WO2000078342A1 - Compositions and methods for the prevention, treatment and detection of tuberculosis and other diseases - Google Patents
Compositions and methods for the prevention, treatment and detection of tuberculosis and other diseases Download PDFInfo
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- WO2000078342A1 WO2000078342A1 PCT/US2000/016679 US0016679W WO0078342A1 WO 2000078342 A1 WO2000078342 A1 WO 2000078342A1 US 0016679 W US0016679 W US 0016679W WO 0078342 A1 WO0078342 A1 WO 0078342A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0001—Archaeal antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1217—Phosphotransferases with a carboxyl group as acceptor (2.7.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1223—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
- C12Y102/99—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with other acceptors (1.2.99)
- C12Y102/99002—Carbon-monoxide dehydrogenase (acceptor) (1.2.99.2)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
- C12Y203/01008—Phosphate acetyltransferase (2.3.1.8)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
- C12Y207/02001—Acetate kinase (2.7.2.1)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
Definitions
- This invention generally relates to compositions and methods for detecting, preventing and treating infectious diseases such as M. tuberculosis (“M. TB”), M. pneumonia (“M. TP”), and to new classes of antibiotics effective against anaerobic and facultative anaerobic microorganisms.
- M. tuberculosis M. TB
- M. pneumonia M. pneumonia
- new classes of antibiotics effective against anaerobic and facultative anaerobic microorganisms.
- One embodiment of the invention is a method for the prevention of an infectious disease in a human subject, comprising the steps of providing a pharmaceutical composition comprising at least one substance selected from the group consisting of CODH antigen, acetate kinase antigen, phosphotransacetylase antigen, CODH antisense nucleic acid, acetate kinase antisense nucleic acid and phosphotransacetylase antisense nucleic acid; and administering the composition to the patient in an form that allows uptake by cells of the subject.
- Another embodiment of the invention is a method for the treatment of an infectious disease in a human subject, comprising the steps of providing a pharmaceutical composition comprising at least one substance selected from the group consisting of CODH antigen, acetate kinase antigen, phosphotransacetylase antigen, CODH antisense nucleic acid, acetate kinase antisense nucleic acid and phosphotransacetylase antisense nucleic acid; and administering the composition to the patient in an form that allows uptake by cells of the subject.
- a pharmaceutical composition comprising at least one substance selected from the group consisting of CODH antigen, acetate kinase antigen, phosphotransacetylase antigen, CODH antisense nucleic acid, acetate kinase antisense nucleic acid and phosphotransacetylase antisense nucleic acid
- the invention is a pharmaceutical composition for the prevention of an infectious disease in a human subject, comprising at least one substance selected from the group consisting of CODH antigen, acetate kinase antigen, phosphotransacetylase antigen, CODH antisense nucleic acid, acetate kinase antisense nucleic acid and phosphotransacetylase antisense nucleic acid.
- CODH antigen acetate kinase antigen
- phosphotransacetylase antigen CODH antisense nucleic acid
- acetate kinase antisense nucleic acid phosphotransacetylase antisense nucleic acid
- M. tuberculosis might contain an enzyme - carbon monoxide dehydrogenase -which, under various circumstances, could affect health by producing carbon monoxide, nitric oxide, carbon dioxide, and/or nitrous oxide.
- the inventors determined that the effects of these toxic compounds account for many of the heretofore unexplained symptoms of tuberculosis.
- the inventors conducted Southern blots in 1997, but obtained inconclusive data.
- the genome of M. tuberculosis was published, it was found that the genome, indeed, contained carbon monoxide dehydrogenase.
- inhibiting this enzyme by, for example exposure to chemical inhibitor(s) or antibodies, disables the mycobacterium and alleviates problems of this scourge. Furthermore, the inventors discovered that this enzyme is involved in producing symptoms of Lyme disease and that blood serum from the western fence lizard has one or more binding factors that, in an analogous way, inhibit lyme disease in that animal.
- M. tuberculosis contains all three enzymes, whereas the published genome of T. pallidum indicates that this other organism contains phosphotransacetylase and acetate kinase, but not CODH.
- acetate kinase and phosphotransacetylase are critical to the metabolism of the microorganisms in which they are found, the inventors further realized that inhibiting these two other enzymes or disabling CODH, in the presence of CODH or independent of it, presents a novel way of disabling CODH or, beyond that, the entire microorganism, thereby treating the related disease.
- preferred embodiments of the invention utilize knowledge of the structure(s) of one or more of the enzymes, inhibitors of the enzymes, and/or methods for inhibition of the enzymes to prevent and/or treat disease.
- knowledge of the structure of each enzyme permits the development of new and more efficacious medical compositions and methods for inhibiting the enzyme, as well as methods for creating antibodies directed against the enzymes ⁇ thereby preventing the growth and proliferation of TB and other microorganisms.
- the strategy of determining the structure of such enzyme and then deriving or finding inhibitors of the enzyme represents a unique departure from the concept of traditional antibiotic treatment.
- compositions and methods are outlined and described in further detail in section B: “Implementation of the Discovery” below.
- the inventors have prepared monoclonal antibodies directed against all subunits of CODH, which are useful for the detection, treatment and prevention of M. tuberculosis and other diseases related to anaerobic microorganisms in which CODH might figure. These new medical agents inhibit their target microorganisms and represent a new class of medically active substances hereinafter termed "Microbe Inhibiting Agent. " Although their initial discovery was pertained to CODH, the invention also features agents that interfere with other enzymes needed for an aerobic and cumulative anaerobic organism such as M. tuberculosis
- Figure 1 shows the Primary Structure (amino acid sequence) of acetate Kinase.
- Figure 2 shows the Primary Structure (amino acid sequence) of Phosphotransacetylase.
- Figure 3 presents 3-dimensional structure data coordinates for Acetate Kinase from
- Methanosarcina thermophila Enzyme according to an embodiment of the invention.
- Figure 4 shows Sequences of the five subunits comprising the Co dehydrogenase/acetyl-
- Figure 5 presents representative biochemical information pertaining to carbon monoxide dehydrogenase.
- compositions and methods for detection, prophylaxis, and treatment of TB and other disease states that arise from activity of CODH include, for example, symptoms of AIDS.
- screen tests for TB often are not reliable in immune-suppressed patients (AIDS patients and others) and some symptoms attributed to "AIDS" arise from undetected TB, whose symptoms mimic those of AIDS.
- one embodiment of the invention is an AIDS therapy.
- a TB CODH enzyme is targetted by a "Microbe Inhibiting Agent. "
- an enzyme of the organism which is not found naturally in the human is crystallized and a 3 dimensional struture is obtained.
- the obtained structure is used to design a "Microbe Inhibiting Agent" that is active against the organism, but substantially not active against human cells. That is, in preferred embodiments Microbe Inhibiting Agents are prepared and used that act by interfering with a target Enzyme of a microorganism.
- the microorganism may be anaerobic or facultative anaerobic, and preferably is the M.TB organism.
- the Microbe Inhibiting Agent is a molecule that is obtained from or modelled after a lyme disease inhibiting substance, and preferably an antibody, from the western fence lizard.
- compositions and methods that employ an "Enzyme” according to the present invention.
- Enzyme refers in its broadest sense to any enzyme made by TB or a related organism that is distinguished from a corresponding enzyme in humans. In most embodiments, “Enzyme” refers to acetate kinase, phosphotransacetylase, and carbon monoxide dehydrogenase of the TB organism. In the preferred embodiment where antibodies are made against the enzyme, immunologically cross-reactive peptides and proteins are included within this meaning. Most preferably the enzyme is carbon monoxide dehydrogenase.
