Poly(Arginine) Derived Cancer-Targeting Peptides for the Development of a Cancer-Targeted Gene Therapy Approach in HepG2 Liver Cancer Cells

Cancer is a disease that has eluded medicinal approaches for many years and as a result new and improved therapeutic approaches are in constant demand. Although chemotherapy and radiation treatments have assisted in suppressing the growth of tumors, their poor selectivity and efficacy are major limitations for effective therapy en route towards the development of a cure for the cancer epidemic. With the mission of conquering cancer at heart, researchers have pursued a new form of cancer therapy, aptly named, a cancer targeting approach. This method revolves around the selection of a suitable biomarker, typically a cell surface receptor overexpressed or selectively localized on the surface of cancer cells but not on normal tissues. This biomarker forms the basis of a cancer targeting approach, in which high affinity and specific binding ligands (e.g. peptides) have been selected by phage display bio-panning methods or combinatorial chemistry to target cancer cells in-vivo. Building on this approach, the identification of a cyclic peptide, Pep42, H2N-CTVALPGGYVRVC-CONH2, has been selected as a specific and high-affinity binding ligand of the cell surface receptor, Glucose Regulated Protein 78 (GRP78). GRP78 is a member of the heat shock family of chaperone proteins, assisting in protein folding events under physiological stress induced conditions that are mitigated by the unfolded protein response (UPR) mechanism. In cancer, GRP78 is overexpressed and cell surface localized where it functions as a hub for cell signaling pathways that lead to cancer cell initiation, proliferation and resistance towards chemotherapy. Thus, GRP78 has been classified as a valid biomarker for the development of targeted anti-cancer approaches. Towards this goal, an Fmoc-based solid phase peptide synthesis (Fmoc-SPPS) method has been optimized on a polyethylene glycol (PEG) resin for the production of poly(arginine) derived Pep42 sequences in good yields (14-46%) and purities (\u3e95%) following RP-LCMS. The effect of the length (0-12) and stereochemistry (L/D) of the poly(arginine) sequences on Pep42 structure and stability were next evaluated by CD spectroscopy. Interestingly, peptides displayed varying folded conformations, transitioning between helical and turn structures, that were found to be contingent on the poly(arginine) sequence, solvent and disulfide bond formed within the Pep42 motif. Moreover, the peptide folds were found to be resilient towards thermal denaturation due to the covalent disulfide bond. Structure-activity relationships were next assessed in HepG2 hepatoblastoma cells, in which the cancer-targeting peptides were found to bind to the GRP78 receptor and internalize within the HepG2 cells. This discovery has led to their applications in cancer-targeted gene therapy, from which a GRP78-silencing short-interfering RNA (siRNA) was effectively transfected within the HepG2 cells for potent oncogene knockdown effects (50-60%, 40 pmol siRNA) while triggering modest cell death effects (5-10%). This thesis will highlight my efforts and contribution in collaboration with Drs. Blake and Bitsaktsis research groups towards the development of an effective cancer-targeted gene therapy approach