Methods for developing virus protein-specific capture agents, capture agents, and methods of using the capture agents

Abstract

A method for developing capture agents for target proteins employs a compound library to find cyclic peptide sequences that bind the target protein. The target protein is also reacted with a clickable group-provider reagent to provide the protein with clickable groups. The compounds in the library are provided with complementary clickable groups that bind the clickable group on the target protein when the peptide sequences bind the target protein. In some embodiments, the cyclic peptide sequences that bind the target protein are incorporated into constructs having one or more arms that can serve as capture agents or potential treatments against the pathogens from which the target protein is derived. Some embodiments provide pharmaceutical compositions for immunoassays, diagnostics, therapeutics or the like, that employ the constructs.

What is claimed is: 
     
         1 . A construct molecule having an affinity to the Chikungunya virus (CHIKV) comprising:
 one or more polymer arms having a peptide sequence selected from the group consisting of: Tryptophan-Isoleucine-Tyrosine-Tyrosine-Isoleucine (SEQ ID NO: 1), Tyrosine-Tryptophan-Histidine-Tryptophan-Serine (SEQ ID NO: 2), Isoleucine-Tyrosine-Leucine-Arginine-Tyrosine (SEQ ID NO: 3), Phenylalanine-Tryptophan-Glutamine-Isoleucine-Leucine (SEQ ID NO: 4), and the D-amino and L-amino acid versions of these sequences.   
     
     
         2 . The construct molecule of  claim 1 , wherein the one or more polymer arms comprise polyethylene oxide, polypropylene oxide, copolymers thereof, or any combination thereof. 
     
     
         3 . The construct molecule of  claim 2 , wherein the polymer in the one or more arms is of the form: 
       
         
           
           
               
               
           
         
       
       where n is greater than or equal to 1. 
     
     
         4 . The construct molecule of  claim 1 , wherein there is a plurality of arms. 
     
     
         5 . The construct molecule of  claim 4 , wherein the peptide sequences of the plurality of arms are the same or different from one another. 
     
     
         6 . The construct molecule of  claim 4 , further comprising:
 a solid support; and   at least one linker connecting the plurality of arms, directly or indirectly, to the solid support.   
     
     
         7 . The construct molecule of  claim 1 , wherein the construct is a one-arm construct selected from the group consisting of constructs having the following structure: 
       
         
           
           
               
               
           
         
         wherein R 1  is selected from the group consisting of H, OH, NH 2 , —CONH 2 , COOH, OC 2 H 5 , a solid support, a detection label, a linker connected to a solid support, and a linker connected to a detection label, and wherein X 1  is selected from the peptide sequences, and 
         salts, PEGylation products, tautomers, prodrugs, and stereoisomers, including enantiomers, thereof, wherein the integer n is greater than or equal to 1. 
       
     
     
         8 . The construct molecule of  claim 1 , wherein the construct is a multi-arm construct selected from the group consisting of constructs having the following structure: 
       
         
           
           
               
               
           
         
         wherein R 1  is selected from the group consisting of H, OH, NH 2 , —CONH 2 , COOH, OC 2 H 5 , (OC 2 H 4 ) k —OH, (OC 2 H 4 ) k —NH 2 , (OC 2 H 4 ) k —CONH 2 , (OC 2 H 4 ) k —COOH, (OC 2 H 4 ) k —OC 2 H 5 , (OC 2 H 4 ) k —X 4 , a solid support, a detection label, a linker connected to a solid support, and a linker connected to a detection label, wherein R 2  is selected from the group consisting of H, OH, NH 2 , —CONH 2 , COOH, OC 2 H 5 , (OC 2 H 4 ) k —OH, (OC 2 H 4 ) k —NH 2 , (OC 2 H 4 ) k —CONH 2 , (OC 2 H 4 ) k —COOH, (OC 2 H 4 ) k —OC 2 H 5 , and (OC 2 H 4 ) h —X 3 , wherein n, m, k, and h are integers that are equal to or greater than one, and wherein X 1 , X 2 , X 3 , and X 4  are selected from the peptide sequences, and 
         salts, PEGylation products, tautomers, prodrugs, and stereoisomers, including enantiomers, thereof. 
       
