DOI
https://doi.org/10.25772/WK7F-1G56
Author ORCID Identifier
0000-0001-7970-7815
Defense Date
2022
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Pharmaceutical Sciences
First Advisor
Shijun Zhang
Abstract
Neuroinflammation has proven to play an essential role in the pathogenesis of neurodegenerative diseases by encouraging the maturation and secretion of pro-inflammatory mediators. Studies have shown the upregulation of pro-inflammatory cytokines interleukin (IL)-1β and IL-18 in neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), traumatic brain injury (TBI), and many more. The NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome is the key regulator in the maturation and release of these inflammatory cytokines and is therefore an attractive target for suppressing inflammation. Upon assembly and activation of the multimeric NLRP3 inflammasome complex comprised of NLRP3, ASC protein, and pro-caspase-1, autoproteolytic cleavage of pro-caspase-1 occurs. Activated caspase-1 is then able to cleave pro-IL-1β and pro-IL-18 and release their biologically active forms, IL-1β and IL-18, consequently leading to an inflammatory cell death known as pyroptosis. Glyburide, an FDA-approved drug that treats diabetes, has recently been found to inhibit the NLRP3 inflammasome, and our lab has recently undergone a medicinal chemistry campaign to design and optimize the potency and selectivity of glyburide which has led to a series of novel small molecule NLRP3 inflammasome inhibitors. Our lead compound, YQ128, has been shown to inhibit IL-1b secretion from J774A.1 mouse macrophages with an IC50of 0.3 µM, 33x more potent than glyburide, and has shown both in vitro and in vivo activity and selectivity. Here, we further elucidate the mechanism of action, target, and potential binding of YQ128.
YQ128 is able to inhibit caspase-1 and GSDMD cleavage as well as ASC speck formation in J774A.1 mouse macrophages; however, it has no effect on the NLRP3 expression, suggesting its target is upstream of inflammasome formation and caspase-1 but downstream of priming. FRET studies showed that fluorescence lifetime of NLRP3 fluorescent tags was shortened in the presence of YQ128’s fluorescent tag, inferring intracellular binding. Through target identification studies using a photoaffinity labeling technique, we found that YQ128 specifically interacts with NLRP3, but not other components of the NLRP3 inflammasome complex. Further degradation band analysis suggests YQ128 may target the NACHT domain of NLRP3. Competition studies with the parent compound and other known NLRP3 inhibitors suggested the specific binding of YQ128 to NLRP3. Biophysical assays with YQ128 and recombinant NLRP3 confirmed the strong binding interaction with a dissociation constant of 117 nM. The results from other biological assays proposed its binding to the NACHT domain of NLRP3. These results show YQ128 is a selective and potent inhibitor of NLRP3 inflammasome, and further studies are currently underway to improve these inhibitors as potential therapeutics for neuroinflammation in neurodegenerative diseases.
Rights
© Hallie Morgan Blevins
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
5-10-2022