WO2001021652A1

WO2001021652A1 - Methods for identifying an agent that corrects defective protein folding

Info

Publication number: WO2001021652A1
Application number: PCT/US2000/026193
Authority: WO
Inventors: Peter L. Pedersen; Young Hee Ko
Original assignee: The Johns Hopkins University School Of Medicine
Priority date: 1999-09-24
Filing date: 2000-09-22
Publication date: 2001-03-29
Also published as: AU7712000A

Abstract

The present invention relates to a method of identifying an agent that corrects a defective three dimensional conformation of a polypeptide, 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 containing the defective conformation; and determining that the test agent decreases the defective conformation of the peptide. 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.

Description

METHODS FOR IDENTIFYING AN AGENT THAT CORRECTS DEFECTIVE PROTEIN FOLDING

This invention was made in part with government support under Grant No. DK43962 awarded by the National Institutes of Health. The government may have certain rights in this invention.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION 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.

BACKGROUND INFORMATION 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 (CF), for example, 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. In approximately seventy percent of CF cases, a single amino acid deletion occurs at position 508 of the 1480 ammo acid cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide 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 Interestingly, the mutant CFTR polypeptide appears to have the ability to function in the same way as the normal CFTR polypeptide, although with less efficiency. However, 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.

It has been suggested that the mutation in CFTR may affect proper folding of

CFTR such that the mutant CFTR is marked by the cell as defective and directed to a protein degradation pathway, rather than properly translocating to the cell membrane. Thus, efforts have been made to treat CF associated with a mutant CFTR using chemical agents that can facilitate protein folding. While such efforts have had variable success, these approaches suffer from a lack of knowledge of the actual defect in CFTR. Thus, the identification of potentially useful agents for treatment of CF requires cell based assays, which are expensive and tedious to perform and are not suitable for large scale examination of potentially useful agents. Thus, a need exists for assays that are adaptable for high throughput screening to identify agents that can correct a structural defect in a protein such that the protein can function m a more normal manner The present invention satisfies this need and provides additional advantages.

SUMMARY OF THE INVENTION 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 α-ketoacid dehydrogenase complex, p53, type I procollagen pro-α, LDL receptor, αl-antitrypsin, β-hexosamimdase, rhodopsin, an insulin receptor, a pπon protein, β-amyloid, transthyretm, and a crystalhn polypeptide.

In one embodiment, 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 In another embodiment, 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

In still another embodiment, 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. Optionally, the method can further include quantitating the amount of said agent that corrects the defective three dimensional conformation of the polypeptide

In such a method of the invention, 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. In addition, 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. Such 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 (D₂0), 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-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); and galactosyl-lignoceroyl cerebroside (24:0). 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. In one embodiment, 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. In another embodiment, 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.

DETAILED DESCRIPTION OF THE INVENTION

Various pathologic conditions are associated with proteins having a defective three dimensional conformation in comparison to that of the normal wild-type protein. As a result of the defective three dimensional conformation, 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.

As used herein, the term "three dimensional conformation" or "conformation," when used in reference to a protein, polypeptide or a peptide portion thereof, refers to the secondary, tertiary or quaternary structure of the polypeptide. In addition, the term "folding" is used herein to refer to a secondary structure of a polypeptide, for example, an α-helix, β-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.

The term "wild-type" is used herein to refer to a polypeptide that assumes a particular conformation that can be found naturally in a healthy subject. For purposes of the present disclosure, a wild-type polypeptide can be compared to a polypeptide that exhibits a defective three dimensional conformation. As used herein, 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 α-helix, in comparison to the corresponding wild-type polypeptide or peptide portion thereof, which forms a stable α-helix; by determining that the mutant polypeptide forms a different secondary structure than the wild-type polypeptide, for example, forms a β-sheet, in comparison to the corresponding wild-type polypeptide or portion thereof, which forms an α-helix; or the like.

It should be recognized that a wild-type polypeptide is defined, in part, in that it can be found naturally in a healthy individual. As such, 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. For example, a prion protein can exist in two states, a non-infective state comprising an α-helix, and an infective state comprising a β-sheet. For purposes of the present disclosure, a non-infective prion polypeptide comprising an α-helix is considered to be the wild-type prion protein, whereas an infective prion polypeptide comprising the β-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. As used herein, 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). For example, where the defect is a propensity of a α-helical region of the polypeptide to destabilize, a method of the invention provides a means to identify an agent that stabilizes the α-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).

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 (ΔF508), 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 (see Example 1). However, based on the present disclosure, it will be readily apparent that other 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 α-ketoacid dehydrogenase complex, p53, type I procollagen pro-α, LDL receptor, αl -anti trypsin, β-hexosaminidase, rhodopsin, an insulin receptor, a prion protein, β-amyloid, transthyretin, and a crystallin polypeptide.

The term "peptide portion of a polypeptide" or "peptide" is used broadly herein to mean two or more amino acids linked by a peptide bond. Generally, 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. As such, 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. It should be recognized that a peptide 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. The terms "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. The term "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. Although 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. Examples of such agents include, for example, a small organic molecule such as deuterated water (D₂0), 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 (e.g., NBF2); or cholesterol, or a phosphoglyceride or sphingolipid such as 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); and galactosyl- lignoceroyl cerebroside (24:0). Such agents also can be used as a starting material for preparing derivatives or modified forms thereof, which can be examined using a method of the invention to identify novel agents that can correct a defective three dimensional conformation of a polypeptide.

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. Thus, 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. 5,264,563, which is incorporated herein by reference); a peptidomimetic library (Blondelle et al., Trends Anal. Chem. 14:83-92, 1995; a nucleic acid library (O'Connell et al., supra, 1996; Tuerk and Gold, supra, 1990; Gold et al., supra, 1995; U.S. Pat. No. 5,750,342; each of which is incorporated herein by reference); an oligosaccharide library (York et al, Carb. Res.. 285:99-128, 1996; Liang et al., Science. 274: 1520-1522, 1996; Ding et al., Adv. Expt. Med. Biol. 376:261-269, 1995; each of which is incorporated herein by reference); a lipoprotein library (de Kruif et al., FEBS Lett.. 399:232-236, 1996, which is incorporated herein by reference); a glycoprotein or glycolipid library (Karaoglu et al., J. Cell Biol..

130:567-577, 1995, which is incorporated herein by reference); or a chemical library containing, for example, drugs or other pharmaceutical agents (Gordon et al., J. Med. Chem.. 37: 1385-1401. 1994: Ecker and Crooke. Bio/Technology. 13:351-360, 1995; each of which is incorporated herein by reference).

Various methods are disclosed herein or otherwise known in the art for detecting that a test agent corrects the three dimensional conformation of a polypeptide (see Example 3). For example, 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.

Alternatively, 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 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. Such 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 α-helix, but not to a corresponding 25 amino acid peptide, which is representative of the ΔF508 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).

Methods of obtaining antibodies that selectively bind to a peptide having one conformation, for example, a wild-type conformation, but not a corresponding peptide having a different conformation are routine in the art. As used herein, the term "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

The term "binds specifically" or "specific binding" or the like, when used in reference to an antibody means that an interaction of the antibody and a particular epitope has a dissociation constant of at least about 1 x 10 ⁶, generally at least about 1 x 10 ⁷, usually at least about 1 x 10 ⁸, and particularly at least about 1 x 10 ⁹ or 1 x 10 ¹⁰ or less As such, 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

The term "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 Such 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. 2d ed (Oxford University Press 1995), each of which is incorporated herein by reference) 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. Where 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. Various other carrier molecules and methods for coupling a hapten to a carrier molecule are well known in the art (see, for example, by Harlow and Lane, supra, 1988).

If desired, a kit incorporating an antibody useful in a method of the invention can be prepared. Such 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. Such detectable reagents useful for labeling or otherwise identifying the antibody are described herein and known in the art.

Methods for raising polyclonal antibodies, for example, in a rabbit, goat, mouse or other mammal, are well known in the art (see, for example, Green et al., "Production of Polyclonal Antisera," in Immunochemical Protocols (Manson, ed.,

Humana Press 1992), pages 1-5; Coligan et al., "Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters," in Curr. Protocols Immunol. (1992), section 2.4.1; each or which is incorporated herein by reference). In addition, monoclonal antibodies can be obtained using methods that are well known and routine in the art (Harlow and Lane, supra, 1988). For example, spleen cells from a mouse immunized with a desired peptide can be fused to an appropriate myeloma cell line such as SP/02 myeloma cells to produce hybridoma cells. 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.

Methods of preparing monoclonal antibodies well known (see, for example, Kohler and Milstein, Nature 256:495, 1975, which is incorporated herein by reference; see, also, Coligan et al., supra, 1992, see sections 2.5.1-2.6.7; Harlow and Lane, supra, 1988). Briefly, 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. 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. Optionally, 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.. USA 89:7958-7962, 1992; Chien et al., Proc. Natl. Acad. Sci. USA 88:9578-9582, 1991; Young, Biol. Reprod. 58:302- 311(1998), each of which is incorporated herein by reference), the reverse two hybrid assay (Leanna and Hannink, Nucl. Acids Res. 24:3341-3347, 1996, which is incorporated herein by reference), the repressed transactivator system (U.S. Pat. No. 5,885,779, which is incorporated herein by reference), the phage display system (Lowman, Ann. Rev. Biophys. Biomol. Struct. 26:401-424, 1997, which is incorporated herein by reference), or other such assays that can correlate structure and function of a polypeptide (see, for example, Mathis, Clin. Chem. 41 : 139-147, 1995 Lam. Anticancer Drug Res. 12: 145-167, 1997: Phizicky et al.. Microbiol. Rev. 59:94- 123, 1995; each of which is incorporated herein by reference).

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. For example, 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. However, the peptide also can comprise a portion of a polypeptide such as β-amyloid, which, under certain conditions, can form a β-sheet structure that leads to aggregate formation as occurs in Alzheimer's disease. By determining that a test agent alters the three dimensional conformation of the peptide such that it more closely approximates the conformation of a corresponding wild-type protein, an agent that modulates the three dimensional conformation of protein is identified.

In one embodiment, 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. For example, 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. In another embodiment, 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. For example, a method of the invention can identify an agent that destabilizes the β-sheet conformation of an infectious prion protein such that the prion protein forms an alternative α-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. If desired, the method can further include quantitating the amount of said agent that corrects the defective three-dimensional conformation of the polypeptide.

The polypeptide exhibiting the defective conformation can be any polypeptide, as discussed above. Such 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. For example, 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 α-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-α 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 β-amyloid; familial amyloidosis is associated with aggregation of transthyretin or of lysozyme; cataracts are associated with aggregation of crystallins; familial hypercholesterolemia is associated with mislocalization due to misfolding of LDL receptor; αl -anti trypsin deficiency is associated with mislocalization due to misfolding αl-antitrypsin; Tay-Sachs disease is associated with mislocalization due to misfolding of β-hexosaminidase; retinitis pigmentosa is associated with mislocalization due to misfolding of rhodopsin; and leprechaunism is associated with mislocalization due to misfolding of insulin receptor (see U.S. Pat. No. 5,900,360; Thomas et al., supra, 1995). Thus, an agent identified according to a method of the invention can be used as a medicament to ameliorate the severity of a pathologic condition.

Cystic fibrosis (CF) affects approximately 1 in 2000 people in the United

States and Canada and frequently results in death of the patient before the age of 30. Approximately 70% of CF cases are associated with a deletion of phenylalanine at position 508 in the cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide. CFTR is expressed in many different tissues including the lungs, pancreas, sweat ducts, reproductive tract, salivary glands, duodenum, and liver. CFTR is a member of a superfamily of proteins known as ABC (ATP-binding cassette) transporters or traffic ATPases, and is believed to function physiologically as a chloride ion channel to help regulate conduction pathways for chloride ion and sodium ion in epithelial cells. More specifically, 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.

Deletion of Phe508 (ΔF508) 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). Although the reason that ΔF508 CFTR does not leave the ER and traffic normally to the plasma membrane has not been clearly established, it has been suggested that the deletion mutation causes a folding defect, which prevents the protein from adopting its proper three dimensional conformation essential for normal trafficking (Thomas et al., J. Biol. Chem.

267:5727-5730, 1992; Welch and Brown, Cell Stress. Chap. 1, pages 109-115, 1996; Brown et al., Cell Stress. Chap. 1, pages 117-124, 1996; Brown et al., J. Bioenerg. Biomemb. 29:491-502, 1997). Studies on wild-type and ΔF508 peptide segments of CFTR revealed that the mutation induces a marked instability in the ΔF508 peptide which is accompanied by a significant structural change (Thomas et al., Science 251:555-557, 1991; Thomas et al., supra, 1992).

Blockage of the ΔF508 CFTR in the ER appears to be relieved if ΔF508 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, 1996λ These results, in addition to circular dichroism (CD) and stability studies of long peptide segments (>60 amino acid residues) of NBF1 (Thomas et al., supra, 1992) suggest that deletion of Phe508 leads to a misfolded protein, which cannot be transported to the cell membrane. Nevertheless, the ΔF508 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 ΔF508 region. The expressed ΔF508 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 α-hehcal region (Bianchet et al., J. Bioenerg. Biomemb 29:503-524, 1997, which is incorporated herein by reference)

In order to understand how CFTR works, much attention has been focused on all domains of the protein. Both nucleotide domains (NBF1 and NBF2) have the capacity to bind and hydrolyze ATP (see, for example, Smit et al., Proc. Natl. Acad Sci.. USA 90:9963-9967, 1993; Ko et al., J. Biol Chem. 268:24330-24338, 1995, Randak et al., FEBS Lett 363: 189-194, 1995). For optimal channel opening and closing, these functions are dependent on phosphorylation of the regulatory domain (Tabcharam et al., Nature 352:628-6631, 1991) The 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

(approximately 0.2 to 1 sec^"1)

Over 500 mutations/alterations distributed throughout the five domains of CFTR are known to cause CF (Tsui, 1997, CF Genetic Analysis Consortium; see http://l 99.0.26.114/). Many of these mutations e withm or near the two nucleotide binding folds The most common mutation is ΔF508, which is located near the center of NBF 1. About 90%> of CF patients, including nearly 30,000 in the United States, have at least one ΔF508 CFTR allele (Karem et al., New En l J Med 323- 1517- 1522, 1990) The high incidence of CF caused by the ΔF508 mutation has resulted m many investigations of the mutant protein since the gene encoding CFTR was identified, cloned, and sequenced. Such studies have focused on identifying the cellular, physiological, and biochemical changes that result from the F508 mutation; understanding the structural basis of the disease; and devising novel strategies to correct the problem.

