Resveratrol and compounds related to it, known as resveralogues, have been shown to extend healthy lifespan in a number of species. A diverse set of molecular mechanisms have been proposed to account for these effects, including the activation of the NAD+-dependent histone deacetylase SIRT1. However, both resveratrol and some of its derivatives also display potentially detrimental activities including direct DNA damage, toxicity and the induction of cellular senescence. Accordingly, the overall aim of this thesis was to enable the generation of better compounds with minimal detrimental activity and maximum beneficial activity. The data presented in this thesis show my development of a new, convenient, one-pot stereo-selective synthesis of resveratrol and other trans-stilbene derivatives. This synthesis has been used to prepare more than 40 compounds, of which 20 are completely novel and a further 12 previously uncharacterised with regard to their biological effects. This study have evaluated these resveralogues with respect to their cytotoxicity in human fibroblasts using a range of widely-recognised assays. Data on the compounds’ toxicity are presented, together with evidence that two of the assays commonly used to measure cytotoxicity in vitro (the MTT and neutral red assays) are unsuited for cytotoxicity testing with this class of compound. Twenty-two of the (lowest toxicity) compounds have also been assayed for their effects on growth and senescence using indirect immunostaining for the proliferation marker pki-67 and catalytic histochemical visualisation of SA-β-galactosidase activity, respectively. The results indicate that the growth inhibitory activity of resveratrol is abrogated by the removal of the trans-stilbene double bond (no reduction of growth fraction in dihydroresveralogues at 100 μM). Interestingly, at low doses (5-10 μM), many resveralogues induce a significant increase in proliferation compared to control untreated cells. SIRT1 activity in the presence of each compound was measured using an in-vitro deacetylation assay and, whilst most resveralogues are SIRT1 activators, none of the compounds examined produced a significant increase in SIRT1 activation compared to the parent compound. SIRT1 activation was, for the first time, found to be independent of the presence of a trans-stilbene double bond. Some initial characterisation of the ability of the resveralogues to alter pro-inflammatory cytokine expression was also undertaken by using ELISA for interleukin 6. Some difficulties with this assay, with respect to background signal interference, were encountered but parallel data from collaborator (Professor Lynne Cox, University of Oxford) using a human specific IL6 antibody are available. Collabrator data, evaluating a selection of resveralogues, indicate that inhibition of IL6 release is independent of SIRT1 activation, demonstrating that not all potential beneficial effects are SIRT1 mediated. Finally, this work has identified three novel lead compounds (V24, V31, and V34) that have less detrimental activities with retention in SIRT1 activity.
|Date of Award||Mar 2016|