Chemical tools to validate N-myristoyltransferase as a new target in cancer therapy

• Main Author: Thinon, Emmanuelle
• Other Authors: Tate, Edward ; Mann, David
• Published: Imperial College London 2013
• Subjects: 540
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.676751

N-myristoylation is the irreversible attachment of myristate, a C14-lipid, to the N-terminal glycine of a protein. This modification is catalysed by myristoyl CoA: protein N-myristoyltransferase (NMT). N-myristoylation has been shown to be essential for the viability and survival of many organisms, including plants, parasites and humans. In humans, two isoforms of NMT, HsNMT1 and HsNMT2, were identified and it was suggested that they possess overlapping substrate specificities. NMT was identified as a potential chemotherapeutic target for cancer 18 years ago and subsequent studies showed that NMT was up-regulated in several cancers. However, no study demonstrated that NMT could be a therapeutic target in cancer therapy. In this study, chemically-tagged analogues of myristic acid were synthesised and applied to cancer cell lines to study protein N-myristoylation. The analogues can be incorporated on the N-terminal glycine of NMT substrates. The tag in this system enables a second extremely selective and high-yielding chemical ligation to any reporter of choice, for instance a dye label. Visualisation of the tagged proteins was carried out by in-gel fluorescence. Tagged-proteins could also be enriched and analysed by mass spectrometry-based proteomics. The combination of chemical proteomics with a potent NMT inhibitor is described in this thesis as a new method to identify with high confidence a large number of NMT substrates. In search for novel and potent human NMT inhibitors, various compounds were tested using an in vitro assay and a cell cytotoxicity assay. On-target inhibition of NMT in cells was assessed by in-gel fluorescence using analogues of myristic acid. The best inhibitors were employed as tools to study NMT inhibition in cancer cells. Western blot analysis and flow cytometry were used to assess the phenotype of NMT inhibition. Finally, the substrate specificity of the two isoforms was studied in cells using siRNAs against HsNMT1 or HsNMT2, and in vitro by screening for activity libraries of peptides against NMT1 or NMT2.