Peptide probes for diagnostics and therapeutics

Abstract

Disclosed are agents and methods that may be used to diagnose and treat a variety of diseases associated with conformationally-altered proteins. The agents and methods may be used to identify and deliver drugs useful for treating diseases associated with conformationally-altered proteins.

What is claimed is: 
     
         1 . A method for identifying a target protein present in a specific state of self-aggregation in a sample, comprising:
 (a) contacting the sample with a peptide probe for the target protein, wherein the peptide probe preferentially binds to the target protein in a specific state of self-aggregation; and   (b) detecting any binding between the peptide probe and any target protein present in the specific state of self-aggregation, thereby identifying any target protein present in the specific state of self-aggregation.   
     
     
         2 . The method of  claim 1 , wherein the peptide probe preferentially binds to the target protein in a specific state of self-aggregation selected from the group consisting of monomers, soluble oligomers, and insoluble self-aggregates. 
     
     
         3 . The method of  claim 2 , wherein the peptide probe preferentially binds to insoluble self-aggregates of the target protein selected from the group consisting of insoluble amorphous self-aggregates, protofibrils, and fibrils. 
     
     
         4 . The method of  claim 1 , wherein target protein is selected from the group consisting of amyloid islet polypeptide precursor protein, amyloid beta protein or Aβ peptide, serum amyloid A, insulin, amylin, non-amyloid beta component, prions, hemoglobin, immunoglobulins or fragments thereof β 2 -microglobulin, α-synuclein, rhodopsin, α1-antichymotrypsin, cystallins, tau, p53, presenilins, low-density lipoprotein receptor, apolipoproteins, superoxide dismutase, neurofilament proteins, transthyretin, procalcitonin or calcitonin, atrial natriuretic factor, gelsolin, cystic fibrosis transmembrane regulator, Huntington's disease protein, fibrinogen alpha-chain, phenylalanine hydroxylase, collagen, beta-hexosaminidase, and cystatin C protein. 
     
     
         5 . The method of  claim 1 , wherein the peptide probe further comprises a detectable label. 
     
     
         6 . The method of  claim 1 , wherein the peptide probe comprises an amino acid sequence selected from SEQ ID NO:36 and SEQ ID NO:45. 
     
     
         7 . The method of  claim 1 , wherein the peptide probe is immobilized on a solid support. 
     
     
         8 . An in vivo method for identifying a target protein present in a patient in a specific state of self-aggregation, comprising:
 (a) administering to the patient a peptide probe for the target protein, wherein the peptide probe preferentially binds to the target protein in the specific state of self-aggregation and wherein the peptide probe is labeled with a detecable label; and   (b) scanning the subject for labeled peptide probe localized at target protein present in the patient,   thereby identifying target protein present in the patient in the specific state of self-aggregation.   
     
     
         9 . The method of  claim 8 , wherein the peptide probe preferentially binds to the target protein in a specific state of self-aggregation selected from the group consisting of monomers, soluble oligomers, and insoluble self-aggregates. 
     
     
         10 . The method of  claim 8 , wherein target protein is selected from the group consisting of amyloid islet polypeptide precursor protein, amyloid beta protein or Aβ peptide, serum amyloid A, insulin, amylin, non-amyloid beta component, prions, hemoglobin, immunoglobulins or fragments thereof β 2 -microglobulin, α-synuclein, rhodopsin, α1-antichymotrypsin, cystallins, tau, p53, presenilins, low-density lipoprotein receptor, apolipoproteins, superoxide dismutase, neurofilament proteins, transthyretin, procalcitonin or calcitonin, atrial natriuretic factor, gelsolin, cystic fibrosis transmembrane regulator, Huntington's disease protein, fibrinogen alpha-chain, phenylalanine hydroxylase, collagen, beta-hexosaminidase, and cystatin C protein. 
     
     
         11 . A method for preventing the formation of protein aggregates of a target protein, comprising contacting the target protein with a peptide probe for the target protein, wherein the peptide probe preferentially binds to the target protein in a specific state of self-aggregation, thereby preventing the formation of higher order protein aggregates of the target protein. 
     
