganisms and treating bacterial infections. [0016]
  • Similarly, since the glycogen synthase in many pathogenic bacteria is different than the glycogen synthase in mammals, that enzyme also provides an excellent target for inhibition of bacterial growth and treating bacterial infections. [0017]
  • In one aspect, the present invention is directed to a method for treating a microorganism infection by administering an effective amount of a compound capable of inhibiting the production and/or utilization of ADP-glucose. [0018]
  • It is another aspect of the invention to provide a method for treating a microorganism infection by administering an effective amount of a compound capable of inhibiting the activity of ADP-glucose pyrophosphorylase and/or glycogen synthase. [0019]
  • It is a further aspect of the invention to provide a method of identifying a compound capable of inhibiting the growth of pathogenic microorganisms which comprises identifying a compound which inhibits an enzyme important in the catabolism and metabolism of energy storage pathways, particularly a compound that inhibits the activity of ADP-glucose pyrophosphorylase and/or glycogen synthase.[0020]
  • BRIEF DESCRIPTION OF THE FIGURES
  • FIGS. 1-5 are graphs plotting the results of experiments testing the inhibiting effect of ADP-glucose borano analogs on an ADP-glucose Ppase enzyme in in vitro enzyme tests.[0021]
  • DETAILED DESCRIPTION OF THE INVENTION
  • The present inventor has discovered that while certain important pathogenic microorganisms require the activity of ADP-glucose pyrophosphorylase (EC 2.7.7.27) to produce ADP-glucose, that enzyme is not present in mammals, particularly not in humans. In addition, the present inventor has recognized that the glycogen synthase (EC 2.4.1.21) in many pathogenic bacteria as the ADPGlucose dependent glucan chain lengthening enzyme is not present in mammals, particularly not in humans. As a result, the present inventor has first determined that inhibition of ADP-glucose pyrophosphorylase (EC 2.7.7.27) and/or glycogen synthase (EC 2.4.1.21) provide excellent targets for inhibiting the growth of pathogenic microorganisms, while not inhibiting any important biosynthetic pathway in humans. As noted above, in plants and most bacteria, ADP-glucose pyrophosphorylase catalyzes the reaction of α-glucose-1-phosphate with ATP to produce ADP-glucose. In mammals and in eukaryotic microorganisms, the “corresponding” reaction is catalyzed by UDP-glucose pyrophosphorylase by transferring a glucosyl residue from UDP-glucose, as shown below: [0022]
  • UTP+glucose-1-phosphate→UDP-glucose (UDPGlc)+PPi  (1)
  • UDPGlc+α-1,4-glucan→UDP+α-1,4-glucosyl-α-1,4-glucan.  (2)
  • Step (1) in this pathway in mammals, including humans, is catalyzed by UDP-glucose pyrophosphorylase. Recognizing the difference between the critical use of UDPGlc PPase in mammals as compared to the use of ADPGlc PPase in certain pathogenic bacteria, the present inventor first recognized that such bacteria could be selectively killed by inhibiting the activity of ADPGlc PPase, without adversely affecting any mammal so infected with the bacteria. This selectivity provides a basis and target for a novel and important new class of antibiotics, antibiotics which are inhibitors of energy storage and utilization enzymes, namely ADPGlc PPase inhibitors. [0023]
  • Step (2) in the above is catalyzed in humans by glycogen synthase (EC 2.4.1.11), whereas in many pathogenic bacteria the corresponding enzyme is EC 2.4.1.21 because the sugar substrate is ADP-based, not UDP-based. Again, this provides a target for a novel and important new class of antibiotics which inhibit glycogen synthase (EC 2.4.1.21). [0024]
  • As is well known, antibiotics are currently used to treat a wide range of bacterial infections, ranging from minor to life threatening infections. Broad spectrum antibiotics treat a variety of gram-positive and gram-negative organisms, while mild spectrum antibiotics only cover limited types of bacterial organisms and are useful for curing infections with known bacterial strains. [0025]
  • But it has recently been noted that pathogenic bacteria and fungi increasingly exhibit resistance to existing classes of antibiotics, such as penicillin, vancomycin and erythromycin. According to the Center for Disease Control, pathogenic resistance has significantly increased mortality rates, maling infectious disease the third largest cause of death in the United States. The rates of antibiotic resistant bacteria have particularly increased recently with respect to [0026] S. aureus, Enterococcus strains, S. pneumoniae and M. tuberculosis.
  • The mechanism of action for most antibiotics is the inhibition of bacterial cell wall completion, or DNA or protein synthesis. Sulfonamides and trimethoprin act by inhibiting an essential metabolic step, namely folate synthesis. But there is a great need for new antibiotics with different targets, especially in light of the ever increasing problem of resistant strains. [0027]
  • The present inventor has found that compounds which act as inhibitors of ADP-glucose pyrophosphorylase and/or glycogen synthase offer another class of antibiotics which inhibit an essential bacterial metabolic step, namely a pathway essential for bacterial energy storage and utilization. A first aspect of the invention relates to a method for identifying compounds capable of inhibiting the growth of pathogenic microorganisms which comprises: [0028]
  • a. identifying an enzyme that is important to energy storage or utilization in the pathogenic microorganism, which enzyme is not present in mammals, particularly not in humans; and [0029]
  • b. identifying a compound that inhibits that enzyme in the pathogenic microorganism. [0030]
