Hewawasam, Nirun Videesha (2015) Developing novel pharmacological inhibitors of Rab7-GTPase to promote [Beta]-cell expansion for diabetes therapy. (MSc(R) thesis), Kingston University, .

Abstract

Loss of insulin producing [Beta]-cells is central to the pathogenesis of both type 1 and type 2 diabetes mellitus and thus developing novel treatment promoting [Beta]-cell expansion will be of profound benefit to diabetes patients. Rab7 is a member of the Rab family of GTPases that promotes intracellular trafficking between late endosomes and the lysosome. Specifically Rab7 regulates the trafficking of internalised receptors to the lysosomes to be degraded. Previous studies conducted at Kingston University showed that reducing Rab7 expression in [Beta]-cells using siRNA enhances growth factor receptor density, increases growth factor signalling and promotes [Beta]-cell growth. Therefore, Rab7 inhibition is a promising novel approach to promote [Beta]-cell expansion. A library of seven small molecules, based on the published novel Rab7 inhibitor CID1067700 with subtle modifications was developed by medicinal chemists at Kingston University. The aim of this project was to assess these compounds for their cytotoxicity, cell permeability, binding to Rab7 and ability to competitively inhibit binding of GTP to Rab7 as well as for their effect on IGF-1 signalling in INS-1 [Beta]-cells. This is the first study assessing a panel of novel pharmacological inhibitors of Rab7 in cells. The study employed a range of technologies including Promega CytoTox96 non-radioactive cytotoxicity assay, Sulphorhodamine B (SRB) assay, Differential Scanning Fluorimetry (DSF), Flow Cytometry and Nuclear Magnetic REsonance (NMR) spectroscopy. The ability to promote [Beta]-cell expansion of the selected compounds was tested using Western blotting. In particular, NMR was developed as an approach to detect cellular uptake of the compounds. The results showed that the modifications conferred an improved permeability to the derivatives compared to CID1067700 (3 to 4 fold increase). Although a weak binding to Rab7 was observed, the compound did not compete with GTP for the binding pocket on Rab7. We found that CID1067700 enhanced GTP binding to Rab7 suggesting that the drug activated Rab7. This was further confirmed by the reduction in IGF-1 induced ERK 1/2 activation in the presence of this drug and many of the novel derivatives. We have therefore characterised CID1067700 as a potential enhancer of GTP binding to Rab7. This has a potential application for the inhibition of tumour growth since Rab7 regulates growth factor receptor trafficking. In addition, we report the use of NMR as a novel approach for the determination of drug uptake in cells.

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