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Title: Synthesis and evaluation of receptors for phosphatidylinositol phosphates
Author: Whyte, Gillian
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Phosphatidylinositol phosphates (PIPs) are signalling phospholipids with a diverse set of cellular functions. The most important of these PIPs are phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) and phosphatidylinositol (3,4,5) trisphosphate (PI(3,4,5)P3). These two are responsible for a number of cellular events which regulate cell growth, proliferation and apoptosis. Deregulation of PIP levels disrupts these pathways and can therefore lead to uncontrolled cell growth and which can lead to tumourigenesis. Control of PIP levels has been identified as a potential target for diagnosis and intervention in diseases including Alzheimer’s disease, cancer, and the genetic disorder Lowe Syndrome. The use of small molecules that bind PIPs has been shown by our group to interfere with protein-PIP interactions. By preventing proteins from binding to PIPs, the effective concentration of the target PIP is reduced, proteins are not recruited to the membrane for activation and downstream signalling pathways are attenuated. