Year

2020

Degree Name

Master of Philosophy

Department

School of Chemistry and Molecular Bioscience

Abstract

Nitric oxide (NO) is a lipophilic, freely-diffusing, reactive (free radical) gas that acts as a signalling molecule in the cardiovascular, nervous and immune systems. Since the discovery that NO acts as a powerful vasodilator, several clinically useful NO-donor drugs have been developed in cardiovascular medicine; for example, nitroglycerin and isosorbide dinitrate for the treatment of acute angina. However, all the drugs developed to date have been spontaneous NO donors that non-specifically release NO in physiological milieu, leading to systematic exposure of host tissues to NO. Thus, there is an unmet opportunity to create innovative NO technologies that selectively target NO to specific tissue locations in the body. Antibody- directed enzyme prodrug therapy (ADEPT) is one promising strategy that could enable such tissue-specific NO delivery. In ADEPT, an antibody or antibody fragment that recognises a tissue-specific antigen is linked to an enzyme capable of activating some prodrug. Administration of the antibody-enzyme bioconjugate localises the enzyme activity to the tissue site bound by the attached antibody and administration of the prodrug then leads to selective revelation of active drug at the tissue site. A major advantage of ADEPT is its ability to reduce unwanted side-effects. In this Masters research project, an new ADEPT strategy was explored that uses an antibody-β-lactamase bioconjugate in combination with a cephalosporin-3′- diazeniumdiolate (C3D) NO-donor prodrug, a class of highly drug-like compounds that rapidly release NO upon reaction with β-lactamases, as a strategy for targeted NO therapy.

A possible application for a targeted NO therapy of this type is as an emergency intervention during heart attacks caused by atherosclerotic plaques, where the plaque occludes a coronary vessel and causes myocardial ischemia. Patients suffering from this type of heart attack could be given ADEPT NO therapy by paramedics that triggers vasodilation directly at the site of the occlusion. The increased cardiac perfusion and reduced myocardial ischemia could preserve the cardiac tissue and buy time for patients to undergo emergency surgery to remove the blockage. ADEPT under this scenario could use an atheroma-targeting antibody (e.g. antiLIBS scFv, Prof. Karlheinz Peter) β-lactamase conjugate in combination with a C3D. My Masters project aimed to provide the first molecular proof-of-concept towards this goal by constructing a model antibody-β-lactamase conjugate and demonstrating that such a conjugate can cause NO release from a C3D. The specific aims were: 1) design, clone and express a suitable TEM-1 β-lactamase, 2) conjugate the TEM-1 β-lactamase to the model antibody fragment Fab' 528, 3) synthesise and purify a C3D (DEA-C3D 1) and 4) confirm NO release from DEA-C3D 1 is triggered by the Fab' 528-β-lactamase conjugate.

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.