As disclosed herein, two dimensional (2D) 1H nuclear magnetic resonance (NMR) studies at 600 MHz of a 26 amino acid peptide (Met498 to Ala523 of CFTR, representative of the Phe508-containing region) in 10% DMSO (pH 4.0) at 25°C showed a continuous, but labile, helix from Gly500 to Lys522, based on both

NH- H - _;+n and

Overhauser effects (NOEs; see Example 1).

Phe508 within this helix showed only short-range (i, i+2) NOEs. In comparison, a corresponding 25 amino acid peptide, which lacks Phe508 and is representative of the ΔF508 region of CFTR, also forms a labile helix from Gly500 to Lys522. However, the relative intensities of the NH-NH(/ + \ )/ αH-NH + ) NOEs, fewer intermediate-range NOEs, and downfield αH and NH chemical shifts indicated a lower helical propensity of the 25-mer between residues 505 and 517, surrounding the missing residue, Phe508 (Example 1).

2D 1H NMR studies of both peptides in saturating (43%) TFE revealed stable α-helices from Gly500 to Lys522, based on NH-NH z+i _s2,3V ^αH-NH(/_{j Z}-+2 3 4), αH-βH( /+3), and weak αH-NH(/₍/+ι ) NOEs. However, downfield shifts of the

H resonances from residues Gly500 to Ile507 and fewer intermediate-range NOEs were indicative of a less stable α-helix in the 25-mer even in saturating TFE (Example 1). These results demonstrate that the Phe508-containing region of CFTR has a propensity to form an α-helix, which is destabilized by the ΔF508 mutation found in most CF patients. This observation provides a means to identify mutations associated with defective folding of a protein, and further provides a means to identify agents that can correct such a defect.

Prior to the present disclosure, the primary defect in ΔF508 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 ΔF508 region, and excluded a critical part of the region. As disclosed herein, an in vitro assay has been developed for readily identifying agents that correct the basic defect in ΔF508 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, ΔF508 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 ΔF508 region. Both wild-type CFTR and ΔF508 CFTR are degraded with similar half-lives, although the mutant protein is eventually completely degraded, whereas about 20-50%> of the wild-type protein escapes this fate and exits the ER, suggesting that the events involved in the retention of ΔF508 CFTR and those involved in its degradation are separate and distinct, and that the degradation events do not discriminate between wild type and mutant forms, whereas those involved in retention do so discriminate. 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). In addition, 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. 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 ΔF508 CFTR in the ER, including the ubiquitin-proteasome pathway. Introduction of ΔF508 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)

These observations suggest that ΔF508 CFTR can fold in the ER into a nearly wild-type molecule, with only a localized region, the ΔF508 region within NBF1, being misfolded, and that this structural defect prevents the ATP-dependent conversion from a protease-sensitive form to a protease-resistant form essential for continuing on in the trafficking pathway Failure to undergo this ATP-dependent step, results in retention of the mutant protein in the ER, where the degradation machinery, operating, at least in part, from the cytoplasmic surface with the aid of one or more chaperones, degrades the mutant protein

The use of cellular osmolytes or other known protein stabilizing agents has been successful m facilitating the trafficking of ΔF508 CFTR to the plasma membrane (Welch and Brown, supra, 1996,, Sato et al , J Biol Chem 271 7261- 7264, 1996, Brown et al , J Bioenerg Biomemb 29 491-501, 1997) Among these osmolytes, the best studied is glycerol, which facilitated trafficking of ΔF508 CFTR to the plasma membrane in a form that mediates cAMP-stimulated chloride ion conductance Prior to the present disclosure, however, it had not been demonstrated that glycerol or other chemical chaperones such as D₂O, DMSO, or tπmethylamine N-oxide promote trafficking of mutant CFTR by correcting a structural defect in the ΔF508 region of CFTR For example, cellular osmolytes are known to transcπptionally up-regulate genes, and DMSO has been shown effect cell differentiation by increasing the amount of AF508 CFTR in the plasma membrane (Bebok et al , Am J Phvsiol 275 C599-C607, 1998)

As disclosed herein, a peptide (P26) corresponding to the predicted α-hehcal region containing Phe508 as defined by the 3D modeling studies (Bianchet et al , supra, 1997), and a corresponding peptide lacking Phe508 (P25), were synthesized, and their solution structures were determined by ^XH NMR (see Massiah et al , in "42d Ann Meeting Biophys Soc , Biophys J " 76 A428, Abs W-Pos -155, 1999, which is incorporated herein by reference). 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. As disclosed in Example 1, 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. First, the relative intensity ratios of the sequential NH-NH( /+i )/aH-NH( _z-+i) NOEs, which is a measure of the helical/extended contributions to conformation, were significantly lower (0.707±0.158) for residues 505 to 514 of P25 than for the corresponding residues of P26. Second, P26 showed 29 intermediate-range NOEs characteristic of helices, while P25 showed only 21. Third, in water, significant downfield chemical shifts of the αH and NH resonances of residues 505 to 517 occurred in P25 in comparison with those of P26, consistent with a lower helicity of P25 in the region surrounding the missing residue. This difference in helical propensity was confirmed by the computed NMR structures of P26 and P25 in water.

The lower helicity of P25 compared to P26 in water was surprising because P25 lacks a Phe residue, which is an amino acid residue that normally has an unfavorable intrinsic helix-forming propensity of +0.67 kcal/mol in alanine-rich peptides (Chakrabarty et al., Prot. Sci. 3:843-852, 1994). Helical regions of peptides and proteins containing Phe residues are believed to be stabilized by side chain interactions. Hence, the loss of side chain interactions involving Phe508 can lead to destabilization of the helix in P25. This explanation is supported by the observed decrease in helicity of P25 in water, between residues 505 and 517, which surrounds position 508.

In TFE, both P26 and P25 formed α-helices from residues Thr501 to Lys522 that mimic the folded state of this region in intact CFTR. Although the P25 ΔF508 mutant peptide formed an α-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. The α-hehcal structure of P26 in TFE revealed a hydrophobic i to ι+4 side chain-side chain interaction between Glu504 and Phe508 that may contribute to the net stabilization of the helix by Phe508 In P26, a weak NOE was found between Phe508 δH and Glu504 βH, indicating the proximity of these two residues in the α-hehcal form of this peptide. Such proximity in intact CFTR can also provide a hydrophobic environment, which would increase the pK_a of Glu504.

