Author ORCID Identifier

https://orcid.org/0000-0002-1898-1551

Semester

Fall

Date of Graduation

2023

Document Type

Dissertation

Degree Type

PhD

College

Eberly College of Arts and Sciences

Department

Chemistry

Committee Chair

Werner Geldenhuys

Committee Co-Chair

Justin Legleiter

Committee Member

Brian Popp

Committee Member

Bjorn Soderberg

Committee Member

Patrick Callery

Abstract

MitoNEET belongs to the CDGSH Iron-Sulfur Domain (CISD)-gene family of proteins and is a [2Fe-2S] cluster-containing protein found on the outer membrane of mitochondria. The specific functions of mitoNEET/CISD1 remain to be fully elucidated, but the protein is involved in regulating mitochondrial bioenergetics in several metabolic diseases. Unfortunately, drug discovery efforts targeting mitoNEET to improve metabolic disorders are hampered by the lack of ligand-binding assays for this mitochondrial protein. We have developed a protocol amenable for high-throughput screening (HTS) assay, by modifying an ATP fluorescence polarization method to facilitate drug discovery targeting mitoNEET. Based on our observation that adenosine triphosphate (ATP) interacts with mitoNEET, ATP-fluorescein was used during assay development. We established a novel binding assay suitable for both 96- or 384-well plate formats with tolerance for the presence of 2% v/v dimethyl sulfoxide (DMSO). We determined the IC50-values for a set of benzenesulfonamide derivatives and found the novel assay reliably ranked the binding-affinities of compounds compared to radioactive binding assay with human recombinant mitoNEET. The developed assay platform is crucial in identifying novel chemical probes for metabolic diseases, as it will accelerate drug discovery targeting mitoNEET and potentially other members of the CISD gene family. Furthermore, we carried out ITC studies of the interactions between mitoNEET and thiazolidinediones (TZDs), a group of compounds/drugs that bind to peroxisome proliferator-activated receptor-gamma (PPAR-γ) to manage and treat type 2 diabetes mellitus. The binding affinities or KDs as well as the thermodynamic parameters such as enthalpy (ΔH), entropy (ΔS), and Gibbs free energy (ΔG) were determined for each thiazolidinedione compound employed in this work. Moreover, the reaction stoichiometry (n) and c values were also determined for each interaction between a thiazolidinedione and mitoNEET. These studies may offer insightful perspective into designing compounds that may have higher affinity for mitoNEET in drug discovery efforts.

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