Dual-action kinase inhibitors influence p38α MAP kinase dephosphorylation

Abstract

Reversible protein phosphorylation directs essential cellular processes including cell division, cell growth, cell death, inflammation, and differentiation. Because protein phosphorylation drives diverse diseases, kinases and phosphatases have been targets for drug discovery, with some achieving remarkable clinical success. Most protein kinases are activated by phosphorylation of their activation loops, which shifts the conformational equilibrium of the kinase toward the active state. To turn off the kinase, protein phosphatases dephosphorylate these sites, but how the conformation of the dynamic activation loop contributes to dephosphorylation was not known. To answer this, we modulated the activation loop conformational equilibrium of human p38α ΜΑP kinase with existing kinase inhibitors that bind and stabilize specific inactive activation loop conformations. From this, we identified three inhibitors that increase the rate of dephosphorylation of the activation loop phospho-threonine by the PPM serine/threonine phosphatase WIP1. Hence, these compounds are "dual-action" inhibitors that simultaneously block the active site and promote p38α dephosphorylation. Our X-ray crystal structures of phosphorylated p38α bound to the dual-action inhibitors reveal a shared flipped conformation of the activation loop with a fully accessible phospho-threonine. In contrast, our X-ray crystal structure of phosphorylated apo human p38α reveals a different activation loop conformation with an inaccessible phospho-threonine, thereby explaining the increased rate of dephosphorylation upon inhibitor binding. These findings reveal a conformational preference of phosphatases for their targets and suggest a unique approach to achieving improved potency and specificity for therapeutic kinase inhibitors.

Keywords: allostery; drug mechanism; kinase; phosphatase.

Fig. 1.

p38α inhibitors modulate dephosphorylation of p38α by WIP1 phosphatase. (A) Schematic depicting p38α inhibitors changing activation loop (A-loop) conformation (apo orange and blue drug bound), leading to a change in phosphatase activity on phosphorylated p38α MAP Kinase. Phosphorylation sites are depicted as yellow spheres on activation loop cartoon. (B) Fold change in single turnover WIP1 phosphatase activity [k_obs (min⁻¹)] in the presence of inhibitors relative to a DMSO treated control (error is the propagated error of the fit). A previously validated construct encoding the WIP1 catalytic domain (1-420) was used for these and all subsequent assays (15). Data for the compounds that caused the largest effects are shown in (C). Observed rates were obtained from single turnover reactions [k_obs (min⁻¹)] of p38α (0.25 µM) dephosphorylation at pT180 by WIP1 (2.5 µM) in the absence or presence of excess p38α inhibitors (all inhibitors were used at 1.25 µM except for imatinib which was 1 mM). Data are fit to an exponential decay (Methods) (error on datapoints are ±1 SD from an n = 3). The Inset depicts the first 20 min of the reaction. (D) k_obs from panel C shown as bar graphs (k_obs were averaged from an n = 3 ± 1 SD).

Fig. 2.

Dual-action p38α inhibitors induce an activation loop conformation that exposes pT180 for dephosphorylation. (A) Overlay of X-ray crystal structure of human apo p38α-2p (orange; PDB: 9CJ2) and pexmetinib bound human p38α-2p (dark blue; PDB: 9CJ3) emphasizing activation loop (A-loop, solid colors) and phosphorylation site rearrangements (shown as sticks). Left zoom shows pexmetinib-mediated A-loop conformation and coordination of pY182. Note that pT180 is unresolved, likely due to flexibility. Right zoom shows apo p38α-2p A-loop and coordination of pT180 and pY182. Light blue dotted lines indicate hydrogen bonds within 4 Å to the phosphate group in both zooms. (B) Activation loop of p38α-2p bound pexmetinib (dark blue). Light blue dotted lines indicate hydrogen bonding within 4 Å of pexmetinib to D168 (DFG motif). (C) Activation loop of p38α-2p (orange) overlayed with pexmetinib from the bound structure to emphasize the clash of pexmetinib with F169. In all structures, oxygen, nitrogen, phosphorous, and fluorine atoms are colored red, blue, orange, and light green, respectively. Crystal contacts are illustrated in SI Appendix, Fig. S7.

