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 p38α 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 (Aβ) 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 p38α 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 p38α 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 p38α MAPK pathway is quantitatively important in the Aβ-induced production of proinflammatory cytokines in hippocampus, and brain p38α MAPK is a viable molecular target for future development of potential disease-modifying therapeutics in AD and related neurodegenerative disorders.

Background: Hyperphosphorylation and aggregation of tau protein are the pathological hallmarks of Alzheimer’s disease and related tauopathies. We previously demonstrated that the microglial activation induces tau hyperphosphorylation and cognitive impairment via activation of p38 mitogen-activated protein kinase (p38 MAPK) in the hTau mouse model of tauopathy that was deficient for microglial fractalkine receptor CX3CR1.Method: We report an isoform-selective, brain-permeable, and orally bioavailable small molecule inhibitor of p38α MAPK (MW181) and its effects on tau phosphorylation in vitro and in hTau mice.Results: First, pretreatment of mouse primary cortical neurons with MW181 completely blocked inflammation-induced p38α MAPK activation and AT8 (pS199/pS202) site tau phosphorylation, with the maximum effect peaking at 60–90 min after stimulation. Second, treatment of old (~20 months of age) hTau mice with MW181 (1 mg/kg body weight; 14 days via oral gavage) significantly reduced p38α MAPK activation compared with vehicle-administered hTau mice. This also resulted in a significant reduction in AT180 (pT231) site tau phosphorylation and Sarkosyl-insoluble tau aggregates. Third, MW181 treatment significantly increased synaptophysin protein expression and resulted in improved working memory. Fourth, MW181 administration reduced phosphorylated MAPK-activated protein kinase 2 (pMK2) and phosphorylated activating transcription factor 2 (pATF2), which are known substrates of p38α MAPK. Finally, MW181 reduced the expression of interferon-γ and interleukin-1β.Conclusions: Taken together, these studies support p38α MAPK as a valid therapeutic target for the treatment of tauopathies.

Brain p38α mitogen-activated protein kinase (MAPK), a potential therapeutic target for cognitive dysfunction based on the neuroinflammation-synaptic dysfunction cycle of pathophysiology progression, offers an innovative pharmacological strategy via inhibiting the same activated target in both glia and neurons, thereby enhancing the possibility for efficacy. The highly selective, brain-penetrant p38αMAPK inhibitor MW150 attenuates cognitive dysfunction in two distinct Alzheimer’s disease (AD)-relevant models and avoids the problems encountered with previous mixed-kinase inhibitor drug candidates. Therefore, it is essential that the glial effects of this CNS-active kinase inhibitor be addressed in order to anticipate future use in clinical investigations. We explored the effects of MW150 on glial biology in the AD-relevant APP/PS1 knock-in (KI) mouse model where we previously showed efficacy in suppression of hippocampal-dependent associative and spatial memory deficits. MW150 (2.5 mg/kg/day) was administered daily to 11–12-month-old KI mice for 14 days, and levels of proinflammatory cytokines IL-1β, TNFα, and IL-6 measured in homogenates of mouse cortex using ELISA. Glial markers IBA1, CD45, CD68, and GFAP were assessed by immunohistochemistry. Microglia and amyloid plaques were quantified by immunofluorescence staining followed by confocal imaging. Levels of soluble and insoluble of Aβ40 and Aβ42 were measured by ELISA. The studies of in vivo pharmacodynamic effects on markers of neuroinflammation were complemented by mechanistic studies in the murine microglia BV2 cell line, using live cell imaging techniques to monitor proliferation, migration, and phagocytosis activities. Intervention with MW150 in KI mice during the established therapeutic time window attenuated the increased levels of IL-1β and TNFα but not IL-6. MW150 treatment also increased the IBA1+ microglia within a 15 μm radius of the amyloid plaques, without significantly affecting overall microglia or plaque volume. Levels of IBA1, CD45, CD68, GFAP, and Aβ40 and Aβ42 were not affected by MW150 treatment. MW150 did not significantly alter microglial migration, proliferation, or phagocytosis in BV2 cells. Our results demonstrate that MW150 at an efficacious dose can selectively modulate neuroinflammatory responses associated with pathology progression without pan-suppression of normal physiological functions of microglia.