  • According to the present invention, an enzyme in an energy storage or utilization pathway which is important to continued growth and viability of a pathogenic microorganism but which is absent in humans provides a unique, specific target for compounds which can inhibit infections of such pathogenic microorganisms without causing undesirable side effects or toxicity to a mammalian patient. Various biosynthetic pathways have been identified in the literature for various microorganisms and for mammals, and those pathways, which include an important enzyme present in pathogenic microorganisms but absent in mammals, provide a unique target for screening for compounds useful for inhibiting pathogenic microorganism infections. [0031]
  • According to the present invention, the present inventor has specifically identified ADP-glucose pyrophosphorylase (EC 2.7.7.27) and glycogen synthase (EC 2.4.1.21) as enzymes present in a biosynthetic pathway important for energy storage and utilization in pathogenic microorganisms, but absent in mammals, specifically absent in humans. Since the biosynthetic pathway is important for energy storage or utilization in pathogenic microorganisms, inhibition of this pathway significantly decreases the viability of pathogenic microorganisms, leading ultimately to death of the microorganism, either by action of the inhibitor alone, or in combination with the patient's own immunological systems for resisting infections, or in combination with other antibiotics. [0032]
  • In some instances, inhibition of the biosynthetic pathways and/or inhibition of ADP-glucose pyrophosphorylase (EC 2.7.7.27) or glycogen synthase (EC 2.4.1.21) may not per se kill the bacteria, but will render the microorganisms non-infective or non-pathogenic. It has been known for quite some time that complex carbohydrates can act as virulence factors in bacteria responsible for invasive infections (Glazer, A. N. and Nikaido, H. Microbial Biotechnology. Fundamentals of Applied Microbiology (1995) W. H. Freeman and Co., pgs. 266-272). Specific to glycogen biosynthesis, Uttaro, A. D., and Ugalde, R. A. Gene 150: 117-122 (1994) and J. E. Ugalde et al. J. Bact. 180: 6557-64 (1998) have shown that mutations that prevent gene expression of the gig operon render [0033] Agrobacterium tumefaciens non-infective. Illiffe-Lee and McClarty, Mol. Microbiology 38(1): 20-30 (2000) have shown that growth of Chlamydia trachomatis, under conditions that limit glycogen, severely reduces the number of infectious bodies.
  • Although not considered a limiting list, the present inventor has specifically identified a number of important pathogenic microorganisms which require ADP-glucose pyrophosphorylase (EC 2.7.7.27) and glycogen synthase (EC 2.4.1.21), including [0034] Chlamydia pneumoniae, Chlamydia trachomatis, Esherichia coli O157, Haemophilus influenzae, Mycobacterium leprae, Mycobacterium tuberculosis, Salmonella typhimurium and Vibrio cholerae, Streptococcus pneumoniae, Yersinia pestis, Bacillus subtilus and Bacillus anthracis.
  • The above listed bacteria comprise some of the most important pathogenic microorganisms which account for significant numbers of disease patients in the United States and around the world. The following table summarizes the prevalence and current treatments available for these pathogenic microorganisms. [0035]
    TABLE 1
    Prevalence and Current Treatments
    Incidence Prevalence
    (estimated (estimated number of
    number of new people currently
    Microorganism Disease(s) cases/yr) infected) Treatment
    Chlamydia Acute and chronic
    pneumoniae respiratory diseases
    including: pneumonia,
    pharyngitis, bronchitis,
    sinusitis, otitis media,
    COPD, asthma, Reiter
    syndrome, and
    sarciodosis.
    Chlamydia STD and blindness 3 million/yr 89 million/yr STD; 400 Doxycycline,
    trachomatis (trachoma). STD million partially blind, 6 tetracycline,
    million totally blind. chloramphenicol,
    refampicin,
    fluroquinones,
    erythromycin, and
    azithromycin
    Escherichia coli Abdominal cramps,
    O157 (food non-bloody diarrhea,
    poisoning) hemorrhagic colitis, and
    haemolytic-uraemic
    syndrome.
    Haemophilus Bacteremia, acute 3.5 million/yr Ampicillin,
    influenzae bacterial meningitis, cephalosporin,
    otitis media, sinusitis, chloramphenicol,
    and pneumonia. tetracycline, sulfa
    drugs, and amoxicillin
    Mycobacterium Leprosy (Hansen's 250 new 12 million world-wide. Dapsone, refampin,
    leprae disease) cases/yr in the It is a public health ethionamide
    U.S., 600,000 problem in 72 countries,
    new cases/yr 19 of which account for
    world-wide. 90% of all the cases in
    the world.
    Mycobacterium Tuberculosis >20,000 in the 16 million world-wide. Isoniazid, rifampin,
    tuberculosis U.S. According to the WHO, ethambutol, and
    tuberculosis is the pyrazinamide.
    number one killer among
    infectious diseases in the
    world. TB kills more
    people than AIDS,
    malaria, and tropical
    diseases combined.
    Salmonella Salmonellosis, >50,000/yr Ampicillin,
    typhimurium abdominal cramps, non- world-wide chloramphenicol,
    bloody diarrhea. streptomycin,
    sulphonamides, and
    tetracycline
    Vibrio cholerae Cholera >50,000/yr; Over 1,000,000 reported
    mostly in cases throughout the
    southeast asia. world. Usually epidemic
    or pandemic.
  • Description of Method for Inhibition Screening [0036]
  • As noted above, one aspect of the present invention is a method for the identification of a compound capable of inhibiting the growth of pathogenic microorganisms by interfering with the activity of glycogen synthase and/or ADP-glucose pyrophosphorylase. Compounds can be identified by growing bacteria on defined media in the presence or absence of a test compound, and assessing the effect on glycogen synthesis by iodine staining of colonies (Govons, S. et al (1969) [0037] J. Bacteriol. 97, 970-972). More quantitatively, the amount of glucan accumulated in the absence or presence of test compounds can be assessed by collection of the glycogen from the culture and quantitatively converting it to glucose with glucoamylase and α-amylase (Preiss, J. et al (1975) J. Biol. Chem. 250: 7631-7638) Compounds capable of inhibiting glycogen synthase and/or ADP-glucose pyrophosphorylase can also be identified by means of in vitro experiments by exposing a substrate comprising glycogen synthase and/or ADP-glucose pyrophosphorylase to a plurality of test compounds and identifying those compounds which inhibit the tested enzyme according to known catalytic measurement techniques.
