Conformationally dynamic peptides

Abstract

Disclosed are novel peptides that are useful, for example, for detecting target proteins having a β-sheet secondary structure which may be associated with a disease, and for diagnosing and treating such a disease. Related methods and kits also are disclosed.

1 . A peptide probe for a target protein capable of exhibiting a conformation change associated with an amyloidogenic disease, wherein the peptide probe is a peptide or peptide mimic that (i) consists of from 10 to 50 amino acid residues comprising an amino acid sequence that is a variant of a reference sequence consisting of an amino acid sequence of a β-sheet forming region of the target protein, (ii) is capable of adopting both a random coil/alpha-helix conformation and a β-sheet conformation, and (iii) undergoes a change in conformation that generates a detectable signal upon binding to target protein, wherein
 the variant sequence comprises one or more amino acid additions, substitutions or deletions relative to the reference sequence, such that   (A) the random coil/alpha-helix conformation of the variant sequence is more stable in an oxidizing environment than a probe consisting of the reference amino acid sequence; and/or   (B) the distance between the N-terminus and the C-terminus of the variant sequence in a random coil/alpha-helix conformation differs from the distance between the N-terminus and the C-terminus of the variant sequence in a β-sheet conformation; and/or   (C) the variant sequence adopts a β-sheet conformation upon binding to target protein exhibiting a β-sheet conformation more efficiently than the reference sequence; and/or   (D) the variant sequence adopts a less ordered conformation upon binding to target protein exhibiting a β-sheet conformation; and/or   (E) the β-sheet structure of the variant sequence is less thermodynamically strong than that of the reference sequence; and/or   (F) the variant sequence has increased stability and/or decreased reactivity than the reference sequence; and/or   (G) the variant sequence has an increased hydrophilicity and/or solubility in aqueous solutions than the reference sequence; and/or   (H) the variant sequence has an additional Aβ binding motif than the reference sequence; and/or   (I) the variant sequence has an enhanced ability to form aggregates.   
     
     
         2 . The peptide probe of  claim 1 , wherein the peptide probe is labeled with a detectable label at the N-terminus, the C-terminus, both termini, or at one or more positions that generate a signal when the peptide undergoes a conformation change upon binding to target protein. 
     
     
         3 . The peptide probe of  claim 2 , wherein the peptide probe is labeled with two or more labels, wherein the distance between two or more labels on the peptide probe when the peptide probe is bound to target protein is different than the distance when the peptide probe is not bound to target protein. 
     
     
         4 . The peptide probe of  claim 3 , wherein the signal generated by the detectable label when the peptide probe is bound to target protein is different than the signal generated when the peptide probe is not bound to target protein. 
     
     
         5 . The peptide probe of  claim 3 , wherein the peptide probe is labeled with a detectable label pair selected from an excimer pair, a FRET pair and a fluorophore/quencher pair. 
     
     
         6 . The peptide probe of  claim 5 , wherein the peptide probe is labeled with an excimer pair and emits an excimer signal when the peptide probe exhibits a β-sheet conformation. 
     
     
         7 . The peptide probe of  claim 6 , wherein the detectable label comprises a pyrene moiety. 
     
     
         8 . The peptide probe of  claim 5 , wherein the peptide probe is labeled with a FRET pair and emits a fluorescence resonance transfer (FRET) signal when the peptide probe exhibits a (β-sheet conformation. 
     
     
         9 . The peptide probe of  claim 8 , wherein the FRET pair is selected from the group consisting of DACIA-I/NBD, Marina Blue/NBD, Dansyl/Trp, and EDANS/FAM. 
     
     
         10 . The peptide probe of  claim 5 , wherein the peptide probe is labeled with a fluorophore/quencher pair and wherein the fluorphore signal is quenched when the peptide probe exhibits a β-sheet conformation. 
     
     
         11 . The peptide probe of  claim 10 , wherein the fluorphore/quencher pair is selected from the group consisting of pyrene/Dabcyl, EDANS/Dabcyl and FAM/Dabcyl. 
     
     
         12 . The peptide probe of  claim 1 , wherein the one or more amino acid additions, substitutions or deletions are made at an internal portion of the reference sequence. 
     
     
         13 . The peptide probe of  claim 1 , wherein the one or more amino acid additions, substitutions or deletions are made at the N-terminus or C-terminus of the reference sequence. 
     
