Targeting Human Central Nervous System Protein Kinases: An Isoform Selective p38αMAPK Inhibitor that Attenuates Disease Progression in Alzheimer\u27s Disease Mouse Models

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\u27s 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

Development of Novel In Vivo Chemical Probes to Address CNS Protein Kinase Involvement in Synaptic Dysfunction

Serine-threonine protein kinases are critical to CNS function, yet there is a dearth of highly selective, CNS-active kinase inhibitors for in vivo investigations. Further, prevailing assumptions raise concerns about whether single kinase inhibitors can show in vivo efficacy for CNS pathologies, and debates over viable approaches to the development of safe and efficacious kinase inhibitors are unsettled. It is critical, therefore, that these scientific challenges be addressed in order to test hypotheses about protein kinases in neuropathology progression and the potential for in vivo modulation of their catalytic activity. Identification of molecular targets whose in vivo modulation can attenuate synaptic dysfunction would provide a foundation for future disease-modifying therapeutic development as well as insight into cellular mechanisms. Clinical and preclinical studies suggest a critical link between synaptic dysfunction in neurodegenerative disorders and the activation of p38αMAPK mediated signaling cascades. Activation in both neurons and glia also offers the unusual potential to generate enhanced responses through targeting a single kinase in two distinct cell types involved in pathology progression. However, target validation has been limited by lack of highly selective inhibitors amenable to in vivo use in the CNS. Therefore, we employed high-resolution co-crystallography and pharmacoinformatics to design and develop a novel synthetic, active site targeted, CNS-active, p38αMAPK inhibitor (MW108). Selectivity was demonstrated by large-scale kinome screens, functional GPCR agonist and antagonist analyses of off-target potential, and evaluation of cellular target engagement. In vitro and in vivo assays demonstrated that MW108 ameliorates beta-amyloid induced synaptic and cognitive dysfunction. A serendipitous discovery during co-crystallographic analyses revised prevailing models about active site targeting of inhibitors, providing insights that will facilitate future kinase inhibitor design. Overall, our studies deliver highly selective in vivo probes appropriate for CNS investigations and demonstrate that modulation of p38αMAPK activity can attenuate synaptic dysfunction

Targeting Human Central Nervous System Protein Kinases: An Isoform Selective p38αMAPK Inhibitor That Attenuates Disease Progression in Alzheimer’s Disease Mouse Models

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

Discovery synthetic scheme for title compounds.

<p>Reagents and conditions: i) Hydrazine sulphate, H₂O, 100°C; ii) Pyridin-4-ylboronic acid; 1,2-dimethoxyethane (DME)/water, Na₂CO₃, Pd(PPh₃)₄, 110°C; iii) phosphorus oxychloride/acetonitrile, 90°C; iv) Ethanol, amine, reflux; v) 1- or 2- Naphthylboronic acid, DME/water, Na₂CO₃, Pd(PPh₃)₄, 110°C; vi) concentrated HCl, anhydrous isopropanol, 80°C.</p

Data collection and phasing statistics. Values in parentheses are for highest resolution shell.

<p>Data collection and phasing statistics. Values in parentheses are for highest resolution shell.</p

Localized conformational change in the hinge region of human p38αMAPK when a non-selective inhibitor is bound.

<p><b>A.</b> Electron density maps (2mFo-Dfc; colored in light gray) contoured at 1.5 rmsd for the hinge region of p38αMAPK near Gly110 for 4F9W (in complex with MW108) and for 4FA2 (in complex with SB239063) are shown. Differences in the electron density path are seen in the area around Gly110, indicated by arrows. <b>B.</b> A superposition of 4F9W and 4FA2 reveals the differences around the peptide bond at Met109-Gly110, referred to as a glycine flip for structures such as 4FA2.</p

Attenuation of Aβ₄₂-induced synaptic and cognitive dysfunction by MW108 administration.

<p><b>A.</b> MW108 ameliorated the LTP deficit in Aβ₄₂-treated slices (n = 7–8 slices/group; F(1,13) = 17.25, p = 0.0011, compared to slices treated only with Aβ₄₂). Horizontal bar = time of application of Aβ₄₂ and MW108. <b>B.</b> MW108 ameliorated the reference memory deficit in Aβ₄₂-infused mice (n = 10–14 mice/group; F(1,24) = 1.827, p = 0.0001 comparing Aβ-infused vs. Aβ-infused+compound). <b>C.</b> MW108 ameliorated the contextual fear memory deficit in Aβ₄₂-infused mice (n = 16-18 mice/group; p = 0.009 comparing Aβ-infused vs. Aβ-infused+compound). <b>D-E.</b> No difference was detected between groups when tested for visual-motor-motivational deficits with the visible platform test; speed and time to the platform are shown in D and E, respectively (n = 10–14 mice/group). <b>F.</b> The same animals that underwent contextual fear conditioning testing were assessed for cued learning 24 hrs after the contextual learning. No difference was detected between groups (n = 16–18 mice/group). <b>G.</b> Sensory threshold was not affected regardless of treatment (n = 13–17 mice/group). <b>H-I.</b> No differences in exploratory behavior, as shown by a similar percentage of time spent in the center compartment (H) and the number of entries into the center compartment (I), were observed (n = 13–17 mice/group).</p

Characterization of selective p38αMAPK inhibitors MW108 (panel A) and MW181 (panel B).

<p>The title compounds were characterized in terms of their physical characteristics, target selectivity, and pharmacological properties.</p

Synthesis of p38αMAPK inhibitor analogs and initial screens for differential p38αMAPK vs CK1δ inhibition and for improvement in high dose tolerance.

<p>The affinity of compounds for p38αMAPK and CK1δ is shown, as well as the highest dose at which no adverse events are observed in dose escalation studies.</p