     
     
         9 . The construct molecule of  claim 1 , wherein the construct is a multi-arm construct selected from the group consisting of constructs having the following structure:
 wherein R 1  is selected from the group consisting of H, OH, NH 2 , —CONH 2 ,   
       
         
           
           
               
               
           
         
       
       COOH, OC 2 H 5 , (OC 2 H 4 ) k —OH, (OC 2 H 4 ) k —NH 2 , (OC 2 H 4 ) k —CONH 2 , (OC 2 H 4 ) k —COOH, (OC 2 H 4 ) k —OC 2 H 5 , (OC 2 H 4 ) k —X 4 , a solid support, a detection label, a linker connected to a solid support, and a linker connected to a detection label, wherein R 2  is selected from the group consisting of H, OH, NH 2 , —CONH 2 , COOH, OC 2 H 5 , (OC 2 H 4 ) k —OH, (OC 2 H 4 ) k —NH 2 , (OC 2 H 4 ) k —CONH 2 , (OC 2 H 4 ) k —COOH, (OC 2 H 4 ) k —OC 2 H 5 , and (OC 2 H 4 ) h —X 3 , wherein i, n, m, n i , m i , k, and h are integers that are equal to or greater than one, and wherein X 1 , X 2 , X 1(i) , X 2(i) , X 3 , and X 4  are selected from the peptide sequences, and
 salts, tautomers, PEGylation products, prodrugs, and stereoisomers, including enantiomers, thereof. 
 
     
     
         10 . The construct molecule of  claim 1 , wherein the construct is a multi-arm construct selected from the group consisting of constructs having the following structure: 
       
         
           
           
               
               
           
         
         wherein R 1  is selected from the group consisting of H, OH, NH 2 , —CONH 2 , COOH, OC 2 H 5 , (OC 2 H 4 ) k —OH, (OC 2 H 4 ) k —NH 2 , (OC 2 H 4 ) k —CONH 2 , (OC 2 H 4 ) k —COOH, (OC 2 H 4 ) k —OC 2 H 5 , (OC 2 H 4 ) k —X 4 , a solid support, a detection label, a linker connected to a solid support, and a linker connected to a detection label, wherein n, m, k, and h are integers that are equal to or greater than one, and wherein X 1 , X 2 , X 3 , and X 4  are selected from the peptide sequences, and 
         salts, tautomers, PEGylation products, prodrugs, and stereoisomers, including enantiomers, thereof. 
       
     
     
         11 . The construct molecule of  claim 10 , wherein X 1 , X 2 , X 3 , and X 4  each comprise a variable portion consisting of the corresponding polypeptide of each of X 1 , X 2 , X 3 , and X 4 , and wherein the corresponding polypeptide of each of X 1 , X 2 , X 3 , and X 4  are selected from the peptide sequences. 
     
     
         12 . The construct molecule of  claim 6 , wherein the solid support is selected from the group consisting of beads, multi-well ELISA plates, and BRAP chips. 
     
     
         13 . The construct molecule of  claim 1 , wherein the peptide sequence is at the end of the one or more polymer arms. 
     
     
         14 . The construct molecule of  claim 1 , wherein the the peptide sequence binds to the CHIKV E2 protein. 
     
     
         15 . A capture agent for assaying comprising:
 a construct molecule according to  claim 1 ; and   an attachment configured to enable an assaying technique.   
     
     
         16 . The capture agent of  claim 15 , wherein the attachment comprises H, OH, NH 2 , COOH, OC 2 H 5 , —C(O)NH 2 , an amino acid, polymer-based beads, dyed polymer-based beads, fluorescent beads, quantum dots, nanoparticles, magnetic beads, metals, silicon, graphene, polymer, detection label, or fluorophore. 
     
     
         17 . A method of assaying the Chikungunya virus (CHIKV) comprising:
 providing a construct molecule according to  claim 1 ; and   exposing a sample comprising CHIKV to the construct molecule.   
     
     
         18 . A method of assaying the Chikungunya virus (CHIKV) comprising:
 providing a reagent compound comprising a peptide sequence selected from the group consisting of: Tryptophan-Isoleucine-Tyrosine-Tyrosine-Isoleucine (SEQ ID NO: 1), Tyrosine-Tryptophan-Histidine-Tryptophan-Serine (SEQ ID NO: 2), Isoleucine-Tyrosine-Leucine-Arginine-Tyrosine (SEQ ID NO: 3), Phenylalanine-Tryptophan-Glutamine-Isoleucine-Leucine (SEQ ID NO: 4), and the D-amino and L-amino acid versions of these sequences; and   exposing a sample comprising CHIKV to the reagent compound.   
     
     
         19 . A set of peptide sequences having an affinity to the Chikungunya virus (CHIKV) consisting of: Tryptophan-Isoleucine-Tyrosine-Tyrosine-Isoleucine (SEQ ID NO: 1), Tyrosine-Tryptophan-Histidine-Tryptophan-Serine (SEQ ID NO: 2), Isoleucine-Tyrosine-Leucine-Arginine-Tyrosine (SEQ ID NO: 3), Phenylalanine-Tryptophan-Glutamine-Isoleucine-Leucine (SEQ ID NO: 4). 
     
     
         20 . The set of peptide sequences of  claim 19 , where the sequences include D-amino and/or L-amino acid versions of these sequences.