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WO1996035804A1 - Nouvelle interaction biochimique dans des bacteries - Google Patents

Nouvelle interaction biochimique dans des bacteries Download PDF

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Publication number
WO1996035804A1
WO1996035804A1 PCT/US1996/006224 US9606224W WO9635804A1 WO 1996035804 A1 WO1996035804 A1 WO 1996035804A1 US 9606224 W US9606224 W US 9606224W WO 9635804 A1 WO9635804 A1 WO 9635804A1
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WO
WIPO (PCT)
Prior art keywords
interaction
ftsz
novel
pbp3
protein
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.)
Ceased
Application number
PCT/US1996/006224
Other languages
English (en)
Inventor
David L. Pompliano
David Bramhill
Barry R. Cunningham
Mohamed El-Sherbeini
A. Brian Jones
Dorina Trusca
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.)
Merck and Co Inc
Original Assignee
Merck and Co Inc
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 Merck and Co Inc filed Critical Merck and Co Inc
Publication of WO1996035804A1 publication Critical patent/WO1996035804A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material

Definitions

  • the FtsZ protein is highly conserved in bacteria and appears to play a cytoskeletal role during cell division. FtsZ is located in the bacterial cytoplasm during most of the cell cycle. As division begins, FtsZ relocalizes to a ring at the inner surface of the inner membrane at the division site, and remains at the leading edge of the inward growth of the septum until division is completed.
  • PBP3 the septum-specific, membrane-spanning penicillin binding protein is encoded by the cell division gene FtsI, and is essential for septum formation.
  • One hydrophobic helix of PBP3 spans the inner membrane connecting a 23 amino acid cytoplasmic domain to the transglycosylase and transpeptidase domains located in the periplasm.
  • FtsZ is a cytoplasmic protein and PBP3 is a transmembrane protein with its N- terminus residing in the cytoplasm, the only way these proteins could interact in the cell would be through the N-terminal amino acid residues of PBP3.
  • FIG. 2 represents theoretically the FtsA-FtsZ-PBP3 complex spanning the inner membrane.
  • the present invention relates to a novel interaction between bacterial (E. coli) cell division proteins.
  • the present invention also relates to a novel target for antibiotic development. Additionally, the present invention relates to the use of the yeast two-hybrid screen, as well as an in vitro flourescence assay, to screen for agents which would inhibit the novel interaction.
  • the essential Escherichia coli cell division protein FtsZ shares biochemical properties and amino acid similarities with tubulins, suggesting a cytoskeletal role for FtsZ. Both have GTPase activity and undergo GTP-dependent polymerization to form structures comprised of protofilaments.
  • the yeast two-hybrid protein interaction screen was used to identify associations between FtsZ (the "bait") and other cell division proteins.
  • the yeast two-hybrid system is an in vivo method for genetically identifying interacting proteins using reconstitution of activity of a transcriptional activator (GAL4). This reconstitution makes use of chimeric genes which express hybrid proteins.
  • GAL4 transcriptional activator
  • Two types of hybrid proteins are prepared. The first hybrid contains the DNA- binding domain of a transcriptional activator fused to the first test protein. The second hybrid protein contains a transcriptional activation domain fused to the second test protein. If the two test proteins are able to interact, they bring into close proximity the two domains of the transcriptional activator. This proximity is sufficient to cause transcription of a reporter gene which contains a binding site for the DNA-binding domain.
  • FtsZ polymerization was measured at optimal and sub- optimal levels of GTP, to determine whether the PBP3 peptide stabilized or destabilized the FtsZ polymer. No effect on polymerization was observed. Neither was there an effect on the GTPase activity of FtsZ in the presence of the PBP3 peptide. Polymerization of FtsZ did not significantly alter the PBP3-FtsZ interaction.
  • PBP3 cytoplasmic domain peptide (MKAAAKTQKPKRQEEHANFISWR-NH 2 ) (SEQ ID NO:— -) was prepared by standard Fmoc synthesis.
  • the N-dansylated derivative was synthesized by reacting the peptide with dansyl chloride prior to deprotection and removal from the support.
  • Peptides were purified by reverse phase C18 HPLC and their structures confirmed by mass spectrometry.
  • FtsZ was prepared as described in Bramhill et al., Proc. Nat'l Acad. Sci. 91 , 5813-7 (1994). Ligand binding experiments were conducted at room temperature (19-21 °C).
  • Pairs of plasmids each pair consisting of a DNA binding domain and an activation domain fusion, were introduced into the Saccharomyces cerevisiae reporter strain SFY526 (Bartel et al., Bio Techniques 14, 920-924 (1993) and tested for their ability to activate lacZ expression driven from a GAL4 responsive promoter .
  • the results of the assay are shown below i-j Table 1.
  • RNA division genes were cloned by PCR using DNA of the Escherichia coli K12 strain R477 as template. PCR primers (CGG AGA GAA ACT ATG TTT GGA TCC ATG GAA CTT ACC (SEQ ID NO:— -)and CGA AAC CCA AAC TGC AGT CAA TTC TTA ATC AGC (SEQ ID NO:—) for FtsZ, GGC ACA GGC
  • AGA ACA ACA ATG ATG AAT TCG ACG G (SEQ ID NO: — ) and CCG TAA CAT CGT CGG CTG CAG AAA AAT TAA AAC TC (SEQ ID NO: — ) for FtsA
  • CAA AAA TAA GGA TAA ACG GAA TTC ATG AAA GCA GCG GC (SEQ ID NO: — ) and CCC ACG GAG CAA GAA GGA TCC GCA AAT TAC GAT CTG CC (SEQ ID NO — ) for PBP3) introduced appropriate restriction endonuclease sites for in- frame fusion of each gene to the GAL4 activator or DNA-binding domain in the vectors pGAD424 and pGBT9, respectively.
  • Plasmid DNAs were prepared from Escherichia coli K12 strain DH5 ⁇ , (Hanahan, D., J. Mol. Biol., 166 557-580 (1983), and transformed into yeast strain SFY526 in the combinations indicated. Yeast transformants were selected on media lacking leucine and tryptophan. ⁇ -galactosidase activity on two independent transformants was determined as previously described, and is expressed as Miller units. Activation domain plasmids containing either no fusion protein or fused with T-antigen were used as controls.
  • FIG. 2 diagrams the interaction linking the cytoplasmic and periplasmic components of the division apparatus. If the FtsZ protein is serving as a guide for septum synthesis, and if it is a cytoskeleton acting to overcome the outward osmotic pressure of about four atmospheres, then the fewest number of links connecting FtsZ to the peptidoglycan synthesis complex will provide the strongest structure.
  • FtsZ and PBP3 are relatively low. However, this may not be a limiting parameter given the extremely high local concentration of FtsZ at a division site. Also, there may be other components in the cell which stabilize the FtsZ- PBP3 complex. At least four other Fts proteins appear to span the inner membrane and may also interact with FtsZ.
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Peptides Or Proteins (AREA)

