In addition to drug-target affinity, drug-target residence time is becoming recognized as a critical parameter influencing drug efficacy. Desired compound action can only take place when a drug is interacting with its target protein, which is governed both by the affinity of the compound for the target, as well as the dissociative off-rate of the compound from the drug-target complex. A compound with a slower dissociative off-rate may require reduced dosing schedule relative to a compound with a rapid off-rate, and effective target specificity may be increased if the off-rate for the desired drug-target complex is greater than that for off-target complexes. With respect to kinase inhibitors, a slow off-rate for Tykerb relative to the related EGFR inhibitors Tarceva and Iressa has been proposed to explained longer lasting effects in cell culture. Currently, off rates for kinases can be measured by surface plasmon resonance (SPR)-based instruments or by detailed enzyme kinetics experiments. While SPR yields highly quantitative data, it is low-throughput and requires costly instrumentation. Furthermore, it often involves covalent conjugation of protein targets to surfaces, which can lead to loss of protein function. Off-rate measurements by classical enzyme kinetics methods are established, but fairly labor-intensive. We have developed and will present data on a fully homogenous (in-solution) method for determining kinase-compound off-rates that is simple to perform in a standard fluorescence-based plate reader. In this method, a drug-kinase complex is first formed at supra-Kd concentrations of drug, and then rapidly diluted into a solution containing a fluorescent, ATP-site directed small molecule probe. As the compound dissociates from the kinase the fluorescent probe associates with the kinase, causing an increase in the fluorescence signal that can be monitored in real time.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5496.