The 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 β-sheet region m P67 that became unstable in P66, resulting in random coil formation. The differences in secondary structure likely reflect the different conditions used m the two studies (90% H₂O/10% DMSO, pH 4.0, in this study; 100% H₂O, pH 5.5, in the earlier study); or can be due to the lack of Tyr517 to Ala523 at the C-termim of the P67 and P66 peptides. These amino acids, which are present m the P25 and P26 peptides, are predicted from three dimensional modeling studies (Bianchet et al., supra, 1997) to contain the entire α-hehcal region of CFTR that includes Phe508. It has long been known that truncations of helices can destabilize them. Further m this regard, 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 α helix, in agreement with 3D modeling studies based on the structures of FI ATPase and the RecA protein (Bianchet et al., supra, 1997)

The results disclosed herein demonstrate that the Phe508 region of CFTR has a propensity to form an α-hehx in solution, and that deletion of this single aromatic residue destabilizes the helix. These results are relevant to the earlier finding that, in CF caused by the F508 mutation, ΔF508 CFTR is retained m the endoplasmic reticulum and targeted for degradation. Thus, the structural changes that occur upon deletion of Phe508 appear to prevent CFTR from undergoing the conformational transition believed to be essential for its normal trafficking to the plasma membrane, while alerting the quality control proteins that target the mutant protein for degradation

A number of chemiosmolytes that are known to stabilize proteins also can promote normal trafficking of ΔF508 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 Thus, the P26 and P25 peptides, which are representative of the Phe508 and F508 regions of the wild-type and ΔF508 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.

In one embodiment, 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. Alternatively, 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. 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). Where the antibody is contained in a kit useful for practicing a method of the invention, 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. For example, 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" α-helical structure or a "defective" β-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. In particular, such 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. 274:325-37, 1997; Matsumoto et al., J. Biol. Cheπ 270: 19524-31, 1995: Sali et al.. J. Biol. Chem. 268:9023-34. 1993; Sali, Molec. Med. Today 1 :270-7, 1995a; Sali, Curr. Opin. Biotechnol. 6:437-51, 1995b; Sali et al., Proteins 23: 318-26, 1995c; Sali. Nature Struct. Biol 5: 1029-1032. 1998; U.S. Pat. No. 5,933,819; U.S. Pat. No. 5,265,030, each of which is incorporated herein by reference).

The crystal structure coordinates of a polypeptide that has a conformational defect and is associated with a pathologic condition (see Thomas et al., supra, 1995) 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. Such 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).

Methods of using crystal structure data to design agents that can interact with a peptide portion of a polypeptide, thereby modulating its conformation, are known in the art. For example, the coordinates of a polypeptide can be superimposed onto other available coordinates of similar polypeptides, including polypeptides treated with an agent previously shown by a method of the invention to correct a conformational defect, to provide an approximation of the way the agent affects the conformation of the polypeptide. Computer programs employed in the practice of rational drug design also can be used to identify such agents.

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 Databases™ - an information retrieval program accessing chemical databases such as BioByte Master File, Derwent WDI and ACD; Catalyst/HYPO™ - generates models of compounds and hypotheses to explain variations of activity with the structure of drug candidates; Ludi™ - fits molecules into the active site of a protein by identifying and matching complementary polar and hydrophobic groups; and Leapfrog™ - "grows" new ligands using a genetic algorithm with parameters under the control of the user.

Various general purpose machines can be used with such programs, or it may be more convenient to construct a more specialized apparatus to perform the operations.

Generally, 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. In any case, 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. As used herein, the term "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.

Typically 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. A skilled artisan can readily appreciate that any one of the currently available computer systems are suitable.

In one embodiment, 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. In some embodiments, 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). In some embodiments, 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. In addition, 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.

Where it is desired to identify an agent that can correct a defective three dimensional conformation of a polypeptide, 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.

Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interaction. Examples of programs designed for such uses include Gaussian 92, revision C (Frisch, Gaussian, Inc., Pittsburgh PA, 1992); AMBER, version 4.0 (Kollman, University of California at San Francisco, 1994); QUANT A/CHARMM (Molecular Simulations, Inc., Burlington MA, 1994); and Insight II/Discover (Biosysm Technologies Inc., San Diego CA, 1994). These programs may be implemented using, for example, a Silicon Graphics workstation, IRIS 4D/35 or IBM RISC/6000 workstation model 550. Other hardware systems and software packages will be known to those skilled in the art of which the speed and capacity are continually modified.

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. In a first step, a virtual representation of a peptide portion of a polypeptide is performed. Thus, in one embodiment, 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. As discussed above, 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 appropπate. 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.

If a determination is made that a virtual test agent corrects the defective conformation of the virtual target peptide, 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. Once the name of the identified test agent is displayed to the user, 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. However, if more test agents exist in the database, then 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. In this manner, the new agent is examined for the ability to correct the defective conformation of the virtual target peptide. As such, 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.

As disclosed herein, 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. Such pathologic conditions, which are associated with expression of a polypeptide having a defective three dimensional conformation, are exemplified herein otherwise known in the art (see, for example, Thomas et al., supra, 1995; U.S. Pat. No. 5,900,360). As used herein, 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 characteπstic of a particular pathologic condition and will be well known to skilled clinician, as will the methods for monitoπng the signs and symptoms of the condition For example, where the pathologic condition is cystic fibrosis, the skilled clinician can monitor the amount of chloπde ion in the patient's sweat, the patient's ability to breathe, or other clinical sign or symptom associated with CF. Where 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.

Where 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. It is recognized that some peptide agents identified using a method of the invention can be relatively large and, therefore, may not readily traverse a cell membrane. However, 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 characteπstics of the target cell, into which the peptide is to be introduced. For example, where the target cells, or a few cell types including the target cells, express a receptor, which, upon binding a particular ligand, is internalized into the cell, 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)).

In addition, 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. Similarly, one or more peptide bonds in the peptide can be modified. In addition, 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. Of course, 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.

Where 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). If desired, such a polynucleotide 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. 25:37-42, 1993; Kirshenbaum et al., J. Clin. Invest. 92:381-387, 1993; each of which is incorporated herein by reference). A tetracycline (tet) inducible promoter can be particularly useful for driving expression of a polynucleotide in a cell. 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-

990, 1992; Anderson et al., Nature 392:25-30 Suppl., 1998; Verma and Somia, Nature 389:239-242, 1997; Wilson. New Engl. J. Med. 334: 1 1 5-1 1 7 (1996), each of which is incorporated herein by reference).

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. Introduction of 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). Where 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. Such 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. One skilled in the art would know that the choice of 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. 5,882,679; 5,395,619; and 5,225,212, each of which is incorporated herein by reference) are an example of such encapsulating materials particularly useful for preparing a pharmaceutical composition, and other "masked" liposomes similarly can be used, such liposomes extending the time that the agent remains in the circulation. Cationic liposomes, for example, also can be modified with specific receptors or ligands (Morishita et al., J. Clin. Invest.. 91 :2580-2585 (1993), which is incorporated herein by reference). In addition, 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, for example, are not particularly useful when administered orally because they can be degraded in the digestive tract. However, 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. Furthermore, 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. In general, 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. In addition 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).

