Date of Award

2010

Degree Type

Thesis

Degree Name

Master of Science in Pharmaceutical Sciences

Department

Biomedical and Pharmaceutical Sciences

First Advisor

Matthew A. Strae

Abstract

The synthesis of bile acids is the major biological mechanism for cholesterol removal in the human body. Strict regulation of both cholesterol and bile acid levels is necessary to maintain a healthy balance and to prevent health problems. Bile acids are natural ligands for famesoid x receptor (FXR), a nuclear receptor that controls gene expression for multiple proteins involved in maintenance of bile acid homeostasis. Many endogenous and exogenous chemical ligands have been found to activate FXR; chenodeoxycholic acid (CDCA) is the most well characterized endogenous ligand. This study identifies a synthetic indole-acetamide, FGIN-1-27, as a new FXR agonist. FGIN-1-27 is already a known ligand of the translocator protein 18 kDa (TSPO), a mitochondrial cholesterol transporter.

FXR regulates target gene transcription through binding to special inverted repeat-I (IR-1) consensus DNA elements. Ligand binding to FXR was measured by inserting an IR-1 sequence upstream of a firefly luciferase detector gene that increased transcription of luciferase pr:oportional to ligand binding in a human hepatoma cell line (HuH-7). Results show that FGIN-1-27 is a partial agonist of FXR that activates FXR alone at 10 μM, but decreases activation from CDCA at 100 μM when cotreated. Two other well-known ligands of TSPO, FGIN-1-43 and PKl 1195 were investigated also for their effects on FXR mediated transcription. Both compounds acted as antagonists, decreasing the activity of CDCA (100 μM) while showing no activation of FXR alone at 10 μM treatment.

Agonist ligand binding to FXR increases the expression of the target gene, bile salt export pump (BSEP), and another nuclear receptor, small heterodimer partner (SHP). Through real time RT-PCR DNA amplification of both genes, we found FGIN-1-27 treatment in HuH-7 cells and primary human hepatocytes increased both BSEP and SHP gene expression. Additionally, expression of cholesterol 7a-hydroxylase (CYP7 Al), an enzyme involved in bile acid synthesis, is negatively regulated by FXR; we show that FGIN-1-27 decreased the expression ofCYP7A1.

In addition to in vitro studies, we investigated in silica molecular modeling of the binding of these TSPO ligands to FXR and demonstrated that these synthetic compounds fit into the ligand-binding pocket of FXR with favorable energy measurements. We identified key amino acids involved in agonist ligand binding in silica, and through mutation assays we confirmed that H447 is the major amino acid responsible for FXR interaction with an agonist ligand.

Taken together, FGIN-1-27 binding to and modulating two of the proteins involved in bile acid synthesis indicates there is overlap in the role of TSPO and FXR. FGIN-1-27 and related indole-acetamides may be potential therapeutic drugs beneficial to populations with enzyme deficiencies that cause high cholesterol levels. Further investigation of the role of mitochondria in bile acid synthesis will lead to a better understanding of the regulation of cholesterol and bile acid homeostasis.

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