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Cited by 225Alzheimer's disease research and treatmentsNeuroinflammation and Neurodegeneration MechanismsNeuroscience and Neuropharmacology Research

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A novel p38α MAPK inhibitor suppresses brain proinflammatory cytokine up-regulation and attenuates synaptic dysfunction and behavioral deficits in an Alzheimer's disease mouse model

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Munoz, Lenka, Ranaivo, Hantamalala Ralay, Roy, Saktimayee M, Hu, Wenhui, Craft, Jeffrey M, McNamara, Laurie K, Chico, Laura Wing, Van Eldik, Linda J, Watterson, D Martin·2007·Journal of Neuroinflammation
DOI 10.1186/1742-2094-4-21PMC PMC2014744
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Abstract

BACKGROUND: An accumulating body of evidence is consistent with the hypothesis that excessive or prolonged increases in proinflammatory cytokine production by activated glia is a contributor to the progression of pathophysiology that is causally linked to synaptic dysfunction and hippocampal behavior deficits in neurodegenerative diseases such as Alzheimer's disease (AD). This raises the opportunity for the development of new classes of potentially disease-modifying therapeutics. A logical candidate CNS target is p38 alpha MAPK, a well-established drug discovery molecular target for altering proinflammatory cytokine cascades in peripheral tissue disorders. Activated p38 MAPK is seen in human AD brain tissue and in AD-relevant animal models, and cell culture studies strongly implicate p38 MAPK in the increased production of proinflammatory cytokines by glia activated with human amyloid-beta (A beta) and other disease-relevant stressors. However, the vast majority of small molecule drugs do not have sufficient penetrance of the blood-brain barrier to allow their use as in vivo research tools or as therapeutics for neurodegenerative disorders. The goal of this study was to test the hypothesis that brain p38 alpha MAPK is a potential in vivo target for orally bioavailable, small molecules capable of suppressing excessive cytokine production by activated glia back towards homeostasis, allowing an improvement in neurologic outcomes. METHODS: A novel synthetic small molecule based on a molecular scaffold used previously was designed, synthesized, and subjected to analyses to demonstrate its potential in vivo bioavailability, metabolic stability, safety and brain uptake. Testing for in vivo efficacy used an AD-relevant mouse model. RESULTS: A novel, CNS-penetrant, non-toxic, orally bioavailable, small molecule inhibitor of p38 alpha MAPK (MW01-2-069A-SRM) was developed. Oral administration of the compound at a low dose (2.5 mg/kg) resulted in attenuation of excessive proinflammatory cytokine production in the hippocampus back towards normal in the animal model. Animals with attenuated cytokine production had reductions in synaptic dysfunction and hippocampus-dependent behavioral deficits. CONCLUSION: The p38 alpha MAPK pathway is quantitatively important in the A beta-induced production of proinflammatory cytokines in hippocampus, and brain p38 alpha MAPK is a viable molecular target for future development of potential disease-modifying therapeutics in AD and related neurodegenerative disorders.

