Residence time and kinetic efficiency analysis of extracellular signal-regulated kinase 2 inhibitors

The Ras/Raf/MEK/ERK signal transduction cascade plays an important role in the regulation of critical cellular processes such as cell proliferation, migration, and differentiation. The up-regulation of this pathway can negatively affect cell homeostasis and is responsible for the development of various forms of Cancer and inflammation processes. Therefore, there is a strong interest in pursuing drug programs targeting some of the enzymes involved in this pathway. In addition to the determination of Ki, Kd, IC50, and/or EC50, a more thorough kinetic analysis can provide useful information for the selection of the best lead series during the early stage of the drug discovery process. This study describes a medium-throughput fluorescent probe displacement assay for the rapid determination of the k(off) constant, residence time, and kinetic efficiency for ERK (extracellular signal-regulated kinase) inhibitors. Using this method, we have identified several inhibitors that we have subjected to further kinetic analysis by comparing k(off) constants determined for these time-dependent inhibitors using either the active or inactive form of ERK2.

ERK2; Kinetic efficiency; Off-rate; Residence time; Structure–kinetic relationship; Time-dependent inhibition.