Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Chemistry

Collaborative Specialization

Molecular Imaging

Supervisor

Luyt, Leonard G.

Abstract

The growth hormone secretagogue receptor-1a (GHS-R1a) is expressed in many human tissues, most notably the hypothalamus, and causes an increase in appetite upon activation by its endogenous ligand, ghrelin. GHS-R1a is differentially expressed in malignant compared to benign tumours. Therefore, there is an interest in developing GHS-R1a-targeted peptides as novel drugs to modulate signaling for diseases such as cancer cachexia and obesity and to image the receptor for disease diagnosis and following progression.

Chapter 2 discusses a fluorescently labelled ghrelin analogue for imaging GHS-R1a in ex vivo biopsy analysis and in vivo distribution studies. The analogue was created through side-chain cyclization resulting in an improved affinity and stability compared to natural ghrelin. This stapled peptide was used as a cancer cell-specific fluorescent stain.

G7039, a peptidomimetic ghrelin agonist (IC50 5.2 nM/EC50 0.18 nM), underwent structure-activity relationship studies to generate improved ligands and positron emission tomography (PET) agents. The first generation peptidomimetic (Chapter 3) [1-Nal4,Lys5 (4-fluorobenzoyl-4-FB)]G7039 (IC50 69 nM/EC50 1.1 nM) was radiolabelled with 18F in a radiochemical yield of 48%, radio purity of ≥ 99%, and molar activity of ≥ 34 GBq/μmol. Despite success in radiolabelling, its solubility (cLogP = 8.76) and binding affinity needed improvement.

The second generation peptidomimetic (Chapter 4) [Tyr4,Lys5(2-fluoropropionyl (2-FP)]G7039 (IC50 0.28 nM/EC50 0.12 nM) had improved binding and lipophilicity (cLogP = 4.36). Labeling of this ligand was low yielding, however, a unique H-bond interaction was identified with molecular docking.

The third generation (Chapter 5) required a modified prosthetic group (2-FP to ammonium methyltrifluoroborate-AMBF3) to radiolabel in higher yields, resulting in [Tyr4,Lys5(AMBF3)]G7039 (IC50 0.85 nM). This compound was radiolabelled in a single step and with improved radiochemical data.

Finally, in Chapter 6 a homobivalent G7039 ligand (IC50high 0.43 nM:IC50low 0.42 pM /EC50 1.8-2.1 nM) was found to bind to GHS-R1a homo-oligomer and was designed to study GHS-R1a homo-oligomerization. Differential signalling with the GHS-R1a homo-oligomer was observed by measuring cellular signals such as b-arrestin, ERK, and gene reporters. A successful series of GHS-R1a targeting probes have been synthesized and characterized with applications driven towards imaging and therapy.

Summary for Lay Audience

Within health care there is a growing need to better diagnose and monitor human diseases to have the best possible outcome for the patient. Improvements in disease diagnosis and monitoring is due to new and emerging biomarkers (human proteins) that can be targeted by fluorescent and/or radioactive pharmaceuticals. These various pharmaceuticals can also allow us to study a biomarker from a molecular pharmacology standpoint (i.e. signalling and regulation). The pharmaceuticals that are designed to image human diseases can also have the dual purpose in treating the human disease by targeting and regulating the biomarkers.

This thesis describes the development of chemical tools that target a very attractive G-protein coupled receptor (a specific family of human proteins): the growth hormone secretagogue receptor-1a (GHS-R1a). This is a protein that is found on the cellular membrane of human tissues such as the heart, lungs, and brain. GHS-R1a is an attractive protein target because of its wide range of physiological functions in the human body including metabolism, cardiac output, depression, and neuroprotection. This protein is also regulated differently when there is disease present. Within malignant prostate, ovarian, lung, and uterine cancer the GHS-R1a has greater expression. The upregulation or downregulation of GHS-R1a can change the outcome of human diseases, leading to worse or better prognosis. GHS-R1a is also highly promiscuous as it couples (hetero-oligomerizes) with many other GPCRs (i.e. dopamine, melanocortin), as well as it has been shown to couple (homo-olgiomerize) with itself. When it couples with itself or other GPCRs there are changes to the normal human cellular events leading to different physiological outcomes in humans. This can alter the human disease to make the prognosis either worse or better.

Overall in my PhD I was able to develop several different styles of drug molecules targeting GHS-R1a. Each of these drugs was modified in such a way as to have applications in either imaging or therapy. This work opens the doors to further drug development to study this protein in human physiology and disease.

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