     
         14 . The peptide probe of  claim 1 , wherein variant sequence further comprises the addition of a lysine residue at the C-terminus. 
     
     
         15 . The peptide probe of  claim 1 , wherein the variant sequence comprises the substitution of a methionine residue with a residue resistant to oxidation. 
     
     
         16 . The peptide probe of  claim 15 , wherein the methionine residue is substituted with an alanine residue. 
     
     
         17 . The peptide probe of  claim 1 , wherein the variant sequence comprises the substitution or addition of at least three consecutive residues of the reference sequence with alanine residues. 
     
     
         18 . The peptide probe of  claim 1 , wherein the one or more amino acid additions, substitutions or deletions introduces a salt bridge into the variant sequence. 
     
     
         19 . The peptide probe of  claim 18 , wherein the variant sequence comprises a salt bridge between residues selected from the group consisting of a glutamic acid residue and a histidine residue, a glutamic acid residue and an arginine residue, and a glutamic acid residue and a lysine residue. 
     
     
         20 . The peptide probe of  claim 1 , wherein the one or more amino acid additions, substitutions or deletions introduces an Aβ binding motif into the peptide probe. 
     
     
         21 . The peptide probe of  claim 20 , wherein the Aβ binding motif comprises GXXEG (SEQ ID NO:25). 
     
     
         22 . The peptide probe of  claim 1 , wherein the variant sequence adopts a less ordered conformation upon binding to target protein exhibiting a β-sheet conformation. 
     
     
         23 . The peptide probe of claim of  claim 22 , wherein the target protein is Aβ protein, and the variant sequence comprises one or more substitutions selected from the group consisting of G29H, G29R, G29K, and G33E. 
     
     
         24 . The peptide probe of  claim 22 , wherein the β-sheet structure of the variant sequence is less thermodynamically strong than that of the reference sequence. 
     
     
         25 . The peptide probe of claim of  claim 24 , wherein the target protein is Aβ protein, and the variant sequence comprises one or more substitutions selected from the group consisting of 132S, F19S, S26D, H29D, 131D, L34D, and L34P. 
     
     
         26 . The peptide probe of  claim 1 , wherein the variant sequence has an increased hydrophilicity and/or solubility in aqueous solutions than the reference sequence. 
     
     
         27 . The peptide probe of  claim 26 , wherein the variant sequence comprises one or more amino acid additions or substitutions that introduce a glutamic acid residue and/or a d-arginine residue. 
     
     
         28 . The peptide probe of  claim 26 , wherein the variant sequence is conjugated to a hydrophilic moiety. 
     
     
         29 . The peptide probe of  claim 28 , wherein the hydrophilic moiety comprises a soluble polyethylene glycol moiety. 
     
     
         30 . The peptide probe of  claim 2 , wherein a detectable label is conjugated to a side chain of a terminal lysine residue. 
     
     
         31 . The peptide probe of  claim 2 , wherein a detectable label is conjugated to a side chain of an internal lysine residue. 
     
     
         32 . The peptide probe of  claim 1 , wherein the peptide probe is conjugated to a biotin moiety. 
     
     
         33 . The peptide probe of  claim 32 , wherein the peptide probe is conjugated to a biotin moiety through a peptide linker. 
     
     
         34 . The peptide probe of  claim 32 , wherein the peptide linker is selected from the group consisting of a flexible linker, a helical linker, a thrombin site linker and a kinked linker. 
     
     
         35 . The peptide probe of  claim 34 , wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:56-60. 
     
     
         36 . The peptide probe of  claim 32 , wherein the peptide probe is conjugated to a biotin moiety through a side chain of an internal lysine residue. 
     
     
         37 . The peptide probe of  claim 1 , wherein the target protein is Aβ protein. 
     
     
         38 . The peptide probe of  claim 37 , wherein the variant sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-55. 
     
     
         39 . The peptide probe of  claim 37 , wherein the variant sequence comprises the amino acid sequence of SEQ ID NO:2 (Peptide 22). 
     