The following examples are intended to illustrate but not limit the invention.

EXAMPLE 1 NORMAL. BUT NOT MUTANT. CFTR PEPTIDE FORMS AN α-HELIX This example demonstrates that a peptide portion of CFTR, which is mutated in many cystic fibrosis (CF) patients, forms an α-helix, whereas a peptide representing the mutant CFTR region does not form an α-helix (see, also, Massiah et al., supra, 1998). Peptide Synthesis and Purification

The 26-residue peptide (P26):

Met-Pro-Gly-Thr-Ile-Lys-Glu-Asn-Ile-Ile-Phe-Gly-Val-Ser-Tyr-Asp-Glu-Tyr- Arg-Tyr-Arg-Ser-Val-Ile-Lys-Ala (SEQ ID NO: 1),

which includes Met498 to Ala523 of CFTR, and the 25-residue peptide (P25):

Met-Pro-Gly-Thr-Ile-Lys-Glu-Asn-Ile-Ile-Gly-Val-Ser-Tyr-Asp-Glu-Tyr-Arg- Tyr-Arg-Ser-Val-Ile-Lys-Ala (SEQ ID NO: 2),

which lacks Phe508 of CFTR (ΔF508), were synthesized on a PE Biosystems Model 430A Peptide Synthesizer by the solid phase method of Merrifield (J. Am. Chem. Soc. 85:2149-2154, 1963) using F-moc (9-fluorenylmethyloxycarbonyl) chemistry to protect the α-amino group. The general methodology for preparing structured peptides has been previously described in detail (Garboczi et al., J. Biol. Chem. 263:812-816, 1988; Arora et al., J. Biol. Chem. 265:5324-5328, 1990; each of which is incorporated herein by reference).

Synthesized peptides were purified by semi-preparative HPLC chromatography utilizing a Vydac Protein & Peptide Cι₈ (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. To avoid oxidation of cysteine residues, where appropriate, 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. In addition, matrix-assisted LTV laser desorption/ionization (MALDI) mass spectrometry was performed using a Kratos Compact Maldi IV to confirm purity and identity. A 3 μg sample of a peptide was mixed with an UV absorbing matrix, ACHA (α-cyano-4-hydroxycιnnamιc acid plus free HCN, Sigma), which absorbs the laser energy to evaporate and ionize the sample.

NMR Spectroscopy

The NMR samples consisted of 1.4 mM solutions of the 25-mer and 26-mer peptides in 90% H2θ/10% perdeuterated dimethylsulfoxide (DMSO→fø pH 4.0).

Identical NMR experiments were performed in 43% tπfluoroethanol-ii_j (TFE) in water (pH 4.0). NMR experiments were recorded at 25°C on a Vaπan UmtyPlus 600 MHz spectrometer equipped with a pulse field gradient unit, four independent RF channels, and a Vaπan 5 mm triple resonance probe with an actively shielded z-gradient. The data were recorded using the States-time-proportional phase incrementation (TPPI) method (Marion et al., Biochemistry 28:6150-6156, 1989, which is incorporated herein by reference) in all indirect dimensions, with a relaxation delay of 1.5 sec Water suppression for the nuclear Overhauser effect (NOE) spectroscopy (NOESY; Jeener et al., J. Chem. Phys. 71 -4546-4553, 1979; Macura and Ernst, Mol Phys. 41 :95-117, 1980; each of which is incorporated herein by reference) and total correlation spectroscopy (TOCSY; Braunschweiler and Ernst, J. Magnetic Res. 53:521-528, 1983; Davis and Bax, J. Am. Chem. Soc. 107:2820-2821, 1985; each of which is incorporated herein by reference) experiments was achieved with a WATERGATE pulse train (Piotto et al., J. Biomol. NMR 2-661, 1992, which is incorporated herein by reference) attached at the end of each pulse sequence immediately before data acquisition.

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°-shιfted sinebell square (ss) window function to the 1024 points of the FIDs, followed by Fourier transformations and polynomial baseline corrections. Apodization with an 80°-sιnebell-shιfted window function was applied followed by zero-filling m the tj dimension to 1024 points to yield final matrix sizes of 1024 ( ) x 1024 (/?) real data points, the observed ^H chemical shift was referenced with respect to the water signal, which was taken as 4.773 ppm downfield from external sodium 3-(tπ- methylsilyl) propιonate-2,2,3,3,-d (TSP) at 25°C. Subsequent data analysis including measurements of intensities of H-NH(_{Z t}+\ ) and NH-NH(_{Z z}+ι ) NOEs was done using the NMR VIEW 2.1 software package (Johnson and Blevins, J. Biomol NMR 4:603, 1994, which is incorporated herein by reference)

Distance restraints of 1.8-2.8, 1.8-3.2 and 1.8-5.0 A were employed for NOE cross-peaks of strong, medium and weak intensity, respectively, observed in the

2D ^H-NOESY spectra obtained with 200 ms mixing times. An additional 1.0 A was added for NOEs involving methyl protons, and a correction of 2.3 A was added to restraints for NOEs involving degenerate H or H protons of the tyrosines and phenylalanme For the structures presented here, no hydrogen bond or dihedral angle restraints were employed

Distance geometry, simulated annealing, and refinement calculations were performed with X-PLOR 3.8 (Brunger, X-PLOR (Version 3.2) (Yale University Press 1992), which is incorporated herein by reference) operating on an 8-processor Silicon Graphics R10000 Power Challenge computer. All atoms of the P25 and P26 peptides were included in the structure calculations. For both P25 and P26 in water and in TFE, a set of 50 embedded substructures was generated using distance geometry and regularized by energy minimization against a distance geometry energy term (the sub-embed protocol in X-PLOR). After template fitting, 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. During the refinement, 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. At the final stages of refinement, a square- well potential energy function was used for the NOEs with a force constant of 50 kcal mol^'l A^~2. Of the 50 embedded substructures, 40 converged to acceptable structures with NOE violations 0.35 A Of these, the best structures with NOE violations 0.35 A and total energies 70 kcal/mol were selected. Superpositionmg of computed structures and calculations of rmsd values were performed with the MidasPlus software package.

