Assay for exo-site binding molecules

Abstract

Methods for the identification of agents the bind to exo-sites of proteins are provided. Agents identified by the methods described herein and pharmaceutical compositions comprising the identified agents are also provided. Methods of using an identified agent for the treatment or prevention of a disease, disorder, or condition are also provided, including methods of treating or preventing a disease associated with reduced, elevated, or ectopic expression or aberrant activity of a protein comprising an exo-site.

What is claimed is: 
     
         1 . A method of identifying an agent that binds an exo-site of a protein, the method comprising:
 providing a first variant of the protein, wherein the protein comprises an exo-site;   providing a second variant of the protein, wherein the exo-site of the second variant comprises at least one different amino acid than the exo-site of the first variant;   contacting a library of candidate agents with each of the first and second variants;   determining an enrichment-dependent parameter of each candidate agent to each of the first and second variants by a library binding or enrichment assay;   comparing, for each candidate agent, the binding to the first variant with the binding to the second variant, wherein if the enrichment-based parameter using the first protein variant is greater than the enrichment-based parameter using the second protein variant, then the candidate agent is identified as an agent that binds an exo-site of the protein.   
     
     
         2 . The method of  claim 1 , wherein the exo-site comprises a binding pocket that modulates the interactions of the protein with one or more substrates, one or more metabolites, or one or more native partners of the protein when an agent is bound to the exo-site. 
     
     
         3 . The method of  claim 1  or  2 , wherein the exo-site comprises a binding pocket that modulates the substrate selectivity or binding preferences of the protein when an agent is bound to the exo-site. 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein the exo-site comprises a binding pocket defined by amino acids that are at least about 5 angstroms away from the amino acids of the protein catalytic site. 
     
     
         5 . The method of any one of  claims 1 - 3 , wherein the exo-site comprises a binding pocket defined by amino acids that are at least about 10 angstroms away from the amino acids of the protein catalytic site. 
     
     
         6 . The method of any one of  claims 1 - 3 , wherein the exo-site comprises a binding pocket defined by amino acids that are at least about 15 angstroms away from the amino acids of the protein catalytic site. 
     
     
         7 . The method of any one of  claims 1 - 3 , wherein the exo-site comprises a binding pocket defined by amino acids that are at least about 20 angstroms away from the amino acids of the protein catalytic site. 
     
     
         8 . The method of any one of  claims 1 - 3 , wherein the exo-site comprises a binding pocket defined by amino acids that are at least about 25 angstroms away from the amino acids of the protein catalytic site. 
     
     
         9 . The method of any one of  claims 1 - 3 , wherein the exo-site comprises a binding pocket defined by amino acids that are at least about 30 angstroms away from the amino acids of the protein catalytic site. 
     
     
         10 . The method of any one of  claims 1 - 3 , wherein the exo-site comprises a binding pocket defined by amino acids that are at least about 35 angstroms away from the amino acids of the protein catalytic site. 
     
     
         11 . The method of any one of  claims 1 - 3 , wherein the exo-site comprises a binding pocket defined by amino acids that are at least about 40 angstroms away from the amino acids of the protein catalytic site. 
     
     
         12 . The method of any one of  claims 1 - 3 , wherein the exo-site comprises a binding pocket defined by amino acids that are at least about 50 angstroms away from the amino acids of the protein catalytic site. 
     
     
         13 . The method of any one of  claims 4 - 12 , wherein the amino acids of the protein catalytic site are the amino acids that participate in chemical steps at the protein active site. 
     
     
         14 . The method of any one of  claims 1 - 13 , wherein the exo-site comprises a binding pocket defined by amino acids that can be replaced with different amino acids without significantly altering the activity of the protein. 
     
     
         15 . The method of  claim 14 , wherein the activity of the protein is not significantly altered if the K m  or V max  is altered by a factor of between about 1.1-fold and about 10-fold for the same substrate and under the same assay conditions. 
     
     
         16 . The method of any one of  claims 1 - 15 , wherein the first variant is a wild-type protein or a truncated domain of a wild-type protein. 
     
     
         17 . The method of any one of  claims 1 - 16 , wherein the second variant comprises a mutant of the first variant, wherein one amino acid of the exo-site in the first variant is replaced with a different amino acid in the second variant. 
     
     
         18 . The method of  claim 17 , wherein the replacement amino acid in the second variant comprises a side chain with a higher number of non-hydrogen atoms than the replaced amino acid in the first variant. 
     
     
         19 . The method of any one of  claims 1 - 18 , wherein the first and second variants are two different isoforms of the protein, or truncated domains from two different isoforms. 
     
     
         20 . The method of any one of  claims 1 - 19 , wherein the library is a library of small molecules. 
     