- Enzyme homolog refers to a homolog, particularly of CODH enzyme but optionally one of the other enzymes, as exemplified by the sequence shown in Figure 3, naturally occurring enzyme, or active fragments thereof, which are encoded by mRNAs transcribed from cDNA coding for the enzyme.
- Enzyme refers to those forms of Enzyme which retain biologic and/or immunologic activities of any naturally occurring Enzyme.
- “Naturally occurring Enzyme” refers to Enzyme produced by human cells that have not been genetically engineered and specifically contemplates various Enzymes arising from post-translational modifications of the polypeptide including but not limited to acetylation, carboxy lation, glycosylation, phosphorylation, lipidation and acylation.
- Derivative refers to polypeptides derived from naturally occurring Enzyme by chemical modifications such as ubiquitination, labeling (e.g., with radionuclides, various enzymes, etc.), pegy lation (derivatization with polyethylene glycol), or by insertion (or substitution by chemical synthesis) of amino acids (aa) such as ornithine, which do not normally occur in human proteins.
- chemical modifications such as ubiquitination, labeling (e.g., with radionuclides, various enzymes, etc.), pegy lation (derivatization with polyethylene glycol), or by insertion (or substitution by chemical synthesis) of amino acids (aa) such as ornithine, which do not normally occur in human proteins.
- Merobe Inhibiting Agent refers to an active compound or other molecule that destroys or inhibits a bacterium by interfering with the activity of one or more Enzymes of that bacterium.
- the preferred Enzymes in this context are carbon monoxide dehydrogenase
- CODH acetate kinase and phosphoacetyltransferase. CODH is most preferred.
- the bacterium preferably is an anaerobic or facultative anaerobic bacterium and most preferably M. tuberculosis
- Recombinant variant refers to any polypeptide differing from naturally occurring Enzyme by aa insertions, deletions, and substitutions, created using recombinant DNA techniques.
- Guidance in determining which aa residues may be replaced, added or deleted without abolishing activities of interest, such as cell adhesion and chemotaxis, may be found by comparing the sequence of the particular Enzyme with that of homologous molecules and minimizing the number of aa sequence changes made in regions of high homology.
- aa “substitutions” are the result of replacing one aa with another aa having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine, i.e., conservative aa replacements.
- "Insertions” or “deletions” are typically in the range of about 1 to 5 aa. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of aa in an Enzyme molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.
- a polypeptide "fragment,” “portion, “ or “segment” is a stretch of aa residues of at least about 5 aa, often at least about 7 aa, typically at least about 9 to 13 aa, and, in various embodiments, at least about 17 or more aa.
- any Enzyme polypeptide must have sufficient length to display biologic and/or immunologic activity on their own or when conjugated to a carrier protein such as keyhole limpet hemocyanin.
- oligonucleotide or polynucleotide “fragment”, “portion,” or “segment” is a stretch of nucleotide residues which is long enough to use in polymerase chain reaction (PCR) or various hybridization procedures to amplify or simply reveal related parts of mRNA or DNA molecules.
- PCR polymerase chain reaction
- One or both oligonucleotide probes will comprise sequence that is identical or complementary to a portion of Enzyme where there is little or no identity or complementarity with any known or prior art molecule.
- the oligonucleotide probes will generally comprise between about 10 nucleotides and 50 nucleotides, and preferably between about 15 nucleotides and about 30 nucleotides.
- Activated monocytes refers to the activated, mature monocytes or macrophages found in immunologically active tissues.
- Animal as used herein may be defined to include human, domestic or agricultural (cats, dogs, cows, sheep, etc) or test species (mouse, rat, rabbit, etc).
- Recombinant may also refer to a polynucleotide which encodes Enzyme and is prepared using recombinant DNA techniques.
- the DNAs which encode Enzyme may also include allelic or recombinant variants and mutants thereof.
- Nucleic acid probes are prepared based on the cDNA sequences which encode Enzyme provided by the present invention. Nucleic acid probes comprise portions of the sequence having fewer nucleotides than about 6 kb, usually fewer than about 1 kb. After appropriate testing to eliminate false positives, these probes may be used to determine whether mRNAs encoding Enzyme are present in a cell or tissue and to isolate similar nucleic acid sequences from chromosomal DNA extracted from such cells or tissues as described by Walsh PS et al (1992, PCR Methods Appl 1.-241-250).
- Probes may be derived from naturally occurring or recombinant single- or double- stranded nucleic acids or be chemically synthesized. They may be labeled by nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. Probes of the present invention, their preparation and/or labeling are elaborated in Sambrook J et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY; or Ausubel FM et al (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York City, both incorporated herein by reference.
- recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the "redundancy" in the genetic code.
- Various codon substitutions such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system.
- Mutations may also be introduced to modify the properties of the polypeptide, including but not limited to ligand-binding affinities, interchain affinities, polypeptide degradation and turnover rate.
- ligand-binding affinities include interchain affinities, polypeptide degradation and turnover rate.
- One example involves inserting a stop codon into the nucleotide sequence to limit the size of Enzyme so as to provide a binding, non-activating ligand of smaller molecular mass which would serve to block the activity of the natural Enzyme.
- Active substance homologs with modified specificity can be synthesized readily by substituting the non-cysteine residues of the conserved pentapeptide. Such recombinant mutants are well known in the art; substitution of the four aa with other natural and synthetic aa is readily performed. Activity is tested by the methods disclosed in the cited references.
- Embodiments of the present invention utilize purified Enzyme polypeptides from natural or recombinant sources, or cells transformed with recombinant nucleic acid molecules encoding Enzyme.
- Narious methods for the isolation of the Enzyme polypeptides may be accomplished by procedures well known in the art.
- such polypeptides may be purified by immunoaffmity chromatography by employing the antibodies provided by the present invention.
- Narious other methods of protein purification well known in the art include those described in Deutscher M (1990) Methods in Enzymology, Nol 182, Academic Press, San Diego Calif ; and Scopes R (1982) Protein Purification: Principles and Practice. Springer-Nerlag, New York City, both incorporated herein by reference.
- nucleotide sequences encoding Enzyme have numerous applications in techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use in the construction of oligomers for PCR, use for chromosome and gene mapping, use in the recombinant production of Enzyme, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like. Uses of nucleotides encoding Enzyme disclosed herein are exemplary of known techniques and are not intended to limit their use in any technique known to a person of ordinary skill in the art.
- nucleotide sequences disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, e.g. , the triplet genetic code, specific base pair interactions, etc.
- nucleotide sequences which encode Enzyme and/or its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring Enzyme under stringent conditions, it may be advantageous to produce nucleotide sequences encoding Enzyme or its derivatives possessing a substantially different codon usage. Codons can be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accordance with the frequency with which particular codons are utilized by the host.
- RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
- Nucleotide sequences encoding Enzyme may be joined to a variety of other nucleotide sequences by means of well established recombinant DNA, techniques (cf
- vectors of interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and the like.
- vectors of interest may contain an origin of replication functional in at least one organism, convenient restriction endonuclease sensitive sites, and selectable markers for the host cell.
- Another aspect of the subject invention is to provide for Enzyme-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences encoding Enzyme. Such probes may also be used for the detection of similar Enzyme encoding sequences and should preferably contain at least 50% of the nucleotides from the conserved region or active site.