     
         12 . The method of  claim 11 , wherein the peptide probe preferentially binds to the target protein in a specific state of self-aggregation selected from the group consisting of monomers, soluble oligomers, and insoluble self-aggregates. 
     
     
         13 . The method of  claim 11 , wherein target protein is selected from the group consisting of amyloid islet polypeptide precursor protein, amyloid beta protein or Aβ peptide, serum amyloid A, insulin, amylin, non-amyloid beta component, prions, hemoglobin, immunoglobulins or fragments thereof β 2 -microglobulin, α-synuclein, rhodopsin, α1-antichymotrypsin, cystallins, tau, p53, presenilins, low-density lipoprotein receptor, apolipoproteins, superoxide dismutase, neurofilament proteins, transthyretin, procalcitonin or calcitonin, atrial natriuretic factor, gelsolin, cystic fibrosis transmembrane regulator, Huntington's disease protein, fibrinogen alpha-chain, phenylalanine hydroxylase, collagen, beta-hexosaminidase, and cystatin C protein. 
     
     
         14 . A method of delivering a therapeutic agent to a target protein, comprising combining the therapeutic agent with a peptide probe for the target protein,
 wherein the peptide probe comprises an amino acid sequence corresponding to a region of the target protein that undergoes a conformational shift from an alpha-helical conformation to a beta-sheet conformation, and the peptide probe undergoes a conformational shift from an alpha-helical conformation to a beta-sheet conformation and wherein the peptide probe does not comprise the full-length sequence of the target protein, and   administering the peptide probe-therapeutic agent combination to a patient in need thereof.   
     
     
         15 . The method of  claim 14 , wherein the therapeutic agent has anti-amyloid activity. 
     
     
         16 . The method of  claim 14 , wherein the peptide probe preferentially binds to the target protein in a specific state of self-aggregation. 
     
     
         17 . The method of  claim 16 , wherein the peptide probe preferentially binds to the target protein in a specific state of self-aggregation selected from the group consisting of monomers, soluble oligomers and insoluble aggregates. 
     
     
         18 . A method of assessing an agent's ability to inhibit aggregation of a target protein, comprising:
 (A) contacting a fusion protein and a test agent, the fusion protein comprising:
 (i) a peptide probe for the target protein, wherein: (a) the peptide probe comprises an amino acid sequence corresponding to a region of the target protein that undergoes a conformational shift from an alpha-helical conformation to a beta-sheet conformation, (b) the peptide probe undergoes a conformational shift from an alpha-helical conformation to a beta-sheet conformation, and (c) the peptide probe does not comprise the full-length sequence of the target protein; and 
 (ii) a label which generates a signal dependent on the aggregative state of the fusion protein; 
   (B) detecting a signal generated by the label; and   (C) correlating the signal with the ability of the agent to inhibit aggregation of the target protein.   
     
     
         19 . A method of assessing an agent's ability to inhibit aggregation of a target protein, comprising:
 (A) contacting the target protein, a fusion protein, and a test agent, the fusion protein comprising:
 (i) a peptide probe for the target protein, wherein: (a) the peptide probe comprises an amino acid sequence corresponding to a region of the target protein that undergoes a conformational shift from an alpha-helical conformation to a beta-sheet conformation, (b) the peptide probe undergoes a conformational shift from an alpha-helical conformation to a beta-sheet conformation, and (c) the peptide probe does not comprise the full-length sequence of the target protein; and 
 (ii) a label which generates a signal dependent on the aggregative state of the fusion protein; 
   (B) detecting a signal generated by the label; and   (C) correlating the signal with the ability of the agent to inhibit aggregation of the target protein.   
     