  • One particular in vitro method for assessing the activity of an inhibitor to purified ADPGlc PPase is the following: [0038]
  • Assaying for ADPGlc synthesis activity involves measuring the amount of 14-C labeled Glc-1-P converted to ADPGlucose (Preiss, J., Shen, L., Greenberg, E., and Gentner, N. (1966) Biochemistry 5, 1833-1845). Briefly, unreacted Glc-1-P is separated from product by the following steps: 1) digestion with alkaline phosphatase (thus removing the negatively charged phosphate); 2) spotting an aliquot of the reaction mixture on to positively charged DE-81 filters (Whatman); and 3) washing the filters with water (thus removing the now neutral C-14 glucose). [0039]
  • To assess the effect of a putative inhibitor, enzyme assays are performed at a subsaturating concentration of substrate (depending on the enzyme, ATP=0.2-1 mM, Glc-1-P=0.5 mM) under standard conditions in the absence and presence of the major activator for each enzyme (1-5 mM depending on enzyme). In this way, the effect of the inhibitors can be evaluated under the range of the expected in vivo conditions. Initial screening of a putative inhibitor typically includes testing at two concentrations (˜25 μM and 1 mM)±major activator with appropriate controls and blanks for a total of 9 assays/inhibitor/enzyme (2 control assays—appropriate enzyme concentrations in the absence of inhibitor; 4 experimental assays; 1 blank in the absence of inhibitors; 2 blanks in the presence of inhibitor (no activator required). [0040]
  • A quantitative assay to measure the activity of an inhibitor to bacterial (ADPG dependent) Glycogen Synthase (EC 2.4.1.21) involves the measurement of incorporated 14-C-labeled ADPGlc into a glucan molecule (a specific glycogen, or amylopectin) that serves as a primer wherein the labeled glucan can easily be separated from unincorporated ADPGlc by a precipitation step (methanol insoluble polysaccaride). Useful specific procedures are as described in Furakawa, K., Tagaya, M., Inouye, M., Preiss, J., and Fukui, T. (1990) “Identification of Lys-15 at the Active Site in [0041] E. coli Glycogen Synthase,” J. Biol. Chem. 265, 2086-2090; and Thomas, J. A., Schlender, K. K., and Lamer, J. (1968) Anal. Biochem. 25, 486-499.
  • Useful inhibitors can also be identified, and potential inhibitors assessed, by in vitro treatment of bacteria in, for example, culture tubes or petri dish samples. Such assessments can be performed, for example, by spreading a measured aliquot of a diluted bacterial culture into nutrient agar plates, both treated and control, and counting the number of visible cells. Detailed procedures are well known to those in the art, as shown for example in Miller, [0042] Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, 1972.
  • Compounds which inhibit ADPGlc PPase or glycogen synthase can also be assessed in an in vivo animal model test. Such tests can be conducted in an animal which is susceptible to infection by the pathogenic microorganism of interest. For example, the effect of the test compound on the virulence of [0043] H. influenzae is assessed by comparing the survival rates of animals which have been administered the test compound with the survival rate of animals which have not been administered the test compound, wherein a higher survival rate of animals administered the test compound is an indication that the test compound has an effect on the virulence of H. influenzae.
  • To determine the effect of a test compound on colonization of the mucosal surface or on invasiveness and/or virulence of [0044] H. influenzae, the test compound is administered to the animals either prior to, at the time of, or after inoculation of the animals with H. influenzae. The test compound may be administered directly into the nasopharynx, or may be administered by any other route including any one of the traditional modes (e.g., orally, parenterally, transdermally or transmucosally), in a sustained release formulation using a biodegradable biopolymer, or by on-site delivery using micelles, gels and liposomes, or rectally (e.g., by suppository or enema). Precise formulations and dosages will depend on the nature of the test compound and may be determined using standard techniques, by a pharmacologist of ordinary skill in the art.
  • There are essentially two types of in vivo models in which a compound may be tested for antibiotic activity directed against [0045] S. pneumoniae. In the first model, colonization of the mucosal surface of the nasopharynx by S. pneumoniae in the presence or absence of the test compound is assessed. Measurement of colonization of the mucosal surface of the nasopharynx by S. pneumoniae may be conducted in an animal model essentially as described in Weiser et al. (1994, supra).
  • To assess colonization, briefly, an amount of [0046] S. pneumoniae, generally about 10 mu.l of phosphate buffered saline (PBS)-washed mid log phase organisms adjusted to the desired density, is inoculated into the left anterior naris of the animal. Colony counts are performed to ensure that the inocula are of the desired density and phenotype. The nasopharynx is cultured for the presence of viable S. pneumoniae by the slow instillation of 20 to 40 μl of sterile PBS into the left naris and withdrawal of the initial 10 μl from the right naris. This procedure ensures that the fluid has passed through the nasopharynx. The quantity of organisms recovered is then assessed in a well known culture assay.