     
         21 . The method of any one of  claim 1 - 19 , wherein the library is library of polynucleotides, polypeptides, or polysaccharides. 
     
     
         22 . The method of any one of  claims 1 - 21 , wherein the library is an encoded library. 
     
     
         23 . The method of  claim 22 , wherein the step of contacting comprises incubating the encoded candidate agents with each of the first and second variants in parallel. 
     
     
         24 . The method of  claim 22 , wherein the step of contacting comprises incubating the encoded candidate agents with each of the first and second variants in series. 
     
     
         25 . The method of any one of  claims 22 - 24 , wherein the library is a DNA-encoded library. 
     
     
         26 . The method of  claim 25  further comprising amplifying the DNA codons of DNA encoded agents with PCR, optionally wherein the PCR primers are encoded for the first or second variant. 
     
     
         27 . The method of  claim 26  further comprising sequencing the PCR amplified DNA. 
     
     
         28 . The method of  claim 27 , wherein the step of determining binding comprises measuring the relative or absolute library member sequence abundance for the candidate agent DNA coding sequences. 
     
     
         29 . The method of  claim 28 , wherein the step of determining binding comprises measuring an enrichment of postselection sequence abundance for the candidate agent codons over preselection sequence abundance. 
     
     
         30 . The method of  claim 29 , wherein the step of comparing comprises comparing the measured enrichment in sequence abundance for each candidate agent after incubation with the first variant with the measured enrichment in sequence abundance after incubation with the second variant. 
     
     
         31 . The method of any one of  claims 1 - 30 , wherein the protein is insulin degrading enzyme (IDE). 
     
     
         32 . The method of  claim 31 , wherein the second variant is IDE with a mutation at Ala479, Leu201, Glu205, Tyr302, Thr316, Val360, Gly361, Gly362, Leu364, Ala198, Trp199, Phe202, Tyr314, Ile374, or a combination thereof. 
     
     
         33 . The method of  claim 31 , wherein the second variant is IDE with the mutation A479L, V360Q, G362Q, A198T, W199F, F202R, Y314F, I374Q, or a combination thereof. 
     
     
         34 . An agent that binds an exo-site of a protein identified by the method of any one of  claims 1 - 33 . 
     
     
         35 . A pharmaceutical composition comprising an agent of  claim 34 , or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. 
     
     
         36 . A method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent of  claim 34 , or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of  claim 35 . 
     
     
         37 . The method of  claim 36 , wherein the disease is associated with reduced, elevated, or ectopic expression of a protein comprising an exo-site. 
     
     
         38 . The method of  claim 36 , wherein the disease is associated with aberrant activity of a protein comprising an exo-site. 
     
     
         39 . A method of modulating the substrate selectivity and processivity of a protein comprising an exo-site in a biological sample, the method comprising contacting the biological sample with an agent of  claim 34 , or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of  claim 35 . 
     
     
         40 . A method of modulating the substrate selectivity and processivity of a protein comprising an exo-site in a subject, the method comprising administering to the subject an agent of  claim 34  or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of  claim 35 . 
     
     
         41 . A method of inhibiting the activity on one or more substrates by a protein comprising an exo-site in a biological sample, the method comprising contacting the biological sample with an agent of  claim 34 , or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of  claim 35 . 
     
     
         42 . A method of inhibiting the activity on one or more substrates by a protein comprising an exo-site in a subject, the method comprising administering to the subject an agent of  claim 34 , or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of  claim 35 . 
     
     
         43 . An agent that binds an exo-site of IDE identified by the method of any one of  claims 31 - 33 . 
     
     
         44 . A pharmaceutical composition comprising an agent of  claim 43 , or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. 
     
     
         45 . A method of treating a metabolic disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent of  claim 34 , or pharmaceutically acceptable salt thereof, or a pharmaceutical composition of  claim 35 . 
     
     
         46 . The method of  claim 45 , wherein the metabolic disorder is diabetes. 
     
     
         47 . The method of  claim 45 , wherein the metabolic disorder is hyperglycemia, impaired glucose tolerance, obesity, sodium imbalance, or hypertension. 
     
     
         48 . A method of inhibiting the degradation of insulin in a biological sample, the method comprising contacting the biological sample with an agent of  claim 34 , or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of  claim 35 . 
     
     
         49 . A method of inhibiting the degradation of insulin in a subject, the method comprising administering to the subject an agent of  claim 34 , or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of  claim 35 . 
     
     
         50 . The method of  claim 48  or  49 , wherein inhibiting the degradation of insulin is selective over inhibiting the degradation of glucagon. 
     
     
         51 . The method of  claim 50 , wherein the selectivity is between about 2-fold and about 10-fold, between about 10-fold and about 50-fold, between about 50-fold and about 100-fold, between about 100-fold and about 500-fold, or between about 500-fold and about 1000-fold.