+

WO2003003012A1 - Identification de molecules d'interaction - Google Patents

Identification de molecules d'interaction Download PDF

Info

Publication number
WO2003003012A1
WO2003003012A1 PCT/AU2002/000856 AU0200856W WO03003012A1 WO 2003003012 A1 WO2003003012 A1 WO 2003003012A1 AU 0200856 W AU0200856 W AU 0200856W WO 03003012 A1 WO03003012 A1 WO 03003012A1
Authority
WO
WIPO (PCT)
Prior art keywords
molecule
target molecule
tissue
proteins
pool
Prior art date
Application number
PCT/AU2002/000856
Other languages
English (en)
Inventor
Robert James
Lawrence Eddie
Jan Kazenwadel
Susan O'connor
Pasquale Razzino
David Ward
Original Assignee
Medimolecular Pty. Ltd.
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 Medimolecular Pty. Ltd. filed Critical Medimolecular Pty. Ltd.
Publication of WO2003003012A1 publication Critical patent/WO2003003012A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/56—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C59/40—Unsaturated compounds
    • C07C59/58—Unsaturated compounds containing ether groups, groups, groups, or groups
    • C07C59/64—Unsaturated compounds containing ether groups, groups, groups, or groups containing six-membered aromatic rings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures

Definitions

  • the present invention relates to methods for identifying candidate proteins that interact with target substrates.
  • the present invention also relates to candidate proteins identified using such methods, and to target molecules that can be used to identify candidate proteins.
  • the identification of the interactions between molecules is central to the understanding of how biologically active molecules exert their effect. In most cases, the molecules to which a biologically active molecule binds are not known. For this reason, the identification of the interacting partner molecule is an important step in the understanding of how a particular molecule exerts its effect. For example, many drugs have been identified for which the mechanism of action has not been determined. The identification of the molecule (or molecules) with which the drug interacts may allow further understanding of how the drug works, and therefore lead to the design or discovery of new drugs with enhanced efficacy.
  • the identification of molecules that interact with a target may be achieved in a number of ways. For example, in vitro methods such as affinity purification may be used. Alternatively, in vivo interaction assays may be used to identify interacting molecules. When the interacting molecules are proteins, genetic analyses may also reveal the identity of interacting proteins.
  • affinity purification methods generally rely on the principal of immobilisation of a target molecule on a solid support, passing a mixture of molecules over the solid support, washing the solid support so as to remove molecules that do not bind, and isolating the molecules that remain bound to the immobilised target.
  • Molecules that remain bound to the target are generally ones that bind to the target molecule with a higher affinity, and are therefore more likely to be of biological relevance.
  • the lower affinity binding molecules that are removed by the selected washing treatment are usually less likely to be of biological relevance.
  • targets include small organic molecules, cofactors, nucleic acids, small peptides, proteins, oligosaccharides and lipids.
  • Affinity purification methods may generally be used to isolate any type of molecule that interacts with a target molecule of interest. As many biologically active molecules exert their effects by binding to proteins, affinity purification methods have been used to isolate proteins that bind to a specific target. In such cases, the candidate binding protein may often need to be isolated from a complex mixture of proteins.
  • Methods have become available whereby very complex pools of candidate binding molecules may be generated for the purposes of screening. This may be important in cases where a candidate binding molecule is likely to be present at a very low abundance. In such a case, the likelihood of finding an appropriate binding molecule will increase with increasing complexity of the mixture to be screened.
  • Methods for generating complex mixtures of potential binding molecules include, for example, the chemical synthesis of short random peptides and the chemical synthesis of short random nucleic acid molecules (aptamers).
  • Complex mixtures of proteins may be generated by the cloning of a large number of individual cellular DNAs into expression systems to form a library of proteins expressed from the cloned DNA molecules.
  • a pool of complementary DNAs cDNAs
  • cDNAs complementary DNAs
  • the proteins expressed from such pools or libraries may be used as the source of potential binding proteins for screening by affinity purification.
  • phage display One method for screening complex mixtures of proteins using an affinity purification approach is the method referred to as "phage display”.
  • various DNAs are cloned in a viral nucleic vector.
  • the DNAs are inserted into a viral gene that normally expresses a protein that is found on the surface of the virus.
  • viral particles When viral particles are produced, the protein expressed from the particular DNA inserted into the virus DNA will be displayed on the surface of the virus.
  • a library of DNA molecules is cloned into the viral vector, each virus produced will display a different protein from the library on its surface.
  • the complex mixture of viral particles so produced may be passed over a target molecule immobilised to a solid support.
  • the solid support is washed and the viral particles that remain bound are collected.
  • Phage display methods are useful for the screening of potential candidate binding proteins, because once the viral particles binding to the target are isolated, the viral particles may be allowed to infect new cells and produce a new enriched population of viral particles.
  • the resultant new mixture of viral particles may then be re-applied to the immobilised target molecule and the process reiterated. In this way, a population of viral particles may be successively enriched for those viral particles expressing candidate binding proteins on their surface.
  • the present invention relates to an improved method for isolating proteins from complex mixtures, by providing means to improve the likelihood that the proteins identified are biologically relevant to the desired activity or function of the target molecule.
  • the present invention provides a method for identifying a protein capable of binding to a target molecule, the method including the steps of:
  • the present invention provides a method for identifying a protein capable of binding to a target molecule, the method including the steps of:
  • the present invention provides a method for identifying a protein capable of binding to target molecule, the method including the steps of: (a) providing a first pool of candidate proteins;
  • the present invention provides a method for identifying a protein capable of binding to a target molecule, the method including the steps of:
  • the present invention relates to a method for identifying proteins that are able to bind to a target molecule, the target molecule preferentially being a biologically active molecule.