  • The effect of the test compound on colonization of the nasopharynx by [0047] S. pneumoniae is evaluated by comparing the degree of colonization of the nasopharynx in animals which have been administered the test compound with the degree of colonization in animals which have not been administered the test compound, wherein a lower degree of colonization in animals administered the test compound is an indication that the test compound inhibits colonization of the nasopharynx by S. pneumoniae.
  • The invasive capability of [0048] S. pneumoniae may also be measured in the same animal model. Essentially, bacteria which have entered the blood stream following inoculation of the nasopharynx are detected by culturing the same in a sample of blood obtained from the animal. Again, the effect of the test compound on the invasive capacity of S. pneumoniae is assessed by comparing the number of organisms found in the blood stream in animals which have been administered the test compound with the number of organisms in the blood stream in animals which have not been administered the test compound, wherein a lower number of organisms in the blood stream of animals administered the test compound is an indication that the test compound has an effect on the invasive capacity of S. pneumoniae.
  • In a second in vivo model, the virulence of [0049] S. pneumoniae is assessed in animals as described in Berry et al. (1995, Infect. Immun. 63:1969-1974). Essentially, cultures of S. pneumoniae are diluted to a density of 2.times. 10.sup.6 colony forming units per ml, and volumes of 0.1 ml are injected intraperitoneally into groups of animals. The survival time of the animals is recorded and the differences in median survival time between groups may be analyzed by the Mann-Whitney U test (two-tailed). Differences in the overall survival rate between groups may be analyzed by the x.sup.2 test (two tailed).
  • The effect of the test compound on the virulence of [0050] S. pneumoniae is assessed by comparing the survival rates of animals which have been administered the test compound with the survival rate of animals which have not been administered the test compound, wherein a higher survival rate of animals administered the test compound is an indication that the test compound has an effect on the virulence of S. pneumoniae.
  • To determine the effect of a test compound on colonization of the mucosal surface or on invasiveness and/or virulence of [0051] S. pneumoniae, the test compound is administered to the animals either prior to, at the time of, or after inoculation of the animals with S. pneumoniae. The test compound may be administered directly into the nasopharynx, or may be administered by any other route including any one of the traditional modes (e.g., orally, parenterally, transdermally or transmucosally), in a sustained release formulation using a biodegradable biopolymer, or by on-site delivery using micelles, gels and liposomes, or rectally (e.g., by suppository or enema). Precise formulations and dosages will depend on the nature of the test compound and may be determined using standard techniques, by a pharmacologist of ordinary skill in the art.
  • The present invention further provides a method for treating pathogenic microorganism infections in a patient by administering to the patient an effective amount of an inhibitor against glycogen synthase and/or ADP-glucose pyrophosphorylase, wherein an effective amount of the inhibitor will inhibit the activity of the enzyme so as to decrease viability of and/or kill the microorganism. The inhibitor utilized in the treatment may be one identified by one of the methods described above, or inhibitors may be identified by any other method. One such inhibitor is the nucleoside α-P-boranodiphosphoglucose (particularly its triethylammonium salt), which is a borane analog of glucose-conjugated nucleoside diphosphate, described by Lin and Shaw, [0052] Tetrahedron Letters 41 (2000) 6701-6704. The particular compound, adenosine α-P-boranodiphosphoglucose, is a boran analog of ADP-glucose generating two sterioisomers and has the following structures:
    Figure US20040242532A1-20041202-C00003
  • wherein A is adenosine. [0053]
  • Adenosine boranophosphate exists as two stereoisomers, the preparation of which is described below: [0054]
    Figure US20040242532A1-20041202-C00004
  • As an analog of ADP-glucose, the compound of formula (I) is a useful inhibitor of ADPGlc PPase according to the present invention. [0055]
  • Other known regulators and inhibitors of ADPGlc PPase are the following: [0056]
    TABLE 2
    Regulatory properties of ADP-glucose pyrophosphorylase
    in different organisms
    ADP-glucose pyrophosphorylase
    Allosteric regulators
    Organism Activator Inhibitor
    Prokaryotes
    Enterobacteria
    Escherichia coli Fructose-1,6-bisP AMP
    Salmonella typhimurium
    Enterobacter aerogenes
    Aeromonas formicans Fructose-1,6-bisP AMP
    Micrococcus luteus Fructose-6-P ADP
    Mycobacterium smegmatis
    Serratia mercescens None AMP
    Enterobacter hafniae
    Clostridium pasteurianum
    Agrobacterium tumefaciens Pyruvate AMP
    Arthrobacter viscosus Fructose-6-P ADP
    Chromatium vinosum
    Rhodobacter capsulata
    Rhodomicrobium vannielii
    Rhodobacter gelatinosa Pyruvate AMP
    Rhodobacter globiformis Fructose-6-P Pi (inorganic
    phosphate)
    Rhodobacter sphaeroides Fructose-1,6-bisP
    Rhodospirillum rubrum Pyruvate
    Rhodospirillum tenue
    Rhodocyclus purpureus
    Cyanobacteria 3-P-glycerate Pi
    Synechococcus 6301
    Synechocysits 6803
    Anabaena 7120
    Eukaryotes 3-P-glycerate Pi
    Green algae
    Chlorella fusca
    Chlorella vulgaris
    Chlamydomonas reinhardtii
    Higher Plants
    Photosynthetic tissues 3-P-glycerate Pi
    (leaves of spinach, Arabidopsis,
    wheat, maize, rice)
    Non-photosynthetic tissues 3-P-glycerate Pi
    (Potato tubers, maize endosperm)
  • EXAMPLE 1 Effect of ADPG Borano Analogs on Rhodobacter Sphaeroides (RB.S.) ADPG Ppase
  • The APDGlc Ppase from [0057] Rhodobacter sphaeroides has been used as a model enzyme for initial testing of inhibitors as it has been cloned, sequenced, and expressed in the inventor's laboratory (Meyer et al (1999) Arch. Biochem. Biophys. 372, 179-188;
