Förslaget inkom 2004-02-06
Biacore technology, synthetic inhibitor binding interactions and kinetics
OBS! ANSÖKNINGSTIDEN FÖR DETTA EXJOBB HAR LÖPT UT.
The project aim is to study small synthetic inhibitor binding interactions and kinetics to a cytosolic tyrosine kinase. The focus on the practical work will be using the Biacore technology. A biochemical phosphorylation TRF assay (Time-Resolved Fluorescence) will also be used. The Biacore surface plasmon resonance (SPR) technology provides information-rich data on biomolecular interactions between a receptor (protein or compound) bound to a chip and a ligand that passes over the chip. The difference in mass bound to the chip produces a shift in the angle of the reflected light. The binding and dissociation kinetics, equilibrium constant as well as mass bound can be monitored using SPR. Two Biacore 3000 protocols are developed for the kinase. (1) A DBA (Direct Binding Assay) protocol where the kinase is captured to the chip by a bound antibody and low MW inhibitors are passed over the kinase surface has been used. In this mode both on- and off-rates as well as dissociation constant KD can be determined. (2) An ISA (Inhibition in Solution Assay) where a small inhibitor is bound to the chip surface is also developed. In this mode the kinase and different concentrations of inhibitors, with similar binding mode as the bound inhibitor, are mixed. When passed over the surface, the free kinase concentration can be determined when captured to the inhibitor on the chip and hereby the KD can be calculated. In this mode it is not possible to measure the binding kinetics (on- and off-rates) of the inhibitor/kinase interaction.
Issues to focus on for this project are (1) Optimization of the Biacore DBA protocol to prolong the limited stability of the kinase chip at room temperature. (2) To determine binding mode and competitivity with ATP for a number of ATP mimetic kinase inhibitors. (3) Study the ATP binding kinetics to the kinase using Biacore and determine the Mg2+/Mn2+ dependency for this and also see whether Mg2+/Mn2+ is necessary for the inhibitors binding to the kinase. (4) Does ATP bind differently to phosphorylated (activated) kinase compared with inactive (unphosphorylated) kinase. Compared with these findings, determine whether the selected inhibitors bind to the two kinase forms in a different manner.
The applicant must be well-trained in experimental lab work. Christina Jungar will be main responsible supervisor for the Biacore work and Björn Johansson for the biochemical assay as well as theoretical kinase issues.
Supervising team: Biological Sciences, Primary Screening Team
Supervisors: Björn Johansson and Christina Jungar