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Figure 1 Synthetic scheme for synthesis of MW01-2-069A-SRM. Reactions, conditions and yields: a) POCl3, reflux, 2 hrs, 99%; b) NaOH, H2O, reflux, 2 hrs, 67%; c) 1-(2-pyrimidyl)piperazine, 1-butanol, 130°C, 26 hrs, 98%; d) POCl3, reflux, 3 hrs, 99%; e) 4-pyridylboronic acid, Pd(PPh3)4, K2CO3, DME, 110°C, 15 hrs, 74%.
Figure 1 Synthetic scheme for synthesis of MW01-2-069A-SRM. Reactions, conditions and yields: a) POCl3, reflux, 2 hrs, 99%; b) NaOH, H2O, reflux, 2 hrs, 67%; c) 1-(2-pyrimidyl)piperazine, 1-butanol, 130°C, 26 hrs, 98%; d) POCl3, reflux, 3 hrs, 99%; e) 4-pyridylboronic acid, Pd(PPh3)4, K2CO3, DME, 110°C, 15 hrs, 74%.CC BY · PubMed Central
Figure 2 Design of MW01-2-069A-SRM. A) Pharmacophore model showing the potential for 069A to make selective interactions in the active site of p38α MAPK. The pyridine ring nitrogen has the potential to make the critical interaction with the hydrogen of the amide bond formed between Met109 and Gly110. This interaction and the potential to occupy the nearby hydrophobic pocket are important interactions for p38α MAPK selective inhibitors. The "gatekeeper residue" in p38α and p38β is Thr106. Its small size compared to the larger Met in p38δ and p38γ isoforms allows bulkier groups in the compound to access the pocket, thereby providing isoform selectivity and potential affinity. B) Amino acid sequence alignment of p38 MAPK isoforms in the region containing key amino acids implicated in selective kinase-inhibitor interactions.
Figure 2 Design of MW01-2-069A-SRM. A) Pharmacophore model showing the potential for 069A to make selective interactions in the active site of p38α MAPK. The pyridine ring nitrogen has the potential to make the critical interaction with the hydrogen of the amide bond formed between Met109 and Gly110. This interaction and the potential to occupy the nearby hydrophobic pocket are important interactions for p38α MAPK selective inhibitors. The "gatekeeper residue" in p38α and p38β is Thr106. Its small size compared to the larger Met in p38δ and p38γ isoforms allows bulkier groups in the compound to access the pocket, thereby providing isoform selectivity and potential affinity. B) Amino acid sequence alignment of p38 MAPK isoforms in the region containing key amino acids implicated in selective kinase-inhibitor interactions.CC BY · PubMed Central
Figure 3 Inhibition of p38α MAPK enzyme activity and validation of design for selective interactions. A) MW01-2-069A-SRM inhibits p38α MAPK enzyme activity in a concentration-dependent manner. B) In contrast, the 069A analogs are > 100-fold less active. These include the starting scaffold (MW01-3-183WH), an analog (MW01-4-119SRM) with a different orientation of the pyridine ring nitrogen such that potential H-bond interactions are minimized, and an analog (MW01-6-189WH) with the pyridine ring at a different position on the scaffold. The in vitro phosphorylation of the standard protein substrate, myelin basic protein, by purified p38α MAPK was measured as described in Methods, in the absence or presence of increasing concentrations of compound. Data are expressed as percent of the maximal enzyme activity, where enzyme activity in the presence of solvent only (absence of inhibitor) is taken as 100%. Data are a representative example of five experiments.
Figure 3 Inhibition of p38α MAPK enzyme activity and validation of design for selective interactions. A) MW01-2-069A-SRM inhibits p38α MAPK enzyme activity in a concentration-dependent manner. B) In contrast, the 069A analogs are > 100-fold less active. These include the starting scaffold (MW01-3-183WH), an analog (MW01-4-119SRM) with a different orientation of the pyridine ring nitrogen such that potential H-bond interactions are minimized, and an analog (MW01-6-189WH) with the pyridine ring at a different position on the scaffold. The in vitro phosphorylation of the standard protein substrate, myelin basic protein, by purified p38α MAPK was measured as described in Methods, in the absence or presence of increasing concentrations of compound. Data are expressed as percent of the maximal enzyme activity, where enzyme activity in the presence of solvent only (absence of inhibitor) is taken as 100%. Data are a representative example of five experiments.CC BY · PubMed Central
Figure 4 Oral bioavailability, brain uptake, safety and metabolic stability of 069A. Compound bioavailability (A and B) was determined by administration of 069A (2.5 mg/kg) by oral gavage to C57Bl/6 mice, processing of blood and brain at different times after administration, and measuring compound levels in plasma and brain homogenate extracts by HPLC as described in Methods. Compound 069A rapidly appears in plasma (A) and brain (B) within 5 min, and then slowly declines to basal levels by 60 min. Data are the mean ± SEM from 5 mice at each time point. Lack of idiopathic tissue injury (C) after chronic administration of compound was assessed by treating C57Bl/6 mice (5 mice per group) with either vehicle (Veh) or 069A (Cmpd; 2.5 mg/kg) by oral gavage once daily for two weeks. Histological liver injury was determined by a scoring system as described in Methods. There is no significant liver toxicity after chronic two-week administration of compound compared to vehicle, the same dose and paradigm used in efficacy testing. To screen for potential metabolic instability (D), compound 069A, the clinical CNS drug Minaprine, or the drug Minozac were incubated with human liver microsomes, and the amount of compound remaining was analyzed by HPLC as described in Methods. Significantly different from time 0 (**p < 0.01; ***p < 0.001).
Figure 4 Oral bioavailability, brain uptake, safety and metabolic stability of 069A. Compound bioavailability (A and B) was determined by administration of 069A (2.5 mg/kg) by oral gavage to C57Bl/6 mice, processing of blood and brain at different times after administration, and measuring compound levels in plasma and brain homogenate extracts by HPLC as described in Methods. Compound 069A rapidly appears in plasma (A) and brain (B) within 5 min, and then slowly declines to basal levels by 60 min. Data are the mean ± SEM from 5 mice at each time point. Lack of idiopathic tissue injury (C) after chronic administration of compound was assessed by treating C57Bl/6 mice (5 mice per group) with either vehicle (Veh) or 069A (Cmpd; 2.5 mg/kg) by oral gavage once daily for two weeks. Histological liver injury was determined by a scoring system as described in Methods. There is no significant liver toxicity after chronic two-week administration of compound compared to vehicle, the same dose and paradigm used in efficacy testing. To screen for potential metabolic instability (D), compound 069A, the clinical CNS drug Minaprine, or the drug Minozac were incubated with human liver microsomes, and the amount of compound remaining was analyzed by HPLC as described in Methods. Significantly different from time 0 (**p < 0.01; ***p < 0.001).CC BY · PubMed Central
Figure 5 Compound 069A suppresses proinflammatory cytokine up-regulation in AD mouse model. A schematic of the experimental paradigm is shown. Treatment with 069A resulted in significant suppression of the Aβ-induced increase in the hippocampal levels of the proinflammatory cytokines IL-1β (A), TNFα (B) and S100B (C). Mice were infused ICV with either the diluent control (C) or the injurious oligomeric form of human Aβ1–42 (I) for 4 weeks. Starting at 3 weeks after the start of Aβ infusion, mice were administered by daily oral gavage either 069A (2.5 mg/kg; I + Cmpd) or vehicle (I + Veh) for two weeks. Spontaneous alternation of mice was measured for 10 days in the Y-maze, a hippocampus-dependent spatial learning task, beginning at day 50 after the start of Aβ infusion. Mice were sacrificed on day 60, and hippocampal extracts analyzed by ELISA. Data are means ± SEM of n = 5 mice per group. Significantly different from Aβ-injured: *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 5 Compound 069A suppresses proinflammatory cytokine up-regulation in AD mouse model. A schematic of the experimental paradigm is shown. Treatment with 069A resulted in significant suppression of the Aβ-induced increase in the hippocampal levels of the proinflammatory cytokines IL-1β (A), TNFα (B) and S100B (C). Mice were infused ICV with either the diluent control (C) or the injurious oligomeric form of human Aβ1–42 (I) for 4 weeks. Starting at 3 weeks after the start of Aβ infusion, mice were administered by daily oral gavage either 069A (2.5 mg/kg; I + Cmpd) or vehicle (I + Veh) for two weeks. Spontaneous alternation of mice was measured for 10 days in the Y-maze, a hippocampus-dependent spatial learning task, beginning at day 50 after the start of Aβ infusion. Mice were sacrificed on day 60, and hippocampal extracts analyzed by ELISA. Data are means ± SEM of n = 5 mice per group. Significantly different from Aβ-injured: *p < 0.05, **p < 0.01, ***p < 0.001.CC BY · PubMed Central
Figure 6 Compound 069A attenuates synaptic protein loss and behavioral deficits in AD mouse model. Treatments, behavioral testing, and preparation of hippocampal extracts were done as described in Figure 5. Treatment with 069A significantly attenuated Aβ-induced loss of synaptophysin (A) and the behavioral deficit in the Y-maze (B). Data are means ± SEM of n = 5–12 mice per group. Significantly different from Aβ-injured: *p < 0.05, **p < 0.01.
Figure 6 Compound 069A attenuates synaptic protein loss and behavioral deficits in AD mouse model. Treatments, behavioral testing, and preparation of hippocampal extracts were done as described in Figure 5. Treatment with 069A significantly attenuated Aβ-induced loss of synaptophysin (A) and the behavioral deficit in the Y-maze (B). Data are means ± SEM of n = 5–12 mice per group. Significantly different from Aβ-injured: *p < 0.05, **p < 0.01.CC BY · PubMed Central

