Botulinum neurotoxin-specific capture agents, compositions, and methods of using and making
AssigneesCalifornia Institute of Technology, Regeneron Pharmaceuticals, Inc.
InventorsBlake Farrow, James Heath, Heather Dawn Agnew
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Abstract
The present application provides stable peptide-based Botulinum neurotoxin (BoNT) serotype A capture agents and methods of use as detection and diagnosis agents and in the treatment of diseases and disorders. The application further provides methods of manufacturing BoNT serotype A capture agents using iterative on-bead in situ click chemistry.
Claims28
A stable, synthetic capture agent that specifically binds botulinum neurotoxin serotype A protein, wherein the botulinum neurotoxin serotype A protein comprises a heavy chain and a light chain, wherein the light chain comprises an enzymatic active site, wherein the capture agent comprises a anchor ligand and a secondary ligand and wherein the ligands specifically bind the botulinum neurotoxin serotype A protein.
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A method of synthesizing a capture agent to a target peptide comprising the steps of:
- a. identifying an anchor ligand and a secondary ligand that bind to the same target peptide at distinct epitopes;
- b. identifying the binding sites of the anchor ligand and the secondary ligand on the target peptide;
- c. calculating the distance between the binding site of the anchor ligand and the secondary ligand;
- d. selecting a linker to connect the anchor ligand to the secondary ligand, thereby generating a capture agent, wherein the dissociation constant of the capture agent for binding to the target protein is lower than the dissociation constant of either the anchor ligand or the secondary ligand for binding to the target protein;
- and
- e. synthesizing the capture agent comprising the anchor ligand and secondary ligand of step (a) and the linker of step (d).
A method of synthesizing a capture agent for inhibiting SNAP-25 cleavage mediated by botulinum neurotoxin serotype A protein in one or more neurons, wherein the botulinum neurotoxin serotype A protein comprises a heavy chain and a light chain, wherein the light chain comprises an enzymatic active site, comprising
- a. selecting an anchor ligand that specifically binds to the enzymatic active site of the botulinum neurotoxin serotype A protein;
- b. selecting a secondary ligand that specifically binds at a distinct epitope from the anchor ligand within 5-10 angstroms on the botulinum neurotoxin serotype A protein;
- and
- c. linking the anchor ligand and secondary ligand together,
- wherein, the secondary ligand must bind to an epitope so that it specifically binds to an occluded conformation of the botulinum neurotoxin serotype A holotoxin, thereby synthesizing the capture agent for inhibiting SNAP-25 cleavage mediated by botulinum neurotoxin serotype A protein in one or more neurons.
Dependencies are inferred from the claim text.