Date of Award

4-27-2026

Embargo Period

4-27-2031

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Regenerative Medicine and Cell Biology

College

College of Graduate Studies

First Advisor

Robin Muise-Helmericks

Second Advisor

Stephen Duncan

Third Advisor

Don C. Rockey

Fourth Advisor

Kenneth D. Tew

Fifth Advisor

Christopher Davies

Sixth Advisor

Mindy Engevik

Abstract

Homozygous familial hypercholesterolemia (HoFH) is a rare autosomal genetic disorder characterized by markedly elevated levels of low-density lipoprotein cholesterol (LDL-C), resulting in severe hypercholesterolemia and premature atherosclerotic cardiovascular disease. Patients with HoFH are often refractory to standard lipid-lowering therapies due to a loss of functional LDL receptor activity. Although recently developed therapeutics for HoFH can reduce circulating lipid levels, many are associated with significant hepatotoxicity, limited accessibility, or both. These limitations highlight an ongoing need for mechanistically distinct and safer therapeutic strategies.

Previously, we identified a class of small molecules, triazine thiols, that effectively reduce apolipoprotein B-100 (APOB) secretion in hepatocytes and lower serum cholesterol, triglycerides, APOB-containing lipoproteins, and lipoprotein(a) in humanized mouse models. However, the molecular target and mechanism of action of these compounds remained unknown. Therefore, the central aim of this dissertation was to identify the target for triazine thiols and determine the mechanism underlying their effects on hepatic lipoprotein secretion.

In this study, we used affinity-based mass spectrometry to identify carboxylesterase 1 (CES1) as a direct binding partner of triazine thiols. We further characterized this interaction using biochemical assays and demonstrated that triazine thiols act as slow-binding, covalent, allosteric inhibitors of CES1. Molecular modeling identified a putative binding pocket in close proximity to cysteine 390, and site-directed mutagenesis confirmed that substitution of this residue confers resistance to triazine thiol-mediated inhibition, supporting a cysteine-dependent mechanism of action. Additionally, hepatocyte-like cells derived from CES1-deficient induced pluripotent stem cells exhibited markedly reduced ApoB secretion, phenocopying the effects observed with triazine thiol treatment.

Collectively, these findings establish carboxylesterase 1 as the molecular target of triazine thiols and provide insight into the mechanism by which they regulate lipoprotein secretion. This work further demonstrates that pharmacologic or genetic disruption of CES1 function reduces APOB secretion, highlighting CES1 inhibition as a potential LDL receptor-independent therapeutic strategy for hypercholesterolemia, including HoFH.

Rights

Copyright is held by the author. All rights reserved.

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