Identification and pharmacological profile of SPP1, a potent, functionally selective and brain penetrant agonist at muscarinic M1 receptors.

BACKGROUND AND PURPOSE: We aimed to identify and develop novel, selective muscarinic M1 receptor agonists as potential therapeutic agents for the symptomatic treatment of Alzheimer's disease. EXPERIMENTAL APPROACH: We developed and utilized a novel M1 receptor occupancy assay to drive a structure activity relationship in a relevant brain region while simultaneously tracking drug levels in plasma and brain to optimize for central penetration. Functional activity was tracked in relevant native in vitro assays allowing translational (rat-human) benchmarking of structure-activity relationship molecules to clinical comparators. KEY RESULTS: Using this paradigm, we identified a series of M1 receptor selective molecules displaying desirable in vitro and in vivo properties and optimized key features, such as central penetration while maintaining selectivity and a partial agonist profile. From these compounds, we selected spiropiperidine 1 (SPP1). In vitro, SPP1 is a potent, partial agonist of cortical and hippocampal M1 receptors with activity conserved across species. SPP1 displays high functional selectivity for M1 receptors over native M2 and M3 receptor anti-targets and over a panel of other targets. Assessment of central target engagement by receptor occupancy reveals SPP1 significantly and dose-dependently occupies rodent cortical M1 receptors. CONCLUSIONS AND IMPLICATIONS: We report the discovery of SPP1, a novel, functionally selective, brain penetrant partial orthosteric agonist at M1 receptors, identified by a novel receptor occupancy assay. SPP1 is amenable to in vitro and in vivo study and provides a valuable research tool to further probe the role of M1 receptors in physiology and disease.

Combination of resveratrol and 5-azacytydine improves osteogenesis of metabolic syndrome mesenchymal stem cells.

Endocrine disorders have become more and more frequently diagnosed in humans and animals. In horses, equine metabolic syndrome (EMS) is characterized by insulin resistance, hyperleptinemia, hyperinsulinemia, inflammation and usually by pathological obesity. Due to an increased inflammatory response in the adipose tissue, cytophysiological properties of adipose derived stem cells (ASC) have been impaired, which strongly limits their therapeutic potential. Excessive accumulation of reactive oxygen species, mitochondria deterioration and accelerated ageing of those cells affect their multipotency and restrict the effectiveness of the differentiation process. In the present study, we have treated ASC isolated from EMS individuals with a combination of 5-azacytydine (AZA) and resveratrol (RES) in order to reverse their aged phenotype and enhance osteogenic differentiation. Using SEM and confocal microscope, cell morphology, matrix mineralization and mitochondrial dynamics were assessed. Furthermore, we investigated the expression of osteogenic-related genes with RT-PCR. We also investigated the role of autophagy during differentiation and silenced PARKIN expression with siRNA. Obtained results indicated that AZA/RES significantly enhanced early osteogenesis of ASC derived from EMS animals. Increased matrix mineralization, RUNX-2, collagen type I and osteopontin levels were noted. Furthermore, we proved that AZA/RES exerts its beneficial effects by modulating autophagy and mitochondrial dynamics through PARKIN and RUNX-2 activity.

Effects of Hypergravity on Osteopontin Expression in Osteoblasts.

Mechanical stimuli play crucial roles in bone remodeling and resorption. Osteopontin (OPN), a marker for osteoblasts, is important in cell communication and matrix mineralization, and is known to function during mechanotransduction. Hypergravity is a convenient approach to forge mechanical stimuli on cells. It has positive effects on certain markers of osteoblast maturation, making it a possible strategy for bone tissue engineering. We investigated the effects of hypergravity on OPN expression and cell signaling in osteoblasts. Hypergravity treatment at 20 g for 24 hours upregulated OPN expression in MC3T3-E1 cells at the protein as well as mRNA level. Hypergravity promoted OPN expression by facilitating focal adhesion assembly, strengthening actin bundles, and increasing Runx2 expression. In the hypergravity-triggered OPN expression pathway, focal adhesion assembly-associated FAK phosphorylation was upstream of actin bundle assembly.

The bile acid sensor farnesoid X receptor is a modulator of liver immunity in a rodent model of acute hepatitis.

Immune-mediated liver diseases including autoimmune and viral hepatitis are a major health problem worldwide. In this study, we report that activation of the farnesoid X receptor (FXR), a member of the ligand-activated nuclear receptor superfamily and bile sensor highly expressed in the liver, attenuates liver injury in a model of autoimmune hepatitis induced by Con A. We found that FXR gene ablation results in a time-dependent increase of liver expression (up to 20-fold in a 9-mo-old mouse) of osteopontin, a NKT cell-derived extracellular matrix protein and immunoregulatory cytokine. In comparison to wild-type, FXR(-/-) mice are more susceptible to Con A-induced hepatitis and react to Con A administration by an unregulated production of osteopontin. Administering wild-type mice with a synthetic FXR agonist attenuated Con A-induced liver damage and liver expression of the osteopontin gene. By in vitro studies, we found that FXR is expressed by primarily isolated NKT cells and its ablation favors ostepontin production in response to Con A. Chromatin immunoprecipitation assay and coimmunoprecipitation experiments demonstrate that the short heterodimer partner (SHP), a nuclear receptor and FXR target, was expressed by NKT cell hybridomas and increased in response to FXR activation. FXR activates SHP that interacts with and inhibits c-Jun binding to the osteopontin promoter. These data indicate that in NKT cells, FXR activation causes a SHP-mediated inhibition of osteopontin production. These data support the notion that the bile acid sensor FXR regulates the activation of liver NKT cells.