- the hybridization probes of the subject invention may be derived from the nucleotide sequences known for the Enzymes or from genomic sequences including promoters, enhancer elements and/or possible introns of the respective naturally occurring Enzymes.
- Hybridization probes may be labeled by a variety of reporter groups, including radionuclides such as .sup.32 P or .sup.35 S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
- reporter groups including radionuclides such as .sup.32 P or .sup.35 S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
- PCR as described U.S. Pat. Nos. 4,683,195; 4,800,195; and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequence which encodes Enzyme.
- probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both and comprise a discrete nucleotide sequence for diagnostic use or a degenerate pool of possible sequences for identification of closely related genomic sequences.
- RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides.
- the nucleotide sequence can be used in an assay to detect inflammation or disease associated with abnormal levels of expression of Enzyme.
- the nucleotide sequence can be labeled by methods known in the art and added to a fluid or tissue sample from a patient under hybridizing conditions. After an incubation period, the sample is washed with a compatible fluid which optionally contains a dye (or other label requiring a developer) if the nucleotide has been labeled with an enzyme. After the compatible fluid is rinsed off, the dye is quantified and compared with a standard. If the amount of dye is significantly elevated, the nucleotide sequence has hybridized with the sample, and the assay indicates the presence of inflammation and/or disease.
- the nucleotide sequence for Enzyme can be used to construct hybridization probes for mapping that gene.
- the nucleotide sequence provided herein may be mapped to a particular chromosome or to specific regions of that chromosome using well known genetic and/or chromosomal mapping techniques. These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries, flow-sorted chromosomal preparations, or artificial chromosome constructions YAC, PI or BAC constructions.
- the technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York City.
- Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265: 198 If). Correlation between the location of Enzyme on a physical chromosomal map and a specific disease (or predisposition to a specific disease) can help delimit the region of DNA associated with that genetic disease.
- the nucleotide sequence of the subject invention may be used to detect differences in gene sequence between normal and carrier or affected individuals.
- Nucleotide sequences encoding Enzyme may be used to produce purified Enzyme using well known methods of recombinant DNA technology. Among the many publications that teach methods for the expression of genes after they have been isolated is Goeddel (1990) Gene Expression Technology, Methods and Enzymology, Vol 185, Academic Press, San Diego Calif. Enzyme may be expressed in a variety of host cells, either prokaryotic or eukaryotic. Host cells may be from the same species in which Enzyme nucleotide sequences are endogenous or from a different species. Advantages of producing Enzyme by recombinant DNA technology include obtaining adequate amounts of the protein for purification and the availability of simplified purification procedures.
- Enzyme produced by a recombinant cell may be secreted or may be contained intracellular ly, depending on the Enzyme sequence and the genetic construction used. In general, it is more convenient to prepare recombinant proteins in secreted form. Purification steps vary with the production process and the particular protein produced.
- fragments of Enzyme may be produced by direct peptide synthesis using solid-phase techniques (cf Stewart et al (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco Calif ; Merrifield J (1963) J Am Chem Soc 85:2149-2154.
- In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Foster City, California Calif.) in accordance with the instructions provided by the manufacturer.
- Various fragments of Enzyme may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
- Enzyme for antibody induction does not require biological activity; however, the protein must be immunogenic.
- Peptides used to induce specific antibodies may have an aa sequence consisting of at least five aa, preferably at least 10 aa. They should mimic a portion of the aa sequence of the protein and may contain the entire aa sequence of a small naturally occurring molecule such as Enzyme. Short stretches of Enzyme aa may be fused with those of another protein such as keyhole limpet hemocyanin and the chimeric molecule used for antibody production.
- Antibodies specific for Enzyme may be produced by inoculation of an appropriate animal with the polypeptide or an antigenic fragment.
- An antibody is specific for Enzyme if it is produced against an epitope of the polypeptide and binds to at least part of the natural or recombinant protein.
- Antibody production includes not only the stimulation of an immune response by injection into animals, but also analogous steps in the production of synthetic antibodies or other specific-binding molecules such as the screening of recombinant immunoglobulin libraries (cf Orlandi R et al (1989) PNAS 86:3833-3837; Huse WD et al (1989) Science 256: 1275-1281) or the in vitro stimulation of lymphocyte populations.
- Current technology (Winter G and Milstein C (1991) Nature 349:293-299) provides for a number of highly specific binding reagents based on the principles of antibody formation. These techniques may be adapted to produce molecules specifically binding Enzymes.
- An additional embodiment of the subject invention is the use of Enzyme specific antibodies, inhibitors, receptors or their analogs as bioactive agents to treat activated monocyte disorders, such as inflammatory bowel disease, insulin-dependent diabetes mellitus, rheumatoid arthritis, septic shock and similar pathologic problems.
- Bioactive compositions comprising agonists, antagonists, receptors or inhibitors of Enzyme may be administered in a suitable therapeutic dose determined by any of several methodologies including clinical studies on mammalian species to determine maximal tolerable dose and on normal human subjects to determine safe dose. Additionally, the bioactive agent may be complexed with a variety of well established compounds or compositions which enhance stability or pharmacological properties such as half-life. It is contemplated that the therapeutic, bioactive composition may be delivered by intravenous infusion into the bloodstream or any other effective means which could be used for treating problems involving Enzyme production and function. An antisense strand coding for an Enzyme can be used either in vitro or in vivo to inhibit expression of the protein.
- probes can be designed at various locations along the nucleotide sequence.
- the gene of interest By treatment of cells or whole test animals with such antisense sequences, the gene of interest can effectively be turned off. Frequently, the function of the gene can be ascertained by observing behavior at the cellular, tissue or organismal level (e.g. lethality, loss of differentiated function, changes in morphology, etc.).
- modifications of gene expression can be obtained by designing antisense sequences to intron regions, promoter/enhancer elements, or even to trans-acting regulatory genes.
- inhibition can be achieved using Hogeboom base-pairing methodology, also known as "triple helix" base pairing.
- Enzyme may be accomplished by subcloning the cDNAs into appropriate expression vectors and transfecting the vectors into appropriate expression hosts.
- the cloning vector previously used for the generation of the tissue library also provide for direct expression of the included Enzyme sequence in E. coli. Upstream of the cloning site, this vector contains a promoter for .beta.-galactosidase, followed by sequence containing the amino terminal Met and the subsequent 7 residues of .beta.-galactosidase. Immediately following these eight residues is an engineered bacteriophage promoter useful for artificial priming and transcription and a number of unique restriction sites, including Eco RI, for cloning.
- the Enzyme cDNA can be shuttled into other vectors known to be useful for expression of protein in specific hosts. Oligonucleotide amplimers containing cloning sites as well as a segment of DNA sufficient to hybridize to stretches at both ends of the target cDNA (25 bases) can be synthesized chemically by standard methods. These primers can then used to amplify the desired gene segments by PCR. The resulting new gene segments can be digested with appropriate restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternately, similar gene segments can be produced by digestion of the cDNA with appropriate restriction enzymes and filling in the missing gene segments with chemically synthesized oligonucleotides. Segments of the coding sequence from more than one gene can be ligated together and cloned in appropriate vectors to optimize expression of recombinant sequence.
- Suitable expression hosts for such chimeric molecules include but are not limited to mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells, insect cells such as Sf9 cells, yeast cells such as Saccharomyces cerevisiae, and bacteria such as E. coli.
- a useful expression vector may also include an origin of replication to allow propagation in bacteria and a selectable marker such as the .beta.- lactamase antibiotic resistance gene to allow selection in bacteria.
- the vectors may include a second selectable marker such as the neomycin phosphotransferase gene to allow selection in transfected eukaryotic host cells.
- Vectors for use in eukaryotic expression hosts may require RNA processing elements such as 3' polyadenylation sequences if such are not part of the cDNA of interest.
- the vector may contain promoters or enhancers which increase gene expression.
- promoters are host specific and include MMTV, SV40, or metallothionine promoters for CHO cells; trp, lac, tac or T7 promoters for bacterial hosts; or alpha factor, alcohol oxidase or PGH promoters for yeast.
- Transcription enhancers such as the rous sarcoma virus (RSV) enhancer, may be used in mammalian host cells.
- RSV rous sarcoma virus
- the Enzyme protein may be lethal to certain cell types, care should be given to the selection of a suitable host species.
- the protein can be expressed in the inactive form, such as in inclusion bodies.
- the inclusion bodies can be separated from the cells, the protein solubilized and refolded into active form.
- Enzyme may be expressed as a chimeric protein with one or more additional polypeptide domains added to facilitate protein purification.
- purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine- tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle Wash.).
- the inclusion of a cleavable linker sequence such as Factor XA or enterokinase (Invitrogen, San Diego Calif.) between the purification domain and the Enzyme sequence may be useful to facilitate expression of Enzyme.
- denatured protein from the reverse phase HPLC separation is obtained in quantities up to 75 mg.
- This denatured protein can be used to immunize mice or rabbits using standard protocols; about 100 micrograms are adequate for immunization of a mouse, while up to 1 mg might be used to immunize a rabbit.
- the denatured protein can be radioiodinated and used to screen potential murine B-cell hybridomas for those which produce antibody. This procedure requires only small quantities of protein, such that 20 mg would be sufficient for labeling and screening of several thousand clones.
- the amino acid sequence of Enzyme is analyzed to determine regions of high immunogenicity. Oligopeptides comprising appropriate hydrophilic regions, as shown in FIG. 3, are synthesized and used in suitable immunization protocols to raise antibodies. Analysis to select appropriate epitopes is described by Ausubel FM et al (supra).
- the optimal amino acid sequences for immunization are usually at the C-terminus, the N-terminus and those intervening, hydrophilic regions of the polypeptide which are likely to be exposed to the external environment when the protein is in its natural conformation.
- selected peptides typically, about 15 residues in length, are synthesized using an Applied Biosystems Peptide Synthesizer Model 431 A using fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH, Sigma) by reaction with M-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS; cf. Ausubel FM et al, supra). If necessary, a cysteine may be introduced at the N-terminus of the peptide to permit coupling to KLH. Rabbits are immunized with the peptide-KLH complex in complete Freund's adjuvant.
- the resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1 % BSA, reacting with antisera, washing and reacting with, labeled (radioactive or fluorescent), affinity purified, specific goat anti-rabbit IgG.
- Hybridomas may also be prepared and screened using standard techniques. Hybridomas of interest are detected by screening with labeled Enzyme to identify those fusions producing the monoclonal antibody with the desired specificity.
- wells of plates FAST; Becton-Dickinson, Palo Alto, Calif.
- affinity purified, specific rabbit-anti-mouse antibodies or suitable anti-species Ig
- the coated wells are blocked with 1 % BSA, washed and exposed to supernatants from hybridomas. After incubation the wells are exposed to labeled Enzyme, 1 mg/ml.
- Clones producing antibodies will bind a quantity of labeled Enzyme which is detectable above background. Such clones are expanded and subjected to 2 cycles of cloning at limiting dilution (1 cell/3 wells). Cloned hybridomas are injected into pristine mice to produce ascites, and monoclonal antibody is purified from mouse ascitic fluid by affinity chromatography on Protein A. Monoclonal antibodies with affinities of at least 10e8 Me-1, preferably 10e9 to lOelO or stronger, will typically be made by standard procedures as described in Harlow and Lane (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor NY; and in Goding (1986) Monoclonal Antibodies: Principles and Practice, Academic Press, New York City, both incorporated herein by reference.
- Enzyme antibodies are useful for the diagnosis of prepathologic conditions, and chronic or acute diseases which are characterized by differences in the amount or distribution of Enzyme. To date, Enzyme has been found only in the activated
- THP-1 library is thus associated with abnormalities or pathologies which activate monocytes.
- Diagnostic tests for Enzyme include methods utilizing the antibody and a label to detect Enzyme in human body fluids, tissues or extracts of such tissues.
- the polypeptides and antibodies of the present invention may be used with or without modification. Frequently, the polypeptides and antibodies will be labeled by joining them, either covalently or noncovalently, with a substance which provides for a detectable signal.
- labels and conjugation techniques are known and have been reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. Patents teaching the use of such labels include U.S. Pat. Nos.
- recombinant immunoglobulins may be produced as shown in U.S. Pat. No. 4,816,567, incorporated herein by reference.
- a variety of protocols for measuring soluble or membrane-bound Enzyme, using either polyclonal or monoclonal antibodies specific for the respective protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
- ELISA enzyme-linked immunosorbent assay
- RIA radioimmunoassay
- FACS fluorescent activated cell sorting
- a two-site monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non- interfering epitopes on Enzyme is preferred, but a competitive binding assay may be employed. These assays are described, among other places, in Maddox, DE et al (1983, J Exp Med 158: 1211).
- Native or recombinant Enzyme can be purified by immunoaffinity chromatography using antibodies specific for Enzyme.
- an immunoaffinity column is constructed by covalently coupling the anti-Enzyme antibody to an activated chromatographic resin.
- Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated Sepharose (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
- a chromatographic resin such as CnBr-activated Sepharose
- Such immunoaffinity columns are utilized in the purification of Enzyme by preparing a fraction from cells containing Enzyme in a soluble form. This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, soluble Enzyme containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown.
- a soluble Enzyme-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of Enzyme (eg, high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody /Enzyme binding (e.g., a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion), and Enzyme is collected.
- a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion
- This invention is particularly useful for screening compounds by using Enzyme polypeptide or binding fragments thereof in any of a variety of drug screening techniques.
- the Enzyme polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
- One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays.
- One may measure, for example, the formation of complexes between Enzyme and the agent being tested. Alternatively, one can examine the diminution in complex formation between Enzyme and its target cell, the monocyte or macrophage, caused by the agent being tested.
- the present invention provides methods of screening for drugs, natural inhibitors or any other agents which can affect disease. These methods comprise contacting such an agent with a Enzyme polypeptide or fragment thereof and assaying 1) for the presence of a complex between the agent and the Enzyme polypeptide or fragment, or 2) for the presence of a complex between the Enzyme polypeptide or fragment and the cell, by methods well known in the art.
- the Enzyme polypeptide or fragment is typically labeled. After suitable incubation, free Enzyme polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to Enzyme or to interfere with the Enzyme and agent complex.
- Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the Enzyme polypeptide and is described in detail in European Patent Application 84/03564, published on September 13, 1984, incorporated herein by reference. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with Enzyme polypeptide and washed. Bound Enzyme polypeptide is then detected by methods well known in the art. Purified Enzyme can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support.
- This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding Enzyme specifically compete with a test compound for binding to Enzyme polypeptides or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with Enzyme.
- Microbe Inhibiting Agents can provide different effects when administered therapeutically.
- Microbe Inhibiting Agent will be formulated in a nontoxic, inert, pharmaceutically acceptable aqueous carrier medium preferably at a pH of about 5 to 8, more preferably 6 to 8, although the pH may vary according to the characteristics of the antibody, inhibitor, or antagonist being formulated and the condition to be treated.