  • Igarashi, R. Y. and Meyer, C. R. (2000) [0058] Arch. Biochem. Biophys. 376, 47-58) and the purified recombinant enzyme is readily available. Further, this particular enzyme has the most complex regulation (having partial overlap with nearly every type of ADPG Ppase) and has sufficient homology (ranging from ˜40-70% similarity) to the enzyme primary sequence from the mentioned pathogenic bacteria.
  • 1. OVERVIEW: 2 analogs (ANALOG; stereoisomers I, II) of adenosine-α-P-boranodiphosphoglucose were tested for inhibiting activity against the ADPG Ppase enzyme from [0059] Rhodobacter sphaeroides (Rb.s.) ADPG Ppase.
  • 2. Materials [0060]
  • PREMIX: A: 100 mM HEPES (pH 7), 0.5 mg/mL BSA, 0.5 mM Glc-1-P, 0.25 mM ATP subsaturating (near S0.5 value), 5 mM MgC12, (0.2 U Pyrophosphatase)) [0061]
  • PREMIX B: A+1 mM F6P [0062]
  • (170 μL premix will allow 20 μL for analog vol., 10 μL of E) [0063]
  • II. ANALOG CONC. 5, 50, 500 μM (STOCK CONC.=5 mM) in 50 mM HEPES, pH 7, note that 20 μL of 5 mM in 200 μL assay will yield 500 μM final concentration to obtain 5 mM aliquots: ANALOG I: add 625 μL of HEPES buffer, mix thoroughly [0064]
  • ANALOG II: add 500 μL of HEPES buffer, mix thoroughly [0065]
  • 1:10 DILUTION ALIQUOTS OF 5 mM STOCKS are prepared in order to generate stock solutions that will give 50 and 5 μM with the addition of 20 μL. 100 μL aliquots are stored at. −20 C [0066]
  • RBS ADPG PPASE WT ENZYME CONC.=17 mg/mL (use 1:8000 dilution in Dilution Buffer) [0067]
  • The Following Assay Set of 11 are Utilized for each Analog [0068]
  • 1—BLANK (Premix A) [0069]
  • 2—BLANK+50 μM ANALOG (A) [0070]
  • 3—BLANK+500 μM ANALOG (A) [0071]
  • 4—E CON (A) [0072]
  • 5—E CON+F6P (B) [0073]
  • 6—E+5 μM (A) [0074]
  • 7—E+5 μM (B) [0075]
  • 8—E+50 μM (A) [0076]
  • 9—E+50 μM (B) [0077]
  • 10—E+500 μM(A) [0078]
  • 11—E+500 μM (B) [0079]
  • Purified [0080] Rb.s ADPG Ppase was assayed under standard conditions (0.25 mM ATP [˜S0.5 value], 0.5 mM Glc-1-P, 5 mM Mg, 100 mM HEPES, pH7) in the presence and absence of the activator F6P (1 mM) at 3 concentrations of the analogs: 5, 50, and 500 μM. Analog stock solutions were made in 50 mM HEPES, pH 7). These concentrations should be regarded as estimates as there could have been some breakdown of the compounds during 2 years of storage at −20° C. The analogs had no effect on the background cpm of the assays.
  • Results [0081]
  • The detailed experimental data results are set forth in Table 3, and those results are graphed in FIGS. 1-5. [0082]
  • Analog I successfully inhibited the [0083] Rb.s. ADPG Ppase. While the response may be biphasic, the results showed about 75% inhibition by 500 μM in the absence of the activator F6P. The inhibition was less in the presence of F6P (˜50% at 500 μM). Analog II also successfully inhibited the Rb.s. enzyme with about 70% inhibition at 500 μM in both the presence and absence of F6P.
    TABLE 3
    Inhibition Study with ADPGlucose Borano compounds Analog I and II
    Analog I Analog II
    Blank 60.33 56.33
    Blank + 50 Um 76.33 51.00
    Blank + 500 uM 53.67 53.33
    825 Cpm/nmol
    Volume dil. Corr
    Description Dil (uL) CPM cor. CPM nmol *2.2 nmol/min nmol/min/uL nmol/min/uL Units/mg
    E Con (A) 8000 10 492.00 431.67 0.52 1.151 0.115 0.0115 92.090 5.42
    E Con + F6P (B) 8000 10 8526.00 8465.67 10.26 22.575 2.258 0.2258 1806.010 106.24
    E + 5 uM (A) 8000 10 380.67 320.34 0.39 0.854 0.085 0.0085 68.339 4.02
    E + 5 uM (B) 8000 10 7371.33 7311.00 8.86 19.496 1.950 0.1950 1559.680 91.75
    E + 50 uM (A) 8000 10 359.33 283.00 0.34 0.755 0.075 0.0075 60.373 3.55
    E + 50 uM (B) 8000 10 7041.00 6964.67 8.44 18.572 1.857 0.1857 1485.796 87.40
    E + 500 uM (A) 8000 10 160.00 106.33 0.13 0.284 0.028 0.0028 22.684 1.33
    E + 500 uM (B) 8000 10 4306.67 4253.00 5.16 11.341 1.134 0.1134 907.307 53.37
    E Con (A) 8000 10 491.00 434.67 0.53 1.159 0.116 0.0116 92.730 5.45
    E Con + F6P (B) 8000 10 8629.00 8572.67 10.39 22.860 2.286 0.2286 1828.836 107.58
    E + 5 uM (A) 8000 10 477.00 420.67 0.51 1.122 0.112 0.0112 89.743 5.28
    E + uM (B) 8000 10 7536.00 7479.67 9.07 19.946 1.995 0.1995 1595.663 93.86
    E + 50 uM (A) 8000 10 399.67 348.67 0.42 0.930 0.093 0.0093 74.383 4.38
    E + 50 uM (B) 8000 10 6165.33 6114.33 7.41 16.305 1.630 0.1630 1304.390 76.73
    E + 500 uM (A) 8000 10 194.33 141.00 0.17 0.376 0.038 0.0038 30.080 1.77
    E + 500 uM (B) 8000 10 2724.00 2670.67 3.24 7.122 0.712 0.0712 569.743 33.51
  • Inhibitors useful for the treatment of pathogenic bacteria and microorganisms can be administered by a variety of means and dosage forms well known to those skilled in the art. When used as an antimicrobial agent in the treatment of microorganism infections, the present compounds are administered, for example, orally in the form of a tablet, capsule, powder, syrup, etc., or parenterally such as intravenous injection, intramuscular injection, or intrarectal administration. [0084]
  • The suitable administration forms as mentioned above may be prepared by mixing an active ingredient with a conventional pharmaceutically acceptable carrier, excipient, binder, stabilizer, etc. When administered in the form of an injection, a pharmaceutically acceptable buffering agent, solubilizer, isotonic agent, etc. may be added thereto. The active compound may be administered per se, or in the form of a pharmaceutically acceptable salt thereof, or in the form of a pro-drug, such as an ester. [0085]
  • The dosage of the compound varies according to the conditions, ages, weights of the patient, the administration form, the frequency of the administration, etc., but it is usually in the range of 100 to 3000 mg per day for an adult, which is administered once or divided into several dosage units. [0086]
  • All of the publications referred to herein, are hereby specifically incorporated by reference. [0087]
  • The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims. [0088]