     
         40 . A peptide probe for a target protein capable of exhibiting a conformation change associated with an amyloidogenic disease, wherein the peptide probe is a peptide or peptide mimic that (i) consists of from 10 to 50 amino acid residues comprising an amino acid sequence that is a variant of a reference sequence consisting of an amino acid sequence of a β-sheet forming region of the target protein, (ii) is capable of adopting both a random coil/alpha-helix conformation and a β-sheet conformation, and (iii) adopts a less ordered conformation upon binding to target protein. 
     
     
         41 . The peptide probe of  claim 40 , wherein the peptide probe is labeled with a detectable label at the N-terminus, the C-terminus, both termini, or at one or more positions that generate a signal when the peptide undergoes a conformation change upon binding to target protein. 
     
     
         42 . The peptide probe of  claim 41 , wherein the peptide probe is labeled with a detectable label pair selected from an excimer pair, a FRET pair and a fluorophore/quencher pair. 
     
     
         43 . The peptide probe of  claim 42 , wherein the peptide probe is labeled with an excimer pair and emits an increased excimer signal when the peptide probe is not bound to target protein and emits an increased self signal when the peptide probe is bound to target protein. 
     
     
         44 . The peptide probe of  claim 43 , wherein the excimer pair comprises two pyrene moieties. 
     
     
         45 . The peptide probe of  claim 42 , wherein the peptide probe is labeled with a FRET pair and emits an increased a fluorescence resonance transfer (FRET) signal when the peptide probe is not bound to target protein and emits a non-FRET fluorophore signal when the peptide probe is bound to target protein. 
     
     
         46 . The peptide probe of  claim 42 , wherein the peptide probe is labeled with a fluorophore/quencher pair and emits a decreased or quenched signal when the peptide probe is not bound to target protein and emits a fluorophore signal when the peptide probe is bound to target protein. 
     
     
         47 . The peptide probe of  claim 40 , wherein the target protein is Aβ protein. 
     
     
         48 . The peptide probe of  claim 47 , wherein the variant sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5, 14-15 and 28-35. 
     
     
         49 . The peptide probe of  claim 1 , wherein the target protein is selected from the group consisting of amyloid islet polypeptide precursor protein, amyloid beta protein, Aβ peptide, serum amyloid A, insulin, amylin, non-amyloid beta component, prions, hemoglobin, immunoglobulins or fragments thereof β2-microglobulin, α-synuclein, rhodopsin, α1-anti chymotrypsin, 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. 
     
     
         50 . A method for detecting target protein in a test sample, wherein the target protein exhibits a β-sheet conformation associated with an amyloidogenic disease, comprising:
 (i) contacting the sample with the peptide probe of  claim 1  to form a test mixture; and   (ii) detecting any binding between the peptide probe and any target protein present in the sample.   
     
     
         51 . The method of  claim 50 , wherein the peptide probe is labeled with a detectable label at the N-terminus, the C-terminus, both termini, or at one or more positions that generate a signal when the peptide undergoes a conformation change upon binding to target protein. 
     
     
         52 . The method of  claim 51 , wherein the peptide probe is labeled with two or more labels, wherein the distance between two or more labels on the peptide probe when the peptide probe is bound to target protein is different than the distance when the peptide probe is not bound to target protein. 
     
     
         53 . The method of  claim 52 , wherein the signal generated by the detectable label when the peptide probe is bound to target protein is different than the signal generated when the peptide probe is not bound to target protein. 
     
     
         54 . The method of  claim 52 , wherein the peptide probe is labeled with a detectable label pair selected from an excimer pair, a FRET pair and a fluorophore/quencher pair. 
     
     
         55 . The method of  claim 54 , wherein the peptide probe is labeled with an excimer pair and emits an excimer signal when the peptide probe exhibits a β-sheet conformation. 
     
     
         56 . The method of  claim 55 , wherein the detectable label comprises a pyrene moiety. 
     
     
         57 . The method of  claim 54 , wherein the peptide probe is labeled with a FRET pair and emits a fluorescence resonance transfer (FRET) signal when the peptide probe exhibits a β-sheet conformation. 
     
     
         58 . The method of  claim 54 , wherein the peptide probe is labeled with a fluorophore/quencher pair and wherein the fluorphore signal is quenched when the peptide probe exhibits a β-sheet conformation. 
     
     
         59 . The method of  claim 51 , wherein step (ii) comprises detecting any signal generated by the peptide probe undergoing a conformational change upon binding to a target protein. 
     
