A fluorescence-based assay for p38α recruitment site binders: identification of rooperol as a novel p38α kinase inhibitor

Abstract

A new p38α inhibitor: Using a D-recruitment site (DRS) probe for p38α which exploits covalent interaction with Cys119 and alkyne-azide "click" chemistry to identify small molecules that recognize the p38α DRS, the anti-inflammatory natural product rooperol was identified as a novel p38α inhibitor.

Figure 1

(A) ESI-MS spectrum of unphosphorylated p38α. (B) ESI-MS spectrum of p38α incubated in the presence of 2 for 12 h at 37 °C. (C) structure of compound 2.

Figure 2

Structure of p38α (from PDB 1DI9) illustrating the ATP binding pocket (orange), DFG loop (purple), MAPK insert (green), and the D-site recognition docking site (red surface) which includes Cys119 (sulfur atom in yellow). The location of the other Cys residues are shown in yellow.

Figure 3. Design of DAim

3 as an In vitro and in-cell recruitment site probe for p38α

Figure 4

SDS-PAGE analysis of p38α treated with 3 followed by “click” addition of Alexa594-azide: lane 1, p38α (2 μg) alone; lane 2. p38α (2 μg) subjected to “click” reagents; lane 3. p38α (0.4 μg) incubated with 3 followed by “click” reaction. (A): gel visualized by fluorescence. (B): Coomassie blue staining of the same gel.

Figure 5

Determination of the pseudo-first-order rate of the covalent adduction of p38α by 3.

Figure 6

In-gel fluorescence SDS-Page analysis of HEK 293T cells over-expressing Flag-tagged p38α treated with 3 and then subjected to lysis, immunoprecipitation, and click reaction with Alexa594-azide. Lane 1. Cells treated with vehicle (0.05 % DMSO); 2. Cells treated with 1 μM 3. Lane 3. Cells treated with 5 μM 3. Lane 4. Cells treated with 50 μM 3. Lane 5. Isolated flag-tagged p38α (~0.6 μg) treated with 50 μM 3 in vitro followed by click reaction.

Figure 7

Adduction of p38α by 3 is selectively blocked by recruitment site ligands. p38α was incubated in the presence of 3 (100 μM) alone (Control) or (A): with an equimolar concentration of the MKK3 D-site peptide (MKK3) or the Ste7 D-site peptide (Ste7) or (B) with an equimolar concentration of the ATP-binding site inhibitors SB 203580 or BIRB-796 for 16 h followed by click reaction and by in-gel fluorescence SDS-PAGE analysis.

Figure 8

Inhibition of p38α adduct formation with 3 by a collection of natural products and analogs. Inhibitors were assayed at 200 μM concentration (54 μg/ml in the case of African potato extract (APE)).

Figure 9

Inhibition of p38α phosphorylation of ATF2 by rooperol. The rate of phosphorylation of ATF2 (12.5 μM) by active p38α (10 nM) in the presence of 500 μM ATP was determined in the presence of 0 to 432 μM rooperol.

Scheme 1

Structure of the p38α inhibiting alkynylimidazole 1, which forms covalently adduct p38α, and the proposed aza-Bergman rearrangement of 1 to afford diradical and carbene intermediates