Developing Novel Therapeutic Agents Targeting NLRP3 Inflammasome Inhibition and Oxidative Stress Mitigation for Epilepsy and Neurodegenerative Disorders
Vikas Singh1, Pritam Thapa1, Inamul Haque1, Ram Sharma1, Mukut Sharma1
1Neurobiology and Neurobehavior Research Lab, Kansas City VA Medical Center, Department of Neurology
Objective:
Design and screening of small chalcone derivative based compounds for targeting NLRP3 inflammasome and ROS inhibition.
Background:
NLRP3 activation and its adverse downstream effects contributes to neuroinflammation and epileptogenesis. Studies using various epilepsy models have demonstrated suppression of neuroinflammation, attenuation of neuronal loss and decrease in occurrence of spontaneous recurrent seizures with NLRP3 inhibition.
Design/Methods:
IL-1β inhibition: SIM-A9 cells seeded at 1.5X105 cells/well and incubated overnight. Cells primed with LPS, 1.0 mg/ml for 6 h and treated with DMSO as vehicle (0.1 – 1.5%, v/v) or different compounds (10 µM) for 1.5 h followed by ATP (5 mM) for 45 min. Supernatant centrifuged at 300 x g for 5 min at 4 oC to remove cell debris and stored at -80 oC until analysis. Sandwich-based ELISA assay used to determine the amount of secreted IL-1b as per manufacturer’s instructions.
ROS inhibition: Intracellular ROS and mitochondrial superoxide measured after DCFDA and MitoSOX staining of live cells. 5 × 104 cells/well were incubated at 37 oC in 5% CO2 for 24 h to allow adherence; treated with different compounds at 10 µM concentration. After 1 h, cells primed with 1 µg/ml of LPS for 1 h and subsequently treated with ATP (5mM) for 30 min. Next, cells were washed using 1× PBS, exposed to CM-H2DCFDA (10 µM) for 45 min or mitoSOX (5µM) for 30 min at 37 oC in humidified 5% CO2 incubator in the dark. DCFDA fluorescence recorded using excitation at 495 nm and emission at 532 nm; MitoSOX fluorescence excitation at 510 nm and emission at 580 nM.
Results:
Compounds Ch-PT-8 and Ch-PT-10 showed significant inhibition of IL-1β (59% and 65% at 10 µM) and ROS (79% and 72% at 10 µM).
Conclusions:
Two compounds with significant anti inflammatory activity were identified with ongoing studies to determine IC50 values and further compound optimization.
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