Application Notes

Competitive assay approach: binding of small molecules to the active form of p38

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2 Fig. 2 Competitive approach using a fluorescently-labeled tracer (grey with red star). Adding unlabeled compounds (blue) that compete for the same binding site or allosterically induce conformational changes of the tracer binding site will lead to competive removal of the tracer ending up with free labeled tracer. The affinity of the non-labeled small molecule to the unlabeled protein can be determined precisely by using the equation In this equation K d is the dissociation coefficient of the tracer and protein, P 0 is the concentration of the protein at 0 % binding, I 50 is the concentration of free ligand at 50 % binding, L 50 the concentration of the free tracer at 50 % binding. K i is the affinity of the non-labeled small molecule for the unlabeled protein. Please note, this type of assay can also be performed with a labeled peptide, nucleic acid or even a protein. By doing so it is not only possible to analyze if a molecule is binding, but also to infer its activity (e.g. if a compound is able interrupting a protein-protein interaction). Results First the binding of tracer199 to the active form of the kinase p38α was evaluated. The results are shown in Fig. 3. Fig. 3 Binding of tracer199 to p38α It shows a decreasing MST signal with increasing p38 concentration (starting at 805 units, decreasing to 738 units) and a sigmoidal behavior that allows deducing a K d of about 80 nM. With the experiment shown above, the tracer- protein system is well characterized and a competition experiment can be performed. A prepared stock solution of the tracer199-p38 complex (see Materials and Methods) was mixed with a serial dilution of the small molecule SB203580 (MW = 377.4 Da) starting at 4 μM (see Fig. 4). This molecule is known to have a high affinity to the protein p38 IC 50 = 34 nM in vitro and 600 nM in cells. Fig. 4 Competitive binding of SB203580 to p38α loaded with fluorescently marked tracer199 The signal starts at about 748 units which indicates that most of the tracer is in complex with the protein. When increasing the concentration of SB203580, the MST signal increases to about 805 units, which is exactly the signal level we expect for free tracer199 thermophoresis. The signal allows to determine an IC 50 of 100 nM and thus a K d of 26 nM (according to equation 1) in good accordance with literature values (Davies et al. 2000). Conclusion This competitive approach using a labeled molecule that is replaced allows measuring a dissociation constant at essentially label-free conditions. It also enables a site specific detection of the binding since a MST signal of the respective amplitude is only observed when the titrated small molecule binds to the same site as the tracer or allosterically influences this specific binding site. In addition to these advantages, this approach also allows to setup larger screening projects on the Monolith instrument: In case the protein is labeled and no tracer is used, the signal direction and amplitude will differ depending on the chemistry of the small molecule, its binding site and the conformational changes induced. This is different when a tracer is used. The MST signal direction (increasing or decreasing) is independent of the small molecule and its binding mode/site. The amplitude only depends on the amount of tracer that has been released by the compound. This allows screening for binders by using just few different concentrations and defining a certain amplitude cut-off value that defines which molecules are interesting for more detailed

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