Claims (21)

1-31. (canceled)
32. A method of identifying a compound that inhibits glycogen synthesis in pathogenic bacteria, comprising:
a. exposing a test compound to an enzyme that is part of a glycogen biosynthesis pathway in a pathogenic bacteria, wherein the enzyme exhibits a catalytic activity not found in mammals, and wherein the exposure occurs under conditions that, in the absence of an inhibitor, allow the enzyme to exhibit the catalytic activity; and
b. determining if the test compound inhibits the catalytic activity of the enzyme, and, if so, identifying the test compound as an inhibitor of glycogen synthesis in pathogenic bacteria.
33. A method according to claim 32 wherein the pathogenic bacteria is selected from the group consisting of Chlamydia pneumoniae, Chlamydia trachomatis, Esherichia coli O157, Haemophilus influenzae, Mycobacterium leprae, Mycobacterium tuberculosis, Salmonella typhimurium and Vibrio cholerae, Streptococcus pneumoniae, Yersinia pestis, Bacillus subtilus, and Bacillus anthracis.
34. A method according to claim 32 wherein the enzyme is selected from the group consisting of ADP-glucose pyrophosphorylase and glycogen synthase.
35. A method according to claim 32 wherein the enzyme is ADP-glucose pyrophosphorylase.
36. A method according to claim 35 wherein the catalytic activity is EC. 2.7.7.27.
37. A method according to claim 32 wherein the enzyme is glycogen synthase.
38. A method according to claim 37 wherein the catalytic activity is EC. 2.4.1.21.
39. A method according to claim 32 that is performed in vitro.
40. A method according to claim 32 that is performed while culturing the pathogenic bacteria under growth conditions.
41. A method according to claim 32 further comprising determining whether the inhibitor of glycogen synthesis can be used to treat an infection in a mammal caused by the pathogenic bacteria, whereby the inhibitor is administered to a non-human animal having an infection caused by the pathogenic bacteria and progress of the infection is monitored.
42. An inhibitor of glycogen synthesis in pathogenic bacteria, or a pharmaceutically acceptable salt thereof, wherein the inhibitor is identified by a method according to claim 1 and the inhibitor inhibits an enzyme having a catalytic activity not found in mammals.
43. An inhibitor according to claim 42, wherein the inhibitor inhibits the catalytic activity of an enzyme selected from the group consisting of ADP-glucose pyrophosphorylase and glycogen synthase.
44. An inhibitor according to claim 43 that is an ADP-glucose borano analog.
45. An inhibitor according to claim 44 wherein the ADP-glucose borano analog is adenosine α-P-boranodiphosphoglucose.
46. A method of inhibiting of inhibiting glycogen synthesis in pathogenic bacteria, comprising exposing a pathogenic bacteria to an inhibitor according to claim 42.
47. A method according to claim 46 wherein the inhibitor inhibits the catalytic activity of an enzyme selected from the group consisting of ADP-glucose pyrophosphorylase and glycogen synthase.
48. A method according to claim 47 wherein the inhibitor is an ADP-glucose borano analog or a pharmaceutically acceptable salt thereof.
49. A method according to claim 46 wherein the pathogenic bacteria is a source of an infection in a mammal.
50. A method according to claim 46 wherein inhibition of glycogen synthesis in the pathogenic bacteria effects treatment of the infection.
51. A method according to claim 50 wherein the mammal is a human.
US10/867,959 2002-01-22 2004-06-14 Method for the treatment of microorganism infections by inhibiting energy storage and utilization Abandoned US20040242532A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US10/867,959 US20040242532A1 (en) 2002-01-22 2004-06-14 Method for the treatment of microorganism infections by inhibiting energy storage and utilization