INTRODUCTION: Neuropsychiatric syndromes such as anxiety and agitation are clinical presentations common to diverse neurodegenerative diseases and brain injury sequelae. They are a concern due to the impact on cognition, social interactions, and non‐pharmacological treatments. Cognitive or behavioral disturbances occur at early disease stages and increase with disease progression. Coincident pathologies include the loss of serotonin (5‐HT) neurons and appearance of neurofibrillary tangles in the raphe nucleus. Brain 5‐HT _2b receptor (5‐HT _2b R) levels are increased in Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and post‐stroke morbidity. HTR2B gene variants are implicated in psychiatric disorders. 5‐HTRs are associated with atypical neurotropic drug mechanisms and behavioral dysfunction in drug abuse. The accumulating body of evidence suggests that selective 5‐HT _2b R inhibition might mitigate neuropsychiatric syndromes and the associated cognitive dysfunction. Atypical neurotropic drugs interact with a variety of monoamine receptors and outcomes are viewed as a combination of 5‐HT and dopamine D2 receptor mediated actions. Clearly, there is a need for insight into precision 5‐HT _2b R inhibition as a potential pharmacological mechanism for treatment of neuropsychiatric syndromes and cognitive dysfunction associated with dementia. METHODS: Strategic optimization of an atypical neurotropic drug was used to develop MW073, a highly selective and orally bioavailable inhibitor of 5‐HT _2b R activity and β‐arrestin‐1 recruitment that is devoid of dopamine receptor recognition and risk of 5‐HT _2b R agonist activity. RESULTS: MW073 ameliorates amyloid and tau induction of behavioral dysfunction in preventive or disease stage intervention paradigms. Using MW073 as a standard of comparison, risperidone was shown to be a dose‐dependent inhibitor 5‐HT _2b R activity and β‐arrestin‐1 recruitment. DISCUSSION: Selective inhibition of 5‐HT _2b R activity is a viable mechanism for the treatment of neuropsychiatric syndromes with synaptic dysfunction as a root cause and is a previously unrealized pharmacodynamic mechanism potentially embedded in current neurotherapeutics. Highlights: A new highly selective 5‐HT _2b R antagonist, MW073, is described and used as a reference standard. MW073 attenuates synaptic and behavioral dysfunctions an animal models of neuropsychatric syndromes. Risperidone is a dose dependent inhibitor of 5‐HT _2b R activity and arrestin recruitment.

Chronic neuroinflammation and cholinergic dysfunction are major contributors to cognitive decline in Alzheimer’s disease. Here, we report a series of pyridazine-based multitarget-directed ligands that simultaneously inhibit human butyrylcholinesterase (hBChE) and p38α mitogen-activated protein kinase (p38α MAPK). Pyridazines 8 and 21 were identified as potent dual inhibitors with submicromolar inhibitory potencies against target enzymes and high selectivity over acetylcholinesterase. X-ray crystallographic analyses revealed the experimental binding modes of 8 and 21 in hBChE and p38α MAPK, and identified conserved π–π, π–cation, and hinge region interactions that rationalize dual-target engagement. Both compounds were blood-brain barrier permeable, exhibited low cytotoxicity, and significantly attenuated lipopolysaccharide-induced proinflammatory responses and apoptosis in BV2 microglia. In vivo, compound 21 enhanced cognitive performance in mouse models of scopolamine-induced amnesia and LPS-stimulated neuroinflammatory cognitive impairment. Collectively, these results document the potential of structure-assisted design to achieve selective dual-target pharmacology within a single molecular entity.

1. Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder and the leading cause of dementia worldwide. Progressive synaptic and neuronal loss underlies the decline in cognition and daily functioning, often accompanied by behavioral and psychological symptoms. Among these, neuropsychiatric disturbances such as agitation and aggression affect 20–65% of patients and represent a major source of caregiver burden. The serotonin receptor antagonist MW073 has recently emerged as a potential therapeutic candidate, showing efficacy in counteracting Aβ- and tau-induced synaptic and memory deficits in AD mouse models. Here, we investigated whether MW073 also mitigates aggressive behavior in Tg2576 mice, a widely used AD model that also displays heightened aggressiveness. Our findings demonstrate that MW073 significantly reduced aggressive tendencies in Tg2576 mice, suggesting that serotonergic modulation may represent a promising strategy to address both cognitive and neuropsychiatric symptoms of AD.