Establishment of Conditions for NMR Studies

The NMR solution studies of both the P26 and P25 peptides were performed with 10% DMSO-d_g- in water (pH 4.0) at 25°C because these peptides tended to form a highly viscous solution over time in D O, and to become much less soluble at higher pH. Ten percent deuterated DMSO in water was therefore selected for both

P26 and P25 because the peptides appeared to be both stable and highly soluble m this solvent mixture at pH 4.0. The deuterated DMSO also provided a deuterium lock for the NMR samples. NMR Analysis of the Structure of P26 in H^O. 10% DMSO

The assignments of the backbone and side chain proton resonances of P26 were made from the 2D NOESY and TOCSY spectra collected at 200 and 65 ms mixing times, respectively. Well-resolved NH-NHΛ /+1 ) and αH-NH(/ i+\\ sequential NOEs provided unambiguous connectivities from Pro499 to Lys522. Due to exchange broadening, the NH and αH resonances of amino-terminal Met498 and carboxyl-terminal Ala523 were not observed, precluding the assignment of their side chain proton resonances. The side chain proton resonances of all other residues were unambiguously identified from the 2D TOCSY. In addition, only a limited number of medium-range (M+2), and i,i+3) NOEs were observed, and no αH-NH - /+4) or

NOEs between sequential residues were detected. The assignment of the amino acid type was also aided by the presence of aromatic residues in the sequence, which have distinct δH and εH shifts (see, also, Massiah et al., supra, 1998).

Based on the intense NH-NH₍7 _r+j) and αH-NH(7 ;+ι Λ sequential NOEs and the lack of medium-range sequential NOEs, P26 appears to form a continuous but labile helix. The intensities of the NOEs providing the

connectivities between Gly500 and Ile521 are on average 57% of the intensities of the αH-NHΛ- _j+\\ NOEs. If the peptide assumed uniform conformations, then this value would suggest that it resides in an extended conformer 64%> of the time and in a helix 36% of the time, although these are approximations. However, on the basis of the presence of i,i+2, and i,i+3 NOEs, it appears that residues Tyr512 to Lys522 exist in a more stable helical conformation than do residues of the preceding half of the protein.

NMR Analysis of the Structure of P25 in H^O. 10% DMSO

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 αH-NH(₇ ;+

NOE connectivities from Pro499 to Ala523. The NH and αH resonances of the amino-terminal Met498 were not observed, precluding the assignment of its other resonances. The assigned amide NH resonances of Ser51 1 and Tyr512 and of Lys522 and Ala523 were close to the diagonal. Thus, unambiguous sequential connectivities for these pairs of residues were determined from αH-NH - ^+j \ NOEs, which also established their NH-NHΛ _j+i \ connectivities. In a similar manner, the αH resonances of Val520 and Ile521 overlapped and definitive connectivity between these residues was provided by the NH-NHΛ-^+ I) NOEs. The side chain proton resonances of all amino acid residues were easily identified in the 2D TOCSY which were well-resolved. Like the backbone connectivities of P26, no aH-NH(/ +4) or

NOEs were observed for P25 (see, also, Massiah et al., supra, 1998).

As found with P26, 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 αH-NH₍7 /+ι \ 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. However, as in P26, residues Tyr512 to Val520 of P25 appear to form a more stable helical structure than do residues Gly500 to Ser511 (Figure 4).

Helical Propensities of P25 and P26 in water

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(/ +ι αH-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)/αH-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.

Independent evidence for a decrease in helical propensity in P25 surrounding the missing residue was the downfield chemical shifts of the αH and NH resonances of P25 compared to those of P26 from residues 505 to 517 (Figure 6B) (30). For the αH resonances, the average Δδ over this region was 0.05 ppm and the range was 0.02 to 0.13 ppm. For the NH resonances, the average Δδ was 0.037 ppm and the range was -0.01 to 0.15 ppm. Although these small changes could have resulted in part from the loss of ring current shielding by Phe508, their uniform direction argues against this being the sole explanation.

Computed Structures of P26 and P25 in H2O For P26, 10 of the 50 computed structures had no NOE violations greater than

0.35 A and were of low energy (see Massiah et al., supra, 1998; Table 1). While all of the structures showed helicity between residues Phe508 and Tyr517, they did not superimpose well as was generally found for peptides. For P25, 27 of the 50 computed structures were acceptable by the above criteria, and also did not superimpose well. While some turns were present in P25, all of the structures of P25 showed much less helicity than those of P26 in water, confirming the analysis of the primary NMR data (see Massiah et al., supra, 1998; Figures 7 and 8).

Comparison of P26 and P25 in TFE The structures of P26 and P25 were also determined in trifluoroethanol (TFE), a solvent known to promote helix formation of small polypeptides which have helix- forming propensities. Preliminary circular dichroism (CD) titrations of both P26 and P25 with TFE showed progressive decreases in molar ellipticity at 222 nm indicative of helix formation, with no further changes above 33% TFE (Massiah et al., supra, 1999). Accordingly, 43%> TFE, deuterated to permit field/frequency locking, was used for the NMR studies of both peptides. In 43 % TFE, the NMR data indicated that both P25 and P26 form stable helices between residues Gly500 and Lys522. Backbone proton connectivities were assigned via high-intensity

and weak to medium intensity αH-NHΛ-_{j Z}-+ι ) NOEs. In addition, extensive intermediate range NH-NH^- ;+2₃3), αH-NH(; _z-+2,3,4) and αH-βH(7 _z+3) NOEs were observed, consistent with both peptides existing in stable α-helical conformations. The chemical shifts of the H resonances as well as the relative intensities of the sequential NH-NH(₇ -+i) to αH-NH₍7 _j+ NOEs were also consistent with residues Gly500 to

Lys522 being in an α-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).

A comparison of the H chemical shifts of P25 and P26 in 43 % TFE indicates that the deletion of Phe508 deshields the H resonances of the preceding residues Gly500 to Ile507 but does not significantly affect those of Tyr512 to Lys522. The αH resonance of Ile507 was the most deshielded, by 0.32 ppm, while residues Asn505 to Lys522 show varying degrees of deshielding (0.09-0.31 ppm). However, the αH resonance of Thr501 was highly shielded, shifting upfield by 0.35 ppm. While these changes can result in part from the loss of ring current effects of Phe508, they can also reflect a subtle change in the peptide backbone conformation. The NH shifts, which are less sensitive to changes in the backbone conformation than the αH shifts, did not show significant differences between corresponding residues of the two peptides, arguing against significant ring current effects of Phe508. The downfield direction of most of the changes in the H chemical shifts of residues Gly500 to Ile507 of P25 toward random coil values, as well as the fewer intermediate-range NOEs of P25 (see Massiah et al., supra, 1998; Figure 9 and Table 1), indicate that this region of P25 does not form as stable an α-helix as does the corresponding region of P26, even at a high concentration of TFE.

Computed Structures of P26 and P25 in TFE For both P26 and P25 in TFE, 13 of the 50 computed structures were acceptable and converged well. Both P26 and P25 showed well-defined α-helices from Thr501 to Lys522, which were supeπmposable onto each other with an rmsd of 2.60+0 58 Angstroms for the backbone atoms

These results demonstrate the Phe508-contaιnmg region of CFTR formed an α-hehx, which is destabilized in the ΔF508 mutant found in most CF patients.

