Biophysical platform for drug development based on energy landscape

A method of selecting an inhibitor of a target protein having an active site, the method comprising

• measuring stability of an induced fit conformation (E*-I) of a candidate agent contacted to the active site of the target protein, wherein the measuring comprises
• performing fast fluorescence binding kinetics of the binding of the candidate agent to the target protein, determining an observed binding rate (kfast) and an induced fit binding rate (kslow) from the fast fluorescence binding kinetics data as a function of candidate agent concentration, extracting from kfast and kslow microscope rate constants for conformational selection steps and induced fit steps in binding of the candidate agent to the target protein, and providing a kinetic binding scheme for the candidate agent to the target protein that includes the conformational selection steps and induced fit steps,
• determining x-ray crystal structures of free target protein and target protein complexed with candidate agent and to determine flexible portions of the target protein which undergo conformational change upon binding of the candidate agent, and
• performing in silico simulation combining the kinetic binding scheme determined by the fast fluorescence binding kinetics and the conformational change in the flexible portions of the target protein determined by the x-ray crystal structures, wherein the in silico simulation includes target protein motions on picosecond, microsecond and millisecond timescales to determine the flexibility of the target protein and the stability of the induced fit conformation (E*-I),
• wherein the candidate agent is selected as the inhibitor of the target protein when the determined stability of the induced fit conformation (E*-I) of the candidate agent contacted to the active site increased by at least about 1 kcal/mol compared to a reference stability.