     
         20 . A method of assessing an agent's ability to inhibit aggregation of a target protein, comprising:
 (A) subjecting a fusion protein to conditions that promote aggregation, the fusion protein comprising:
 (i) a peptide probe for the target protein, wherein: (a) the peptide probe comprises an amino acid sequence corresponding to a region of the target protein that undergoes a conformational shift from an alpha-helical conformation to a beta-sheet conformation, (b) the peptide probe undergoes a conformational shift from an alpha-helical conformation to a beta-sheet conformation, and (c) the peptide probe does not comprise the full-length sequence of the target protein; and 
 (ii) a label which generates a signal dependent on the aggregative state of the fusion protein; 
   (B) detecting a first signal generated by the label;   (C) subjecting the fusion protein to conditions that promote aggregation in the presence of a test agent, and detecting a second signal generated by the label; and   (D) assessing the relative intensities of the first and second signals, thereby identifying an agent that inhibits aggregation of the target protein.   
     
     
         21 . A method of assessing an agent's ability to inhibit aggregation of a target protein, comprising:
 (A) contacting a fusion protein and the target protein, wherein the fusion protein comprises:
 (i) a peptide probe for the target protein, wherein: (a) the peptide probe comprises an amino acid sequence corresponding to a region of the target protein that undergoes a conformational shift from an alpha-helical conformation to a beta-sheet conformation, (b) the peptide probe undergoes a conformational shift from an alpha-helical conformation to a beta-sheet conformation, and (c) the peptide probe does not comprise the full-length sequence of the target protein; and 
 (ii) a label which generates a signal dependent on the aggregative state of the fusion protein; 
   (B) detecting a first signal generated by the label;   (C) contacting the fusion protein, the target protein, and a test agent, and detecting a second signal generated by the label; and   (D) assessing the relative intensities of the first and second signals, thereby identifying an agent that inhibits aggregation of the target protein.   
     
     
         22 . A method of identifying a peptide probe for a target protein that exhibits an increased or decreased tendency to form aggregates relative to a reference peptide probe, comprising:
 (A) detecting a first signal generated by a reference fusion protein that comprises:
 (i) a reference peptide probe comprising: (a) an amino acid sequence corresponding to a region of the target protein that undergoes a conformational shift from an alpha-helical conformation to a beta-sheet conformation, (b) wherein the peptide probe undergoes a conformational shift from an alpha-helical conformation to a beta-sheet conformation, and (c) the reference peptide probe does not comprise the full-length sequence of the target protein; and 
 (ii) green fluorescent protein; 
   (B) detecting a second signal generated by a test fusion protein comprising a test peptide probe and green fluorescent protein, wherein the test peptide probe is a mutant of the reference peptide probe that comprises an amino acid insertion, deletion or substitution relative to the amino acid sequence of the reference peptide probe; and   (C) correlating the intensity of the second signal relative to the first signal, thereby identifying a peptide probe for a target protein that exhibits an increased or decreased tendency to form aggregates relative to the reference peptide probe.   
     
     
         23 . A method of identifying a peptide probe specific for a target protein in a specific structural state that falls on a spectrum of structural states ranging from a low end of soluble monomers to a high end of insoluble self-aggregates, comprising:
 (A) subjecting a fusion protein to conditions that promote self-aggregation, the fusion protein comprising:
 (i) a peptide probe for the target protein, wherein: (a) the peptide probe comprises an amino acid sequence corresponding to a region of the target protein that undergoes a conformational shift from an alpha-helical conformation to a beta-sheet conformation, (b) the peptide probe undergoes a conformational shift from an alpha-helical conformation to a beta-sheet conformation, and (c) the peptide probe does not comprise the full-length sequence of the target protein; and 
 (ii) green fluorescent protein; 
   (B) detecting a signal generated by the fusion protein; and   (C) correlating the intensity of the signal with the specificity of the peptide probe for a target protein in a specific structural state, thereby identifying a peptide probe specific for a target protein in a specific structural state.   
     
     
         24 . A method for treating a disease associated with a target protein, comprising contacting the target protein with a fusion protein comprising (i) a peptide probe for the target protein, wherein the peptide probe preferentially binds to the target protein, and (ii) a therapeutic agent.