EXAMPLE 2

MUTANT CFTR PEPTIDE CAN BE INDUCED

TO FORM STABLE α-HELIX This example demonstrates that a chemical agent can induce a mutant peptide portion of CFTR, which exhibits a folding defect, to assume a stable α-hehcal conformation characteristic of a corresponding wild-type CFTR peptide

Circular dichroism was used to examine the structure of the ΔF508 P25 peptide in the presence or absence of deuterated water (D O), which previously has been show to promote ΔF508 CFTR function in intact cells (Brown et al., supra, 1996). In the presence of 10%o D₂O, the ΔF508 P25 peptide formed an α-hehcal structure characteristic of that obtained for the wild-type P26 peptide. This result demonstrates that an agent, which can promote function of ΔF508 CFTR in mtact cells, can correct a conformational defect in a peptide portion of CFTR comprising the ΔF508 mutation.

EXAMPLE 3 SCREENING ASSAY TO IDENTIFY AN AGENT THAT CORRECTS A DEFECT IN THE THREE DIMENSIONAL CONFORMATION

OF A POLYPEPTIDE

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 ΔF508 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.

Detection of Conformational Change Monoclonal antibodies

A monoclonal antibody that specifically binds the wild-type P26 peptide, which forms a stable α-helix, but not the mutant ΔF508 P25 peptide was prepared using standard methods. The P26 and ΔDF508 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 α-helical structure confirmed by CD spectroscopy.

Two monoclonal antibodies that reacted with the P26 peptide, but not the ΔF508 P25 peptide, were identified using an ELISA assay and a dot blot assay. In the ELISA assay, the peptides were attached to the well, followed by addition of monoclonal antibody, then by a secondary antibody coupled to horseradish peroxidase. The reactivity of both monoclonal antibodies against the ΔF508 P25 peptide was equal to that of a control antibody, whereas their reactivity against the P26 peptide was greater than twice that of the control antibody. In dot blot analysis using either PVDF or nitrocellulose membranes and the ECL detection system, reactivity of the P26 monoclonal antibody was also highly specific for the P26 peptide.

Fluorescent label

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,

Biochemistry 5: 1908-1919, 1966; Turner and Brand, Biochemistry 7:3381-339900, 1968; Slavik, Biochim. Biophys. Acta. 694: 1-25, 1982; each of which is incorporated herein by reference). This property makes this family of compounds (anilinonaphthalene sulfonates) sensitive indicators of protein conformational changes. Bis-ANS (Takashi et al., Proc. Natl. Acad. Sci.. USA 74:2334-2338, 1977; Yoo et al., Biochim. Biophys. Acta 1040:66-70, 1990; Secnik et al., Biochemistry 31 :2982-2988, 1992; each of which is incorporated herein by reference) also can be used in the disclosed assays, as it is an excellent probe for non-polar cavities in proteins, and often binds with a very high affinity, particularly for nucleotide binding sites of proteins.

Circular Dichroism (CD)

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 μl 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 β-sheet, α-helix, random coil, or β-turns.

Examples of Agents to be Examined

Four classes of 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, D₂0, 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 ΔF508 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:

IM (F81-L102); FMFYGIFLYLGEVTKAVQPLLL (SEQ ID NO: 3);

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);

TMl (S308-I328); SAFFFSGFFVVFLSVLPYALI (SEQ ID NO: 7);

TM6 (G330-V350); GIILRKIFTTISFCIVLRMAV (SEQ ID NO: 8);

TMl (I860-V880); IFVLIWCLVIFLAEVAASLVV (SEQ ID NO: 9); TM8 (S912-F932); SYYVFYIYVGVADTILLANGFF (SEQ ID NO: 10);

7 2 (ι991-L1011); IFDFIQLLLIVIGAIAVVAVL (SEQ ID NO: 11);

7 Z_2 (Y1014-L1034); YIFVATVPVIVAFIMLRAYFL (SEQ ID NO: 12);

TM11 (II 1 103-G1123); IEFMIFVIFFIAVTFISILTTG (SEQ ID NO: 13); and

TM12 (VI 129-11150); VGIILTLAMNIMSTLQWAVNSI (SEQ ID NO: 14). or derived from 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 α-helical structure in an exposed area. Such a structural organization in this region of HisP predicts a possible interaction of this region with the membrane-spanning domains, HisQ/HisM (Hung et al., supra, 1998). Thus, the F508 region of CFTR may similarly interact with the membrane-spanning domains of CFTR and be stabilized by this interaction. Class 4 agents represent eukaryotic cell membrane components, including phosphoglycerides, cholesterol, and sphingolipids, which are commonly found in the heart and the brain. Examples of such 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.

Screening Assays

Varying concentrations of P25 (1 μM to 100 μM) 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.

Monoclonal Antibody Detection

After treatment of the peptide, as above, 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. The plates are washed three times with PBS, then anti-mAb mouse IgG-horseradish peroxidase antibody (HRP-secondary antibody) is added at a dilution of 1 :2,000 in PBS/0.1% Tween 20, and incubation is continued for 1 hr at 25°C, after which the plates are washed three times with PBS.

Detection of the HRP reaction is performed by adding 100 μl 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. When the color of the reaction solution becomes yellow, 50 μl 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 β-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.

Fluorescence Intensity Detection

Following treatment with an agent as discussed above, approximately 5 μM of P25 is added to 1,8-ANS or bis-ANS (at varying concentrations of 0.5 μM to 100 μM) in PBS (pH 7.4) at 37°C in a 3 ml system. 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.

Detection by Circular Dichroism Spectroscopy

CD spectra of treated P25, at 100 μM, are collected as described above and the amounts of β-sheet, α-helix, random coil, and β-turns are quantified. Similarly, 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.

Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is-

1. A method of identifying an agent that corrects a defective three dimensional conformation of a polypeptide, the method comprising: a) 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 comprising the defective conformation, and b) determining that the test agent decreases the defective conformation of the peptide portion of the polypeptide, thereby identifying an agent that corrects the defective three dimensional conformation of the polypeptide.

2. The method of claim 1, wherein the defective conformation results m misfolding of the polypeptide.

3. The method of claim 2, wherein the misfolding of the polypeptide results in aberrant cellular localization of the polypeptide.

4. The method of claim 2, wherein the misfolding results in aggregation of the polypeptide.

5. The method of claim 1, wherein the polypeptide is a cystic fibrosis transmembrane regulator (CFTR) polypeptide

6. The method of claim 5, wherein the CFTR polypeptide comprises a deletion of Phe508.

7. The method of claim 6, wherein the peptide portion of the CFTR polypeptide has an amino acid sequence as set forth in SEQ ID NO- 2.

8. The method of claim 1, wherein the polypeptide is selected from the group consisting of fibrillin, superoxide dismutase, collagen, a polypeptide of an α-ketoacid dehydrogenase complex, p53, type I procollagen pro-α, an LDL receptor, αl-antitrypsin, β-hexosamimdase, rhodopsin, and an insulin receptor.

9. The method of claim 1, wherein the polypeptide is selected from the group consisting of a prion protein, β-amyloid, transthyretin, and a crystallin.

10. The method of claim 1, wherein the step of determining that the test agent decreases the defective conformation of the peptide portion of the polypeptide comprises contacting the peptide with a fluorescent compound, and detecting a change in fluorescence intensity of the peptide in the presence of the test agent, wherein the change in fluorescence intensity is indicative of a decrease in the defective conformation of the peptide.

