Methods to measure dissociation rates for ligands that form reversible covalent bonds

摘要

The crystal structure of the ligand binding domain of ERR-α in complex with a ligand that forms a reversible thioether bond to Cys325 of ERR-α, methods to measure dissociation rates for ligands that form reversible covalent bonds, and methods to design ligands that form reversible covalent bonds for use as modulators of ERR-α activity are disclosed. The crystal structure and methods provide a novel molecular mechanism for modulation of the activity of ERR-α and provide the basis for rational drug design to obtain potent specific ligands for use as modulators of the activity of this new drug target.

主权项

1. A method to measure the dissociation rate of a ligand that forms a reversible covalent bond with a protein, wherein said protein is an Estrogen Related Receptor alpha (ERR-α), comprising the steps of:
(a) measuring by LC/MS a mass for the protein, a mass for a first ligand that forms a reversible covalent thioether bond with the protein at Cys325, and a mass for a competing second ligand that forms a reversible covalent bond with the protein, wherein the competing second ligand has a different mass than the first ligand; (b) mixing the protein and the first ligand in a solution with the first ligand in molar excess of the protein; (c) incubating the protein and the first ligand in the solution to allow for a protein:ligand complex to form; (d) removing an aliquot of the solution and measuring by LC/MS the mass for the protein:ligand complex; (e) adding the competing second ligand to the solution containing the protein:ligand complex, wherein the competing second ligand is in molar excess to the protein:ligand complex and in molar excess to the first ligand; (f) removing aliquots of the solution at time 0 and at regular time intervals; (g) measuring the time-dependent change in the mass of the protein:ligand complex equal to the mass difference of the first ligand and the competing second ligand; and, (h) determining the dissociation rate for the first ligand.