Title

The Use of Ruthenium Substituted NGR-Tagged Rubredoxin for Targeted Cancer Therapeutic Drug Delivery

Date of Award

6-2022

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Francis E. Jenney, Jr.

Second Advisor

Kimberly Baker

Third Advisor

Lori Redmond

Abstract

Current cancer treatments such as chemotherapy, radiation, and surgical resection lack specificity and may result in extreme side effects such as nausea, vomiting, hair loss, among others. The hyperthermophilic archaeon Pyrococcus furiosus grows at extreme temperatures while also containing thermostable proteins such as rubredoxin (Rd). Rd is of particular interest due to possessing the characteristics of a non-heme iron protein with the ability to unfold, refold and substitute its iron atom cofactor for other metals. The iron core is held together by four cysteinyl sulfur ligands. Therefore, Rd has unique features that allow this protein to be a potential therapeutic tool in the medical field. Previous research has shown specific binding of tumor homing peptides such as NGR (Asparagine-Glycine-Arginine) to an overexpressed amino peptidase receptor (APN) on cell lines such as human fibrosarcoma (HT-1080). Previous studies have also shown the substitution of the core iron atom in NGR-tagged Rd with a cytotoxic metal such as ruthenium (RuRd). Once this Rd-based compound binds to the APN surface cell receptor, cell death can occur while ideally sparing surrounding healthy tissue. The purpose of this research project is to assess the effectiveness of NGR-tagged RuRd as a potential selective anti-cancer therapeutic. NGR-tagged Rd was expressed and purified from the transformation of the appropriate plasmid in E. coli cells. After purification, NGR-tagged Rd underwent substitution of its core iron atom with the cytotoxic metal ruthenium. Purification methods were confirmed through the use of a purification gel, UV-Visible Spectroscopy, and both Electrospray Ionization (ESI) as well as Inductively Coupled Plasma (ICP) Mass Spectrometry. Binding assays and binding competition assays also showed evidence of preferential binding of NGR-tagged Rd to the APN receptor on HT-1080 cells. These results suggest that NGR-tagged RuRd could serve as a targeted anti-cancer drug.

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