  • the identification of proteins that are able to bind to such a target molecule may be important for a number of reasons. For example, it may allow the identification of the proteins that the target molecule binds to in order to exert its biological effect. It may also allow the identification of new proteins that themselves may serve as drugs to inhibit or augment the biological activity of the target molecule.
  • the presence of a second molecule in the binding reaction may diminish the binding to the target molecule of proteins that inhibit the binding of biologically relevant proteins.
  • the use of the second molecule may serve to diminish a number of inhibitory interactions and thereby may allow binding of biologically relevant proteins.
  • the use of reiterated cycles of binding and amplification of viral particles may further augment these effects of the second molecule.
  • the methods according to the present invention allow the identification of candidate proteins that are more likely to interact with a target molecule in a manner that is associated with a desired activity of the target molecule.
  • the methods of the present invention also allow the identification of candidate proteins that interact with a specific region of a target molecule.
  • the method of the present invention allows the identification of candidate proteins that are not only more likely to be biologically relevant, but which are also differentially represented between pools of candidate proteins.
  • the target molecule is either of the non- steroidal antiinflammatory drugs, flurbiprofen or sulindac sulfide or biologically active analogues of either of these.
  • the second molecule may be an analogue of either flurbiprofen or sulindac sulfide that is inactive or has a reduced biological activity relative to the parent compound.
  • the present invention provides an analogue of flurbiprofen, the analogue having the formula (I):
  • R 1 is selected from hydrogen and lower alkyl (C1 to C8);
  • R 2 is YX 2 ((CH 2 )m X 2 )n-, wherein m is 2 to 4, n is 1 to 6, X 2 is selected from O, S and N, and Y is independently selected from hydrogen, lower alkyl, or a suitable heteroatom protecting group;
  • R 3 is selected from one or more of hydrogen, alkyl, aryl, halogen, hydroxy, alkoxy, aryloxy, amino (unsubstituted and substituted) and caroboxy;
  • R 4 is selected from one or more of hydrogen, alkyl, aryl, halogen, hydroxy, alkoxy, aryloxy, amino (unsubstituted and substituted) and caroboxy;
  • - X is selected from fluoro, chloro, bromo and iodo
  • - M is selected from hydroxy, alkoxy, aryloxy, amino, alkylamino (mono- and di-), arylamino (mono- and di-), N-morpholino, hydroxyalkylamino, dialkylaminoalkylamino, aminoalkylamino, polyhydroxyamino, and salts of any of the aforementioned.
  • the present invention provides an analogue of sulindac sulfide, the analogue having the formula (II):
  • - X 1 is selected from sulfide, sulfone and sulfoxide; - R 1 is selected from hydrogen, hydroxy (when X 1 is sulfone or sulfoxide), and lower alkyl (C1 to C8);
  • - R 2 is YX 2 ((CH 2 ) m X 2 )n-, wherein m is 2 to 4, n is 1 to 6, X 2 is selected from O, S and N, and Y is independently selected from hydrogen, lower alkyl, or a suitable heteroatom protecting group;
  • - R 3 is selected from hydrogen, halogen, alkyl, alkoxy, acyloxy, amino, alkylamino (mono- and di-), arylamino (mono- and di-), nitro, cyano, carboxyl;
  • R 4 is selected from hydrogen and lower alkyl (C1 to C8);
  • - M is selected from hydroxy, alkoxy, aryloxy, amino, alkylamino (mono- and di-), arylamino (mono- and di-), N-morpholino, hydroxyalkylamino, dialkylaminoalkylamino, aminoalkylamino, polyhydroxyamino, and salts of any of the aforementioned.
  • the compounds of formula (I) or (II) may be used as target molecules or second molecules to identify proteins that interact with flurbiprofen or sulindac respectively.
  • non-nucleic acid target molecule as used throughout the specification is to be understood to mean any molecule to which a protein may bind, but which is not a nucleic acid molecule.
  • the target molecule may include drug molecules, proteins, peptides, polypeptides, polysaccharides, glycoproteins, hormones, receptors, lipids, small molecules, metabolites, cofactors, transition state analogues and toxins.
  • the second molecule will be a molecule that is structurally similar to the non-nucleic acid target molecule.
  • structurally similar as used throughout the specification is to be understood to mean a molecule that is similar to the non-nucleic acid target molecule in terms of its three dimensional structure.
  • structurally similar molecules may include isomeric molecules such as isomers, geometric isomers, enantiomers, conformers, stereoisomers, structural isomers, molecules that substitute one or more chemical groups in a molecule with other chemical groups, or molecules that are substantially similar in the three dimensional structure of one or more parts of the molecule.
  • protein as used throughout the specification is to be understood to mean any polypeptide consisting of two or more constituent amino acids.
  • the polypeptide may also contain one or more side chains derived from a modified amino acid.
  • viral particle as used throughout the specification is to be understood to mean any virus with a protein coat, wherein the virus genome contains a gene for a coat protein that will allow the display of a subject protein on the surface of the protein, when the DNA encoding the subject protein is inserted into the gene for an appropriate coat protein.
  • the viral particle according to the present invention may include bacteriophage particles.
  • selectable moiety as used throughout the specification is to be understood to mean any chemical group that is linked to the target molecule and which may be used to substantially purify the target molecule.
  • the present invention provides a method for identifying a protein capable of binding to a target molecule, the protein so identified being more likely to be interact with the target molecule in a manner that is associated with a desired activity of the target molecule.
  • the target molecule according to the methods of the present invention may be any molecule to which a protein may bind, but which is not in itself a nucleic acid molecule.
  • the target molecule may include drug molecules, proteins, peptides, polypeptides, polysaccharides, glycoproteins, hormones, receptors, lipids, small molecules, metabolites, cofactors, transition state analogues and toxins.
  • the target molecule will have an activity or function that is of interest, referred herein to as "a desired activity" of the target molecule.
  • the activity of the target molecule may be an activity associated with one or more biological effects of the target molecule on a biological system.
  • the desired activity of the target molecule may be associated with its ability to control the proliferation of neoplastic cells.
  • the desired activity may also be any activity that is associated with a particular region (or regions) of a target molecule.
  • the desired activity may be the activity associated with a region or regions of a drug molecule or a specific protein that has, or is likely to have, biological activity.
  • the target molecule is a drug molecule. More preferably, the drug molecule is flurbiprofen or sulindac sulfide.
