Comparison Of Biochemical Activity Assay Formats For Kinases And Study Of Molecular Mechanism Of Sorafenib

It is over10years since non-radioactivity and homogeneous biochemical assays have been applied for kinase inhibitor/activator screening. However, none of them may analyze all the518kinases since the diverse of the kinome. It is hard for enzymologists to choose because none of them is perfect and it is also an intricate task on the combination of more than two assay formats because of the difference of enzyme utility, ATP concentration, assay condition even the distinct theories.14kinases in house with8well known kinase inhibitors were selected for this research, by handling with5non-radioactivity and homogeneous biochemical assay formats:ADP-Glo, Htrf, LanthaScreen Binding, Mobility Shift and Z-lyte, which have been applied widely in the world. First of all, we prove that the Z factors of all the5kinase assay formats were over0.5, which were meant the fitness of pharmaceutical research, esp. on high throughput screening. Full length instead of kinase domain should be selected for activity assays if possible and meanwhile appropriate peptide is preferred to lower the kinase per well utility. Apparent ATP Km is in a range nearby the real ATP regardless of the assay formats you selected. The inhibition data were relatively comparable of all the5assays, esp. on the Mobility Shift and Z-lyte, which were all in3-fold difference confidence. All the assays are good for high throughput screening except Mobility Shift due to the slow read speed. Mobility shift or ADP-Glo can be used for mechanism study. For kinase panel screening, ADP-Glo, Mobility Shift or Z-lyte is more comprehensive while LanthaScreen Binding can be alternative choice for low/non-activity kinases. Sorafenib (BAY43-9006, Nexavar(?)) is a novel oral kinase inhibitor that targets multiple tyrosine kinases in vivo and in vitro. Main targets are receptor tyrosine kinase pathways frequently deregulated in cancer such as the raf-ras pathway, vascular endothelial growth factor (VEGF) pathway, and FMS-like tyrosine kinase3(FLT3). Sorafenib was approved by the FDA in fast track for advanced renal cell cancer and hepatocellular cancer and shows good clinical activity in thyroid cancer. Multiple clinical trials are undertaken to further investigate the role of sorafenib alone or in combination for the treatment of various tumor entities. As critical components of cellular signal transduction cascades, protein kinases have become the most important target family for drug development. This report is to validate the important function of receptor tyrosine kinases RET and vascular endothelial growth factor receptor2(KDR), as well as the RAF/MEK/ERK kinase signaling cascades in cancer treatment with sorafenib, to reveal the molecular mechanism and provide more evidences for therapeutic approaches with sorafenib. In the present studies, sorafenib inhibited the activity of cRAF kinase with an IC5020.9nM,0.4nM for RET and4nM for KDR. Moreover, sorafenib inhibited the activity of cRAF, KDR and RET through non ATP-competitive, ATP-competitive and mixed-type inhibition mode respectively. Sorafenib displayed moderate cytotoxicity inhibition to cell proliferation does-dependently. Based on the present studies, it is confirmed that those protein kinases indeed play a crucial role in the effective mechanism of sorafenib, and they are important effective targets for specific therapeutic approaches, moreover, we found that sorafenib inhibited cell proliferation first mainly through blocking post-translational modifications which then fed back to regulate related gene expression. Therefore, this study provides more evidences for the important role of kinases involved in the pharmacological mechanism of sorafenib in cancer treatment, and partially revealed the detailed alteration at bimolecular level.