Patent ID: 7470543

Claim:
A method of identifying a ligand to a target molecule, the method comprising: providing a reference compound that binds to a target molecule with a binding affinity in the micromolar range; collecting a 1D 1 H nuclear magnetic resonance spectrum of a plurality of solutions of the reference compound in the presence of the target molecule while varying a concentration of one of the reference compound and the target molecule within the plurality of solutions; providing a test sample, the test sample comprising at least one competitive test compound; collecting a 1D 1 H nuclear magnetic resonance spectrum of a solution of the reference compound in the presence of the test sample and the target molecule; comparing the spectrum of the solution of the reference compound in the presence of the target molecule to the spectrum of the solution of the reference compound in the presence of the test sample and the target molecule to determine a change in one or more of the reference compound resonances, wherein the change in one or more of the reference compound resonances comprises an increase in signal intensity in at least one reference resonance; identifying the at least one competitive test compound that interacts with the target molecule based on the change in one or more of the reference compound resonances, wherein the at least one competitive test compound displaces the reference compound; calculating a ratio of target molecule bound reference compound concentration [EL] to total reference compound concentration [L TOT ] for each of the 1D 1 H nuclear magnetic resonance spectra for the plurality of solutions while using the equation, [EL]/[L TOT ]=([ E TOT ]+[L TOT ]+K D −sqrt(([ E TOT ]+[L TOT ]+K D ) 2 −4 [E TOT ][L TOT ])/2 [L TOT ], where K D is a separately measured dissociation constant of the reference compound and the target molecule and [E TOT ] is a total concentration of the target molecule; correlating a measured parameter within each 1D 1 H nuclear magnetic resonance spectrum for the plurality of solutions to the ratio of target molecule bound reference compound concentration to total reference compound concentration for each 1D 1 H nuclear magnetic resonance spectrum; determining a value for the 1D 1 H nuclear magnetic resonance parameter for the test compound in the presence of the reference compound and the target molecule and correlating the value to a particular target molecule bound reference compound concentration; determining an apparent dissociation constant for the particular target molecule bound reference compound concentration in the presence of the test compound using the equation, K D app =([ E TOT ][L TOT ]−[E TOT ][EL]+[EL] 2 −[L TOT ][EL ])/[ EL]; and determining the dissociation constant of the competitive test compound K I using the equation K I =[I]K D/(K D app −K D ), where [I] equals a concentration of the competitive test compound.