  • flurbiprofen or sulindac sulfide.
  • an analogue of flurbiprofen that may be used as an active target molecule is the (f?)-stereoisomer of a molecule with the following chemical formula:
  • R 1 is selected from hydrogen and lower alkyl (C1 to C8);
  • R 2 is YX 2 ((CH 2 ) m X 2 )rr, wherein m is 2 to 4, n is 1 to 6, X 2 is selected from O, S and N, and Y is independently selected from hydrogen, lower alkyl, or a suitable heteroatom protecting group;
  • R 3 is selected from one or more of hydrogen, alkyl, aryl, halogen, hydroxy, alkoxy, aryloxy, amino (unsubstituted and substituted) and caroboxy;
  • R 4 is selected from one or more of hydrogen, alkyl, aryl, halogen, hydroxy, alkoxy, aryloxy, amino (unsubstituted and substituted) and caroboxy;
  • - X is selected from fluoro, chloro, bromo and iodo
  • - M is selected from hydroxy, alkoxy, aryloxy, amino, alkylamino (mono- and di-), arylamino (mono- and di-), N-morpholino, hydroxyalkylamino, dialkylaminoalkylamino, aminoalkylamino, polyhydroxyamino, and salts of any of the aforementioned.
  • X is preferably fluoro and most preferably substituted meta to the alkylcarboxylate group.
  • R 1 is a lower alkyl group, and is most preferably a methyl group.
  • R 2 is an alkyleneoxy or polyoxyalkylene chain, more preferably having between 1 and 4 alkyleneoxy repeating units. Suitable alkyleneoxy repeating units include ethyleneoxy and propyleneoxy. In one particularly preferred form of the invention, R 2 is a triethylene glycol group.
  • the R 2 0- group is substituted at a position para to the aryl substituent.
  • both R 3 and R 4 are hydrogen.
  • M is preferably hydroxy or a salt thereof.
  • the present invention provides a compound of formula (III):
  • the (S)-stereoisomer of compounds of formula (I) or (III) may be inactive or have a reduced activity relative to the (F?)-stereoisomer and therefore the (S)-stereoisomer may be suitable as a second molecule in the methods of the present invention.
  • the stereoisomers of compounds of formula (I) may be separated by any of the techniques used for that purpose in the art, including chromatography for example.
  • Compounds of formula (I) may be produced by any suitable synthetic method, including those known methods for the production of flurbiprofen.
  • An example of a suitable synthetic method is shown in Scheme 1 , in which the method includes the steps of hydroxylation of the unsubstituted phenyl ring in flurbiprofen, followed by substitution of the free phenol with the triethylene glycol group.
  • an analogue of sulindac sulfide that may be used as an active target molecule is a molecule with the following chemical formula:
  • - X 1 is selected from sulfide, sulfone and sulfoxide; - R 1 is selected from hydrogen, hydroxy (when X 1 is sulfone or sulfoxide), and lower alkyl (C1 to C8);
  • - R 2 is YX 2 ((CH 2 )m X 2 )n-, wherein m is 2 to 4, n is 1 to 6, X 2 is selected from O, S and N, and Y is independently selected from hydrogen, lower alkyl, or a suitable heteroatom protecting group;
  • - R 3 is selected from hydrogen, halogen, alkyl, alkoxy, acyloxy, amino, alkylamino (mono- and di-), arylamino (mono- and di-), nitro, cyano, carboxyl;
  • R 4 is selected from hydrogen and lower alkyl (C1 to C8);
  • - M is selected from hydroxy, alkoxy, aryloxy, amino, alkylamino (mono- and di-), arylamino (mono- and di-), N-morpholino, hydroxyalkylamino, dialkylaminoalkylamino, aminoalkylamino, polyhydroxyamino, and salts of any of the aforementioned.
  • X 1 is preferably either a sulfone or a sulfide, and is most preferably a sulfide.
  • R 1 is a lower alkyl group, and is most preferably a methyl group.
  • R 2 is an alkyleneoxy or polyoxyalkylene chain, more preferably having between 1 and 4 alkyleneoxy repeating units. Suitable alkyleneoxy repeating units include ethyleneoxy and propyleneoxy. In one particularly preferred form of the invention, R 2 is a triethylene glycol group.
  • R 3 is a halogen group (iodo-, bromo-, chloro- or fluoro-), more preferably a fluoro group, and most preferably a fluoro group ortho to the hydroxy group.
  • R 4 is a lower alkyl group and is most preferably methyl.
  • M is preferably hydroxy or a salt thereof.
  • the invention provides a compound of formula (IV), or a salt thereof:
  • compounds of formula (II) and (IV) may be formed by any one of a number of synthetic routes.
  • the present invention also provides a process for the preparation of compounds of formula (II), the process including the steps of: intramolecular cyclisation of acyl donor (a) to form ketone (b); enolate addition to (b) followed by dehydration to form indene (c); and addition of (c) to aldehyde (d) to form indene (e).
  • the second molecule according to the methods of the present invention is any molecule that is structurally similar to the target molecule, but which does not show a desired activity associated with the target molecule.
  • Structurally similar molecules include isomeric molecules such as isomers, geometric isomers, enantiomers, conformers, stereoisomers, structural isomers, molecules that substitute one or more chemical groups in a molecule with other chemical groups, or molecules that are substantially similar in the three dimensional structure of one or more parts of the molecule.
  • the second molecule may include a drug analogue that shows reduced activity as compared to the biologically active drug.
  • the second molecule may be an analogue of a known drug, such as flurbiprofen or sulindac sulfide.
  • the second molecule may also be a molecule that is structurally similar to the target molecule, but which has been altered in a region (or regions) that is of interest in the target molecule.
  • an analogue of flurbiprofen that may be utilised as a second molecule in the methods of the present invention is the molecule with the following chemical formula:
  • This molecule is the (S)-stereoisomer.
  • the (fi)-stereoisomer shows similar activity to flurbiprofen, while the (S)-stereoisomer is inactive.
  • the source of the pool of candidate proteins in the methods of the present invention may be any source of proteins, including proteins derived from cellular or viral extracts, proteins displayed on the surface of a viral particle by cloning one or more DNAs into a suitable viral vector, the protein products of one or more DNAs cloned into a protein expression vector and expressed in a suitable expression system, the products of in vitro translation of different mRNAs, or chemically synthesized polypeptides or proteins.
  • the pool of candidate proteins may also contain post-translation modifications.
  • the pool of candidate proteins may include one or more glycosylated proteins.
  • the source of the pool of candidate proteins is a pool of proteins expressed from suitable DNA molecules inserted into a viral genome. More preferably, the source of the pool of candidate proteins is a pool of viral particles wherein each of the candidate proteins in the pool is displayed on the surface of a viral particle.
  • the source of the pools of candidate proteins includes cellular extracts derived from cell populations, group of cells, tissues or organs.
  • Cellular extracts may be prepared by suitable methods known in the art.
  • Cellular extracts may be derived from any prokaryotic or eukaryotic organism, including animals or humans.
