Assay for exo-site binding molecules
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.
1. A method of identifying a small molecule 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 each member of a library of small molecules with each of the first and second variants;
determining an enrichment-based parameter for each small molecule with respect to each of the first and second variants by a binding assay;
comparing, for each small molecule, 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 small molecule is identified as an agent that binds an exo-site of the protein;
wherein the exo-site is not an allosteric site; wherein the library is an encoded library; and
wherein when a small molecule binds to the exo-site, the bound exo-site modulates substrate selectivity or binding preferences of an active site of the protein but does not change the catalytic activity of the active site of the protein.
2. The method of claim 1 , wherein the bound exo-site does not induce a conformational change in the active site of the protein.
3. The method of claim 1 , wherein the exo-site comprises a binding pocket defined by amino acids that are distinct from the amino acids of the active site of the protein.
4. The method of claim 1 , wherein the first variant is a wild-type protein or a truncated domain of a wild-type protein.
5. The method of claim 1 , 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.
6. The method of claim 5 , 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.
7. The method of claim 1 , wherein the first and second variants are two different isoforms of the protein, or truncated domains from two different isoforms.
8. The method of claim 1 , wherein the step of contacting comprises incubating each of the encoded small molecules with each of the first and second variants in parallel.
9. The method of claim 1 , wherein the step of contacting comprises incubating each of the encoded small molecules with each of the first and second variants in series.
10. The method of claim 1 , wherein the library is a DNA-encoded library.
11. The method of claim 10 further comprising amplifying the DNA codons of DNA encoded agents with PCR.
12. The method of claim 11 further comprising sequencing the PCR amplified DNA.
13. The method of claim 12 , wherein the step of determining binding comprises measuring the relative or absolute library member sequence abundance for the small molecule DNA coding sequences.
14. The method of claim 13 , wherein the step of determining binding comprises measuring an enrichment of postselection sequence abundance for the small molecule codons over preselection sequence abundance.
15. The method of claim 14 , wherein the step of comparing comprises comparing the measured enrichment in sequence abundance for each small molecule after incubation with the first variant with the measured enrichment in sequence abundance after incubation with the second variant.
16. The method of claim 1 , wherein the protein is insulin degrading enzyme (IDE).
17. The method of claim 11 , wherein PCR primers are used to amplify DNA.