Abstract

On a identifié une nouvelle interaction biochimique entre des protéines bactériennes de division cellulaire. Cette interaction peut représenter une cible thérapeutique potentielle pour une nouvelle catégorie d'antibiotiques qui inhibent cette interaction.
PCT/US1996/006224 1995-05-08 1996-05-03 Nouvelle interaction biochimique dans des bacteries Ceased WO1996035804A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US43725395A 1995-05-08 1995-05-08
US08/437,253 1995-05-08

Publications (1)

Publication Number Publication Date
WO1996035804A1 true WO1996035804A1 (fr) 1996-11-14

Family

ID=23735692

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/006224 Ceased WO1996035804A1 (fr) 1995-05-08 1996-05-03 Nouvelle interaction biochimique dans des bacteries

Country Status (1)

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WO (1) WO1996035804A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998026088A1 (fr) * 1996-12-12 1998-06-18 Isis Innovation Limited Souche bacillus et procede de selection d'antibiotiques
EP1043403A1 (fr) * 1999-04-09 2000-10-11 GPC AG, Genome Pharmaceuticals Corporation Méthode pour l'identification des composés antibactériens
WO2000061793A3 (fr) * 1999-04-09 2001-01-11 Gpc Biotech Ag Nouvelle methode d'identification de composes antibacteriens

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5283173A (en) * 1990-01-24 1994-02-01 The Research Foundation Of State University Of New York System to detect protein-protein interactions
US5409898A (en) * 1990-02-23 1995-04-25 Bristol-Myers Squibb Company Compositions and methods for treating infections caused by organisms sensitive to β-lactam antibiotics

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5283173A (en) * 1990-01-24 1994-02-01 The Research Foundation Of State University Of New York System to detect protein-protein interactions
US5409898A (en) * 1990-02-23 1995-04-25 Bristol-Myers Squibb Company Compositions and methods for treating infections caused by organisms sensitive to β-lactam antibiotics

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CELL, June 1993, Vol. 73, SHAPIRO L., "Protein Localization and Asymmetry in the Bacterial Cell", pages 841-855. *
J. BACTERIOL., December 1992, Vol. 174, No. 23, GUZMAN et al., "FtsL, an Essential Cytoplasmic Membrane Protein Involved in Cell Division in Escherichia Coli", pages 7716-7728. *
MOL. MICROBIOL., 1993, Vol. 9, No. 3, LUTKENHAUS J., "FtsZ Ring in Bacterial Cytokinesis", pages 403-409. *
PROC. NATL. ACAD. SCI. U.S.A., June 1994, Vol. 91, BRAMHILL et al., "GTP-Dependent Polymerization of Escherichia Coli FtsZ Protein to Form Tubules", pages 5813-5817. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998026088A1 (fr) * 1996-12-12 1998-06-18 Isis Innovation Limited Souche bacillus et procede de selection d'antibiotiques
US6255065B1 (en) 1996-12-12 2001-07-03 Isis Innovation Limited Bacillus strain and antibiotic screening method
EP1043403A1 (fr) * 1999-04-09 2000-10-11 GPC AG, Genome Pharmaceuticals Corporation Méthode pour l'identification des composés antibactériens
WO2000061793A3 (fr) * 1999-04-09 2001-01-11 Gpc Biotech Ag Nouvelle methode d'identification de composes antibacteriens

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