ABSTRACT
Considerable research efforts have been dedicated to understanding ovarian and breast cancer mechanisms, but there has been little progress translating the research into effective clinical applications. Hence, personalized/precision medicine has emerged because of its potential to improve the accuracy of tumor targeting and minimize toxicity to normal tissue. Targeted therapy in both breast and ovarian cancer has focused on antibodies, antibody drug conjugates (ADCs), and very recently the introduction of human antibody fusion proteins. Small molecule inhibitors and monoclonal antibodies (mAbs) are used in conjunction with chemotherapeutic drugs as a form of treatment but problems arise from a board expression of the target antigen in healthy tissues. Also, insufficient tumor penetration due to tight binding affinity and macromolecular size of mAbs compromise the efficacy of these ADCs. A more targeted approach is thus needed, and ADCs were designed to meet this need. However, in ADCs the method of conjugation of drug to antibody is >1, altering the structure of the drug which leads to off-target effects. Random conjugation also causes the drug to affect the pharmokinetics and biodistribution of the antibody and may cause nonspecific binding and internalization. Recombinant therapeutic proteins achieve controlled conjugation reactions and combine cytotoxicity and targeting in one molecule. They can also be engineered to extend half-life, stability and mechanism of action, and offer novel delivery routes. SNAP-tag fusion proteins are an example of a theranostic recombinant protein as they provide a unique antibody format to conjugate a variety of benzyl guanine modified labels, e.g. fluorophores and photosensitizers in a 1:1 stoichiometry. On the one hand, SNAP tag fusions can be used to optically image tumors when conjugated to a fluorophore, and on the other hand the recombinant proteins can induce necrosis/apoptosis in the tumor when conjugated to a photosensitizer upon exposure to a changeable wavelength of light. The dual nature of SNAP-tag fusions as both a diagnostic and therapeutic tool reinforces its significant role in cancer treatment in an era of precision medicine.
ABSTRACT
BACKGROUND: The deposition of aggregated ß-amyloid peptide senile plaques and the accumulation of arginine within the astrocytes in the brain of an Alzheimer's patient are classic observations in the neuropathology of the disease. It would be logical, in the aetiology and pathogenesis, to investigate arginine-metabolising enzymes and their intimate association with amyloid peptides. METHODS: Neuronal nitric oxide synthase (nNOS) was isolated, purified and shown, through fluorescence quenching spectroscopy and fluorescence resonance energy transfer (FRET), to interact with structural fragments of Aß(1-40) and be catalytic towards amyloid fibril formation. RESULTS: Only one binding site on the enzyme was available for binding. Two amyloid peptide fragments of Aß(1-40) (Aß(17-28) and Aß(25-35)) had Stern-Volmer values (K(SV)) of 0.111µM(-1) and 0.135µM(-1) indicating tight binding affinity to nNOS and easier accessibility to fluor molecules during binding. The polarity of this active site precludes binding of the predominantly hydrophobic amyloid peptide fragments contained within Aß(17-28) and within two glycine zipper motifs [G-X-X-X-G-X-X-X-G] [Aß(29-37)] and bind to the enzyme at a site remote to the active region. CONCLUSIONS: The interaction and binding of Aß(17-28) and Aß(25-35) to nNOS causes the movement of two critical tryptophan residues of 0.77nm and 0.57nm respectively towards the surface of the enzyme. GENERAL SIGNIFICANCE: The binding of Aß-peptide fragments with nNOS has been studied by spectrofluorimetry. The information and data presented should contribute towards understanding the mechanism for deposition of aggregated Aß-peptides and fibrillogenesis in senile plaques in an AD brain.