11. The method of claim 1 , wherein the step of determining that the test agent decreases the defective conformation of the peptide portion of the polypeptide comprises detecting a change in a nuclear magnetic resonance (NMR) spectrum of the peptide in the presence of the test agent, wherein the change in the NMR spectrum is indicative of a decrease in the defective conformation of the peptide.

12. The method of claim 1, wherein the step of determining that the test agent decreases the defective conformation of the peptide portion of the polypeptide comprises detecting a change in a circular dichroism (CD) spectrum of the peptide in the presence of the test agent, wherein the change in the CD spectrum is indicative of a decrease in the defective conformation of the peptide.

13. The method of claim 1 , wherein the step of determining that the test agent decreases the defective conformation of the peptide portion of the polypeptide comprises detecting specific binding of an antibody to the peptide in the presence of the test agent, wherein the antibody does not specifically binds the peptide in the absence of the test agent, and wherein specific binding of the antibody is indicative of a decrease in the defective conformation of the peptide.

14. The method of claim 1, wherein the peptide portion of the polypeptide, which is representative of a region of the polypeptide comprising the defective conformation, is identified using a method selected from the group consisting of X-ray crystallography, NMR spectroscopy, and CD spectroscopy.

15. A method of identifying an agent that corrects a defective three dimensional conformation of a polypeptide, the method comprising: a) identifying a peptide portion of the polypeptide that is representative of a region of the polypeptide comprising the defective conformation; b) synthesizing a first peptide comprising the peptide identified in step a) as representative of a region of the polypeptide comprising the defective conformation; and a second peptide comprising a corresponding peptide portion of a wild-type polypeptide corresponding to the polypeptide having the defective conformation; c) contacting said first peptide with a test agent; and d) detecting the three dimensional conformation of the first peptide as becoming the three dimensional conformation of the second peptide, thereby identifying an agent that corrects the defective three dimensional conformation of the polypeptide.

16. The method of claim 15, further comprising quantitating the amount of said agent that corrects the defective three-dimensional conformation of the polypeptide.

17. The method of claim 15, wherein step a) comprises examining the polypeptide by a method selected from the group consisting of X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and circular dichroism (CD) spectroscopy.

18. The method of claim 15, wherein step d) comprises detecting a change in fluorescence intensity.

19. The method of claim 15, wherein step d) comprises detecting a change in a spectrum selected from the group consisting of an NMR spectrum or a CD spectrum.

20. The method of claim 15, wherein step d) detecting specific binding to said first peptide of an antibody that specifically binds said second peptide, but does not specifically bind said first peptide in the absence of an agent that corrects a three dimensional conformation of the polypeptide.

21. The method of claim 20, wherein the antibody is a monoclonal antibody.

22. The method of claim 15, wherein the polypeptide is a cystic fibrosis transmembrane regulator (CFTR) polypeptide.

23. The method of claim 22, wherein the CFTR polypeptide comprises a deletion of Phe508.

24. The method of claim 15, wherein the first peptide has an amino acid sequence as set forth in SEQ ID NO: 2.

25. The method of claim 15, wherein the second peptide has an amino acid sequence as set forth in SEQ ID NO: 1.

26. The method of claim 15, wherein the polypeptide is selected from the group consisting of fibrillin, superoxide dismutase, collagen, a polypeptide of an α-ketoacid dehydrogenase complex, p53, type I procollagen pro-α; LDL receptor, αl-antitrypsin, β-hexosaminidase, rhodopsin, and an insulin receptor.

27. The method of claim 15, wherein the polypeptide is selected from the group consisting of a prion protein, β-amyloid, transthyretin, and a crystallin.

28. The method of claim 15, wherein the test agent is selected from the group consisting of deuterated water (D₂0), dimethylsulfoxide, glycerol, trimethylamine N- oxide (TMAO), butyrate, and phenylbutyrate.

29. The method of claim 15, wherein the test agent is an osmolyte.

30. The method of claim 29, wherein the osmolyte is selected from the group consisting of sorbitol, inositol, betaine, glycerophosphoryl choline, arginine, urea, dimethylpropiothetin, and trehalose.

31. The method of claim 15, wherein the test agent is a peptide, or modified form thereof.

32. The method of claim 31, wherein the peptide is selected from the group consisting of:

FMFYGIFLYLGEVTKAVQPLLL (SEQ ID NO: 3);

SIAIYLGIGLCLLFIVRTLLL (SEQ ID NO: 4);

LALAHFVWIAPLQVALLMGLI (SEQ ID NO: 5);

ASAFCGLGFLIVLALFQAGLG (SEQ ID NO: 6); SAFFFSGFFVVFLSVLPYALI (SEQ ID NO: 7);

GIILRKIFTTISFCIVLRMAV (SEQ ID NO: 8);

IFVLIWCLVIFLAEVAASLVV (SEQ ID NO: 9);

SYYVFYIYVGVADTILLANGFF (SEQ ID NO: 10);

IFDFIQLLLIVIGAIAVVAVL (SEQ ID NO: 11); YIFVATVPVIVAFIMLRAYFL (SEQ ID NO: 12);

IEFMIFVIFFIAVTFISILTTG (SEQ ID NO: 13); and

VGIILTLAMNIMSTLQWAVNSI (SEQ ID NO: 14).

33. The method of claim 31, wherein the peptide is derived from CFTR nucleotide binding fold-2 (NBF2).

34. The method of claim 15, wherein the test agent is selected from the group consisting of a phosphoglyceride, cholesterol, and a sphingolipid.

35. The method of claim 34, wherein the test agent is selected from the group consisting of 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); and galactosyl-lignoceroyl cerebroside (24:0).

36. The method of claim 15, wherein the test agent comprises a library of test agents.

37. The method of claim 15, which is a high throughput assay.

38. The method of claim 37, wherein contacting the peptide with the test agent is performed at a range of concentrations of the test agent.

39. The method of claim 37, which is an automated method.

40. The method of claim 15, which is performed in a cell-free system.

41. A method of identifying an agent that corrects a defective three dimensional conformation of a polypeptide, the method comprising: a) contacting a polypeptide having a defective three dimensional conformation with an agent; b) 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 c) 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.

42. The method of claim 41, further comprising quantitating the amount of the agent that corrects the defective three dimensional conformation of the polypeptide.

43. The method of claim 41, wherein the antibody is detectably labeled.

44. The method of claim 43, wherein said antibody is detectably labeled with a label selected from the group consisting of a visible label, a fluorimetric label, a radiometric label, a luminescent label, a colorimetric label, and an enzymatic label.

45. The method of claim 41, wherein the antibody is detected using a second antibody, which specifically binds the first antibody.

46. 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.

47. The virtual representation of claim 46, wherein the polypeptide is a cystic fibrosis transmembrane regulator (CFTR).

48. The virtual representation of claim 47, wherein the peptide comprises

SEQ ID NO: 2.