  • the cellular extract may be derived from cells selected from one or more of the following types of tissue: colorectal tissue, breast tissue, cervical tissue, uterine tissue, renal tissue, pancreatic tissue, esophageal tissue, stomach tissue, lung tissue, brain tissue, liver tissue, bladder tissue, bone tissue, prostate tissue, skin tissue, ovary tissue, testicular tissue, muscle tissue or vascular tissue.
  • tissues may further contain cells that are normal (non-cancerous), pre- cancerous (having acquired some but not all of the cellular mutations required for a cancerous genotype) or cancerous cells (malignant or benign).
  • Such tissues may contain cells that are normal, pre-cancerous or cancerous, any combination of cells that are normal, pre-cancerous or cancerous, or any other form of diseased cell.
  • there are numerous methods well known in the art for determining whether cells are normal, pre- cancerous, cancerous or diseased including histopathology and other phenotypic and genotypic methods of identifying cells.
  • the source of the pool of proteins may also be a pool of proteins expressed from one or more DNAs inserted into a viral genome and which are displayed on the surface of the viral particle when expressed.
  • the DNAs inserted may be complementary DNAs (cDNAs), DNA fragments derived from genomic or viral DNAs, or chemically synthesized DNAs.
  • the DNAs inserted into the viral genome may also make up a library of DNAs, including libraries of cDNAs produced by reverse transcription of cellular mRNAs and genomic DNA fragments.
  • the DNAs inserted into the viral genome may also include one or more random DNA sequences, resulting in the expression of random polypeptides.
  • the random DNAs so inserted may be chemically synthesized DNAs.
  • Suitable viruses for cloning of DNA so as to express proteins on the surface of the viral particle include bacteriophage viruses such as those derived from T7, T4, lambda, lambdoid phage, or filamentous phage, including M13, f1 and fd.
  • the DNAs may be cloned into the vectors derived from these phage by suitable methods known in the art.
  • the insertion of the DNA to be expressed into an appropriate coat protein gene allows the protein encoded by the DNA to be displayed on the surface of a viral particle.
  • Viruses displaying proteins on their surface may then be produced by infection of a suitable host and preparation of viral extracts by suitable methods that are known in the art.
  • the source of the pool of proteins may also be a pool of proteins expressed from one or more DNAs inserted into a vector (plasmid vector or viral genome) and expressed in a suitable expression system.
  • the DNAs inserted may be complementary DNAs (cDNAs), DNA fragments derived from genomic or viral DNAs, or chemically synthesized DNAs.
  • the DNAs inserted into the vector may also make up a library of DNAs, including libraries of cDNAs and genomic DNA fragments.
  • the DNAs inserted into the vector may also include one or more random DNA sequences, resulting in the expression of random polypeptides.
  • the random DNAs so inserted may be chemically synthesized DNAs.
  • the selectable moiety coupled to the non-nucleic acid target molecule is any moiety that allows the target molecule to be substantially purified away from other molecules, including the pool of candidate proteins.
  • the selectable moiety may be a chemical group such as an activated carbonate group that allows the target molecule to be covalently linked to a solid support.
  • covalent coupling of the protein molecule to the solid support also includes coupling to the solid support via primary amines or cysteines by suitable methods that are known in the art.
  • a chemical moiety such as a biotin containing group may be coupled to the target molecule allowing the target molecule to be captured by an avidin or streptavidin group coupled to a solid support.
  • Further examples of methods of immobilisation of the target molecule include coupling of an antigen to the target molecule and capture by an immobilised antibody. Immobilisation of the target molecule may also utilise capture of glutathion-S-transferase-fusion proteins by anti GST antibodies, capture of 6xHis- fusion proteins by anti 6xHis antibodies or a nickel-chelating surface, capture of cAMP or cGMP binding proteins by cAMP or cGMP immobilised on a solid support.
  • the target molecule may be coupled to a protein molecule to be captured, or alternatively, the target molecule may be a protein engineered to be able to be captured by one of these methods.
  • the binding of the pool of candidate proteins to the non-nucleic acid target molecule in the presence of the second molecule in the methods of the present invention may be achieved under conditions suitable to the particular pool of candidate proteins being used.
  • the temperature and solution may be selected depending upon the properties of the pool of candidate proteins being used.
  • the temperature of binding may be within the range from 4°C to 42°C, and the binding achieved in a suitable buffer, including the use of tris-based and/or phosphate buffered solutions.
  • Such solutions may further include appropriate amounts of further components, including salts, detergents and other agents depending upon the properties of the particular pool of candidate proteins being used.
  • the target molecule may be free in solution when binding occurs (and then subsequently captured), or alternatively be immobilised to a solid support during the binding reaction.
  • the second molecule may be free in solution, or alternatively, be immobilised to a solid support.
  • the binding of the candidate proteins to the target molecule in the presence of the second molecule is performed under conditions where the second molecule is present in a molar excess to the target molecule.
  • the second molecule is in a molar excess of at least one hundred fold.
  • the ratio of the second molecule to the target molecule may also be selected so as to obtain binding proteins that bind to the target molecule with varying affinity.
  • the proteins bound to the target molecule may be isolated by a suitable means. If the target molecule is coupled to a fixed solid support, the proteins bound to the target molecule may be isolated by washing the solid support in a suitable buffer to remove any proteins that do not bind to the target molecule. If the target molecule is coupled to a solid support such as beads (for example paramagnetic beads), the beads may first be isolated and the proteins that do not bind removed by washing the beads in a suitable buffer. Alternatively, the target molecule may be captured and thus immobilised on the solid support, and proteins that do not bind them removed by washing.
  • the removal of proteins bound to the target molecule may also be achieved by eluting in a suitable buffer containing free target molecule in substantial molar excess.
  • the free target molecule is in a molar excess of greater than one thousand fold.
  • the eluates may be isolated at different times during the washing procedure to allow for the identification of binding proteins that have different dissociation rates.
