DOI
https://doi.org/10.25772/2GSY-0A65
Defense Date
2018
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Pharmaceutical Sciences
First Advisor
Shijun Zhang
Abstract
Neurodegenerative diseases are a class of conditions that lead to progressive atrophy of different parts of the central nervous system (CNS). These diseases lead to devastating clinical outcomes to patients and give rise to an enormous socio-economical burden on society.1 One commonality among some of the most well-known neurodegenerative disorders, e.g. Alzheimer’s disease (AD), Parkinson’s disease (PD), and multiple sclerosis (MS), is neuroinflammation.2-4 Neuroinflammation stems from interactions of the innate immune system with toxins and insults to the central nervous system. In the case of irremovable or chronic insults and toxins, this leads to chronic damaging inflammation that hastens neuronal degeneration and exacerbates disease pathology.5,6 Recently, inflammasomes of the innate immune system have been indicated in playing essential roles in the observed inflammatory responses. The most studied inflammasome is the nod-like receptor pyrin containing 3 (NLRP3) inflammasome.7–9 Recently our research group has successfully developed sulfonamide-based small molecule inhibitors of the NLRP3 inflammasome, such as JC-21 and JC-171, as potential therapeutics for AD and MS. Our studies established that JC-21 is a selective inhibitor of the NLRP3 inflammasome.10,11 Structural modifications led to the development of JC-171 with improved pharmacokinetic properties. More importantly, our studies demonstrated the in vivo activity of JC-171 to effectively ameliorate the experimental autoimmune encephalomyelitis (EAE), a mouse model of MS.12 Our data also strongly suggested that inhibitors based on this chemical scaffold may directly target the NLRP3 inflammasome.10–12 In this dissertation, we conducted biophysical, biochemical, and modeling studies to further elucidate the mechanistic information of these compounds as inhibitors of the NLRP3 inflammasome. In order to conduct further mechanistic studies, the NLRP3 protein was produced via transfection of HEK 293 cells with a modified plasmid of full-length human NLRP3 protein.13 Furthermore, LC-MS studies were conducted to confirm the blood-brain barrier penetration (BBB) of JC-171. Our studies established that JC-171 directly binds to the NLRP3 protein. The results also suggested that JC-171 may bind to the NACHT domain of NLRP3 while in a site that is distinct from the ATP binding site. This notion is supported by the fact that our compounds do not interfere with the ATPase activity of NLRP3. Docking studies of JC-171 to the homology model of the NACHT domain of NLRP3 also supported this assertion by showing the interaction of JC-171 with residues that are not overlapping with the ATP binding pocket. BBB penetration studies in combination with LC-MS analysis confirmed that JC-171 shows better BBB penetration when compared to MCC950. Collectively, our results strongly support that our compounds function as NLRP3 inflammasome inhibitors by directly binding to the NLRP3 protein, a novel and distinct mechanism of action when compared to the known inhibitors that target the NLRP3 inflammasome pathway. These results strongly encourage further development of such inhibitors as potential therapeutics for neurodegenerative diseases.
Rights
© Ashley G. Boice
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
5-8-2018