Synthesis and Evaluation of Cell Behaviour Modifying Compounds for Cancer Therapy

Abstract

Cell behaviour is a general term for activities of whole cells such as movement, adhesion, proliferation and invasion. A cellular response to external environmental stimuli includes changing the motility, which is driven by the force of the cellular structure called the cytoskeleton. One of the regulators of the cytoskeleton is the Ras superfamily, which consists of the Rho, Rac and CdCl3 subfamilies. ROCK enzyme, a downstream effector of the Rho subfamily pathway, is very specific for cell movement, contraction and metastases, and its overexpression has been found in many types of cancer. YA1 (tert-Butyl(4-(pyridin-4-ylcarbamoyl)benzyl)carbamate), an analogue of the ROCK inhibitor Y27632, has been previously found to inhibit the transformation of Ras transformed murine NIH3T3 cells. This transformation is measured by loss of cell-cell contact growth inhibition, which is determined by the formation of colonies and multi-layer domes. This inhibition was persistent, non-cytotoxic and only observed when the transformed cells were co-cultured with non-transformed NIH3T3 cells. The aim of this research is to evaluate the effect of some structural modifications of YA1 on the activity. The structural features of YA1 that have been modified are the pyridine ring, amide bond, and methyl carbamate. Another objective of this project is to attempt to identify the mechanism of action of YA1 by conducting some biological assays. A library of 18 novel and known analogues of YA1 have been synthesised, characterised and evaluated against foci formation in co-cultures of parent and Ras transformed NIH3T3 cells using the toluidine blue transforming assay. Two analogues showed comparable activity to YA1 or Y27632 with minimal toxicity as indicated by AQ96 and trypan blue proliferation assays. One analogue in the library is modified by N-alkyne substitution of the carbamate fragment in YA1. After it was found to be active, the alkyne analogue was utilised in an azide-alkyne bioconjugation assay to identify the sub-cellular localisation of YA1 within the NIH3T3 cells. This assay was not efficient in determining the localisation of YA1 as there was no evidence of cellular alkyne-azide reaction, which might be caused by poor binding of the drug to its cellular target. An attempt to label YA1 with biotin, to take advantage of the Biotin-Tag assay for target identification, failed. YA2, an analogue of YA1 lacking the (tert-butyloxycarbonyl group), was successfully labelled with biotin and biotin with a long linker. However, these analogues showed decreased activity when compared with YA1, therefore it was not possible to proceed with the biotin tag assay due to weakly interacting molecules. An immunohistochemistry assay was used to evaluate the activity of YA1 and most of the synthesised analogues on YAP protein, compared to Y27632. The YAP protein was studied because it is a key component of the hippo signalling pathway, which has an important role in the regulation of cell contact inhibition and can be a potential target of YA1. The assay was conducted on 6 types of human hepatocyte cell line (HHL) separately or as a co-culture of parent plus Dom.NS4B transfected HHL. YA1 was more active than Y27632 in two types of the transfected HHL. In co-culture, YA1 showed no significant effect on YAP expression relative to Y27632, and this result excludes YAP protein as the target for YA1.

Date of Award	31 Dec 2017
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Sally Freeman (Supervisor) & Lynne Hampson (Supervisor)