The first kinase inhibitor drug approval in 2001 initiated a remarkable decade of tyrosine kinase inhibitor drugs for oncology indications, but a void exists for serine/threonine protein kinase inhibitor drugs and central nervous system indications. Stress kinases are of special interest in neurological and neuropsychiatric disorders due to their involvement in synaptic dysfunction and complex disease susceptibility. Clinical and preclinical evidence implicates the stress related kinase p38αMAPK as a potential neurotherapeutic target, but isoform selective p38αMAPK inhibitor candidates are lacking and the mixed kinase inhibitor drugs that are promising in peripheral tissue disease indications have limitations for neurologic indications. Therefore, pursuit of the neurotherapeutic hypothesis requires kinase isoform selective inhibitors with appropriate neuropharmacology features. Synaptic dysfunction disorders offer a potential for enhanced pharmacological efficacy due to stress-induced activation of p38αMAPK in both neurons and glia, the interacting cellular components of the synaptic pathophysiological axis, to be modulated. We report a novel isoform selective p38αMAPK inhibitor, MW01-18-150SRM (=MW150), that is efficacious in suppression of hippocampal-dependent associative and spatial memory deficits in two distinct synaptic dysfunction mouse models. A synthetic scheme for biocompatible product and positive outcomes from pharmacological screens are presented. The high-resolution crystallographic structure of the p38αMAPK/MW150 complex documents active site binding, reveals a potential low energy conformation of the bound inhibitor, and suggests a structural explanation for MW150's exquisite target selectivity. As far as we are aware, MW150 is without precedent as an isoform selective p38MAPK inhibitor or as a kinase inhibitor capable of modulating in vivo stress related behavior.

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Published in ACS Chemical Neuroscience, v. 6, no. 4, p. 666-680.

Copyright © 2015 American Chemical Society

This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.

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This research was supported in part by NIH Awards U01AG043415 (D.M.W. and O.A.) and R01AG031311 (D.M.W.), ADDF Award 261108 (D.M.W.), and a Thome Memorial Foundation Award (L.J.V.E.).

cn5b00002_si_001.pdf (2042 kB)
Supporting Information

10.1021-acschemneuro.5b00002Figure1.ppt (182 kB)

10.1021-acschemneuro.5b00002Figure1(1).ppt (1580 kB)
Figure 1. Repurposing of a nonkinase CNS experimental therapeutic to CNS kinase inhibitor.

10.1021-acschemneuro.5b00002Figure1(2).ppt (994 kB)
Scheme 1.

10.1021-acschemneuro.5b00002Figure2.ppt (200 kB)
Figure 2. Stereo view of omit map for MW150 bound in the active site of human p38αMAPK. The map represents the difference electron density (mesh) contoured at 2.5σ. The analysis indicates the goodness of fit between the model and experimental data and is consistent with an energetically favorable conformation for MW150 (purple). Key amino acids in p38α MAPK (green) indicated: Met 109, involved in H-bonding with MW150, and Thr 106, gate-keeper for access to the hydrophobic pocket.

10.1021-acschemneuro.5b00002Figure3.ppt (180 kB)
Figure 3. Human p38αMAPK active site occupancy by MW150 (PDB 4R3C). (A) Connolly surface representation of the active site of p38αMAPK containing MW150. (B) Close-up view of the p38αMAPK hinge region near Met109 (left) and the pyridine substituent of MW150 (right) that are involved in hydrogen bond interaction. The gray mesh represents the experimental 2Fo-Fc electron density contoured at 1.2σ. (C) Surface created by amino acids within 5 Å of the naphthyl group of MW150. This perspective highlights the volume that the naphthyl substituent of MW150 occupies within the p38αMAPK hydrophobic pocket that is proximal in space to the hydrogen-bonding region shown in panel (B). The blue mesh surrounding MW150 was built from the experimental 2Fo-Fc electron density contoured at 1.5σ.

10.1021-acschemneuro.5b00002Figure4.ppt (208 kB)
Figure 4. Concentration-dependent cellular activity of MW150. (A) MW150 treatment suppresses the phosphorylation of MK2, a p38αMAPK substrate whose phosphorylation (activation) is increased in response to LPS activation of glia. Serial dilutions were added to BV2 microglial cells stimulated with 100 ng/mL LPS, and the levels of pMK2 at 1 h determined by ELISA analysis. (B) MW150 treatment attenuates the downstream increase in proinflammatory cytokine production, a mechanism of action pharmacodynamic end point. Levels of IL-1β at 16 h were determined by ELISA. Data are expressed as percent of maximal activity (=activity after LPS stimulation + control vehicle treatment) and are representative of at least two independent experiments. Open circle = no LPS + veh; black circle = LPS + veh; gray circle = LPS + MW150.

10.1021-acschemneuro.5b00002Figure5.ppt (147 kB)
Figure 5. CYP inhibition summary.

10.1021-acschemneuro.5b00002Figure6.ppt (464 kB)
Figure 6. MW150 treatment suppresses associative and spatial memory deficit in APP/PS1 Tg mice. Daily oral administration of either saline or MW150 (2.5 mg/kg) was done from age 8 weeks to 3–4 months. Associative and spatial memories were then assessed, respectively, through (A) contextual fear memory and (B) RAWM. Saline treated APP/PS1 mice exhibited cognitive deficits for both types of memory compared to saline treated-type (WT) mice, as evidenced by a significantly lower percent of freezing during assessment of fear memory, and by higher number of errors in the RAWM task. However, treatment of APP/PS1 mice with MW150 resulted in suppression of the deficits, as seen by the percent of freezing and RAWM performance indistinguishable from that of WT mice.

10.1021-acschemneuro.5b00002Figure7.ppt (711 kB)
Figure 7. Control behavioral analyses for MW150 suppression of associative and spatial memory deficits in APP/PS1 transgenic mice. No difference was detected between groups when tested for cued fear memory (A), sensory threshold (B), visual-motor-motivational deficits with the visible platform test (speed and time to the platform are shown in (C) and (D), respectively), and exploratory behavior, as shown by a similar percentage of time spent in the center compartment (E) and the number of entries into the center compartment (F).

10.1021-acschemneuro.5b00002Figure8.ppt (247 kB)
Figure 8. MW150 treatment suppresses spatial memory deficit in APP/PS1 knock-in (KI) mice. MW150 administration (A) to APP/PS1 KI mice (2.5 mg/kg; ip, daily for 14 days; n = 11, gray squares) suppressed cognitive deficits (B) seen in APP/PS1 KI mice treated with vehicle (gray circles, n = 12) and was indistinguishable from WT mice treated with vehicle (black triangles, n = 14). Mice were tested in a 2-day RAWM assay of spatial reference memory starting 3 days after the last treatment. Cognitive deficits in the KI mice treated with vehicle were evidenced by a significantly higher number of errors in RAWM performance compared to KI mice treated with MW150 (#p < 0.05, ##p < 0.005, ###p < 0.001) or WT mice treated with vehicle (*p < 0.05, **p < 0.005, ***p < 0.001).

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