Fluorophore Labeled Kinase Detects Ligands That Bind within the MAPK Insert of p38α Kinase

The vast majority of small molecules known to modulate kinase activity, target the highly conserved ATP-pocket. Consequently, such ligands are often less specific and in case of inhibitors, this leads to the inhibition of multiple kinases. Thus, selective modulation of kinase function remains a major hurdle. One of the next great challenges in kinase research is the identification of ligands which bind to less conserved sites and target the non-catalytic functions of protein kinases. However, approaches that allow for the unambiguous identification of molecules that bind to these less conserved sites are few in number. We have previously reported the use of fluorescent labels in kinases (FLiK) to develop direct kinase binding assays that exclusively detect ligands which stabilize inactive (DFG-out) kinase conformations. Here, we present the successful application of the FLiK approach to develop a high-throughput binding assay capable of directly monitoring ligand binding to a remote site within the MAPK insert of p38α mitogen-activated protein kinase (MAPK). Guided by the crystal structure of an initially identified hit molecule in complex with p38α, we developed a tight binding ligand which may serve as an ideal starting point for further investigations of the biological function of the MAPK insert in regulating the p38α signaling pathway

Data Collection and Refinement Statistics for p38α crystal structures in complex with <b>1b</b> and <b>8b</b>^a.

a<p>For all p38α complex structures, diffraction data from one crystal was used to determine the structure. Values in parenthesis are for the highest resolution shell.</p

Co-crystal structure of p38α and co-evolution analysis.

<p>(A) Crystal structure of wild type p38α (grey) in complex with <b>8b</b> (yellow). (B) Alignment of <b>1b</b> and <b>8b</b> bound to the lipid binding site of MAPK p38α. The 2-phenyl quinazoline scaffolds align nicely. The phenyl ethylene moiety of <b>8b</b> extends further into the hydro-phobic back pocket (brown) resulting in a higher binding affinity. (C, D) p38α in complex with <b>1b</b>. Amino acids showing significant co-evolution are colored according to the strength of the cumulative effect they exert on other residues (C) and of the cumulative effect other residues have on them (D), yellow symbolizing the strongest effect on both panels.</p

Structure activity relationship of 2-arylquinazolines.

<p>Each concentration of compound was tested in triplicate. Ratiometric emission values were plotted against the concentration of compound on a logarithmic scale to generate a binding curve. K_D values of various 2-arylquinazolines were determined bound to p38α.</p

General synthesis of 2-arylquinazolines.

<p>See table in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039713#pone-0039713-g003" target="_blank">Figure 3</a> for identity of substituents (R).</p