Application Notes

1 Protein-Small Molecule Interaction Analysis Application Note NT-MO-003 Competitive Assay Approach: Binding of Small Molecules to the Active Form of p38 Krishna Saxena 1 and Moran Jerabek-Willemsen 2 1 Institute of Organic Chemistry and Chemical Biology Center University of Frankfurt, Germany 2 NanoTemper Technologies GmbH, Munich, Germany Abstract p38α is considered as the key isoform involved in modulating inflammatory response in rheumatoid arthritis and inflammatory pain and is therefore a key target for compound screening in pharmaceutical industry. In this study we demonstrate the use of MicroScale Thermophoresis to perform an essentially label-free measurement of small molecule binding to protein targets by using a competitive approach. This approach therefore allows screening projects aiming to identify and characterize site-specific binders or binders allosterically influencing a specific binding site out of a compound library. Furthermore this approach also can be used to screen for compounds that interrupt protein- protein, protein-peptide or protein-nucleic acid interactions. Introduction p38 is a serine/threonine protein kinase in the mitogen-activated protein kinase (MAPK) family. There are four isoforms of p38 (p38α, p38β, p38γ, and p38δ), and p38α is considered as the key isoform involved in modulating inflammatory response in rheumatoid arthritis and inflammatory pain (Dominuez et al. 2005) Two well characterized small molecule antagonists SB203580 (Davies et al., 2000) and the clinical candidate BIRB-796 (Lee and Dominguez 2005) were used in this study. Whereas SB203580 competes with ATP for the binding site on the kinase, BIRB-796 binds adjacent to the active site in a new allosteric binding pocket and indirectly inhibits enzymatic activity by affecting the conformation of the ATP site (Pargellis et al. 2002). Our experiment describes the competition of a fluorescent tracer molecule (ATP mimetic tracer199, Invitrogen) bound to the MAP kinase p38 by non-labeled small molecules (SB203580, BIRB-796). Fig. 1 Crystal structure of the p38-BIRB-796 complex The competition approach allows for a basically label-free measurement of the interaction between a compound and a protein since all molecules of interest are not labeled. Fig. 2 shows the principle of this approach. A complex of a protein and a fluorescently labeled molecule (grey with red star) and a protein is formed. This complex is mixed with a serial dilution of a small molecule of interest (blue). When the small molecule is binding the fluorescently marked tracer is set free, generating a strong MST signal (i.e. the difference of protein/tracer thermophoresis to thermophoresis of free tracer).

• Cover