     
         60 . The method of  claim 51 , wherein step (ii) comprises detecting complexes comprising the peptide probe and target protein by detecting any signal generated by any detectable label present in the complexes. 
     
     
         61 . The method of  claim 60 , wherein the complexes are insoluble complexes and step (ii) comprises detecting any signal generated by any detectable label present in the insoluble complexes. 
     
     
         62 . The method of  claim 60 , wherein the complexes are soluble complexes and step (ii) comprises detecting any signal generated by any detectable label present in the soluble complexes. 
     
     
         63 . The method of  claim 60 , wherein the method further comprises, prior to step (ii), separating the complexes from the test mixture by a process selected from centrifugation, size exclusion chromatography, and affinity chromatography. 
     
     
         64 . A method for detecting target protein associated with an amyloidogenic disease, wherein the peptide probe in a physiological sample from a subject, comprising:
 (A) contacting the sample with a peptide probe that is a peptide or peptide mimic that (i) consists of from 10 to 50 amino acid residues comprising an amino acid sequence that is a variant of a reference sequence consisting of an amino acid sequence of a β-sheet forming region of the target protein, (ii) is capable of adopting both a random coil/alpha-helix conformation and a β-sheet conformation, and (iii) adopts a less ordered conformation upon binding to target protein; and   (B) detecting any association between said probe and any target protein present in said sample.   
     
     
         65 . The method of  claim 64 , wherein the peptide probe is labeled with a detectable label at the N-terminus, the C-terminus, both termini, or at one or more positions that generate a signal when the peptide undergoes a conformation change upon binding to target protein. 
     
     
         66 . The method of  claim 65 , wherein the peptide probe is labeled with a detectable label pair selected from an excimer pair, a FRET pair and a fluorophore/quencher pair. 
     
     
         67 . The method of  claim 66 , wherein the peptide probe is labeled with an excimer pair and step (ii) comprises detecting any increased self signal or decreased excimer signal. 
     
     
         68 . The method of  claim 66 , wherein the peptide probe is labeled with a FRET pair and step (ii) comprises detecting any increased non-FRET fluorophore signal or decreased FRET signal. 
     
     
         69 . The method of  claim 66 , wherein the peptide probe is labeled with a fluorophore/quencher pair and step (ii) comprises detecting any increased fluorophore signal. 
     
     
         70 . An in vivo method for detecting target protein associated with an amyloidogenic disease in subject, comprising:
 (A) administering to the subject a peptide probe of  claim 1 , wherein the probe is labeled with a detectable label that generates a signal when the probe binds to target protein and   (B) detecting said signal.   
     
     
         71 . The method of  claim 70 , wherein said signal is detected using an imaging technique. 
     
     
         72 . The method of  claim 71 , wherein said imaging technique is selected from the group consisting of positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), radiography, tomography, fluoroscopy, nuclear medicine, optical imaging, encephalography and ultrasonography. 
     
     
         73 . A method of treating subject suffering from or at risk of developing an amyloidogenic disease, comprising administering to the subject a peptide probe of  claim 1 . 
     
     
         74 . The method of  claim 73 , wherein the probe is conjugated to an additional therapeutic agent against said amyloidogenic disease. 
     
     
         75 . A peptide probe for a target protein capable of exhibiting a β-sheet conformation associated with an amyloidogenic disease, wherein the peptide probe
 (i) consists of from 10 to 50 amino acid residues comprising an amino acid sequence that is a variant of a reference sequence consisting of an amino acid sequence of a β-sheet forming region of the target protein, (ii) is capable of adopting both a random coil/alpha-helix conformation and a β-sheet conformation, and (iii) adopts a β-sheet conformation upon binding to target protein exhibiting a β-sheet conformation; wherein   the variant sequence comprises one or more amino acid additions, substitutions or deletions relative to the reference sequence, such that   (A) the random coil/alpha-helix conformation of the variant sequence is more stable in an oxidizing environment than a probe consisting of the reference amino acid sequence and/or   (B) the distance between the N-terminus and the C-terminus of the variant sequence in a random coil/alpha-helix conformation differs from the distance between the N-terminus and the C-terminus of the variant sequence in a β-sheet conformation and/or   (C) the variant sequence adopts a β-sheet conformation upon binding to target protein exhibiting a β-sheet conformation more efficiently than the reference sequence.