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Authors
Munoz, L., Ranaivo, H. R., Roy, S. M., Hu, W., Craft, J. M., McNamara, L. K., Chico, L. W., Van Eldik, L. J., & Watterson, D. M.
Affiliations
Northwestern University (US)
Corresponding author
Watterson, D Martin
ORCID
Venue
Journal of Neuroinflammation
Publisher
Springer Science and Business Media LLC
Published
2007-09-04
Volume
4
Issue
1
Pages
21-21
ISSN
1742-2094 (ISSN-L)
DOI
10.1186/1742-2094-4-21
PMC
PMC2014744
OpenAlex
W2029663297
Cited by
224
References
83
Citations by year
2026: 4 · 2025: 4 · 2024: 11 · 2023: 10 · 2022: 9 · 2021: 12 · 2020: 11 · 2019: 13 · 2018: 9 · 2017: 20 · 2016: 15 · 2015: 13 · 2014: 10 · 2013: 14 · 2012: 21
FWCI
6.26
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Journal of Neuroinflammation (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Northwestern University
  • National Institutes of Health
  • National Institute on Aging (R01AG031311)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
Keywords
Alzheimer's disease, inflammatory cytokines, glial activation, synaptic dysfunction, amyloid-beta, small molecule inhibitors, blood-brain barrier penetration, orally bioavailable drugs, disease-modifying therapy, neuroinflammation
MeSH terms
Alzheimer Disease, Animals, Behavior, Animal, Brain, Disease Models, Animal, Humans, Mice, Inbred C57BL, Pyridazines, Pyrimidines, Synapses, Drugs, Investigational, Up-Regulation, Cytokines, Inflammation Mediators, MAP Kinase Signaling System, Protein Kinase Inhibitors, Mitogen-Activated Protein Kinase 14, Mice

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