Development and Characterization of Selective CB2R Inverse Agonists as a Novel Anti-Inflammatory Therapy for Neuroinflammation – Mechanisms of Action in Murine and Human Microglia Models Provide Valuable Insights into Their Therapeutic Potential
Date of Award
Doctor of Philosophy (PhD)
Bob M. Moore, Ph.D.
Isaac O. Donkor, Ph.D.; Wei Li, Ph.D.; Megan K. Mulligan, Ph.D.; Frank Park, Ph.D.
Anti-Inflammatory, cannabinoid receptor 2, CB2R inverse agonist, Microglia, Neurodegenerative diseases, Neuroinflammation
Introduction: Recently, research has revealed the presence of cannabinoid receptor 2 (CB2R) in the central nervous system (CNS); since then, the receptor has emerged as a promising therapeutic target for treating multiple neurodegenerative diseases (NDD) such as Alzheimer’s (AD), Parkinson’s (PD), Human Immunodeficiency Virus (HIV) and Multiple Sclerosis (MS). The restricted and upregulated expression of CB2R in activated microglia and lack of psychotropic activity make this receptor an attractive target for discovering novel therapy to treat neuroinflammatory diseases for which there is currently no curative treatment. Methodology: A library of compounds were synthesized and evaluated in ACTOne and TANGO-bla functional assays for CB1 and CB2 activity in combination with affinity measurements for the CB1/2 receptors. Moreover, the compounds that demonstrated no activity at functional assays with high binding affinities to CB2R were further assessed in antagonist (competition) studies against the non-selective cannabinoid agonist CP 55,940. Compound 45 demonstrated CB2R inverse agonism at G-protein signaling with high potency, efficacy, and affinity in the binding and functional assays and was selected for further evaluation in murine and human microglia inflammatory models. On cell polarization assays were done to measure microglia M1 and M2 surface markers expression. Scratch assay and ROS measurements were performed to assess the effects of drug treatment on microglia migration and ROS production. Extensive signaling pathway studies were conducted to identify the downstream signaling mechanism for compound 45 and cytoskeleton staining was performed to evaluate the microglia morphological changes upon 45 treatment. A molecular docking study was utilized to explore ligand protein molecular interaction between 45 and CB2R. Additionally, the similarities and differences in the activity of compound 45, relative to standard CB2R agonist and inverse agonist, were examined in the aforementioned assays. Results: Compound 45 demonstrated the highest affinity and selectivity towards CB2R that translated to higher potency in CB2R ACTOne functional assay, whereas no functional activity was detected at CB1R. Moreover, testing compound 45 in the TANGO-bla assay revealed neutral antagonism at β-arrestins signaling, confirming the G-protein functional selectivity of this class of compounds. Treatment with compound 45 triggered a shift in murine and human microglia polarization from a pro-inflammatory (M1) to an anti-inflammatory (M2) phenotype after 24 hours of treatment. The M1 markers were significantly decreased and M2 markers significantly increased relative to LPS stimulus. These results were further confirmed using microscopic examination wherein compound 45 induced a remarkable transformation of microglia into ramified morphology with a small cell body and several elongated cell projections, indicating microglia adopted an anti-inflammatory M2 phenotype. Moreover, compound 45 significantly reduced cell migration, suppressed the M1 associated ROS production which causes neurotoxicity and cell damage. Additionally, the application of compound 45 significantly increased JNK, c-JUN, CREB, and p38 phosphorylation levels and decreased ERK1/2 and NF-κB representing a unique anti-inflammatory mechanism of CB2R inverse agonists in activated microglia. The molecular docking study of compound 45 revealed that the thiophene C ring constrained the toggle switch residue Trp 258 via multiple π-π stacking interactions that stabilized CB2R in the inactive state and led to 45 inverse agonism activity at CB2R. These results were distinctly different when compared to standard CB2R agonists and inverse agonists, suggesting further research is needed to explore more precisely the utility of CB2R based therapeutics in human CNS disorders. Conclusion: The results of these studies provided valuable information that aids in the development of new analogs of the biaryl-aryl scaffold for treating neuroinflammation in human neurodegenerative diseases.
Alghamdi, Sahar Saleh (https://orcid.org/0000-0002-2770-218X), "Development and Characterization of Selective CB2R Inverse Agonists as a Novel Anti-Inflammatory Therapy for Neuroinflammation – Mechanisms of Action in Murine and Human Microglia Models Provide Valuable Insights into Their Therapeutic Potential" (2020). Theses and Dissertations (ETD). Paper 507. http://dx.doi.org/10.21007/etd.cghs.2019.0492.