  • the proteins so isolated in the methods of the present invention may be subject to methods to allow their identification and/or characterisation. For example, if the proteins isolated are sufficiently pure, the amino acid sequence of a binding protein isolated according to the present invention may be determined by a method that includes determination of the amino acid sequence. Alternatively, mass spectrometry may be performed.
  • the identity of the expressed protein may be determined directly by determination of the DNA sequence of the DNA inserted into the viral genome that results in expression of the protein on the surface of the virus. As will be appreciated, determination of the DNA sequence will allow the prediction of the amino acid sequence of the protein that is displayed on the surface of the viral particle.
  • the proteins so isolated that bind to the target molecule may be rebound to the target molecule in the presence of the second molecule. In this way the process may be re-iterated, until a desired population of proteins that bind to the target molecule is achieved.
  • Confirmation of the ability of a binding protein isolated by the methods of the present invention to bind to the target molecule may be by a suitable method known in the art.
  • the protein may be substantially purified and then the binding affinity of the isolated protein to the target molecule be determined by a suitable means, including binding assays, surface plasmon resonance or atomic force microscopy.
  • the binding affinity of a phage particle displaying the binding protein to the drug may be determined by a suitable means, including surface plasmon resonance or atomic force microscopy.
  • the present invention provides a method for identifying a protein capable of binding to a target molecule, the method including the steps of:
  • the source of the pool of proteins is a pool of proteins expressed from one or more DNAs inserted into a viral genome, the proteins so expressed being displayed on the surface of the viral particle.
  • Suitable viruses for cloning of DNA so as to express proteins on the surface of the viral particle include bacteriophage viruses such as those derived from T7, T4, lambda, lambdoid phage, or filamentous phage, including M13, f1 and fd.
  • the DNAs may be cloned into vectors derived from these phage by suitable methods known in the art.
  • the DNAs inserted into such viral vectors may be complementary DNAs (cDNAs), DNA fragments derived from genomic or viral DNAs, or chemically synthesized DNAs.
  • the DNAs inserted into the viral genome may also make up a library of DNAs, including libraries of cDNAs (for example obtained by the reverse transcription of cellular mRNAs) and genomic DNA fragments.
  • the DNAs inserted into the viral genome may also include one or more random DNA sequences, resulting in the expression of random polypeptides.
  • the random DNAs so inserted may be chemically synthesized DNAs.
  • Viruses displaying proteins on their surface may then be produced by infection of a suitable host and preparation of viral extracts by methods that are well known in the art. In this way, a pool of candidate proteins in which each candidate protein is displayed on the surface of the viral particle may be produced.
  • the amplification of viral particle encoding the isolated proteins may be achieved by re-infecting a competent viral host with the isolated viral particles by a suitable procedure.
  • the viral particles so concentrated may be concentrated by a suitable means, so as to allow the process of binding and isolating the proteins that bind to the target molecule to be reiterated.
  • a proportion of the DNA inserts that make up the viral population after amplification may be characterised for insert size and their DNA sequence.
  • the DNA inserted into each viral particle may be isolated by obtaining a pure viral population by way of an isolated plaque and isolating the DNA inserted in that particular viral DNA by polymerase chain reaction using appropriate primers.
  • the size of the DNA inserts may be determined by a suitable method.
  • the DNA sequence of the DNA insert may be determined by a suitable method.
  • the reiteration step of this form of the present invention is continued until a desired level of representation of the DNA inserts is reached in the viral population.
  • the representation of the DNA inserts in the viral population may be determined by a suitable method. Preferably five or more reiterations are performed.
  • the identity of the binding protein may then be determined by determination of the DNA sequence of the DNA inserted into the viral genome that results in expression of the protein on the surface of the virus. As will be appreciated, determination of the DNA sequence will allow the prediction of the amino acid sequence of the protein expressed on the surface of the viral particle.
  • the present invention also provides a method for identifying a protein capable of binding to target molecule, the method including the steps of: (a) providing a first pool of candidate proteins;
  • the identification of proteins that bind to a target molecule and which are differentially represented between two pools of candidate proteins may be achieved.
  • the determination of the level of a protein in the first pool may be achieved by a suitable procedure known in the art, including the determination of the concentration by methods that include the use of antibodies to detect the binding protein.
  • the concentration of the binding protein in the first pool may be achieved with an antibody raised to the binding protein, and the subsequent use of the antibody to visualise the protein by Western analysis or the use of the antibody to immunoprecipitate the protein.
  • the level of the binding protein in a second pool of candidate proteins may then be determined in a similar fashion. In this way, proteins isolated from the first pool of candidate proteins may be compared with a second pool of candidate proteins, so as to identify proteins that are differentially represented between the two pools of binding proteins.
  • the first pool of proteins may be derived from a tissue that contain cells that are normal (non-cancerous), and the second pool of proteins may be derived from cells that are pre-cancerous (having acquired some but not all of the cellular mutations required for a cancerous genotype) or cancerous cells (malignant or benign). Any differences in the level of a binding protein between the pool of proteins derived from a normal tissue and another tissue allows the identification of binding proteins that are differentially expressed between the two different pools of proteins.
  • the present invention also provides a method for identifying a protein capable of binding to target molecule, the method including the steps of:
  • the identification of proteins that bind to a target molecule and which are differentially represented between two pools of candidate proteins may also be achieved.
  • the reactions utilising the first and second pools are performed in parallel experiments under exactly the same conditions.
  • the determination of the level of a protein isolated from the first pool may be achieved by a suitable procedure, including the determination of the concentration by methods that include the use of antibodies to detect the binding protein.
  • concentration of the binding protein isolated from the first pool of candidate proteins may be achieved with an antibody raised to the binding protein, and the subsequent use of the antibody to visualise the protein by Western analysis or the use of the antibody to immunoprecipitate the protein.
  • the level of the binding protein isolated from a second pool of candidate proteins may then be determined in a similar fashion. In this way, proteins isolated from the first pool of candidate proteins may be compared with those isolated from a second pool of candidate proteins, so as to identify isolated proteins that are differentially represented between the two pools of binding proteins.
  • the first pool of proteins may be derived from a tissue that contain cells that are normal (non-cancerous), and the second pool of proteins may be derived from cells that ate pre-cancerous (having acquired some but not all of the cellular mutations required for a cancerous genotype) or cancerous cells (malignant or benign). Any differences in the level of an isolated binding protein between the protein isolated from a normal tissue and that isolated from another tissue allows the identification of binding proteins that are differentially represented between the two different pools of proteins.
  • Sulindac sulfide is a drug that acts to decrease the number of precancerous lesions (adenomas) in the colon both in animals and in humans.
  • sulindac sulfide The structure of sulindac sulfide is as follows:
  • the diester (12) was added to a solution of potassium hydroxide (40.00 g, 0.713 mol) in water (60 cm 3 ) and ethanol (50 cm 3 ) and the mixture refluxed for 16 h.
  • the acid (13) (26.95 g, 0.127 mol) was added to polyphosphoric acid (270 g) at room temperature.
  • the mixture was swirled by hand in an oil bath heated to 90° until homogeneity was achieved.
  • the viscous solution was stirred magnetically for a further 2 h at 90°.
  • the hot mixture was then poured on to crushed ice (500 g), ether (100 cm 3 ) was added and the mixture stirred at room temperature for 15 h. The layers were separated and the aqueous layer washed with more ether
  • the methoxyindanone (14) (23.00g, 0.118 mol) and tetrabutylammonium bromide (3.80 g, 11.8 mmol) were dissolved in 48% aqueous hydrogen bromide (130 ml) and the stirred solution was heated at 115 ° for 5.5 h. Upon cooling, water (300 cm 3 ) and ether (200 cm 3 ) were added and the resultant layers separated. The aqueous layer was washed with more ether (2x50 cm 3 ) and these washings were combined with the original ether layer and extracted with 5% aqueous sodium hydroxide (2x100 cm 3 ).
  • SicapentTM (Merck, 17.8 g; 80% diphosphorus pentoxide, 13.4 g, 94.2 mmol) was added to a solution of the alcohol (17) (18.00 g, 47.1 mmol) in benzene. The mixture was refluxed for 0.5 h, cooled and filtered through flash silica. Elution with more benzene (50 cm 3 ) was followed by elution with ether until the eluate was colourless. The benzene and ether solutions were pooled and evaporated in vacuo to give a mixture of dehydration products (16.29 g, 95%) as a yellow- orange oil. 1 H-NMR indicated that the indene (18) was the major product (ca. 75%). (CDCI3, 300 MHz): ⁇ 0.18 (s, 6H); 1.01 (s, 9H); 1.24 (t, J 7 Hz, 3H); 2.09
  • the silyl ether (18) was added to ethanol (200 cm 3 ) which had been pre-treated with acetyl chloride (4.3 g, 55 mmol) and the solution refluxed for 3 hours. Upon cooling, the volatile components of the mixture were removed in vacuo and the residue was taken up into ether (100 cm 3 ). The ether solution was extracted with 1 M aqueous sodium hydroxide solution (50 cm 3 then 25 cm 3 ) and then discarded. Acidification of the aqueous washings with 25% w/w aqueous sulfuric acid was followed by extraction with ether (1x70 cm 3 then 2x30 cm 3 ).
  • the acid (19) (9.12 g, 41.0 mmol) was added to a solution of concentrated sulfuric acid (2.00 g, 20.4 mmol) in ethanol (100 cm 3 ). The solution was refluxed for 3.5 h, cooled and the ethanol removed in vacuo. The residue was taken up into ether (100 cm 3 ) and the solution washed with water (3x50 cm 3 ) followed by saturated aqueous sodium chloride. The ether solution was dried (MgS ⁇ 4), filtered and the ether evaporated in vacuo to give the ester (20) (9.75 g, 95%) as a beige solid.
  • Diethyl azadicarboxylate (3.05 g, 17.5 mmol) was added slowly to a stirred solution of the alcohol (21) (6.26 g, 15.9 mmol), the phenol (20) (3.99 g, 15.9 mmol) and triphenylphosphine (4.60 g, 17.5 mmol) in dry tetrahydrofuran (70 cm 3 ) at 0 ° .
  • Stirring was continued at 0° for 1 h and then for a further 66 h at room temperature, at which time TLC analysis indicated all of the phenolic starting material had been consumed.
  • a ca. 0.5M solution of sodium methoxide in methanol was made by adding sodium (229 mg, 9.52 mmol) to dry methanol (20 cm 3 ).
  • the indene (22) (2.98 g, 4.76 mmol) and 4-methylthiobenzaldehyde (797 mg, 5.24 mmol) were dissolved in this solution by swirling of the flask by hand.
  • the resultant bright purple solution was refluxed for 1 h. During this time the solution turned orange and an orange oil separated out. Water (20 cm 3 ) was added and reflux continued for a further 0.5 h and the mixture became homogeneous. Water (250 cm 3 ) and ether
  • Example 1.14 Synthesis of 2-(6-fluoro-5- ⁇ 2-[2-(2- hydroxyethoxy)ethoxy]ethoxy ⁇ -2-methyl- 1 -[(4-methylthio ⁇ henyl)methylene]inden-
  • the trityl ether (23) (2.60 g, 3.56 mmol) was dissolved in a mixture of formic acid (40 cm 3 ) and ether (40 cm 3 ). After 16 h at room temperature, the solvents were evaporated in vacuo and the residue was taken up into ether (50 cm 3 ) and 0.2 M aqueous sodium hydroxide (50 cm 3 ).
  • This compound was used as an example of the chemistry that may be used to attach a drug to a solid phase.
  • Example 1.17 - Synthesis of TentaGel bound methyl 2-(6-fluoro-5- ⁇ 2-[2-(2- hydroxyethoxy)ethoxy]ethoxy ⁇ -2-methyl-1-[(4-methylthiophenyl)methylene]inden- 3-yl)acetat ⁇ (27)
  • TentaGel S-NH2 (Fluka, ca. 0.45 mmol N/g resin, particle size 150-200 mm; 50 mg) was added to a solution of the carbonate (26) (20 mg, 30 mmol) and N- methylmorpholine (30 mg, 0.30 mmol) in dimethylformamide (1 cm 3 ). The mixture was shaken at room temperature for 16 h and filtered. The resin was washed with dimethylformamide (3x) followed by methanol (3x) then dried in vacuo lo give 61 mg of the resin (27).
  • IR (potassium bromide disc): v 1734, 1718 cm -1 .
  • the resin (27) (58 mg) was added to a 0.25 M solution of sodium hydroxide in 2:1 ethanol water (4 cm 3 ). The mixture was shaken at room temperature for 4 h and filtered. The resin was washed with water (3x) then 1 M hydrochloric acid (3x), water (3x) and finally methanol (3x). Drying in vacuo gave 58 mg of the modified resin.
  • IR potassium bromide disc
  • the sulindac analogue (IV) was conjugated with glycine and the amine thus produced was coupled to a commercially available biotin derivative to give the biotin labelled compound (29).
  • the biotin derivative was then attached to a solid support by way of the biotin moiety using a biotin:streptavidin coupling technique.
  • biotinylated derivative (30) of sulindac sulfide was also produced by carbodiimide mediated coupling of sulindac sulfide with a commercially available biotin derivative.
  • biotinylated derivatives (31) and (32), were produced via the same chemistry and may be coupled to a solid phase by way of biotin:streptavidin coupling.
  • Flurbiprofen is a drug that acts to decrease the number of precancerous lesions (adenomas) in the colon in animals.
  • the structure of flurbiprofen is as follows:
  • the methyl ester (46) (1.60 g, 3.94 mmol) was dissolved in a solution of sodium hydroxide (520 mg, 13.0 mmol) in water (6.5 cm 3 ) and ethanol (13 cm 3 ). The solution was stirred at room temperature for 2 h and then adjusted to pH 7 with hydrochloric acid. The ethanol was removed in vacuo and the residue extracted with ether. The ether solution was dried (MgS ⁇ 4), filtered and the solvent removed to yield the acid (1.05 g, 68%) as an off-white solid. The aqueous layer was acidified to pH 1 and again extracted with ether; processing of the ether solution as above yielded more of the acid (III) (389 mg, 25%) as a white solid.
  • Phage display libraries were constructed using standard protocols for directional cloning of cDNA.
  • the bacteriophage phage display lambda vectors were prepared as outlined below. The vectors used were T7 Select 1-1 b, T7 Select 10-3, ⁇ fooDc and ⁇ vsx.1.
  • cDNA libraries were made using RNA isolated from human adenomas and normal colonic tissue. The use of two different cDNA libraries not only allows the identification of proteins in a particular sample that bind to a target molecule in the presence of a second molecule, but also allows the identification of any binding proteins that may be differentially represented between the two populations of cells.
  • cDNA for cloning into the T7 Select vector was synthesised using standard procedures with Hindlll random primers and EcoRUHin ⁇ W linkers.
  • cDNA for cloning into ⁇ fooDc was synthesised using EcoR ⁇ random primers (5' TCNNNNNN 3') and Hind ⁇ /EcoR ⁇ linkers (5' ATTCAAGCTTGAAT 3').
  • cDNA for cloning into ⁇ vsx.1 was synthesised using ⁇ /ofl random primers (5' GCNNNNNN 3') and EcoRVNott linkers (5' GGCCGCGAATTCGCGGCC 3').
  • Size selection of cDNA was performed on Size-Sep 400 columns according to standard protocols.
  • Vector arms and cDNA were ligated overnight at 16°C, at a cDNA to arms ratio of 0.5 ⁇ g:25ng in a volume of 10 ⁇ l or less. Ligations were packaged according to standard protocols. The libraries were titred, amplified and stored using standard protocols.
  • bacteriophage DNA was isolated by the plate lysate method, by plating 20 x 150mm dishes for confluent lysis (4 x 10 s pfu/dish) using LE392MP as host cells.
  • the phage was eluted in SM buffer (10ml/dish) for 6 hours, harvested and 1 % v/v CHCI 3 added.
  • the eluate was spun at 3000rpm for 10 minutes and the supernatant recovered.
  • the DNase I concentration was adjusted to 2 ⁇ g/ml and incubated at 37°C for 60 minutes.
  • the mixture was adjusted to 10% w/v PEG8000, 100mM NaCl by dissolution and phage precipitated at 4°C overnight.
  • the mixture was spun at 4500rpm for 20 minutes and the phage pellet resuspended in 4ml SM buffer and transfered to 1.5ml microfuge tubes. A spin of 2 minutes at room temperature was performed, supernatant pooled and TEAE-cellulose pre-equilibrated as follows was added: Wash 1.5 g dry weight resin in 3 x 10 ml SM buffer
  • the cos ends were ligated as follows: Mix 42 ⁇ l ⁇ DNA (0.5 ⁇ g/ ⁇ l)
  • Vector Arms were prepared as follows: ⁇ vsx.1 Digest with EcoR ⁇ and ⁇ /ofl ⁇ fooDc Digest with HindlW and EcoR ⁇
  • Ligation reactions were adjusted to 1 x EcoR ⁇ buffer in a final volume of 200 ⁇ l and included 40 units each of the appropriate enzymes (also include 0.1 mg/ml BSA in the ⁇ vsx.1 digest). Reactions were incubated at 37°C overnight. A further 10 units of each enzyme was added and incubated overnight at 37°C.
  • Vectors were then treated with alkaline phosphatase to reduce background of non-recombinants in the library. Ligation/digestion was monitored by agarose gel electrophoresis. The vector was extracted with buffered phenol/ CHCI 3 and the aqueous phase recovered. DNA was precipitated with 0.1 volumes 3M NaOAc and 2.5 volumes EtOH and resuspend in TE pH8.0. Concentration was adjusted to 0.5 ⁇ g/ ⁇ l.
  • Example 4 Library screening for proteins that bind to a specific drug coupled to a solid support, in the presence of a free drug analogue
  • Resin alone with linker attached, and drug covalenty coupled to coated resin were blocked with 2% skim milk in binding buffer by constant mixing for 1 hour at room temperature.
  • the resins were washed 5 times with 1.5 ml of binding buffer (20 mM Tris HCI pH 7.5, 0.25 M NaCl, 0.1 % Tween 20).
  • the resultant phage supernatant was then incubated with drug-coated resin (20 mg) at room temperature with constant mixing for 2 hours in the presence of 0.5 to 5 mM inactive drug analogue, being the (S)-isomer of the above molecule.
  • the drug-coated resin was washed 5 times with 1.5 ml of binding buffer.
  • the bound phage were eluted from the resin by washes with increasing concentrations of free drug from 10 nM-100 ⁇ M, using ten fold increments in binding buffer over a total period of 2-16 hours at room temperature with constant mixing.
  • T7 phage a further elution of the resin with 1% SDS for 5 minutes at room temperature was required.
  • the resin is stripped with 5% SDS, washed 5 times with binding buffer, and stored at 4°C for next round of selection.
  • Eluted phage were titred, amplified by re-infection of a competent host and the newly enriched pool phage titred using standard protocols.
  • the oligonucleotides used for amplifying inserts from T7 vectors are commercially available.
  • the specific oligonucleotides used for amplifying inserts from ⁇ fooDc are:
  • Oligonucleotides used for amplifying inserts from ⁇ vsx.1 are:
  • the aim of this screening strategy is to not only identify within the one experiment phage that may bind to the drug, but also to identify phage that may bind with differing affinities.
  • Example 6 Library screening for proteins that bind to a specific biotinylated drug in the presence of a drug analogue Binding of proteins displayed on the surface of a phage to the (R)-isomer of the following drug molecule in the presence of the inactive (S)-isomer:
  • the virus particles were washed 5 times with binding buffer (5x volume) at room temperature and phage eluted with 200 ⁇ M drug in binding buffer for 2-16 hours at room temperature.
  • the streptavidin particles may be saturated with biotinylated drug, 0.45 ml of 2 ⁇ M drug per 0.1 ml of particles.
  • the bound phage were then eluted from the paramagnetic particles by washes with increasing concentrations of free drug in binding buffer from 10 nM-100 ⁇ M, using ten fold increments over a total period of 2-16 hours at room temperature with constant mixing.
  • Eluted phage were titred, amplified by re-infection of a competent host and the new enriched pool phage titred using standard protocols.
  • oligonucleotides used for amplifying inserts from T7 vectors are commercially available.
  • the following oligonucleotides are suitable:
  • oligonucleotides used for amplifying inserts from ⁇ fooDc are: 5'-GACCGTTGGGCCAATTGTC and ⁇ '-TAAAACGACGGCCAGTGCC
  • Oligonucleotides used for amplifying inserts from ⁇ vsx.1 are: 5'-AAATTACCGTCACCGCCAGT and 5'-TTTGATGCCTGGCAGTTCC
  • the aim of this screening strategy was to not only identify within the one experiment phage that may bind to the drug, but also to identify phage that may bind with differing affinities.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

L'invention concerne un procédé permettant d'identifier une protéine capable de liaison avec une molécule cible spécifique. Ce procédé consiste à permettre à des protéines candidates de se lier à la molécule cible en présence d'une seconde molécule structurellement similaire à la molécule cible qui n'est pas un acide nucléique, mais déficiente en ce qui concerne une activité voulue de la molécule cible, et à isoler les protéines qui se lient avec la molécule cible. L'invention concerne également des analogues de flurbiprofène et de sulindac en tant que molécules cibles destinées à être utilisées dans les procédés de cette invention.
PCT/AU2002/000856 2001-06-29 2002-06-28 Identification de molecules d'interaction WO2003003012A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPR5986A AUPR598601A0 (en) 2001-06-29 2001-06-29 Identification of interacting molecules
AUPR5986 2001-06-29

Publications (1)

Publication Number Publication Date
WO2003003012A1 true WO2003003012A1 (fr) 2003-01-09

Family

ID=3829953

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2002/000856 WO2003003012A1 (fr) 2001-06-29 2002-06-28 Identification de molecules d'interaction

Country Status (2)

Country Link
AU (1) AUPR598601A0 (fr)
WO (1) WO2003003012A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004099774A3 (fr) * 2003-05-07 2006-01-12 Cellzome Ag Procede d'identification de nouvelles cibles medicales

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HEMMINKI A. ET AL.: "Specificity improvement of a recombinant anti-testosterone Fab fragment by CDRIII mutagenesis and phage display selection", PROTEIN ENGINEERING, vol. 11, no. 4, 1998, pages 311 - 319 *
PARSONS H.L. ET AL.: "Directing phage selections towards specific epitopes", PROTEIN ENGINEERING, vol. 9, no. 11, 1996, pages 1043 - 1049 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004099774A3 (fr) * 2003-05-07 2006-01-12 Cellzome Ag Procede d'identification de nouvelles cibles medicales

Also Published As

Publication number Publication date
AUPR598601A0 (en) 2001-07-19

Similar Documents

Publication Publication Date Title
Matsushita et al. Smart cleavage reactions: the synthesis of benzimidazoles and benzothiazoles from polymer-bound esters
CN104262378B (zh) 一种硅基罗丹明衍生物及其制备方法和应用
CN110938077B (zh) 合成Avapritinib的方法
WO2012083872A1 (fr) Molécule sonde à double fonction de marquage par photoaffinité, procédé de préparation et application de cette molécule
JP7292501B2 (ja) Ｃｄ７３阻害剤の結晶形態
Nazar et al. Synthesis, structure–activity relationship and molecular docking of cyclohexenone based analogous as potent non-nucleoside reverse-transcriptase inhibitors
Abet et al. Biased and unbiased strategies to identify biologically active small molecules
Besong et al. A synthesis of (aR, 7S)-(−)-N-acetylcolchinol and its conjugate with a cyclic RGD peptide
Ernst et al. Switching between bicyclic and linear peptides—the sulfhydryl-specific linker TPSMB enables reversible cyclization of peptides
CN104163799B (zh) 3-氨基-4-氰基异喹啉-1（2h）-酮衍生物的制备方法
WO2023088493A1 (fr) Composé furopyridone et utilisation associée
WO2003003012A1 (fr) Identification de molecules d'interaction
Qiu et al. Stereodivergent total synthesis of chlorofusin and its all seven chromophore diastereomers
CN107400070B (zh) 双胍类探针及其制备方法
Xing et al. Diastereoselective access to 2-aminoindanones via the rhodium (ii)-catalyzed tandem reaction involving O–H insertion and Michael addition
US5977400A (en) Support for synthesis and purification of compounds
CN109824664B (zh) 一组抗肿瘤吲哚类生物碱化合物及其制备方法和应用
CN104098507B (zh) 一种羟吲哚螺环丙烷衍生物及其合成方法
CN112400026A (zh) 用于稀有种类亲和定向富集的组合物和方法
EP3324972B1 (fr) Nouvelles molécules pour l'isolation de polyribosomes, ribosomes, utilisations et kits associés
Devereaux et al. Increasing thyromimetic potency through halogen substitution
CN103524555A (zh) 大黄酸氨基膦酸酯衍生物及其合成方法和应用
JP2014051463A (ja) Ｌｓｔ−１及び／又はｌｓｔ−２によって輸送される化合物
Kuang et al. Synthesis of biotinylated 2-methoxystypandrone and identification of JAK2 and IKK as its targets
CN115300520B (zh) 一种生物素-伊维菌素偶联物及其制备方法和应用

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

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
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

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载