WO2002063015A1 - Aspartyl-protease - Google Patents

Aspartyl-protease Download PDF

Info

Publication number
WO2002063015A1
WO2002063015A1 PCT/GB2002/000478 GB0200478W WO02063015A1 WO 2002063015 A1 WO2002063015 A1 WO 2002063015A1 GB 0200478 W GB0200478 W GB 0200478W WO 02063015 A1 WO02063015 A1 WO 02063015A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
nucleic acid
disease
acid molecule
patient
Prior art date
Application number
PCT/GB2002/000478
Other languages
English (en)
Inventor
Richard Joseph Fagan
Christopher Benjamin Phelps
Andrew Robert Davids
Original Assignee
Inpharmatica Limited
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 Inpharmatica Limited filed Critical Inpharmatica Limited
Publication of WO2002063015A1 publication Critical patent/WO2002063015A1/fr

Links

Classifications

    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6478—Aspartic endopeptidases (3.4.23)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the active sites carry a formal negative charge to effect hydrolysis, and nearby hydrogen bonding residues, Gly 34, Ser35, Gly217 and Thr 218 (pepsin numbering) were modelled as formamide and methanol molecules.
  • a polypeptide according to this aspect of the invention consists of the amino acid sequence as recited in SEQ ID NO:2.
  • the polypeptide having the sequence recited in SEQ ID NO:2 is referred to hereafter as "the APG1 polypeptide".
  • a preferred polypeptide fragment according to part ii) above includes the region of the APG1 polypeptide that is predicted as that responsible for aspartyl protease activity (hereafter, the "APG1 aspartyl protease region"), or is a variant thereof that possesses the catalytic residues (ASP234, and ASP403, or equivalent residues).
  • the APG1 aspartyl protease region is considered to extend between residue 207 and residue 412 of the APG1 polypeptide sequence.
  • the invention provides a purified nucleic acid molecule which hybridizes under high stringency conditions with a nucleic acid molecule of the second aspect of the invention.
  • the invention provides a host cell transformed with a vector of the fourth aspect of the invention.
  • the invention provides a ligand which binds specifically to, and which preferably inhibits the aspartyl protease activity of, a polypeptide of the first aspect of the invention.
  • the present invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in the manufacture of a medicament for the diagnosis or treatment of a disease, such as HIV-1 infection, Alzheimer's disease, pancreatic, gastric, oral and breast cancers, malaria, hypertension (leading to renal failure), candidiasis and gram negative bacterial infection.
  • a disease such as HIV-1 infection, Alzheimer's disease, pancreatic, gastric, oral and breast cancers, malaria, hypertension (leading to renal failure), candidiasis and gram negative bacterial infection.
  • nucleic acid molecules may be used in the methods and applications described herein.
  • the nucleic acid molecules of the invention preferably comprise at least n consecutive nucleotides from the sequences disclosed herein where, depending on the particular sequence, n is 10 or more (for example, 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).
  • Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al., Curr. Opin. Struct. Biol (1996) 6(4), 527-33). Synthetic ribozymes can be designed to specifically cleave mRNAs at selected positions thereby preventing translation of the mRNAs into functional polypeptide. Ribozymes may be synthesised with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively the ribozymes may be synthesised with non-natural backbones, for example, 2'-O-methyl RNA, to provide protection from ribonuclease degradation and may contain modified bases.
  • This expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding the polypeptide, such that the packaging cell now produces infectious viral particles containing the gene of interest.
  • These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo (see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics (1996), T Strachan and A P Read, BIOS Scientific Publishers Ltd).
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR, ligase chain reaction (LCR), strand displacement amplification (SDA), or other amplification techniques (see Saiki et al., Nature, 324, 163-166 (1986); Bej, et al., Crit. Rev. Biochem. Molec. Biol., 26, 301-334 (1991); Birkenmeyer et al., J. Virol. Meth., 35, 117-126 (1991); Van Brunt, J., Bio/Technology, 8, 291-294 (1990)) prior to analysis.
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • the method may comprise the steps of: a) contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule of the invention and the probe; b) contacting a control sample with said probe under the same conditions used in step a); c) and detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels of the hybrid complex in the control sample is indicative of disease.
  • Such diagnostics are particularly useful for prenatal and even neonatal testing.
  • Normal or standard values for polypeptide expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably humans, with antibody to the polypeptide under conditions suitable for complex formation
  • the amount of standard complex formation may be quantified by various methods, such as by photometric means.
  • Diagnostic assays may be used to distinguish between absence, presence, and excess expression of polypeptide and to monitor regulation of polypeptide levels during therapeutic intervention. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials or in monitoring the treatment of an individual patient.
  • a diagnostic kit of the present invention may comprise: (a) a nucleic acid molecule of the present invention
  • FIG. 2B Full list of forward PSI-BLAST results for the search using 1CZI.
  • CAA16015.1 PE protein (APGl) is not identified.
  • CAA16015.1 (APGl) protein sequence is searched against the PFAM database (Protein Family Database of Alignment and hidden Markov models) (see Figure 4).
  • the PFAM-A identifies CAA16015.1 (APGl) as a PE protein as expected.
  • the results also identifiy a PFAM-B match, however, PFAM-B matches confer no functional annotation, only sequence similarity to other functionally unannotated proteins. Thus PFAM does not identify CAA16015.1 (APGl) as an aspartyl protease.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne une nouvelle protéine, appelée CAA16015.1, identifiée ici comme une aspartyl-protéase, ainsi que l'utilisation de cette protéine et d'une séquence d'acide nucléique issue du gène de codage dans le diagnostic, la prévention et le traitement de maladies.
PCT/GB2002/000478 2001-02-05 2002-02-05 Aspartyl-protease WO2002063015A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0102903.2 2001-02-05
GB0102903A GB0102903D0 (en) 2001-02-05 2001-02-05 Novel protein

