title: Residence time and kinetic efficiency analysis of extracellular signal-regulated kinase 2 inhibitors.
abstract: 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.
authors.affiliation: Enzymology and Biophysical Chemistry Group, Takeda California, San Diego, CA 92121, USA.
authors.forename: Darin
authors.initials: D
authors.lastname: Vanderpool
authors.affiliation: Enzymology and Biophysical Chemistry Group, Takeda California, San Diego, CA 92121, USA.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Grimshaw
authors.affiliation: Computational Sciences and Crystallography Group, Takeda California, San Diego, CA 92121, USA.
authors.forename: J David
authors.initials: JD
authors.lastname: Lawson
authors.affiliation: Enzymology and Biophysical Chemistry Group, Takeda California, San Diego, CA 92121, USA. Electronic address: jacques.ermolieff@takeda.com.
authors.forename: Jacques
authors.initials: J
authors.lastname: Ermolieff
mesh_terms: D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D057166:High-Throughput Screening Assays / Q000379:methods*; D007700:Kinetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors* / Q000737:chemistry; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*
chemical_list: D005456:Fluorescent Dyes; D047428:Protein Kinase Inhibitors; D019950:Mitogen-Activated Protein Kinase 1
keywords: ERK2; Kinetic efficiency; Off-rate; Residence time; Structure–kinetic relationship; Time-dependent inhibition