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US10/055,749 US6852485B1 (en) 2002-01-22 2002-01-22 Method for identifying a compound for the treatment of microorganism infections by inhibiting energy storage and utilization in pathogens
US10/867,959 US20040242532A1 (en) 2002-01-22 2004-06-14 Method for the treatment of microorganism infections by inhibiting energy storage and utilization

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US10/055,749 Continuation US6852485B1 (en) 2002-01-22 2002-01-22 Method for identifying a compound for the treatment of microorganism infections by inhibiting energy storage and utilization in pathogens

Publications (1)

Publication Number Publication Date
US20040242532A1 true US20040242532A1 (en) 2004-12-02

Family

ID=33449031

Family Applications (2)

Application Number Title Priority Date Filing Date
US10/055,749 Expired - Fee Related US6852485B1 (en) 2002-01-22 2002-01-22 Method for identifying a compound for the treatment of microorganism infections by inhibiting energy storage and utilization in pathogens
US10/867,959 Abandoned US20040242532A1 (en) 2002-01-22 2004-06-14 Method for the treatment of microorganism infections by inhibiting energy storage and utilization

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US10/055,749 Expired - Fee Related US6852485B1 (en) 2002-01-22 2002-01-22 Method for identifying a compound for the treatment of microorganism infections by inhibiting energy storage and utilization in pathogens

Country Status (1)

Country Link
US (2) US6852485B1 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030232021A1 (en) * 2002-04-12 2003-12-18 Pyro Pharmaceuticals, Inc. Method for the treatment of dental caries caused by streptococci mutans infection by inhibiting energy storage and utilization
WO2007093769A1 (en) * 2006-02-13 2007-08-23 Isis Innovation Limited Oligosaccharide biosynthesis inhibitors
US20080262415A1 (en) * 2005-07-18 2008-10-23 Peyman Gholam A Enhanced wound healing
FR2929958A1 (en) * 2008-04-10 2009-10-16 Centre Nat Rech Scient METHOD FOR SCREENING ANTI-TUBERCULAR COMPOUNDS
WO2011102808A1 (en) 2010-02-19 2011-08-25 Agency For Science, Technology And Research Integrated microfluidic and solid state pyrosequencing systems
WO2022076922A1 (en) * 2020-10-09 2022-04-14 Adarx Pharmaceuticals, Inc. N-ACETYLGALACTOSAMINE(GAlNAc)-DERIVED COMPOUNDS AND OLIGONUCLEOTIDES

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6268471B1 (en) * 1993-07-28 2001-07-31 University Of North Texas Health Science Center At Forth Worth Escherichia coli csrA gene, protein encoded thereby, and methods of use thereof

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4190495A (en) 1976-09-27 1980-02-26 Research Corporation Modified microorganisms and method of preparing and using same
DE4013144A1 (en) 1990-04-20 1991-10-24 Inst Genbiologische Forschung NEW PLASMIDES, CONTAINING DNA SEQUENCES, CHANGES IN CARBOHYDRATE AND PROTEIN CONCENTRATION AND CARBOHYDRATE AND PROTEIN COMPOSITION IN POTATO BULBS, AND CELLS IN A POTATO PLANT PLANT
US5349123A (en) 1990-12-21 1994-09-20 Calgene, Inc. Glycogen biosynthetic enzymes in plants
US5969214A (en) 1990-06-11 1999-10-19 Calgene, Inc. Glycogen biosynthetic enzymes in plants
US5461143A (en) 1991-03-18 1995-10-24 The Scripps Research Institute Oligosaccharide enzyme substrates and inhibitors: methods and compositions
WO1992016640A1 (en) 1991-03-18 1992-10-01 The Scripps Research Institute Oligosaccharide enzyme substrates and inhibitors: methods and compositions
US5824790A (en) 1994-06-21 1998-10-20 Zeneca Limited Modification of starch synthesis in plants
US5753483A (en) 1995-05-05 1998-05-19 University Of Arkansas Purified homogeneous UDP-GlcNAc (GalNAc) pyrophosphorylase
DE19608268A1 (en) 1996-03-05 1997-09-11 Hoechst Ag Enzymes catalysing nucleotide-sugar conjugate synthesis
US5928932A (en) 1996-04-03 1999-07-27 Midwest Research Institute Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica
WO1998012346A1 (en) 1996-09-23 1998-03-26 The Children's Hospital Of Philadelphia COMPOSITIONS AND METHODS FOR TREATMENT OF INFECTION CAUSED BY HAEMOPHILUS INFLUENZAE AND $i(STREPTOCOCCUS PNEUMONIAE)
US5770407A (en) 1996-12-10 1998-06-23 The Scripps Research Institute Process for preparing nucleotide inhibitors of glycosyltransferases