Fig. 3.

pY182 coordination in BIRB796-p38α-2p is structurally similar yet less coordinated than in pexmetinib-p38α-2p. (A) Overlay of BIRB796-p38α-2p (light blue; PDB: 9CJ4) and pexmetinib-p38α-2p (white, pexmetinib in dark blue; PDB: 9CJ3). D168 and F169 are shown as sticks and pY182 is shown as spheres and sticks. (B) Chemical structures of pexmetinib and BIRB796. (C) Interactions of pexmetinib (Left; white and dark blue) and BIRB796 (Right; white and light blue) with D168 and F169 (DFG motif) are shown in sticks with the hydrogen bond within 4 Å of the ligand to D168 shown as light blue dashes and (D) as spheres representing stacking and van der Waals interactions. In all structures, oxygen, nitrogen, phosphorous, and fluorine atoms are colored red, blue, orange, and light green, respectively. Crystal contacts are illustrated in SI Appendix, Fig. S7.

Fig. 4.

Phosphorylation of tyrosine 182 promotes threonine 180 dephosphorylation. (A) Dephosphorylation of p38α^Y182F-pT by WIP1 performed under single turnover conditions: 2.5 µM WIP1 with 0.25 µM single threonine phosphorylated p38α^Y182F-pT in the absence or presence of excess p38α inhibitors (1.25 µM). Data were fit to an exponential decay (Methods, n = 2 ± error of the fit). (B) Bar graph of rates derived from panel A (n = 2 ± error of the fit) with k_obs of p38α-2p pT180 dephosphorylation by WIP1 (Fig. 1D) shown as translucent bars for reference. (C) X-ray crystal structure of unphosphorylated p38α bound to pexmetinib (dark blue; PDB: 9CJ5) overlayed with p38α-2p bound to pexmetinib (white; PDB: 9CJ3), showing a similar activation loop conformation. (D) Zoom-in of pexmetinib-p38α activation loop (white) showing coordination of Y182. (E) Zoom-in of pexmetinib-p38α-2p activation loop (dark blue) showing coordination of pY182. In all structures, blue dashes indicate hydrogen bonding within 4 Å and oxygen, nitrogen, phosphorous, and fluorine atoms are colored red, blue, orange, and light green, respectively. Crystal contacts are illustrated in SI Appendix, Fig. S7.

Fig. 5.

p38α exists in a conformational equilibrium that facilitates dephosphorylation by multiple phosphatases. (A) Comparisons of p38α-2p (0.25 µM) dephosphorylation rates by PPM1A (0.5 µM), DUSP3 (50 µM), and SAP (1 µM) in the presence of excess inhibitors (1.25 µM) as fold change compared to a DMSO control (error displayed is the propagated error of the fit). Comparable relative rates of p38α-2p dephosphorylation by WIP1 (Fig. 1B) are shown as translucent background bars on each plot as a reference. (B) Comparisons of pY182 of p38α^T180A-pY (0.25 µM) dephosphorylation rates by DUSP3 (15 µM) in the presence of excess inhibitor (1.25 µM) compared to a DMSO control [error displayed is the propagated error of the fit (n = 2)]. (C) Model proposing a conformational equilibrium between modification resistant (human apo p38α-2p; PDB: 9CJ2) with inaccessible phosphorylation states, and modification-competent states that represent exposed phosphorylation sites [pexmetinib-p38α-2p (PDB: 9CJ3), HAB1-SnRK2.6, HAB1 not shown (PDB: 3UGJ), and MKK6^DD-p38α^T180V, MKK6^DD not shown (PDB: 8A8M)].