BPS2026 - Accurate determination of drug binding affinities and binding kinetics in NMR-based titrations

Drug discovery efforts frequently produce lead compounds with high hydrophobicity and poor water solubility. Dose response curves often level off prematurely due to poor water solubility, giving a false impression of drug saturation of the protein and calculation of artificially small dissociation constants. In solution NMR spectroscopy, protein signals are broadened by exchange processes between free and bound states, making it possible to extract binding kinetics data that are relatively free from such measurement errors. We titrated 5 different compounds into a chimeric construct for human cardiac troponin. NMR signals during the titration showed characteristic broadening due to drug binding and release. We wrote MATLAB scripts that modelled NMR signal intensity and position during the drug titrations using the Bloch-McConnell equations. There were 4 fitting parameters used: k on , k off , drug solubility, and protein scaling factor. Our MATLAB scripts could precisely determine k off and a protein scaling factor for our NMR-based drug titrations. However, there were a range of values for k on and drug solubility, though their product, a drug association rate, was relatively constant. By assuming similar k on values, it is possible to discern solubilities and relative binding affinities for any family of drug-like small molecules binding to a particular protein target. NMR could become an essential method for drug development based on its ability to determine drug binding kinetics that are not susceptible to binding affinity determination artefacts due to low drug solubility.