- Characteristics of Microbe Inhibiting Agent include solubility of the molecule, half-life and immunogenicity; these and other characteristics may aid in defining an effective carrier.
- Native human proteins are preferred as Microbe Inhibiting Agents, but organic or synthetic molecules resulting from drug screens may be equally effective in particular situations.
- Microbe Inhibiting Agents may be delivered by known routes of administration including but not limited to topical creams and gels; transmucosal spray and aerosol, transdermal patch and bandage; injectable, intravenous and lavage formulations; and orally administered liquids and pills, particularly formulated to resist stomach acid and enzymes.
- routes of administration including but not limited to topical creams and gels; transmucosal spray and aerosol, transdermal patch and bandage; injectable, intravenous and lavage formulations; and orally administered liquids and pills, particularly formulated to resist stomach acid and enzymes.
- the particular formulation, exact dosage, and route of administration will be determined by the attending physician and will vary according to each specific situation. Such determinations are made by considering multiple variables such as the condition to be treated, the Microbe Inhibiting Agent to be administered, and the pharmacokinetic profile of the particular Microbe Inhibiting Agent . Additional factors which may be taken into account include disease state (e.g.
- Microbe Inhibiting Agent formulations might be administered only once per day, or even less often: every 3 to 4 days, every week, or every two weeks depending on half-life and clearance rate of the particular Microbe Inhibiting Agent.
- Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
- Guidance as to particular dosages and methods of delivery is provided in the literature; see U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. It is anticipated that different formulations will be effective for different Microbe Inhibiting Agent and that administration targeting the eosinophil may necessitate delivery in a manner different from that to another organ or tissue.
- tubuculosis may also be treatable with a Microbe Inhibiting Agent.
- composition that contains one or more epitopes of CODH is useful to stimulate antibodies against microbe for practice of the invention.
- Particularly preferred are CODH proteins, peptide fragments and DNA encoding at least part of the CODH and which can be administered to produce an immune response.
- compositions that contains at least one anti-CODH antibody, antibody fragment or other antibody binding site specific to CODH are suitable, particularly as a therapeutic for alleviating and/or curing one or more disease states mentioned herein.
- Microbe Inhibiting Agents Derived or Designed from other M.TBproteins are suitable, particularly as a therapeutic for alleviating and/or curing one or more disease states mentioned herein.
- M.TB has acetate kinase and phosphotransacetylase Enzymes that are homologous to Enzymes from Methanosarcins t. These two Enzymes appear on other bacteria, such as Treponema pallidum.
- Treponema pallidum the inventors note that infection with Treponema pallidum, like infection with M. tuberculosis, is sometimes difficult to detect by screening tests in immune-suppressed patients, who can't mount a conventional antibody response. Moreover, symptoms of TB syphilis and AIDS mimic each other.
- acetate kinase and phosphotransacetylase are found in both TB and TP, medical treatment that inhibits either or both enzymes in an AIDS patient will benefit some cases in which undetected TB or TP plays an adverse clinical role. Because of the availability of these other bacterial enzymes and their uniqueness, an AIDS treatment can be based on any of these other proteins as well.
- the inventors discovered CODH in the genome of M. tuberculosis.
- monoclonal and polyclonal antibodies directed against CODH in the diagnosis treatment and prevention of tuberculosis may be used for disease that is undetectable by conventional screening methods in this, or any, immune-suppressed population.
- M. tuberculosis also, of course, has the two aforementioned "secondary" Enzymes. Identifying acetate kinase or phosphotransacetylase is in itself a potential diagnostic tool in tuberculosis. Furthermore, inhibiting either or both of these two enzymes could treat tuberculosis or syphilis. Although small changes in sequence may exist in CODH found in TB versus other microorganisms in which the enzyme occurs, the enzyme theoretically would be disarmed the same way by a method or material in accordance with the present invention. An important step in the development of materials and method in accordance with the invention is to obtain serum from some tubercular patients and perform gel electrophoresis to purify the CODH found in those patients and to sequence it. Further, the same procedure may be repeated for M. pneumonia.
- DNA vaccines can be made using sequence information from the genome of M. tuberculosis and such sequences readily are available to the skilled artisan. Most preferably a DNA vaccine will contain sequence information from the CODH gene. Likewise, an antisense composition may be used to turn down or turn off synthesis of one or more enzymes needed by the TB organism or one of the other organisms described herein.
- Microbe Inhibiting Agent it is preferred to pick an Enzyme according to the invention.
- the Enzyme, or portions of it, may then be used to formulate a suitable antigen inhibitor, antibody, and the like as described herein.
- Ni is implicated in one or more reactions of CODH. Ni can be removed by chelation and such chelators are specifically contemplated as antibiotics for practice of the invention.
- An example of a nickel chelator is hexahistidine.
- hexahistidine is added to the diet of a patient in a pharmaceutically effective amount.
- the hexahistidine is added with an excipient at more than 10 ug/lOOg of wet food for at least one month.
- the hexahistidine is covalently conjugated to a non-digestible substance, such as a water swellable polymer and added to the food in that form.
- the hexahistidine preferably is excreted by the body, with attached nickel.
- the hexahistidine works by chelating nickel and preventing use of nickel by microorganisms.
- the nickel chelator is a histidine containing compound obtained from a nickel accumulating Alyssum species of the Brassica plant family.
- Inhibition of acetate kinase and phosphotransacetylase also is desired because these enzymes -often found with CODH but common even without CODH - can potentially help disable CODH -but do much more.
- For inhibitors of these two additional enzymes combat a variety of microbial based diseases, many of which are becoming resistant to traditional antibiotic therapy. These resistant strains are becoming alarmingly frequent in recent years prompting an unprecedented search for new classes of antibiotics that target microbial processes distinct from microbial cell wall, DNA, or protein synthesis. Inhibition of these microbial processes has been over-exploited leading to the evolution of resistant strains; thus, a strategy for the development of new antibiotics has been to target microbial processes distinct from those exploited in the past.
- acetyl-CoA is a universal metabolic intermediate found in all living cells, both higher cells (yeast, plants, and animals including humans) and bacteria.
- the enzyme phosphotransacetylase (reaction 1) and acetate kinase (reaction 2) catalyze the production of acetate from acetyl-CoA where a major portion of the energy requirements for the bacteria is obtained through substrate-level phosphorylation of adenosine diphosphate (ADP) producing adenosine triphosphate (ATP).
- ADP adenosine diphosphate
- ATP adenosine triphosphate
- a reversal of reactions 1 and 2 activates acetate to acetyl-CoA for cell biosynthesis in both aerobic and anaerobic bacteria.
- the substrate for acetate kinase acetyl-phosphate (CH 3 CO 2 PO 3 2' ), serves multiple essential roles in bacterial physiology.
- acetyl-phosphate is a phosphoryl donor to the sugar transport system and regulatory proteins effecting transcription.
- acetate kinase directly and indirectly effects several processes that are essential for growth and proliferation of bacteria.
- acetate thiokinase catalyzes the synthesis of acetyl-CoA in one step (reaction 3) producing pyrophosphate (PiPi) and adenosine monophosphate
- AMP phosphotransacetylase
- inhibitors of either acetate kinase or phosphotransacetylase from bacteria are identifiable that will not effect acetyl-CoA synthesis in higher cells, including humans.
