Phenotypic High-Throughput Screening to Identify Small-Molecule Inhibitors of Beta-Cell Glucolipotoxicity
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Small, Jonnell Candice
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CitationSmall, Jonnell Candice. 2022. Phenotypic High-Throughput Screening to Identify Small-Molecule Inhibitors of Beta-Cell Glucolipotoxicity. Doctoral dissertation, Harvard University Graduate School of Arts and Sciences.
AbstractThe global type 2 diabetes (T2D) epidemic continues to grow and by 2045, it is estimated 780 million adults will live with the disease. T2D is characterized by progressive pancreatic b-cell failure and loss of b-cell mass due to several pathophysiological factors including increased levels of circulating glucose and free fatty acids (FFAs). In fact, the growing prevalence of T2D is correlated with rapidly rising obesity rates, especially in low- and middle-income countries. Poor nutrition in early life, combined with overnutrition in later life appear to accelerate the T2D epidemic in these populations experiencing changing food habits and reduced physical activity. The onset of obesity is associated with elevated FFAs which contribute to T2D development by promoting insulin resistance, pancreatic β-cell dysfunction, and pancreatic β-cell death. Exposure to elevated glucose compounds the toxicity of elevated FFAs, leading to β-cell glucolipotoxicity (GLT). GLT is characterized by impaired glucose-stimulated insulin secretion (GSIS), decreased insulin gene transcription, attenuation of β-cell-specific transcription factors like PDX1, and induction of apoptosis through caspase activation. There are currently no therapeutics which address this facet of T2D.
Phenotypic high-throughput screening (HTS) is a target-agnostic therapeutic discovery strategy that strives to preserve the functional cellular context of disease relevant molecular pathways. Unlike target-based drug discovery, phenotypic screening is less biased, allowing the model system to reveal the target(s) crucial to a disease phenotype. Phenotypic HTS is useful for the discovery of novel biological probes and for the repurposing of previously identified drugs to
new disease contexts. Phenotypic HTS relies on the design of model system that near accurately mimic the clinical manifestation of diseases of interest.
In this dissertation, I explore the application of phenotypic screening to the discovery of β-cell GLT-protective small molecules. After optimizing GLT assays in the INS-1E cell line and dissociated human islets, in vivo models of pancreatic β-cells, I screened a total of 20,876 small molecules and validated ten compounds which recovered β-cell viability in both human islets and INS-1E. These compounds were validated in several secondary screening assays highlighting hallmark aspects of GLT including caspase activation, mitochondrial depolarization, calcium influx, and decreased expression of β-cell transcription factor Pdx1.
KD025 was identified as GLT-protective from the phenotypic HTS, and I sought to investigate the beta-cell target responsible for its activity. KD025 is a known Rho-associated kinase 2 (ROCK2) inhibitor and I hypothesized ROCK2 inhibition may mediate its GLT-protectivity. Subsequent experiments revealed Rock2 loss did not affect INS-1E viability in GLT-conditions. Kinase profiling revealed KD025 had potent casein kinase 2 (CK2) α/α’ activity and lead to the validation of CK2α as the mediator of GLT-induced β-cell death. CK2α loss improved INS-1E viability in GLT-conditions and CK2α overexpression ablated KD025 GLT-protectivity. RNA- sequencing analysis additionally revealed KD025 partially reversed the GLT-induced gene expression signature, including recovering the expression of several pancreatic β-cell genes and reducing the expression of genes involved in hypoxia and the inflammation response.
Citable link to this pagehttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37371930
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