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6268471B1 (en) * 1993-07-28 2001-07-31 University Of North Texas Health Science Center At Forth Worth Escherichia coli csrA gene, protein encoded thereby, and methods of use thereof

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030232021A1 (en) * 2002-04-12 2003-12-18 Pyro Pharmaceuticals, Inc. Method for the treatment of dental caries caused by streptococci mutans infection by inhibiting energy storage and utilization
US20060177385A1 (en) * 2002-04-12 2006-08-10 Schechter Alan M Compositions for treating dental caries caused by streptococcus mutans infection
US20080262415A1 (en) * 2005-07-18 2008-10-23 Peyman Gholam A Enhanced wound healing
WO2007093769A1 (en) * 2006-02-13 2007-08-23 Isis Innovation Limited Oligosaccharide biosynthesis inhibitors
FR2929958A1 (en) * 2008-04-10 2009-10-16 Centre Nat Rech Scient METHOD FOR SCREENING ANTI-TUBERCULAR COMPOUNDS
WO2009136086A2 (en) * 2008-04-10 2009-11-12 Centre National De La Recherche Scientifique (C.N.R.S) Method of screening for antitubercular compounds
WO2009136086A3 (en) * 2008-04-10 2010-03-04 Centre National De La Recherche Scientifique (C.N.R.S) Method of screening for antitubercular compounds
WO2011102808A1 (en) 2010-02-19 2011-08-25 Agency For Science, Technology And Research Integrated microfluidic and solid state pyrosequencing systems
US8916347B2 (en) 2010-02-19 2014-12-23 Agency For Science, Technology And Research Integrated microfluidic and solid state pyrosequencing systems
US9790547B2 (en) 2010-02-19 2017-10-17 Agency For Science, Technology And Research Integrated microfluidic and solid state pyrosequencing systems
WO2022076922A1 (en) * 2020-10-09 2022-04-14 Adarx Pharmaceuticals, Inc. N-ACETYLGALACTOSAMINE(GAlNAc)-DERIVED COMPOUNDS AND OLIGONUCLEOTIDES

Also Published As

Publication number Publication date
US6852485B1 (en) 2005-02-08

Similar Documents

Publication Publication Date Title
Hogan et al. A Pseudomonas aeruginosa quorum‐sensing molecule influences Candida albicans morphology
Elwell et al. Antibacterial activity and mechanism of action of 3'-azido-3'-deoxythymidine (BW A509U)
Gangaiah et al. Polyphosphate kinase 2: a novel determinant of stress responses and pathogenesis in Campylobacter jejuni
Xu et al. Phosphorus limitation increases attachment in Agrobacterium tumefaciens and reveals a conditional functional redundancy in adhesin biosynthesis
US20060178320A1 (en) Methods for indentifying compounds that modulate an enzyme involved in thiamine metabolism in a pathogenic microorganism
Honma et al. Role of a Tannerella forsythia exopolysaccharide synthesis operon in biofilm development
Muramatsu et al. Studies on novel bacterial translocase I inhibitors, A-500359s II. Biological activities of A-500359 A, C, D and G
Woodruff et al. Fosfomycin: laboratory studies
CN101636157A (en) Adenylyl cyclases as novel targets for antibacterial interventions
US6852485B1 (en) Method for identifying a compound for the treatment of microorganism infections by inhibiting energy storage and utilization in pathogens
US20150315627A1 (en) Methods and compositions for the detection of functional clostridium difficile toxins
Yuan et al. A novel signaling pathway connects thiamine biosynthesis, bacterial respiration, and production of the exopolysaccharide amylovoran in Erwinia amylovora
Mock et al. Induction of a viable but not culturable (VBNC) state in some Pseudomonas syringae pathovars upon exposure to oxidation of an apoplastic phenolic, acetosyringone
US20060177385A1 (en) Compositions for treating dental caries caused by streptococcus mutans infection
US6955890B2 (en) Method for the identification and treatment of pathogenic microorganisms infections by inhibiting one or more enzymes in an essential metabolic pathway
Rana et al. Single deletion of Escherichia coli K30 group I capsule biosynthesis system component, wzb, is not sufficient to confer capsule-independent resistance to erythromycin
Ojha et al. Ascorbic acid modulates pathogenecity markers of Candida albicans
WO2024096120A1 (en) Microorganism having improved colonization properties in host and method for producing same
Kim et al. ThiL is a valid antibacterial target that is essential for both thiamine biosynthesis and salvage pathway in Pseudomonas aeruginosa
Bischer et al. The Streptococcus mutans Rhamnose-glucose Polysaccharide Plays an Important Role in Oxidative Stress Resistance and Iron Homeostasis
US20060252112A1 (en) Methods for indentifying compounds that modulate an enzyme involved in biotin metabolism in a pathogenic microorganism
Normington The role of biofilms in recurrent Clostridioides difficile infection and the interaction of C. difficile in multispecies biofilms
US20060063224A1 (en) Method for the identification and treatment of pathogenic microorganism infections by inhibiting one or more enzymes in an essential metabolic pathway
Lin et al. Inhibition of Streptococcus mutans growth and biofilm formation through protein acetylation
KANAI et al. TETRAZOLIUM REDUCTION TEST AS A MEASURE TO EXAMINE THE TOTAL VIABILITY OF THE SUSPENSIONS OF TUBERCLE BACILLI II. COMPARISON OF THIS BIOCHEMICAL TEST WITH CULTURE METHOD

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION