ANYL 240 |
| The c-Met tyrosine kinase is an important cancer target. During the drug discovery process it was observed that certain classes of inhibitors that were potent against the recombinant enzyme did not inhibit the cellular form of the enzyme. This was confirmed by the use of antibody based immunoprecipitation (IP) assays thus ruling out a permeability or metabolism issue. Rather, the activation state of the kinase or possibly a weakly interacting cellular protein partner (that was lost during the IP assay) might be the key factor. One way to address this is to analyze the active (phosphorylated) vs. unactive (unphosphorylated) enzyme form to probe for relative inhibitor binding potencies. To probe both forms of the enzyme a ligand-binding assay was developed. This assay utilized an intrinsically fluorescent ligand that had an increased fluorescence upon kinase binding. Interestingly, the affinity of ligand binding was dependent on the concentration of the kinase. NMR spectroscopy was used to assess the possibility of regioisomers or tautomers of the ligand that may have differential binding affinities or intrinsic fluorescence. This presentation will provide experimental details on the development of the ligand-binding assay and the use of NMR to gain further insight into ligand structure. |
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Methods Development for Pharmaceutical Analysis
1:30 PM-5:00 PM, Tuesday, 12 September 2006 Moscone Center -- Room 124, Oral
Division of Analytical Chemistry |