- These inhibitors are particularly effective on anaerobic and facultative anaerobic pathogens such as Treponema pallidum, Escherichia coli, Salmonella typhimurium, and Mycoplasma pneumoniae since they rely heavily on these enzymes to produce energy (ATP) for growth in addition to other essential metabolic processes. This is important since anaerobic bacteria are often difficult to control with currently available antibiotics. Developments facilitating the discovery of inhibitors for acetate kinase and phosphotransacetylase .
- JGF Two discoveries by one of the inventors (JGF) provide inhibitors for acetate kinase and phosphotransacetylase as Microbe Inhibiting Agents.
- the enzymes from M. thermophila have high identity with all other acetate kinases and phosphotransacetylases indicating that any inhibitors identified have a high probability of inhibiting the enzymes from pathogens.
- An important embodiment of the invention is its potentiation of new treatment modalities and substances based on inhibition of an Enzyme used by TP.
- Another embodiment is potentiation of new treatment modalities and substances based on inhibition of the CODH and related Enzymes used by other organisms such as the TB. Accordingly, the inventors specifically intend that the 3-dimensional structures of such Enzymes be obtained and used for rational drug design of inhibitors of the Enzymes. This is a preferred embodiment and the inventors have obtained such data for one Enzyme as described in Fi ure 3.
- the 3-d information is used by an acceptable procedure for drug design and in fact, the 3-D information itself is a valuable tool that allows a drug company to derive an important pharmaceutical simply by possession of the 3-dimensional structurall information of the enzyme. This is particularly helpful when the 3-dimensional structure is of a transition state of the enzyme, because this particular structure shows the type of inhibitor that best interferes with Enzyme function.
- Docking provides a means for using computational tools and available structural data on macromolecules to obtain new information about binding sites and molecular interactions. Docking is the placement of a putative ligand in an appropriate configuration for interacting with a receptor. Docking can be accomplished by geometric matching of a ligand and its receptor, or by minimizing the energy of interaction. Geometric matching is faster and can be based on descriptors or on fragments.
- the invention disclosed herein addresses the need for a structure by providing a model for the three-dimensional structure of an enzyme used by TP.
- the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, e.g. , agonists, antagonists, or inhibitors. Any of these examples can be used to fashion drugs which are more active or stable forms of the polypeptide or which enhance or interfere with the function of a polypeptide in vivo (cf Hodgson J (1991) Bio/Technology 9: 19-21, incorporated herein by reference).
- the three-dimensional structure of a protein of interest, or of a protein-inhibitor complex is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape and charges of the polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of a polypeptide may be gained by modeling based on the structure of homologous proteins. However, the crystal structure of one active substanceprotein is known (Walker, 1994, supra) and can be used as a starting point. In both cases, relevant structural information is used to design analogous Enzyme-like molecules or to identify efficient inhibitors.
- Useful examples of rational drug design include molecules which have different specificity or improved activity or stability as shown by Braxton S and Wells JA (1992 Biochemistry 31:7796-7801) or which act as inhibitors, agonists, or antagonists of native peptides as shown by Athauda SB et al (1993 J Biochem 113:742-746), incorporated herein by reference.
- a target-specific antibody selected by functional assay, as described above, and then to solve its crystal structure.
- This approach in principle, yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore.
- anti-ids anti-idiotypic antibodies
- polypeptide may be made available to perform such analytical studies as X-ray crystallography.
- knowledge of the Enzyme amino acid sequence provided herein will provide guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography.
- a Microbe Inhibiting Agent of the present invention as described above will be administered in a pharmaceutical composition to an individual already showing signs of "AIDS" or other disease described herein or at high risk of such infection.
- Those in the incubation phase or the acute phase of infection can be treated with the immunogenic, immunoactive or antibiotic substance separately or in conjunction with other treatments, as appropriate.
- compositions are administered to a patient in an amount sufficient to elicit an effective B cell and/or T cell response to microbe or to hinder or kill the microbe and to cure or at least partially arrest its symptoms and/or complications.
- An amount adequate to accomplish this is defined as a "therapeutically or prophylactically effective dose” which may be an "immune response provoking amount” or a "lethal dose amount.
- Amounts effective for a therapeutic or prophylactic use will depend on, e.g. , the stage and severity of the disease the age, weight, and general state of health of the patient, and the judgment of the prescribing physician.
- the size of the dose will also be determined by the active substance composition, method of administration, timing and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound(s) and the desired physiological effect. It will be appreciated by one of skill in the art that various conditions or disease states may require prolonged treatment involving multiple administrations.
- Suitable doses and dosage regimens can be determined by conventional range-finding techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages that are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached.
- the present inventive method typically will involve the administration of about 0.1 mg to about 50 mg of one or more of the compounds described above per kg body weight of the individual. For a 70 kg patient, dosages of from about 10 mg to about 100 mg of active substance would be more commonly used, followed by booster dosages from about 0.01 mg to about 1 mg of active substance over weeks to months, depending on a patient's immune response.
- the active substances and compositions of the present invention may generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, in view of the minimization of extraneous substances and the relative nontoxic nature of the active substances, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these active substance compositions.
- the pharmaceutical formulations should provide a quantity of B cell and/or T cell stimulatory active substances of the invention sufficient to effectively treat the patient.
- administration should begin at the first sign of microbe infection or shortly after diagnosis in cases of acute infection, and continue until at least symptoms are substantially abated and for a period thereafter. In well-established and chronic cases, loading doses followed by maintenance or booster doses may be required.
- the pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral or local administration and generally comprise a pharmaceutically acceptable carrier and an amount of the active ingredient sufficient to reverse or prevent the bad effects of microbe infection.
- the carrier may be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the compound, and by the route of administration.
- Examples of pharmaceutically acceptable acid addition salts for use in the present inventive pharmaceutical composition include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, p-toluenesulphonic acids, and arylsulphonic, for example.
- mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids
- organic acids such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, p-toluenesulphonic acids, and arylsulphonic, for example.
- the pharmaceutically acceptable excipients described herein for example, vehicles, adjuvants, carriers or diluents, are well-known to those who are skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one that is chemically inert to the active compounds and one that has no detrimental side effects or toxicity under the conditions of use.
- Such carriers can include immuno- stimulating complexes (i.e. cholesterol, saponin, phospholipid peptide complexes), aluminum hydroxide (alum), heat shock proteins, linkage to synthetic microspheres (polyamino-microspheres) .
- excipient will be determined in part by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention.
- compositions for parenteral administration that comprise a solution of the stimulatory active substances dissolved or suspended in an acceptable carrier suitable for parenteral administration, including aqueous and non-aqueous, isotonic sterile injection solutions.
- the compound may be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethylsulf oxide, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, such as poly (ethylenegly col) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or e
- Oils useful in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils useful in such formulations include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
- Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts
- suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl- beta -aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.
- the parenteral formulations typically will contain from about 0.5 to about 25% by weight of the active ingredient in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.
- HLB hydrophile-lipophile balance
- Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene
- unit-dose or multi-dose sealed containers such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
- sterile liquid excipient for example, water
- Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
- Topical formulations including those that are useful for transdermal drug release, are well-known to those of skill in the art and are suitable in the context of the present invention for application to skin.
- Formulations suitable for oral administration equire extra considerations considering the particular molecular nature of the Microbe Inhibiting Agent and the likely breakdown thereof if such compounds are administered orally without protecting them from the digestive secretions of the gastrointestinal tract.
- Such a formulation can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions.
- Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent.
- diluents such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent.
- Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch.
- Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients.
- Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
- a flavor usually sucrose and acacia or tragacanth
- pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
- the Microbe Inhibiting Agent molecules of the present invention can be made into aerosol formulations to be administered via inhalation.
- the active substances are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of the material are 0.01 %-20% by weight, preferably 1 %-10% .
- the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
- esters or partial esters of fatty acids containing from 6 to 22 carbon atoms such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
- Mixed esters such as mixed or natural glycerides may be employed.
- the surfactant may constitute 0.1 % -20% by weight of the composition, preferably 0.25-5% .
- the balance of the composition is ordinarily propellant.
- a carrier can also be included as desired, e.g., lecithin for intranasal delivery.
- aerosol formulations can be placed into acceptable pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer. Such spray formulations may be used to spray mucosa.
- the compounds and polymers useful in the present inventive methods may be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases.
- bases such as emulsifying bases or water-soluble bases.
- Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.
- the pharmaceutical composition at least one component that primes CTL generally.
- Lipids have been identified that are capable of priming CTL in vivo against viral antigens, e.g., tripalmitoyl-S- glycerylcysteinly-seryl-serine (P sub 3 CSS), which can effectively prime virus specific cytotoxic T lymphocytes when covalently attached to an appropriate immunoactive substance. See, Deres et al., Nature, 342, 561-564 (1989).
- Active substances of the present invention can be coupled to P sub 3 CSS, for example and the lipoprotein administered to an individual to specifically prime a cytotoxic T lymphocyte response to microbe.
- the concentration of the Microbe Inhibiting Agent of the present invention in the pharmaceutical formulations can vary widely, i.e. , from less than about 1 %, usually at or at least about 10% to as much as 20 to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
- a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 100 mg of active substance.
- Actual methods for preparing parenterally administrable compounds will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's
- the compounds of the present inventive method may be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.
- Liposomes serve to target the compounds to a particular tissue, such as lymphoid tissue or microbe-infected cells. Liposomes can also be used to increase the half-life of the active substance composition.
- Liposomes useful in the present invention include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
- the active substance to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, e.g., a receptor, prevalent among lymphoid cells, such as monoclonal antibodies which bind to the antigen, or with other therapeutic or immunogenic compositions.
- liposomes filled with a desired active substance of the invention can be directed to the site of infection, where the liposomes then deliver the selected therapeutic/ immunogenic active substance compositions.
- Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, for example, liposome size and stability of the liposomes in the blood stream.
- a ligand to be incorporated into the liposome can include, for example, antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells.
- a liposome suspension containing an active substance may be administered intravenously, locally, topically, etc. in a dose that varies according to the mode of administration, the active substance being delivered, the stage of disease being treated, etc.
- the invention is directed to vaccines that contain in addition to the Microbe Inhibiting Agent (having desired epitopes) as an active ingredient an immunogenically effective amount of a cytotoxic T-lymphocyte stimulating active substance having a sequence as described herein.
- Other immunomodulators may be added such as interleukin-1, beta (IL-1 beta) peptide and interleukin 12 (IL-12) peptide.
- Active substances may be for example, complexed to cholera toxin B subunit to stimulate mucosal immunity.
- the active substance(s) may be introduced into a patient linked to its own carrier or as a homopolymer or heteropolymer of active units.
- Such a polymer has the advantage of increased immunological reaction and, where different peptides are used to make up the polymer, the additional ability to induce antibodies and/or cytotoxic T cells that react with different antigenic determinants of microbe.
- Useful carriers are well known in the art, and include, e.g., keyhole limpet hemocyanin, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly(D-lysine:D-glutamic acid), and the like.
- the vaccines can also contain a physiologically tolerable (acceptable) diluent such as water, phosphate buffered saline, or saline, and further typically include an adjuvant.
- a physiologically tolerable (acceptable) diluent such as water, phosphate buffered saline, or saline
- adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum or materials well known in the art.
- cytotoxic T lymphocyte responses can be primed by conjugating active substances of the invention to lipids, such as P sub 3 CSS.
- the immune system of the host Upon immunization with an active substance composition as described herein, via injection, aerosol, oral, transdermal or other route, the immune system of the host responds to the vaccine by producing large amounts of cytotoxic T-lymphocytes specific for microbe antigen, and the host becomes at least partially immune to microbe infection, or resistant to developing chronic microbe infection.
- Vaccine compositions containing the active substances of the invention are administered to a patient susceptible to or otherwise at risk of microbe infection to enhance the patient's own immune response capabilities.
- a patient susceptible to or otherwise at risk of microbe infection is defined to be a "immunogenically effective dose” or a “prophylactically effective dose. "
- the precise amounts again depend on the patient's state of health and weight, the mode of administration, the nature of the formulation, etc., but generally range from about 1.0 mg to about 500 mg per 70 kilogram patient, more commonly from about 50 mg to about 200 mg per 70 kg of body weight.
- the active substances of the invention can also be expressed by attenuated viral hosts, such as vaccinia.
- vaccinia This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode at least part of an Enzyme.
- the recombinant vaccinia virus Upon introduction into an microbe-infected host or into a non-infected host, the recombinant vaccinia virus expresses the Microbe Inhibiting Agent and thereby elicits a host cytotoxic T lymphocyte response to microbe.
- Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848.
- BCG Bacille Calmette Guerin
- BCG vectors are described in Stover et al., Nature, 351, 456-460 (1991).
- Salmonella typhi vectors and the like will be apparent to those skilled in the art from the description herein.
- compositions and methods of the claimed invention may be employed for ex vivo therapy, wherein, as described briefly above, a portion of a patient's lymphocytes are removed, challenged with a stimulating dose of a active substance of the present invention, and the resultant stimulated cells are returned to the patient.
- ex vivo therapy concerns the therapeutic or immunogenic manipulations that are performed outside the body on lymphocytes or other target cells that have been removed from a patient. Such cells are then cultured in vitro with high doses of the subject active substances, providing a stimulatory concentration of active substance in the cell medium far in excess of levels that could be accomplished or tolerated by the patient. Following treatment to stimulate the Cells the cells are returned to the host, thereby treating the microbe infection.
- the host's cells also may be exposed to vectors that carry genes encoding the active substances, as described above. Once transfected with the vectors, the cells may be propagated in vitro or returned to the patient. The cells that are propagated in vitro may be returned to the patient after reaching a predetermined cell density.
- DNA, polynucleotide etc. immunization with specific genes has shown promise in several model systems of pathogenic disease, and a few natural systems.
- Use of DNA (or RNA) thus overcomes some of the problems encountered when an animal is presented directly with an antigen.
- DNA segment refers to a DNA molecule that has been isolated free of total genomic DNA of a particular host species. Therefore, a DNA segment encoding a peptide or protein having a desired sequence refers to a DNA segment that contains these coding sequences yet is isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment has been cloned. Included within the term “DNA segment,” are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.
- a DNA segment contemplated here refers to a DNA segment which may include in addition to peptide encoding sequences, certain other elements such as, regulatory sequences, isolated substantially away from other naturally occurring genes or peptide-encoding sequences.
- the term "gene” is used for simplicity to refer to a functional protein, polypeptide or peptide-encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, cDNA sequences and smaller engineered gene segments that express, or may be adapted to express proteins, polypeptides or peptides.
- isolated substantially away from other coding sequences means that the gene of interest, in this case, a gene encoding microbe epitopes forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
- Agents are made from known materials and methods for stimulating formation of antibodies that react with components of TB and/or other Enzymes, found in other anaerobes and facultative anaerobes.