Publications (1)

Publication Number Publication Date
WO2002063015A1 true WO2002063015A1 (fr) 2002-08-15

Family

ID=9908188

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2002/000478 WO2002063015A1 (fr) 2001-02-05 2002-02-05 Aspartyl-protease

Country Status (2)

Country Link
GB (1) GB0102903D0 (fr)
WO (1) WO2002063015A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003106484A1 (fr) * 2002-06-17 2003-12-24 Chr. Hansen A/S Procede de production ameliore d'une protease aspartique dans un organisme hote de recombinaison

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
COLE S T ET AL: "Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence", NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 393, 11 June 1998 (1998-06-11), pages 537 - 544, XP002087941, ISSN: 0028-0836 *
DATABASE EMBL [online] 14 January 1998 (1998-01-14), COLE S.T. ET AL.: "Mycobacterium tuberculosis H37Rv complete genome; segment 110/162", XP002201328, retrieved from EBI Database accession no. AL021185 *
DATABASE GENBANK [online] 11 June 1998 (1998-06-11), COLE S.T. ET AL.: "PE [Mycobacterium tuberculosis H37Rv]", XP002201329, retrieved from NCBI Database accession no. CAA16015 *
DATABASE GENBANK [online] 17 June 1998 (1998-06-17), COLE S.T. ET AL.: "Mycobacterium tuberculosis H37Rv complete genome; segment 89/162.", XP002201330, retrieved from NCBI Database accession no. z74025 *
DATABASE SWALL [online] 1 June 1998 (1998-06-01), COLE S.T. ET AL: "PGRS-Family protein (PE Family protein)", XP002201327, retrieved from EBI Database accession no. O53224 *
RAMAKRISHNAN L ET AL: "GRANULOMA-SPECIFIC EXPRESSION OF MYCOBACTERIUM VIRULENCE PROTEINS FROM THE GLYCINE-RICH PE-PGRS FAMILY", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,, US, vol. 288, 26 May 2000 (2000-05-26), pages 1436 - 1439, XP000979218, ISSN: 0036-8075 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003106484A1 (fr) * 2002-06-17 2003-12-24 Chr. Hansen A/S Procede de production ameliore d'une protease aspartique dans un organisme hote de recombinaison
US7776581B2 (en) 2002-06-17 2010-08-17 Chr. Hansen A/S Method of producing an aspartic protease in a recombinant host organism

Also Published As

Publication number Publication date
GB0102903D0 (en) 2001-03-21

Similar Documents

Publication Publication Date Title
WO2004026904A1 (fr) Proteines precurseurs de peptides associes d'amide rf et peptides d'amide rf
WO2002063015A1 (fr) Aspartyl-protease
WO2002033058A2 (fr) Proteases a cysteine
EP1370584A2 (fr) Domaine de liaison de ligand au recepteur hormonal nucleaire
CA2510066A1 (fr) Proteines de metalloprotease
WO2002029023A1 (fr) Proteine appelee nicastrine
WO2002008393A2 (fr) Serine protease
US20040132038A1 (en) Nuclear hormone receptor ligand binding domain
WO2004101618A2 (fr) Proteines associees au recepteur de progestine yol002c-cgi-45
WO2004003190A1 (fr) Polypeptide identifie comme cytochrome p450
WO2002072805A2 (fr) Thymidylate synthetase
WO2002062976A2 (fr) Proteases a serine
WO2002034901A2 (fr) Proteine kinase
US20040053297A1 (en) Cytokine-like proteins
US20060234218A1 (en) P450 proteins
WO2003066844A2 (fr) Proteine p450 cytochrome
WO2002046380A2 (fr) Proteine kinase
CA2436334A1 (fr) Molecules d'adhesion
WO2003089469A2 (fr) Proteines-canaux cationiques
WO2002029062A2 (fr) Proteines cytokine
WO2008003925A2 (fr) Protéine du complément
WO2007010283A1 (fr) Proteine serine de type beta-lactamase/d-aia carboxypeptidase
WO2002050116A2 (fr) Proteine molecule d'adhesion

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP