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WO2001021652A1 - Procedes d'identification d'un agent corrigeant le repliement defectueux d'une proteine - Google Patents

Procedes d'identification d'un agent corrigeant le repliement defectueux d'une proteine Download PDF

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WO2001021652A1
WO2001021652A1 PCT/US2000/026193 US0026193W WO0121652A1 WO 2001021652 A1 WO2001021652 A1 WO 2001021652A1 US 0026193 W US0026193 W US 0026193W WO 0121652 A1 WO0121652 A1 WO 0121652A1
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polypeptide
peptide
defective
conformation
agent
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Peter L. Pedersen
Young Hee Ko
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Johns Hopkins University
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Johns Hopkins University
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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

Definitions

  • the invention relates generally to screening assays and molecular medicine, and more specifically to methods for identifying an agent that can correct a defective three dimensional conformation of a polypeptide.
  • Proteins which are the primary building blocks of cells, are involved in cell structure and function in an organism. Proteins are encoded by genes, and are produced by a complex process that includes synthesis of the primary amino acid sequence, folding of the synthesized sequence into a preferred three dimensional conformation, and transport of the protein to a particular location in the cell or secretion of the protein from the cell. A defect at any one of the steps in the production of a protein can lead to disease or death of an organism.
  • Cystic fibrosis is an inherited disorder that affects approximately 1 person in 2000 in the United States and Canada. The disease is characterized by lung infections, pancreatic insufficiency, and increased sweat chloride ion concentration. Individuals with severe cases of CF often die before the age of 30 due to chronic pulmonary infections with antibiotic-resistant bacteria.
  • cystic fibrosis transmembrane conductance regulator CFTR
  • the normal CFTR polypeptide is believed to function physiologically as a chloride ion channel to help regulate conduction pathways for chloride ion and sodium ion in epithelial cells
  • the mutant CFTR polypeptide appears to have the ability to function in the same way as the normal CFTR polypeptide, although with less efficiency.
  • the mutant CFTR polypeptide is not transported to its normal location in the cell membrane but, instead, remains inside the cell. As a result, cystic fibrosis occurs.
  • the present invention relates to a method of identifying an agent that corrects a defective three dimensional conformation of a polypeptide
  • a method of the invention can be performed, for example, by contacting in a cell-free system a peptide portion of the polypeptide with a test agent, wherein the peptide is representative of a region of the polypeptide exhibiting the defective conformation, and determining that the test agent corrects the defective conformation of the peptide portion of the polypeptide.
  • the defective three dimensional conformation can result in misfolding of the polypeptide, which, in turn, can result in aberrant cellular localization of the polypeptide or in aggregation of the polypeptide
  • a polypeptide exhibiting a defective three dimensional conformation is exemplified by a cystic fibrosis transmembrane regulator (CFTR) polypeptide, particularly a CFTR polypeptide having a deletion of Phe508, which is the most common mutation found in cystic fibrosis
  • a peptide portion of a CFTR polypeptide that is representative of a region of CFTR having the defective conformation is exemplified by a 25 amino acid peptide having the amino acid sequence set forth in SEQ ID NO 2
  • Additional polypeptides having a defective three dimensional conformation and, therefore, suitable for use in a screening method of the invention include, for example, conformationally defective forms of fibrillm, superoxide dismutase, collagen, a polypeptide of an ⁇ -ket
  • the step of determining whether a test agent corrects the defective conformation of the peptide portion of the polypeptide is performed by contacting the peptide with a fluorescent compound, and detecting a change in fluorescence intensity of the peptide m the presence of the test agent, wherein the change m fluorescence intensity to more closely approximate that of a corresponding wild-type peptide, is indicative of an agent that corrects the defective conformation of a polypeptide comprising the peptide
  • the step of determining whether a test agent corrects the defective conformation of the peptide portion of the polypeptide is performed by detecting a change in a nuclear magnetic resonance (NMR) spectrum or a circular dichroism (CD) spectrum of the peptide in the presence of the test agent, wherein the change in the NMR spectrum or CD spectrum, respectively, is indicative of an agent that corrects the defective conformation of a polypeptide
  • the step of determining whether a test agent corrects the defective conformation of the peptide portion of the polypeptide is performed by detecting specific binding of an antibody to the peptide in the presence of the test agent that corrects the defective conformation, wherein the antibody does not specifically bind the peptide in the absence of the test agent correcting the defective conformation of the peptide, and wherein specific binding of the antibody is indicative of an agent that can correct the defective conformation of a polypeptide.
  • a peptide portion of a polypeptide that is representative of a region of the polypeptide exhibiting the defective conformation can be identified using any method for determining the three dimensional conformation of a peptide, including, for example, X-ray crystallography, NMR spectroscopy, or CD spectroscopy.
  • the present invention also relates to a method of identifying an agent that corrects a defective three dimensional conformation of a polypeptide by identifying a peptide portion of the polypeptide that is representative of a region of the polypeptide exhibiting the defective conformation; synthesizing a first peptide based on the identified peptide, and a second peptide based on a corresponding peptide portion of a wild-type polypeptide that corresponds to the polypeptide exhibiting the defective conformation; contacting said first peptide with a test agent; and detecting that the three dimensional conformation of the first peptide assumes the three dimensional conformation of the second peptide, thereby identifying an agent that corrects the defective three dimensional conformation of the polypeptide.
  • the method can further include quantitating the amount of said agent that corrects the defective three dimensional conformation of the polypeptide
  • the step of identifying a peptide portion of the polypeptide that is representative of a region of the polypeptide exhibiting the defective conformation can be performed using a method such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, or circular dichroism (CD) spectroscopy.
  • a method such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, or circular dichroism (CD) spectroscopy.
  • the step of detecting the three dimensional conformation of the first peptide as assuming the three dimensional conformation of the second peptide can be performed in any of several ways, including, for example, by detecting a change in fluorescence intensity, by detecting a change in an NMR spectrum or a CD spectrum, or by detecting specific binding to the first peptide of an antibody that specifically binds the second peptide, but does not specifically bind the first peptide in the absence of an agent that corrects a three dimensional conformation of the polypeptide.
  • an antibody can be a polyclonal antibody or a monoclonal antibody.
  • a test agent can be any agent that is suspected of being able to affect the three dimensional conformation of a polypeptide, for example, a small organic chemical such as deuterated water (D 2 0), dimethylsulfoxide, glycerol, trimethylamine N-oxide (TMAO), butyrate, or phenylbutyrate; an osmolyte such as sorbitol, inositol, betaine, glycerophosphoryl choline, arginine, urea, dimethylpropiothetin, or trehalose; a peptide, or modified form thereof, such as a portion of a transmembrane domain of CFTR (e.g., SEQ ID NOS: 3 to 14) or a portion of a nucleotide binding fold (NBF) of CFTR (e.g., NBF2); or cholesterol, or a phosphoglyceride or sphingolipid such as palmitoyl-linole
  • a method of the invention can be performed in a cell-free system and, therefore, can readily be adapted for use in high throughput assays, and can readily be adapted to automation. As such, the method can be used to rapidly and efficiently screen a library of test agents, or for screening one or more test agents at a range of concentrations.
  • the present invention further relates to a method of identifying an agent that corrects a defective three dimensional conformation of a polypeptide by contacting a polypeptide having a defective three dimensional conformation with an agent; thereafter contacting the polypeptide with an antibody that specifically binds a wild-type polypeptide corresponding to the polypeptide having a defective three dimensional conformation, wherein the antibody does not specifically bind the polypeptide having a defective three dimensional conformation; and detecting specific binding of the antibody to the polypeptide having a defective three dimensional conformation, thereby identifying an agent that corrects the defective three dimensional conformation of the polypeptide.
  • the method can further include a step of quantitating the amount of the agent that corrects the defective three dimensional conformation of the polypeptide.
  • the antibody can be detectably labeled using, for example, a visible label, a fiuorimetric label, a radiometric label, a luminescent label, a colorimetric label, and an enzymatic label.
  • the binding of the antibody can be detected using a second antibody, which specifically binds to the first antibody.
  • the present invention also relates to a virtual representation of a peptide portion of a polypeptide, wherein the peptide is representative of a region of a polypeptide having a defective three dimensional conformation.
  • the polypeptide can be, for example, a cystic fibrosis transmembrane regulator (CFTR), and the peptide can be, for example, a peptide having the amino acid sequence of SEQ ID NO: 2.
  • CFTR cystic fibrosis transmembrane regulator
  • Various pathologic conditions are associated with proteins having a defective three dimensional conformation in comparison to that of the normal wild-type protein.
  • the proteins cannot perform their normal function due, for example, to a failure to localize to their proper location in a cell, or to a propensity to form aggregates, which, in turn, can further be detrimental to the cell.
  • the present invention provides methods for identifying an agent that corrects a defective three dimensional conformation of a polypeptide. Such agents can be useful for ameliorating the severity of a pathologic condition associated with the expression of a protein that exhibits a defective three dimensional conformation.
  • the term “folding” is used herein to refer to a secondary structure of a polypeptide, for example, an ⁇ -helix, ⁇ -sheet, and the like. It is well known that the three dimensional conformation of a polypeptide is defined primarily by the primary amino acid sequence of the peptide, and that formation of a proper conformation is facilitated by various enzymes and molecular chaperones (Thomas et al., Trends Biol. Sci. 20:456- 459, 1995, which is incorporated herein by reference). Methods for determining the three dimensional conformation of a polypeptide are disclosed herein and well known in the art.
  • wild-type is used herein to refer to a polypeptide that assumes a particular conformation that can be found naturally in a healthy subject.
  • a wild-type polypeptide can be compared to a polypeptide that exhibits a defective three dimensional conformation.
  • reference to a "defective three dimensional conformation” or “defective conformation” means a conformation that is different from that of the corresponding wild-type polypeptide. As a result of the defective conformation, the polypeptide does not function in the same manner as the wild-type polypeptide.
  • a defective three dimensional conformation can be identified, for example, by determining that a polypeptide, or a peptide portion thereof, does not form a stable ⁇ -helix, in comparison to the corresponding wild-type polypeptide or peptide portion thereof, which forms a stable ⁇ -helix; by determining that the mutant polypeptide forms a different secondary structure than the wild-type polypeptide, for example, forms a ⁇ -sheet, in comparison to the corresponding wild-type polypeptide or portion thereof, which forms an ⁇ -helix; or the like.
  • a wild-type polypeptide is defined, in part, in that it can be found naturally in a healthy individual.
  • the definition of a wild-type polypeptide recognizes that some polypeptides can exist in more than a single conformation in nature, wherein one conformation is not associated with a pathology and one or more different conformations are associated with a pathology.
  • a prion protein can exist in two states, a non-infective state comprising an ⁇ -helix, and an infective state comprising a ⁇ -sheet.
  • a non-infective prion polypeptide comprising an ⁇ -helix is considered to be the wild-type prion protein, whereas an infective prion polypeptide comprising the ⁇ -sheet is considered to have a defective three dimensional conformation.
  • a screening assay of the invention provides a means to identify an agent that corrects a defective three dimensional conformation of a polypeptide.
  • the term "correct,” when used in reference to a conformational defect in a polypeptide means that the defect is eliminated (i.e., the polypeptide assumes the same conformation as the corresponding wild-type polypeptide) or the magnitude of the defect is reduced (i.e., the polypeptide assumes a three dimensional conformation that more closely approximates that of the corresponding wild-type polypeptide).
  • a method of the invention provides a means to identify an agent that stabilizes the ⁇ -helix, thereby correcting the defective three dimensional conformation.
  • Methods for identifying that an agent can correct a defective conformation include, for example, detecting specific binding of the region comprising the defect by an antibody that specifically binds the wild-type polypeptide, but not the mutant polypeptide; detecting a change in fluorescence intensity, where the intensity is related to the three dimensional conformation of the polypeptide; or using a method such as circular dichroism (CD) to detect a conformation characteristic of the wild-type polypeptide (see Example 3).
  • CD circular dichroism
  • a method of the invention can be performed, for example, by contacting in a cell-free system a peptide portion of the polypeptide with a test agent, and determining that the test agent corrects the defective conformation of the peptide.
  • the peptide portion of the polypeptide used in a method of the invention is selected such that it is representative of a region of the polypeptide exhibiting the defective conformation, using a method such as X-ray crystallography, NMR spectroscopy, CD spectroscopy, or the like.
  • the contacting generally is performed in vitro in a cell-free system, although, once an agent that corrects a defective conformation of a polypeptide has been identified using an in vitro method, the efficacy of the agent can be further characterized using a cell based system or by testing the agent in an animal model system (see, for example, Ko et al., FEBS Lett. 405:200-208, 1997; U.S. Pat. No. 5,900,360, each of which is incorporated herein by reference).
  • the defective three dimensional conformation can be due to misfolding of the polypeptide, which can result in a failure of the polypeptide to properly localize in a cell or can result in aggregation of the polypeptide.
  • a polypeptide exhibiting a defective three dimensional conformation is exemplified herein by a cystic fibrosis transmembrane regulator (CFTR) polypeptide having a deletion of Phe508 ( ⁇ F508), which is the most common mutation found in cystic fibrosis.
  • CFTR cystic fibrosis transmembrane regulator
  • a peptide portion of a CFTR polypeptide that is representative of a region of CFTR having the defective conformation is exemplified by a 25 amino acid peptide having the amino acid sequence set forth in SEQ ID NO: 2 (see Example 1).
  • polypeptides having a defective three dimensional conformation are suitable for use in a screening method of the invention including, for example, conformationally defective forms of fibrillin, superoxide dismutase, collagen, a polypeptide of an ⁇ -ketoacid dehydrogenase complex, p53, type I procollagen pro- ⁇ , LDL receptor, ⁇ l -anti trypsin, ⁇ -hexosaminidase, rhodopsin, an insulin receptor, a prion protein, ⁇ -amyloid, transthyretin, and a crystallin polypeptide.
  • peptide portion of a polypeptide or "peptide” is used broadly herein to mean two or more amino acids linked by a peptide bond.
  • a peptide of the invention contains at least about six amino acids, usually contains about ten amino acids, and can contain fifteen or more amino acids, particularly twenty or more amino acids such as the 25 and 26 amino acid peptides based on the region of CFTR that exhibits a defective conformation as disclosed herein.
  • the term “peptide” is not used herein to suggest a particular size or number of amino acids comprising the molecule, and, therefore, can contain up to several hundred amino acid residues or more.
  • polypeptide portion of a polypeptide can, but need not, be obtained from a polypeptide, but also can be chemically synthesized, expressed from an encoding polynucleotide, or produced by any other method routine in the art.
  • polypeptide and protein are used herein to refer to an essentially full length molecule as would be expressed in a cell, for example, a CFTR polypeptide or protein or a mutant form thereof, although generally the term “protein” also includes a polypeptide that may, for example, be post-translationally modified during synthesis in a cell.
  • a method of the invention can be performed in a cell-free system and, therefore, can readily be adapted for use in high throughput assays, and can readily be adapted to automation. As such, the method can be used to rapidly and efficiently screen a library of test agents, or for screening one or more test agents at a range of concentrations.
  • test agent is used broadly herein to mean any agent that is being examined for the ability to correct a defective three dimensional conformation of a polypeptide.
  • a method of the invention generally is used as a screening assay to identify previously unknown molecules that can act to correct the three dimensional conformation of a polypeptide
  • the method also can be used to confirm that an agent known to have a particular activity in fact has the activity, for example, in order to standardize the activity of the agent or to use as a control to compare the activity of a test agent.
  • a test agent can be a peptide, a peptidomimetic, a polynucleotide, a small organic molecule, or any other agent.
  • agents include, for example, a small organic molecule such as deuterated water (D 2 0), dimefhylsulfoxide, glycerol, trimethyl amine N-oxide (TMAO), butyrate, phenylbutyrate, or gamma-aminobutyric acid (GABA); an osmolyte such as sorbitol, inositol, betaine, glycerophosphoryl choline, arginine, urea, dimethylpropiothetin, or trehalose; a peptide, or modified form thereof, such as a portion of a transmembrane domain of CFTR (e.g., SEQ ID NOS: 3 to 14) or a portion of a nucleotide binding fold (NBF) of CFTR
  • a screening method of the invention provides the advantage that it can be adapted to high throughput analysis and, therefore, can be used to screen combinatorial libraries of test agents in order to identify those agents that can correct a defective conformation of polypeptide.
  • a combinatorial library of test agents can be prepared de novo, without any prior information regarding the structure of an agent that potentially can correct a defective conformation of a polypeptide, or can be prepared based on the structure of an agent known to have such activity, thereby allowing the identification of an agent having a more desirable characteristic than the known agent, for example, an ability to more readily enter a cell or a greater stability upon exposure to a biological environment.
  • a library of test agents can be made based on the structure of an agent as exemplified above.
  • Methods for preparing a combinatorial library of molecules that can be screened using a method of the invention are well known in the art. Such methods include, for example, methods of making a phage display library of peptides, which can be constrained peptides (see, for example, U.S. Pat. No. 5,622,699; U.S. Pat. No. 5,206,347; Scott and Smith, Science_249:386-390, 1992; Markland et al., Gene 109:13-19, 1991; each of which is incorporated herein by reference); a peptide library (U.S. Pat. No.
  • a test agent corrects the three dimensional conformation of a polypeptide (see Example 3).
  • the nuclear magnetic resonance (NMR) spectrum or circular dichroism (CD) spectrum of the peptide contacted with a test agent can be compared with the respective spectrum of a corresponding wild-type peptide, wherein a change in the spectrum of the treated peptide such that it more closely approximates that of the wild-type peptide indicates that the agent can correct the defective conformation of the polypeptide.
  • the peptide portion of the polypeptide exhibiting a defective conformation can be contacted with a fluorescent compound such as 1,8-ANS (8-anilinonaphthalene-l-sulfonic acid) or bis-ANS, then contacted with a test agent, and the fluorescence intensity of the sample monitored.
  • a fluorescent compound such as 1,8-ANS (8-anilinonaphthalene-l-sulfonic acid) or bis-ANS
  • a change in fluorescence intensity of the peptide in the presence of the test agent such that it more closely approximates the fluorescence intensity of the wild-type peptide indicates that the agent can correct the defective conformation of the polypeptide.
  • a method of detection is particularly suitable for adaptation to high throughput assays, for example, using 96 well trays, microarrays, or the like.
  • Another convenient detection method that also can be readily adapted to a high throughput format utilizes an antibody that specifically binds to the wild-type polypeptide, or peptide portion thereof, but not to the corresponding polypeptide exhibiting the defective conformation, or peptide portion thereof.
  • Such antibodies are exemplified herein by monoclonal antibodies that were raised against and specifically bind a 26 amino acid peptide, which forms stable ⁇ -helix, but not to a corresponding 25 amino acid peptide, which is representative of the ⁇ F508 CFTR mutant region (see Examples 1 and 3).
  • Such antibodies were raised according to standard methods using the P25 and P26 peptides as antigens, monoclonal antibodies were obtained, and monoclonal antibodies that specifically bound P26, but not P25, were isolated (Example 3).
  • an antibody is used in its broadest sense to include polyclonal and monoclonal antibodies, as well as antigen binding fragments of such antibodies
  • An antibody useful in a method of the invention, or an antigen binding fragment thereof is characterized, for example, by having specific binding activity for a polypeptide, or peptide portion thereof, having a particular conformation, for example, a wild-type conformation, but not for a corresponding polypeptide or peptide portion thereof having a different conformation
  • Fab, F(ab') , Fd and Fv fragments of an antibody that retain specific binding activity for an epitope of a polypeptide, or peptide portion thereof, are included withm the definition of an antibody
  • antibody as used herein includes naturally occurring antibodies and non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen- binding fragments thereof
  • non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains (see Huse et al , Science 246 1275-1281 (1989), which is incorporated herein by reference)
  • These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol Today 14 243-246, 1993, Ward et al , Nature 341 544-546, 1989, Harlow and Lane, Antibodies A laboratory manual (Cold Spring Harbor Laboratory Press, 1988), Hilyard et al , Protein Engineering A practical approach (IRL Press 1992), Borrabeck, Antibody Engineering.
  • Antibodies that bind specifically a first peptide having a particular conformation, but not a corresponding second peptide having a different conformation, can be raised using the first peptide as an immunogen and removing antibodies that crossreact with the second peptide.
  • the peptide is non-immunogenic, it can be made immunogenic by coupling the hapten to a carrier molecule such as bovine serum albumin or keyhole limpet hemocyanin, or by expressing the peptide portion as a fusion protein.
  • carrier molecules such as bovine serum albumin or keyhole limpet hemocyanin
  • kits incorporating an antibody useful in a method of the invention can be prepared.
  • a kit can contain, for example, in addition to the antibody, reagents for detecting the antibody, or for detecting specific binding of the antibody to the first peptide, but not the second peptide.
  • detectable reagents useful for labeling or otherwise identifying the antibody are described herein and known in the art.
  • Cloned hybridoma cell lines can be screened using labeled antigen to identify clones that secrete monoclonal antibodies having the appropriate specificity, and hybridomas expressing antibodies having a desirable specificity and affinity can be isolated and utilized as a continuous source of the antibodies.
  • the antibodies can be further screened for the inability to bind specifically to a corresponding peptide having a different conformation than that of the peptide used to raise the antibodies.
  • Such antibodies are useful, for example, for preparing standardized kits for commercial use.
  • a recombinant phage that expresses, for example, a single chain antibody also provides an antibody that can used for preparing standardized kits.
  • monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well established techniques, including, for example, affinity chromatography with Protein-A SEPHAROSE, size exclusion chromatography, and ion exchange chromatography (Coligan et al., supra, 1992, see sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; see, also, Barnes et al., "Purification of Immunoglobulin G (IgG)," in Meth. Molec. Biol. 10:79-104 (Humana Press 1992), which is incorporated herein by reference). Methods of in vitro and in vivo multiplication of monoclonal antibodies is well known to those skilled in the art.
  • Multiplication in vitro can be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages.
  • suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium
  • a mammalian serum such as fetal calf serum or trace elements
  • growth sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages.
  • Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies.
  • Large scale hybridoma cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a continuous stirrer reactor, or in immobilized or entrapped cell culture.
  • Multiplication in vivo can be carried out by injecting cell clones into mammals histocompatible with the parent cells, for example, syngeneic mice, to cause growth of antibody producing tumors.
  • the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal.
  • Additional methods for determining that a peptide representative of a region of a polypeptide having a defective conformation assumes a wild-type conformation include, for example, methods of gel electrophoresis, which can be adapted such that migration of a peptide is indicative of its structure; or methods that involve an interaction of the peptide with a second molecule, wherein the peptide must have the appropriate conformation for interacting, for example, affinity chromatography, the two hybrid system of Fields and Song (Nature 340:245-246, 1989; see, also, U.S. Pat. No. 5,283,173; Fearon et al., Proc. Natl. Acad. Sci..
  • the peptide portion of a polypeptide used in a method of the invention can, but need not, contain a mutation such as an insertion, deletion or substitution as compared to the corresponding peptide portion of a wild-type polypeptide, wherein the mutation contributes to the altered conformation of the polypeptide.
  • the peptide can comprise a R1137P substitution mutation in fibrillin, which produces a mutant fibrillin that fails to properly refold in vitro, and is associated with Marfan syndrome.
  • the peptide also can comprise a portion of a polypeptide such as ⁇ -amyloid, which, under certain conditions, can form a ⁇ -sheet structure that leads to aggregate formation as occurs in Alzheimer's disease.
  • a method of the invention provides a means to identify an agent that stabilizes the three dimensional conformation of a polypeptide, which, in the absence of the agent, exhibits defective folding.
  • a method of the invention can be used to identify an agent that stabilizes a defective three dimensional conformation of a CFTR polypeptide, such that the CFTR polypeptide can properly localize in a cell membrane.
  • a method of the invention provides a means to destabilize a first alternative three dimensional conformation of a polypeptide, thereby inducing the polypeptide to form a second alternative conformation.
  • a method of the invention can identify an agent that destabilizes the ⁇ -sheet conformation of an infectious prion protein such that the prion protein forms an alternative ⁇ -helical conformation, which is not infective.
  • the present invention also provides a method of identifying an agent that corrects a defective three dimensional conformation of a polypeptide by identifying a peptide portion of the polypeptide that is representative of a region of the polypeptide exhibiting the defective conformation; synthesizing a first peptide based on the identified peptide, and a second peptide based on a corresponding peptide portion of a wild-type polypeptide that corresponds to the polypeptide exhibiting the defective conformation; contacting said first peptide with a test agent; and detecting the three dimensional conformation of the first peptide as assuming the three dimensional conformation of the second peptide, thereby identifying an agent that corrects the defective three dimensional conformation of the polypeptide.
  • the method can further include quantitating the amount of said agent that corrects the defective three-dimensional conformation of the polypeptide.
  • polypeptide exhibiting the defective conformation can be any polypeptide, as discussed above.
  • polypeptides are provided as examples of polypeptides that can be the subject of a method of the invention because they are characterized, in part, by a defect in three dimensional conformation or by aberrant interaction with cellular proteins involved in protein folding, and are associated with various pathologies.
  • cystic fibrosis is associated with misfolding of the CFTR polypeptide
  • Marfan syndrome is associated with misfolding of fibrillin
  • amyotrophic lateral sclerosis is associated with misfolding of superoxide dismutase
  • scurvy is associated with misfolding of collagen
  • maple syrup urine disease is associated with misassembly/misfolding of ⁇ -ketoacid dehydrogenase complex
  • various cancers are associated with misfolding of p53 or with an altered interaction of p53 and heat shock protein-70 (Hsp70); osteogenesis imperfecta is associated with misassembly of type I procollagen pro- ⁇ or with altered expression of the Hsp70 homolog, BIP; scrapie, Creutzfeldt- Jakob disease, and fatal familial insomnia are associated with aggregation of prion proteins
  • Alzheimer's disease is associated with aggregation of ⁇ -amyloid
  • familial amyloidosis is associated with aggregati
  • CFTR cystic fibrosis transmembrane conductance regulator
  • CFTR is an ATP hydrolysis-dependent, phosphorylation-regulated chloride ion channel that consists of a single 1480 amino acid polypeptide, which includes two transmembrane spanning domains (TMS 1 and TMS2), two nucleotide binding folds (NBF 1 and NBF2), and a regulatory domain (Riordan et al., Science 245:1066-1073, 1993; Caroll et al., Cell Physiol. Biochem. 3:388-399, 1993).
  • the regulatory domain which is subject to phosphorylation by specific protein kinases, and the channel function, are properties that make CFTR unique among the numerous members of the ABC transporter superfamily.
  • Phe508 ( ⁇ F508) of CFTR is the most common mutation in the CFTR. Phe508 resides in a portion of the sequence identified as the first of the two NBFs. Deletion of Phe508 from CFTR results in a protein that is unable to leave the endoplasmic reticulum (Cheng et al., Cell 63:827-834, 1990) leading to its degradation (Ward et al., £ell 83: 121-127, 1995).
  • Blockage of the ⁇ F508 CFTR in the ER appears to be relieved if ⁇ F508 mutant cells are grown at low temperatures (Denning et al., Nature 358:761-764, 1992) or in the presence of stabilizing agents such as glycerol or water (Brown et al., supra, 1996 ⁇
  • stabilizing agents such as glycerol or water
  • the ⁇ F508 CFTR is functionally active withm the endoplasmic reticulum (Pasyk and Foskett, J. Biol. Chem 270 12347-12350, 1995), suggesting that the misfolded region, which is recognized by local "quality control" mechanisms responsible for retention of a protein, may be confined to a relatively small folding unit of the protein such as the ⁇ F508 region.
  • the expressed ⁇ F508 form of NBF 1 shows significant structure and function, approaching that of wild-type NBF1, and 3D modeling studies of NBF1 predict that the critical Phe508 residue is located withm a short ⁇ -hehcal region (Bianchet et al., J. Bioenerg. Biomemb 29:503-524, 1997, which is incorporated herein by reference)
  • NBF1 and NBF2 nucleotide domains
  • CFTR protein has been isolated, reconstituted with liposomal vesicles, and shown to catalyze both ATPase activity and chloride ion channel activity, which appear, at least in part, to be coupled (Li et al , J. Biol. Chem. 271 "28563-28568, 1996, Bear et al., J Bioenerg Biomemb 29"465- 473, 1997). Both the ATP turnover rate and channel gating rate are slow
  • the primary defect in ⁇ F508 CFTR had not been identified at the structural level due, in part, to the use in earlier studies of peptides (67 and 66 amino acids long) that included much more of NBF 1 than the ⁇ F508 region, and excluded a critical part of the region.
  • an in vitro assay has been developed for readily identifying agents that correct the basic defect in ⁇ F508 CFTR and in other polypeptides that exhibit a defective three dimensional conformation, particularly a defective conformation due to misfolding of the protein.
  • the defective CFTR protein fails to undergo a critical ATP-dependent transition and is completely degraded in cells, despite the fact that the polypeptide has a 99.93%) correct amino acid sequence. Nevertheless, ⁇ F508 CFTR retains its capacity to function as a chloride ion channel within the ER, indicating that it folds into a functional unit, and suggesting that the structural defect is localized, most likely to the ⁇ F508 region.
  • Wild-type CFTR matures in the ER from an unglycosylated form (A) to a core glycosylated form (B), before proceeding to the Golgi to form the more completely glycosylated form (C).
  • B-l and B-2 two distinct forms (B-l and B-2) of core glycosylated CFTR exist in the ER, one protease sensitive (B-l) and the other protease resistant (B-2), suggesting that if CFTR does not proceed from the B-l to the B-form during its maturation, it is directed to the degradation pathway.
  • B-l protease sensitive
  • B-2 protease resistant
  • the conversion from B-l to B-2 requires ATP, which is a substrate for NBF1.
  • Multiple pathways appear to be involved in the degradation of CFTR and ⁇ F508 CFTR in the ER, including the ubiquitin-proteasome pathway.
  • ⁇ F508 CFTR into these pathways may be aided by one or more chaperones like Hsp 70 operating from the cytoplasmic surface (Yang et al , Proc Natl Acad Sci . USA 90 9480-9484, 1993)
  • glycerol which facilitated trafficking of ⁇ F508 CFTR to the plasma membrane in a form that mediates cAMP-stimulated chloride ion conductance
  • glycerol or other chemical chaperones such as D 2 O, DMSO, or t ⁇ methylamine N-oxide promote trafficking of mutant CFTR by correcting a structural defect in the ⁇ F508 region of CFTR
  • cellular osmolytes are known to transc ⁇ ptionally up-re
  • the P26 peptide (SEQ ID NO: 1) includes residues 498 to 523 within the first nucleotide-binding fold (NBF1) of wild-type CFTR, while P25 (SEQ ID NO: 2) lacks Phe508, but is otherwise identical to P26, thus mimicking the predominant mutation found in CF.
  • Phe508 lies within a helical region in the wild-type P26 peptide.
  • Three independent observations demonstrated a significant decrease in the helix-forming propensity of P25 in comparison with P26 in water.
  • both P26 and P25 formed ⁇ -helices from residues Thr501 to Lys522 that mimic the folded state of this region in intact CFTR.
  • the P25 ⁇ F508 mutant peptide formed an ⁇ -helix in TFE, chemical shift and NOE criteria indicate that it is less stable between residues Gly500 and Ile507 (see Massiah et al., Biochemistry 38:7453-7461, 1998, which is incorporated herein by reference; see Table 1). However, this difference was not apparent in the computed NMR structures of P25 and P26.
  • NMR results (Example 2) obtained using P26 and P25 peptides corresponding to Met498 to Ala523 of CFTR, are comparable to results previously obtained by CD using a "wild-type" P67 and mutant P66 peptide corresponding to Arg450 to Arg516 of CFTR, which were consistent with a location of F508 withm a ⁇ -sheet region m P67 that became unstable in P66, resulting in random coil formation.
  • a 1.5 A X-ray structure of the HisP protein (Hung et al , Nature 396'703-707, 1998), which is the ATP -binding subunit of the histidine permease and, like CFTR, is a member of the ABC transporter superfamily (see, for example, Higgms, Ann Rev. Cell Biol. 8-67-113, 1992), the region believed to correspond to the F508 region in CFTR is an ⁇ helix, in agreement with 3D modeling studies based on the structures of FI ATPase and the RecA protein (Bianchet et al., supra, 1997)
  • chemiosmolytes that are known to stabilize proteins also can promote normal trafficking of ⁇ F508 CFTR to the plasma membrane, where it is at least partially functional Although it is assumed that these chemiosmolytes ("chemical chaperones") correct a misfolding problem within the F508 region, this possibility has not previously been demonstrated directly
  • the P26 and P25 peptides which are representative of the Phe508 and F508 regions of the wild-type and ⁇ F508 CFTR proteins, respectively, provide a model system for specifically selecting agents that correct the structural defect caused by the F508 mutation, and for more generally selecting agents that correct a defect in the three dimensional conformation of a polypeptide.
  • a method of identifying an agent that corrects a defective three dimensional conformation of a polypeptide is performed by contacting a polypeptide having a defective three dimensional conformation with an agent, thereafter contacting the polypeptide with an antibody that specifically binds a wild-type polypeptide corresponding to the polypeptide having a defective three dimensional conformation, wherein the antibody does not specifically bind the polypeptide having a defective three dimensional conformation; and detecting specific binding of the antibody to the polypeptide having a defective three dimensional conformation, thereby identifying an agent that corrects the defective three dimensional conformation of the polypeptide
  • the method can further include a step of quantitating the amount of the agent that corrects the defective three dimensional conformation of the polypeptide
  • the antibody used in such a method can be detectably labeled using, for example, a label such as biotm, which can be detected using avidm or streptavidm, a fluorimetric label such as green fluorescent protein, fluorescein, or rhodamine; a radiometric label such as sulfur-35, technicium-99, or tritium; a luminescent label such as luciferin; a colorimetric label, an enzymatic label such as alkaline phosphatase; a paramagnetic spin label such as carbon-13; or the like.
  • the antibody can be an unlabeled antibody that can be detected using a second antibody, which specifically binds to the first antibody, in which case the second antibody can be detectably labeled.
  • a polypeptide such as an antibody
  • Methods of detectably labeling a polypeptide such as an antibody are well known in the art (see, for example, Hermanson, "Bioconjugate Techniques” (Academic Press 1996), which is incorporated herein by reference; see, also, Harlow and Lane, supra, 1988).
  • the reagents for labeling the agent also can be included in the kit, or the reagents can be purchased separately from a commercial source.
  • the present invention also relates to a virtual representation of a peptide portion of a polypeptide, wherein the peptide is representative of a region of a polypeptide having a defective three dimensional conformation.
  • the polypeptide can be a cystic fibrosis transmembrane regulator (CFTR), in which case the peptide can be, for example, a peptide having the amino acid sequence of SEQ ID NO: 2.
  • CFTR cystic fibrosis transmembrane regulator
  • an amino acid sequence of a polypeptide of interest such as a prion protein can be entered into a computer system having appropriate modeling software, and a three dimensional representation of the prion protein, including a "wild-type” ⁇ -helical structure or a "defective" ⁇ -sheet conformation can be produced, similar as to was done for the NBF domains of CFTR (Bianchet et al., supra, 1997).
  • the amino acid sequence can be entered into the computer system, such that the modeling software can simulate portions of a polypeptide, particularly a portion suspected of having a conformational defect.
  • a base line can be predefined by modeling, for example, a peptide portion of a wild-type polypeptide, and identifying the three dimensional conformation of the peptide, such that an abnormal structure then can be identified by comparison to the base line structure.
  • methods of molecular modeling can be used to identify an agent that corrects a defect in the three dimensional structure of a polypeptide, by determining that the conformation of a mutant polypeptide, in the presence of the agent, assumes or more closely approximates the conformation of a corresponding wild-type peptide.
  • Modeling systems useful for the purposes disclosed herein can be based on structural information obtained, for example, by crystallographic analysis, NMR analysis, or the like, or on primary sequence information (see, for example, Dunbrack et al., "Meeting review: the Second meeting on the Critical Assessment of Techniques for Protein Structure Prediction (CASP2) (Asilomar, California, December 13-16, 1996). Fold Des. 2(2): R27-42, (1997); Fischer and Eisenberg, Protein Sci. 5:947-55, 1996; (see, also, U.S. Pat. No. 5,436,850); Havel, Prog. Biophys. Mol. Biol. 56:43-78, 1991; Lichtarge et al., J. Mol. Biol.
  • the crystal structure coordinates of a polypeptide that has a conformational defect and is associated with a pathologic condition can be used to design peptides useful in the methods of the invention.
  • the structure coordinates of the protein can also be used to computationally screen small molecule data bases for agents that can modulate the conformation of a peptide.
  • agents can be identified, for example, by computer fitting kinetic data using standard equations (see, for example, Segel, "Enzyme Kinetics” (J. Wiley & Sons 1975), which is incorporated herein by reference).
  • Computer programs for carrying out the activities necessary to perform a method of the invention are disclosed herein (Example 2) or otherwise known in the art.
  • Examples of such programs include, Catalyst DatabasesTM - an information retrieval program accessing chemical databases such as BioByte Master File, Derwent WDI and ACD; Catalyst/HYPOTM - generates models of compounds and hypotheses to explain variations of activity with the structure of drug candidates; LudiTM - fits molecules into the active site of a protein by identifying and matching complementary polar and hydrophobic groups; and LeapfrogTM - "grows" new ligands using a genetic algorithm with parameters under the control of the user.
  • the embodiment is implemented in one or more computer programs executing on programmable systems each comprising at least one processor, at least one data storage system (including volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device.
  • the program is executed on the processor to perform the functions described herein.
  • Each such program can be implemented in any desired computer language, including, for example, machine, assembly, high level procedural, or object oriented programming languages, to communicate with a computer system.
  • the language may be a compiled or interpreted language.
  • the computer program will typically be stored on a storage medium or device, for example, a ROM, CD-ROM, magnetic or optical media, or the like, that is readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein.
  • the system may also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.
  • Embodiments useful in a method of the invention include systems, for example, internet based systems, particularly computer systems which store and manipulate coordinate information obtained by crystallographic or NMR analysis, or amino acid or nucleotide sequence information, as disclosed herein.
  • computer system refers to the hardware components, software components, and data storage components used to analyze coordinates or sequences as set forth herein.
  • the computer system typically includes a processor for processing, accessing and manipulating the sequence data.
  • the processor can be any well known type of central processing unit, for example, a Pentium II or Pentium III processor from Intel Corporation, or a similar processor from Sun, Motorola, Compaq, Advanced MicroDevices or International Business Machines.
  • the computer system is a general purpose system that comprises the processor and one or more internal data storage components for storing data, and one or more data retrieving devices for retrieving the data stored on the data storage components.
  • the processor and one or more internal data storage components for storing data, and one or more data retrieving devices for retrieving the data stored on the data storage components.
  • the computer system includes a processor connected to a bus, which is connected to a main memory, preferably implemented as RAM, and one or more internal data storage devices such as a hard drive or other computer readable media having data recorded thereon.
  • the computer system further includes one or more data retrieving devices for reading the data stored on the internal data storage devices.
  • the data retrieving device may represent, for example, a floppy disk drive, a compact disk drive, a DVD drive, a magnetic tape drive, or a modem capable of connection to a remote data storage system (e.g., via the internet).
  • the internal data storage device is a removable computer readable medium such as a floppy disk, a compact disk, a magnetic tape, etc. containing control logic and/or data recorded thereon.
  • the computer system may advantageously include or be programmed by appropriate software for reading the control logic and/or the data from the data storage component once inserted in the data retrieving device.
  • the computer system generally includes a display, which is used to display output to a computer user. It should also be noted that the computer system can be linked to other computer systems in a network or wide area network to provide centralized access to the computer system.
  • any of several methods to screen for agents having such activity can be used. This process may begin by visual inspection, for example, of the effect of the agent on conformation on the computer screen. Selected peptide portions of a polypeptide can be examined in a variety of orientations, and docking of an agent with a peptide can be examined. Docking can be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields, such as CHARMM and AMBER.
  • Specialized computer programs can be particularly useful for selecting a peptide portion of a polypeptide or an agent for use in a method of the invention.
  • Such programs include, for example, GRID (Goodford, J. Med. Chem.. 28:849-857, 1985; available from Oxford University, Oxford, UK); MCSS (Miranker and Karplus, Proteins: Structure. Function and Genetics 11:29-34, 1991, available from Molecular Simulations, Burlington MA); AUTODOCK (Goodsell and Olsen, Proteins: Structure. Function, and Genetics 8: 195-202, 1990, available from Scripps Research Institute, La Jolla CA); DOCK (Kuntz, et al., J. Mol. Biol. 161:269-288, 1982, available from University of California, San Francisco CA), each of which is incorporated herein by reference.
  • a molecular modeling process for identifying a peptide portion of a polypeptide that is representative a region composing a conformational defect of a polypeptide, or an agent that can modulate the conformation of a polypeptide having a conformation defect can be performed as disclosed herein.
  • a virtual representation of a peptide portion of a polypeptide is performed.
  • the present invention provides a virtual representation of a peptide portion of a polypeptide, wherein the peptide mimics a conformation of the polypeptide.
  • the virtual representation of the peptide can be displayed or can be maintained in a computer system memory.
  • the process begins at a start state, comprising the virtual peptide, then moves to a state composing a database containing one or more virtual test agents stored to a memory in the computer system.
  • the memory can be any type of memory, including RAM or an internal storage device.
  • the process then moves to a state wherein the ability of a virtual first test agent to correct the defective conformation of the virtual peptide is determined, wherein the database containing the virtual test agent, which can be one of a population of test agents, is opened for analysis of the effect of the virtual test agent on the conformation of the virtual peptide, and the analysis is made.
  • a determination of a conformational change, particularly a change indicative of a correction of the conformational defect can be made based on calculations performed by software maintained in the computer system, or by comparison to a predetermined specific interaction, which can be stored in a memory in the computer system and accessed as approp ⁇ ate.
  • the process then moves to a state wherein, where a conformational change is detected, the virtual test agent is displayed, or is stored in a second database on the computer. If appropriate, the process is repeated for the virtual peptide and a second virtual test agent, a third virtual test agent, and so on, as desired.
  • the identified virtual test agent is moved from the database and can be displayed to the user. This state notifies the user that the agent with the displayed name or structure has the desired parameters within the constraints that were entered.
  • the process moves to a decision state, wherein a determination is made whether more virtual test agents exist in the database or are to be examined. If no more agents exist in the database, then the process terminates at an end state.
  • the process moves to a state, wherein a pointer is moved to the next test agent in the database so that it can be examined for the ability to correct a defective conformation of the polypeptide.
  • the new agent is examined for the ability to correct the defective conformation of the virtual target peptide.
  • the present invention provides a screening method based on molecular modeling to identify an agent that corrects a defective three dimensional conformation of a polypeptide.
  • an agent useful for correcting a defective three dimensional conformation of a polypeptide can be any type of molecule, for example, a polynucleotide, a peptide, a peptidomimetic, peptoids such as vinylogous peptoids, a small organic molecule, or the like, and can be useful for ameliorating the severity of a pathologic condition associated with expression of a polypeptide having a defective conformation. Accordingly, the present invention provides methods for ameliorating the severity of a pathological condition in a subject, wherein the pathologic condition is characterized at least in part by expression of a polypeptide having a defective three dimensional conformation.
  • the term "ameliorate,” when used in reference to the severity of a pathologic condition, means that signs or symptoms associated with the condition are lessened
  • the signs or symptoms to be monitored will be characte ⁇ stic of a particular pathologic condition and will be well known to skilled clinician, as will the methods for monito ⁇ ng the signs and symptoms of the condition
  • the pathologic condition is cystic fibrosis
  • the skilled clinician can monitor the amount of chlo ⁇ de ion in the patient's sweat, the patient's ability to breathe, or other clinical sign or symptom associated with CF.
  • the pathologic condition is Alzheimer's disease
  • the clinician can monitor, for example, the patient's skill in performing a standardized memory test. Relevant clinical tests to monitor other pathologic conditions associated with expression of a polypeptide having a defective three dimensional conformation are well known and routine in the art.
  • the agent is a peptide that acts mtracellularly
  • it can be contacted directly with a cell expressing the defective polypeptide ("target cell"), or a polynucleotide encoding the peptide (or polypeptide) can be introduced into the target cell and the peptide can be expressed therein.
  • target cell a cell expressing the defective polypeptide
  • a polynucleotide encoding the peptide (or polypeptide) can be introduced into the target cell and the peptide can be expressed therein.
  • vanous methods are known for introducing a peptide into a cell The selection of a method for introducing such a peptide into a cell will depend, in part, on the characte ⁇ stics of the target cell, into which the peptide is to be introduced.
  • the peptide agent can be linked to the ligand such that, upon binding of the ligand to the receptor, the linked peptide is translocated into the cell by receptor-mediated endocytosis
  • the peptide agent also can be encapsulated m a hposome or formulated m a lipid complex, which can facilitate entry of the peptide into the cell, and can be further modified to express a receptor (or ligand), as above.
  • the peptide agent also can be introduced into a cell by engineering the peptide to contain a protein transduction domain such as the human immunodeficiency virus TAT protein transduction domain, which facilitates translocation of a peptide a the cell (see Schwarze et al., Science 285:1569-1572 (1999), which is incorporated herein by reference; see, also, Derossi et al.. J. Biol. Chem. 271:18188 (1996)).
  • a protein transduction domain such as the human immunodeficiency virus TAT protein transduction domain
  • a peptide agent can be modified to include a cell compartmentalization domain, such that the peptide localizes to an appropriate location in the cell, for example, the endoplasmic reticulum.
  • Cell compartmentalization domains are well known and include, for example, a plasma membrane localization domain, a nuclear localization signal, a mitochondrial membrane localization signal, an endoplasmic reticulum localization signal, and the like (see, for example, Hancock et al., EMBO J. 10:4033-4039, 1991; Buss et al., Mol. Cell. Biol. 8:3960-3963, 1988; U.S. Pat. No. 5,776,689 each of which is incorporated herein by reference).
  • a peptide agent that is to be administered to a subject also can be modified to contain, for example, one or more D-amino acids in place of a corresponding L-amino acid; or to contain one or more amino acid analogs, for example, an amino acid that has been derivatized or otherwise modified at its reactive side chain, provided the modification does not adversely affect the efficacy of the agent.
  • one or more peptide bonds in the peptide can be modified.
  • a reactive group at the amino terminus or the carboxy terminus or both can be modified.
  • Such peptides can be modified, for example, to have improved stability to a protease, an oxidizing agent or other reactive material the peptide may encounter in a biological environment.
  • the peptides can be modified to have decreased stability in a biological environment, if desired, such that the period of time the peptide is active in the environment is reduced.
  • Such peptides can be useful for ameliorating the severity of a pathologic condition associated with a protein conformational defect in a subject.
  • the agent that corrects a defective three dimensional conformation of a polypeptide is a polynucleotide, or can be encoded by a polynucleotide
  • the polynucleotide can be contacted directly with a target cell, whereupon it can enter the cell and effect its function either directly (a polynucleotide agent) or upon expression (a peptide agent).
  • a polynucleotide agent can be contained in a vector, which can facilitate manipulation of the polynucleotide, including introduction of the polynucleotide into a target cell.
  • the vector can be a cloning vector, which is useful for maintaining the polynucleotide, or can be an expression vector, which contains, in addition to the polynucleotide, regulatory elements useful for expressing the polynucleotide and, where the polynucleotide encodes a peptide, for expressing the encoded peptide in a particular cell.
  • An expression vector can contain the expression elements necessary to achieve, for example, sustained transcription of the encoding polynucleotide, or the regulatory elements can be operatively linked to the polynucleotide prior to its being cloned into the vector.
  • An expression vector (or the polynucleotide) generally contains or encodes a promoter sequence, which can provide constitutive or, if desired, inducible or tissue specific or developmental stage specific expression of the encoding polynucleotide, a poly-A recognition sequence, and a ribosome recognition site or internal ribosome entry site, or other regulatory elements such as an enhancer, which can be tissue specific.
  • the vector also can contain elements required for replication in a prokaryotic or eukaryotic host system or both, as desired.
  • Such vectors which include plasmid vectors and viral vectors such as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus, vaccinia virus, semliki forest virus and adeno-associated virus vectors, are well known and can be purchased from a commercial source (Promega, Madison WI; Stratagene, La Jolla CA; Invitrogen, La Jolla CA) or can be constructed by one skilled in the art (see, for example, Meth. Enzymol.. Vol. 185, Goeddel, ed. (Academic Press, Inc., 1990); Jolly, Cane. Gene Ther. 1 :51-64, 1994; Flotte, J. Bioenerg. Biomemb.
  • a tetracycline (tet) inducible promoter can be particularly useful for driving expression of a polynucleotide in a cell.
  • tetracycline, or a tetracycline analog Upon administration of tetracycline, or a tetracycline analog, to a subject containing a polynucleotide operatively linked to a tet inducible promoter, expression of the encoded peptide is induced, whereby the peptide can effect its activity.
  • the polynucleotide also can be operatively linked to tissue specific regulatory element such that expression is limited to the cells expressing the defective polypeptide.
  • Viral expression vectors can be particularly useful for introducing a polynucleotide into a cell, particularly a cell in a subject. Viral vectors provide the advantage that they can infect host cells with relatively high efficiency and can infect specific cell types. For example, a polynucleotide encoding a peptide that corrects a defective conformation of CFTR can be cloned into an adenovirus vector, which effectively infects lung epithelial cells.
  • Viral vectors have been developed for use in particular host systems, particularly mammalian systems and include, for example, retroviral vectors, other lentivirus vectors such as those based on the human immunodeficiency virus (HIV), adeno-associated virus vectors, herpesvirus vectors, vaccinia virus vectors, and the like (see Miller and Rosman, BioTechniques 7:980-
  • a polynucleotide which can be contained in a vector, can be introduced into a cell by any of a variety of methods known in the art (Sambrook et al., Molecular Cloning: A laboratory manual (Cold Spring Harbor Laboratory Press 1989); Ausubel et al., Current Protocols in Molecular Biology. John Wiley and Sons, Baltimore, MD (1987, and supplements through 1995), each of which is incorporated herein by reference).
  • Such methods include, for example, transfection, lipofection, microinjection, electroporation and, with viral vectors, infection; and can include the use of liposomes, microemulsions or the like, which can facilitate introduction of the polynucleotide into the cell and can protect the polynucleotide from degradation prior to its introduction into the cell.
  • the selection of a particular method will depend, for example, on the cell into which the polynucleotide is to be introduced, as well as whether the cell is isolated in culture, or is in a tissue or organ in culture or in situ.
  • a polynucleotide into a cell by infection with a viral vector is particularly advantageous in that it can efficiently introduce the nucleic acid molecule into a cell ex vivo or in vivo (see, for example, U.S. Pat. No. 5,399,346, which is incorporated herein by reference).
  • an agent that corrects a defective three dimensional conformation of a polypeptide is administered to an individual, it generally is provided as a composition comprising a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters.
  • a pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption of the agent.
  • physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.
  • a pharmaceutically acceptable carrier including a physiologically acceptable compound, depends, for example, on the physico-chemical characteristics of the agent and on the route of administration of the composition, which can be, for example, orally or parenterally such as intravenously, and by injection, intubation, or other such method known in the art.
  • the pharmaceutical composition also can contain a second reagent such as a therapeutic agent specific for the particular pathologic condition.
  • the agent can be incorporated within an encapsulating material such as into an oil-in-water emulsion, a microemulsion, micelle, mixed micelle, liposome, microsphere or other polymer matrix (see, for example, Gregoriadis, Liposome Technology. Vol. 1 (CRC Press, Boca Raton, FL 1984); Fraley, et al., Trends Biochem. Sci.. 6:77 (1981), each of which is incorporated herein by reference).
  • Liposomes for example, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer. "Stealth" liposomes (see, for example, U.S. Pat. Nos.
  • a polynucleotide agent can be introduced into a cell using, for example, adenovirus-polylysine DNA complexes (see, for example, Michael et al., J. Biol. Chem. 268:6866-6869 (1993), which is incorporated herein by reference).
  • the route of administration of the pharmaceutical composition will depend, in part, on the chemical structure of the molecule.
  • Peptides and polynucleotides are not particularly useful when administered orally because they can be degraded in the digestive tract.
  • methods for chemically modifying polypeptides, for example, to render them less susceptible to degradation by endogenous proteases or more absorbable through the alimentary tract are well known (see, for example, Blondelle et al., supra, 1995; Ecker and Crook, supra, 1995; see, also, above).
  • a pharmaceutical composition as disclosed herein can be administered to an individual by various routes including, for example, orally or parenterally, such as intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally, intrarectally, intracisternally or by passive or facilitated absorption through the skin using, for example, a skin patch or transdermal iontophoresis, respectively.
  • the pharmaceutical composition can be administered by injection, intubation, orally or topically, the latter of which can be passive, for example, by direct application of an ointment, or active, for example, using a nasal spray or inhalant, in which case one component of the composition is an appropriate propellant.
  • a pharmaceutical composition also can be administered to the site of a pathologic condition.
  • the total amount of an agent to be administered to an individual can be administered as a single dose, either as a bolus or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol, in which multiple doses are administered over a prolonged period of time.
  • One skilled in the art would know that the amount of the pharmaceutical composition to treat a pathologic condition in a subject depends on many factors including the age and general health of the subject as well as the route of administration and the number of treatments to be administered. In view of these factors, the skilled artisan would adjust the particular dose as necessary.
  • the formulation of the pharmaceutical composition and the routes and frequency of administration are determined, initially, using Phase I and Phase II clinical trials.
  • the pharmaceutical composition can be formulated for oral administration, for example, as a tablet, or a solution or suspension form; or can comprise an admixture with an organic or inorganic carrier or excipient suitable for enteral or parenteral applications, and can be compounded, if desired, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, or other form suitable for use.
  • the carriers in addition to those disclosed above, can include glucose, lactose, mannose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, medium chain length triglycerides, dextrans, and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form.
  • auxiliary, stabilizing, thickening or coloring agents and perfumes can be used, for example a stabilizing dry agent such as triulose (see U.S. Pat. No. 5,314,695).
  • Synthesized peptides were purified by semi-preparative HPLC chromatography utilizing a Vydac Protein & Peptide C ⁇ 8 (250 mm X 10 mm) column. Buffers consisted of 0.1% trifluoroacetic acid (TFA) in water and 0.08 % in acetonitrile. A routinely gradient of 0 to 100% acetonitrile in 600 min with a flow rate of 1.5 ml/min routinely was used to purify peptides. The peptides were separated into fractions each consisting of 750 pi, then each fraction was loaded onto the column to check for peptide purity. The peptide peak was detected by monitoring absorbance at 230 nm.
  • TFA trifluoroacetic acid
  • buffers can be deoxygenated.
  • the amino acid sequences of purified peptides were determined using a PE Biosystems 492 Procise M Protein Sequencer to ensure their fidelities by automated Edman degradation chemistry.
  • matrix-assisted LTV laser desorption/ionization (MALDI) mass spectrometry was performed using a Kratos Compact Maldi IV to confirm purity and identity.
  • a 3 ⁇ g sample of a peptide was mixed with an UV absorbing matrix, ACHA ( ⁇ -cyano-4-hydroxyc ⁇ nnam ⁇ c acid plus free HCN, Sigma), which absorbs the laser energy to evaporate and ionize the sample.
  • ACHA ⁇ -cyano-4-hydroxyc ⁇ nnam ⁇ c acid plus free HCN, Sigma
  • the NMR samples consisted of 1.4 mM solutions of the 25-mer and 26-mer peptides in 90% H2 ⁇ /10% perdeuterated dimethylsulfoxide (DMSO ⁇ f ⁇ pH 4.0).
  • the 2D NOESY spectra were collected as follows 100, 200, and 300 millisecond (ms) mixing times, spectral widths of 5500 Hz (*H, t], 1024 complex points) and 5500 Hz ( ⁇ H, t , 2048 complex points), and 64 transients per hypercomplex t/,t pair.
  • the 2D TOCSY spectra of the peptides were collected with otherwise identical parameters but with a 65 ms spmlock time using the decoupling in the presence of scalar interaction (DIPSI-2; Shaka et al., J Magnetic Res. 77:274-293, 1988, which is incorporated herein by reference) pulse tram.
  • the 2D double quantum-filter correlated spectroscopy (DQF-COSY) spectra were collected with spectral widths of 6000 Hz f l H, tj, 1024 complex points) and 6000 Hz f ⁇ H, t , 2048 complex points) and 64 transients per hypercomplex tj,t2 pair Water suppression for the DQF-COSY was obtained with a 1.5 s presaturation pulse
  • the NMR data were processed on an Silicon Graphics workstation using the Felix 2 3 software package (Biosym Technologies). All of the spectra were processed similarly by first applying a convolution Gaussian window function of 16 to the FIDs to artificially remove the water resonance, then applying a 75°-sh ⁇ fted sinebell square (ss) window function to the 1024 points of the FIDs, followed by Fourier transformations and polynomial baseline corrections.
  • ss sinebell square
  • the embedded substructures were further regularized by simulated annealing with a starting temperature of 1000 K and using 2000 steps in both the annealing and the cooling stages with a time-step of 1 femtosecond (fs).
  • the resulting structures underwent a simulated annealing refinement of the slow-coolmg type with a starting temperature of 2000 K and 1000 steps during the cooling stage with a time-step of 1 fs, followed by 500 steps of energy minimization.
  • the nonbonded interactions were modeled only by a quadratic repulsive energy term, while the attractive components of the Lennard- Jones potential and the electrostatic energy were turned off.
  • the NMR spectra of the P25 peptide were collected using the same parameters as with P26.
  • the ⁇ H chemical shifts of P26 and P25 were different, requiring completely independent assignment of the resonances and sequential connectivities of P25.
  • Well-resolved signals in the amide proton and the alpha proton regions of the 2D NOESY spectra of P25 provided unambiguous NH-NH - ⁇ +I ) and ⁇ H-NH( 7 ;+
  • P25 also forms a continuous but labile helix in water.
  • the intensities of the NH-NH (if+ ⁇ ) NOEs of P25 are on average 51%> of the intensities of the ⁇ H-NH ( 7 /+ ⁇ ⁇ NOEs between residues Gly500 and Val520, suggesting that if the peptide were to assume uniform conformations, it would be in an extended conformation 66% of the time and in a helix 34% of the time, although these are approximations.
  • residues Tyr512 to Val520 of P25 appear to form a more stable helical structure than do residues Gly500 to Ser511 ( Figure 4).
  • a lower helical propensity of P25 compared to P26 in water is indicated by the detection of 21 intermediate-range NOEs characteristic of helices in P25 in comparison with 29 such NOEs in P26 ( Figure 4). Because both P25 and P26 form labile helices in equilibrium with extended conformations, a quantitative estimate of relative contributions of helical to extended conformation for each residue in the two peptides was obtained from the ratios of the integrated intensities of the sequential NH-NH(/ + ⁇ ⁇ H-NH ( 7 z -+i) NOEs. A region of decreased helicity in P25 relative to that in P26 was observed from residues 505-514, surrounding the missing residue, Phe508.
  • the average ratio of NH-NH( ⁇ +i)/ ⁇ H-NH( z - z - + 1 ) NOEs of the 25-mer in comparison with the 26-mer in this region is 0.707 ⁇ 0.158, indicating that the helical propensity of the 25-mer is 1 % of that of the 26-mer between residues 505 and 514.
  • Lys522 being in an ⁇ -helix.
  • the NH and H resonances of the amino-terminal Met498 in both peptides were not observed. Hence its connectivities to Pro499 could not be established.
  • the NH proton of the carboxy-terminal Ala523 was observed in P26 but not in P25 (see, also, Massiah et al., supra, 1998).
  • Circular dichroism was used to examine the structure of the ⁇ F508 P25 peptide in the presence or absence of deuterated water (D O), which previously has been show to promote ⁇ F508 CFTR function in intact cells (Brown et al., supra, 1996).
  • D O deuterated water
  • the ⁇ F508 P25 peptide formed an ⁇ -hehcal structure characteristic of that obtained for the wild-type P26 peptide.
  • an agent which can promote function of ⁇ F508 CFTR in mtact cells, can correct a conformational defect in a peptide portion of CFTR comprising the ⁇ F508 mutation.
  • This example provides various assays useful for identifying an agent that corrects a defect in the three dimensional conformation of a polypeptide
  • Examples of three different assays are provided, wherein a conformational change in the mutant ⁇ F508 P25 peptide to a conformation characteristic of the wild-type P26 peptide is detected using a monoclonal antibody, a fluorescent probe, or a physical method such as circular dichroism (CD) spectroscopy.
  • CD circular dichroism
  • a monoclonal antibody that specifically binds the wild-type P26 peptide, which forms a stable ⁇ -helix, but not the mutant ⁇ F508 P25 peptide was prepared using standard methods.
  • the P26 and ⁇ DF508 P25 peptides were chemically synthesized as described in Example 1, and purified by reverse phase HPLC.
  • the predicted molecular weights of the peptides were confirmed by mass spectrometry; the sequences were confirmed by N-terminal sequence analysis; and the ability of the P26 peptide to form an ⁇ -helical structure confirmed by CD spectroscopy.
  • the P26 peptide but not the P25 peptide, enhances the fluorescence of the probe 1,8-ANS (8-anilinonaphthalene-l-sulfonic acid).
  • ANS is essentially nonfluorescent in water, and only becomes appreciably fluorescent when bound to membranes (quantum yields of approximately 0.25), or more fluorescent when bound to protein (quantum yields of approximately 0.7; McClure and Edelman,
  • CD spectra are collected in a 0.01 mm path length demountable Suprasil cell cuvette on a AVIV 60DS spectropolarimeter at 37°C in a 40 ⁇ l system over a wavelength range of 185 nm to 260 nm.
  • Spectra are deconvoluted using the PROSEC program v2.1, which employs the reference spectra and algorithm of Chang et al. (Anal. Biochem. 91:13-31, 1978, which is incorporated herein by reference), or the like (see, for example, Yang et. al., Meth. Enzymol. 130:208-269, 1986; Bolotina et al., Mol. Biol. 14:902-909, 1980), to quantify the amounts of ⁇ -sheet, ⁇ -helix, random coil, or ⁇ -turns.
  • agents that potentially can affect the folding of a polypeptide are exemplified herein. These agents include organic osmolytes such as those found in the kidney, selected peptide regions of CFTR, and lipids that normally are present in eukaryotic cell membranes.
  • Class 1 agents include, for example, D 2 0, DMSO, glycerol, TMAO, butyrate, and phenylbutyrate. These agents can be obtained, for example, from the Fluka Chemical Corp. (Milwaukee WI).
  • Class 2 agents includes small organic osmolytes such as sorbitol, inositol, betaine, glycerophosphoryl choline, arginine, and urea, which normally are present in the kidney; dimethylpropiothetin, which is an osmolyte in certain marine algae; and trehalose, which is found in insects and fungi.
  • the kidneys of CF patients appear to function normally suggesting that osmolytes in the kidney are involved in aiding a normal processing of ⁇ F508 CFTR.
  • Class 2 reagents are also available from the Fluka Chemical Corp.
  • Class 3 agents are peptides, or modified forms thereof, derived from the twelve predicted transmembrane segments of CFTR (TM1 to TM12), as follows:
  • TM2 (SI 18-L138); SIAIYLGIGLCLLFIVRTLLL (SEQ ID NO: 4); TMl (L195-1215); LALAHFVWIAPLQVALLMGLI (SEQ ID NO: 5);
  • TM4 (A221-G241); ASAFCGLGFLIVLALFQAGLG (SEQ ID NO: 6);
  • TM6 G330-V350
  • GIILRKIFTTISFCIVLRMAV SEQ ID NO: 8
  • TMl (I860-V880); IFVLIWCLVIFLAEVAASLVV (SEQ ID NO: 9); TM8 (S912-F932); SYYVFYIYVGVADTILLANGFF (SEQ ID NO: 10);
  • TM11 (II 1 103-G1123); IEFMIFVIFFIAVTFISILTTG (SEQ ID NO: 13); and
  • TM12 VI 129-11150
  • VGIILTLAMNIMSTLQWAVNSI SEQ ID NO: 14
  • CFTR NBF2 CFTR NBF2
  • the peptide are synthesized, purified and characterized as described in Example 1.
  • Peptide portions of NBF2 have been selected because NBF2 is predicted to interact with NBF1 (Bianchet et al., supra, 1997).
  • Peptide portions of the TM domains have been selected because the crystal structure of the ATP-binding subunit (HisP) of the histidine permease, an ABC transporter from Salmonella typhimurium, at 1.5 A resolution has demonstrated that the region in HisP that is equivalent to the Phe508 region of CFTR, lies within an ⁇ -helical structure in an exposed area.
  • HisP ATP-binding subunit
  • Class 4 agents represent eukaryotic cell membrane components, including phosphoglycerides, cholesterol, and sphingolipids, which are commonly found in the heart and the brain.
  • agents to be examined include palmitoyl-linoleoyl phosphatidylcholine (16:0, 18:2), stearoyl-arachidonyl phosphatidylethanolamine (18:0, 20:4), stearoyl-oleoyl phosphatidylserine (18:0, 18: 1), stearoyl-arachidonyl phosphatidylinositol (18:0, 20:4), stearoyl-sphingomyelin (18:0), galactosyl-lignoceroyl cerebroside (24:0), and cholesterol (Avanti Polar Lipids, Inc., Alabaster AL). These agents have been selected because the Phe508 region of CFTR is predicted to interact with the lipid components of the membrane.
  • Varying concentrations of P25 (1 ⁇ M to 100 ⁇ M) and an agent to be tested (50 to 100 fold excess over P25) will be incubated in microcentrifuge tubes at 37°C. Incubation times can vary from 1 to 4 hours, to overnight, as convenient. Untreated P25 and the wild-type P26 peptide will serve as controls.
  • aliquots are transferred onto wells of a 96 well ELISA plate for binding (1 hr, at 37°C).
  • the wells are then washed with PBS, blocking of nonspecific sites in the well is performed with 3 % BSA in PBS for 1 hr at 25°C, followed by washing three times with PBS, then monoclonal antibody (mAb) specific for P26 is added at various dilutions and incubation continued for 1 hr at 25°C.
  • mAb monoclonal antibody
  • HRP-secondary antibody anti-mAb mouse IgG-horseradish peroxidase antibody
  • Detection of the HRP reaction is performed by adding 100 ⁇ l of a solution containing 50 mM sodium citrate, 50 mM citric acid, 0.1 % o-phenylenediamine dihydrochloride, and 0.006 % hydrogen peroxide to the wells.
  • 50 ⁇ l of 2 M sulfuric acid is added to each well to quench the HRP reaction, and the absorbance at 492 nm is read using a 96 well plate reader (Titertek Multiskan Plus).
  • Untreated P25, P26, Fi catalytic sector of the ATP synthase complex
  • smaller peptides derived from the ⁇ -subunit of F ls and hexokinase which are prepared as described previously (Garboczi et al., supra, 1988; Arora et al., supra, 1990), serve as controls, which can be compared to the results obtained using treated P25.
  • P25 is added to 1,8-ANS or bis-ANS (at varying concentrations of 0.5 ⁇ M to 100 ⁇ M) in PBS (pH 7.4) at 37°C in a 3 ml system.
  • PBS pH 7.4
  • Several buffers, including PBS (pH 7.4), sodium acetate (pH 5.2), MOPS (pH 7.5), HEPES (pH 7.0), and Tris-HCl (pH 8.0) were shown in preliminary experiments to be compatible with this system.
  • the binding of 1,8-ANS or bis-ANS to the treated P25 peptide then is monitored for fluorescence enhancement at an excitation wavelength of 300 nm or 382 nm, respectively, and an emission wavelength of 468 nm or 490 nm, respectively, using a Perkin Elmer LS 50B spectrometer. Any interaction of the fluorescence intensity probe with the test agent will be identified and subtracted from the total fluorescence intensity derived from the interaction between treated P25 and probe.
  • CD spectra of treated P25, at 100 ⁇ M, are collected as described above and the amounts of ⁇ -sheet, ⁇ -helix, random coil, and ⁇ -turns are quantified.
  • the untreated P25 peptide and the wild-type P26 peptide are subjected to CD spectral analysis and their CD spectra are compared with those of treated P25 for the differences in their secondary structures.
  • An agent that corrects the structure of P25 such that it more closely approximates that of P26 can be selected.

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Abstract

L'invention concerne un procédé d'identification d'un agent corrigeant une conformation tridimensionnelle défectueuse d'un polypeptide, ce procédé consistant à mettre en contact, dans un système hors-cellule, une portion peptidique du polypeptide avec un agent d'essai, le peptide représentant une région du polypeptide contenant la conformation défectueuse, et à déterminer si l'agent d'essai diminue la conformation défectueuse du peptide. L'invention concerne également une représentation virtuelle d'une portion peptidique d'un polypeptide, dans laquelle le peptide représente une région d'un polypeptide possédant une conformation tridimensionnelle défectueuse.
PCT/US2000/026193 1999-09-24 2000-09-22 Procedes d'identification d'un agent corrigeant le repliement defectueux d'une proteine Ceased WO2001021652A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6583158B1 (en) 1998-06-01 2003-06-24 Mount Sinai School Of Medicine Of New York University Method for enhancing mutant enzyme activities in lysosomal storage disorders
US11673864B2 (en) 2013-01-31 2023-06-13 Vertex Pharmaceuticals Incorporated Pyridone amides as modulators of sodium channels

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5691306A (en) * 1993-08-26 1997-11-25 National Research Council Of Canada Methods of detection and treatment of protein trafficking disorders and increasing secretory protein production
US5888722A (en) * 1996-11-04 1999-03-30 Institut Curie Stable cell lines expressing the CFTR protein or a mutant of this protein, tool for selecting molecules having an effect on the intracellular transport of these proteins
US5900360A (en) * 1996-04-10 1999-05-04 Welch; William J. Correction of genetic defects using chemical chaperones

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5691306A (en) * 1993-08-26 1997-11-25 National Research Council Of Canada Methods of detection and treatment of protein trafficking disorders and increasing secretory protein production
US5900360A (en) * 1996-04-10 1999-05-04 Welch; William J. Correction of genetic defects using chemical chaperones
US5888722A (en) * 1996-11-04 1999-03-30 Institut Curie Stable cell lines expressing the CFTR protein or a mutant of this protein, tool for selecting molecules having an effect on the intracellular transport of these proteins

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6583158B1 (en) 1998-06-01 2003-06-24 Mount Sinai School Of Medicine Of New York University Method for enhancing mutant enzyme activities in lysosomal storage disorders
US6589964B2 (en) 1998-06-01 2003-07-08 Mount Sinai School Of Medicine Of New York University Method for enhancing mutant enzyme activities in lysosomal storage disorders
US6599919B2 (en) 1998-06-01 2003-07-29 Mount Sinai School Of Medicine Of New York University Method for enhancing mutant enzyme activities in lysosomal storage disorders
US6916829B2 (en) 1998-06-01 2005-07-12 Mount Sinai School Of Medicine Of New York University Method for enhancing mutant enzyme activity in gaucher disease
US7141582B2 (en) 1998-06-01 2006-11-28 Mount Sinai School Of New York University Method for enhancing mutant enzyme activities in Gaucher disease
US7514453B2 (en) 1998-06-01 2009-04-07 Amicus Therapeutics Inc. Method for enhancing mutant protein activity
US7622485B2 (en) 1998-06-01 2009-11-24 Mount Sinai School Of Medicine Of New York University Method of enhancing lysosomal α-galactosidase A
US7812033B2 (en) 1998-06-01 2010-10-12 Mount Sinai School Of Medicine Of New York University Method for increasing the activity of acid-β-galactosidase
US8436018B2 (en) 1998-06-01 2013-05-07 Mount Sinai School Of Medicine Of New York University Method for increasing the activity of lysosomal enzymes
US8633221B2 (en) 1998-06-01 2014-01-21 Mount Sinai School Of Medicine Of New York University Method of enhancing lysosomal α-galactosidase A
US8841322B2 (en) 1998-06-01 2014-09-23 Mount Sinai School Of Medicine Of New York University Method for increasing the activity of lysosomal enzymes
US9265780B2 (en) 1998-06-01 2016-02-23 Icahn School Of Medicine At Mount Sinai Method of enhancing lysosomal α-galactosidase A
US11673864B2 (en) 2013-01-31 2023-06-13 Vertex Pharmaceuticals Incorporated Pyridone amides as modulators of sodium channels

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