- the Agents may also stimulate the formation of an immune response by nucleic acid, or may directly inhibit one or more Enzymes to inhibit a microbe.
- the invention encompasses and utilizes known prior art methods relating to each of these factors and efforts and a skilled artisan in each respective field now can prepare such materials and methods of their use for destroying, inhibiting and detecting TB and syphilis — independently or when they occur, sometimes undetected, in AIDS, as well as appreciate the formation of a new class of antibiotics.
- the invention specifically contemplates and includes such optimization and applications. Nevertheless, the inventors have, in addition to discovered new methods and materials for purifying, studying and utilizing the enzymes implicated in these diseases. A summary of some of their results useful for practice of the invention follows.
- the Polymerase Chain Reaction is used to amplify the coding regions of the ack and pta genes.
- the sequences of the primers for the ack gene are 5'- CATGCATATGAAAGTACTGGTTATA -3' (partially corresponding to nucleotides 1314 - 1331, Fig. 2) and 5'-CAGTGGATCCGAGCAATTTTCGGAC -3' (partially complementary to nucleotides 2734 - 2748, Fig. 2).
- the sequences of the upstream and downstream primers for the pta gene are 5'-GGTGGTCATATGGTAACATTTTTAGAG -3' (partially corresponding to nucleotides 208 - 224, Fig.
- the amplification is performed in a DNA Thermal Cycler (Perkin-Elmer Cetus). Blunt ends are created on the PCR amplification products using T4 DNA polymerase. The purified blunt- ended PCR products are subcloned into the Smal site of a pUC19 vector derivative lacking the Ndel site and the resulting plasmids are purified by cesium chloride density gradient centrifugation. The coding regions are then excised with Ndel and Bam I and the fragments eluted from an agarose gel using the Elutrap.
- E. coli strain TB-1 E. coli strain TB-1.
- the recombinant plasmids are purified by cesium chloride gradient centrifugation.
- E. coli strain BL21(DE3) is transformed with the overexpression pT7-7 plasmids containing the ack or pta genes (designated pML703 and pML702, respectively).
- Transformants are grown at 37oC in Luria-Bertani broth containing 100 ug ml "1 ampicillin and induced with 1 % (final concentration) Bacto-lactose or 0.4 mM (final concentration) IPTG.
- the enzyme eluted from the column between 220 and 270 mM KC1 using a 1800 ml linear gradient of 0 - 1 M KC1 at 6 ml min "1 . Fractions with the highest total activities are pooled and an equal volume of 1.8 M ammonium sulfate in 50 mM Tris (pH 7.2) plus 2 mM DTT is added. A protein sample (100 ml) is loaded onto a Phenyl-Sepharose HiLoad 26/10 column equilibrated with 2 column volumes of 900 mM ammonium sulfate in 50 mM Tris, pH 7.2 with 2 mM DTT.
- the fractions with the highest total activity are pooled, diluted 20-fold with 25 mM Tris (pH 7.6) plus 2 mM DTT, and loaded on the Mono-Q column equilibrated with 5 column volumes of 25 mM Tris, pH 7.6 with 2 mM DTT.
- a 25 mM Tris (pH 7.2) buffer containing 2 mM DTT is used in all steps of the purification unless otherwise noted.
- Cell extract is obtained as described for the purification of acetate kinase except the lysate is centrifuged at 4,000 x g, and no streptomycin sulfate treatment is performed.
- the 4,000 x g pellet is dispersed in 60 ml of buffer and centrifuged as above.
- the washed pellet is dispersed in 10 ml of buffer to give a final volume of 15 ml.
- One volume (15 ml) of 12 M urea is added to the suspension and the mixture is incubated at 13°C for 15 min.
- the solution is diluted to 300 ml with buffer and incubated at 13°C for 5 h.
- the solution is loaded on a Q-Sepharose Fast Flow column (5 cm x 10 cm) equilibrated with 2 column volumes of buffer.
- the column is developed with a 1800 ml linear gradient of 0 - 1 M KCl at 6 ml min "1 .
- Fractions with the highest total activity are pooled, diluted with one volume 50 mM Tris (pH 7.6) plus 2 mM DTT and loaded on a Mono-Q column equilibrated with 5 column volumes of 50 mM Tris (pH 7.6) with 2 mM DTT.
- the purified phosphotransacetylase eluted at 180 mM KCl using a 200 ml linear gradient from 0 - 1 M KCl at 2 ml min "1 .
- the purified material was used to make crystals and X-ray diffraction analysis was used to determine the three dimensional structure of the enzyme by known procedures.
- the coordinates obtained from the X-ray diffraction are provided in Figure 3.
- Figure 3 Using the information from Figure 3, one can derive new inhibitors of the enzyme.
- a preferred method for doing this is "rational drug design" as described above and detailed in U.S. No. 5,808,001, the contents of which are specifically inco ⁇ orated by reference in their entireties.
- Methanosarcina thermophila strain TM-1 is cultured on acetate in a lOOliter pH auxostat.
- the basal medium contains (in grams per liter, final concentration): NH4C1, 1.44; K2HPO4.1.13; KH2PO4, 1.13; NaCl, 0.45; MgSO4.2H2O, 0.09;CaClt ⁇ 2t ⁇ .2HV&20, 0.06; yeast extract (Difco Laboratories), 0.5; Trypticase (BBL Microbiology systems), 0.5; Fe(NH4)2(SO4)2, 0.01; cysteine.HCl, 0.27; Na2S.9H2O, 0.27; Antifoam C, 0.5; and resazurin, 0.001.
- Trace elements and vitamin solutions are each added at a final concentration of 1 % (vol/vol); NiC1.6H2O is added to a final concentration of 0.5 g/liter. Sodium acetate (50 mM) is added as the substrate.
- yeast extract is decreased to 0.1 g/liter, and Ni metal dissolved in nitric acid) is added to a final concentration of 0.5 mM.
- Cells are harvested in a continuous-flow centrifuge (Cepa type LE) under a stream of N2, and the resulting cell paste is frozen and stored in liquid nitrogen.
- the general anaerobic procedures for the preparation of cell extracts and for enzyme assays are as follows.
- Breakage buffer consists of 50 mM potassium N-tris(hydroxymethyl)methyl- 2-aminoethanesulfonate buffer (TES)(pH7.0) containing 10 mM 2-mercaptoethanol, 10 mM MgC12, 5% (vol/vol) glycerol, and 0.015 mg/ml of DNase I (Sigma, St. Louis, MO).
- Buffer A contains 50 mM TES (pH 6.8), 10% (vol/vol) ethylene glycol, and 10 mM MgCl 1 / 2 2.
- Buffer B and buffer C are identical to buffer A except 1.0 M KCl and 0.15 M KCl are added.
- Saturated ammonium sulfate solution in 50 mM TES (pH 6.8) and 10 mM MgCl 1 / 2 2 are added to 10 ml of cell extract to a final concentration of 0.35 saturation.
- a linear gradient from 0.0 to 0.5M KCl is applied at a flow rate of 2.0 ml/min.
- Two peaks of CO dehydrogenase activity elute.
- the second, larger peak is collected and injected again onto the Mono-Q HR 10/10 column equilibrated with buffer A.
- the enzyme is concentrated 10- fold by batch elution with 0.4 M KCl. Aliquots (0.5 ml) of the concentrated protein solution are injected on a Superose-6 (Pharmacia) gel filtration column previously equilibrated with Buffer C.
- the column is developed at a flow rate of 0.4 ml/min.
- Purified CO dehydrogenase is collected and stored in liquid N2.
Abstract
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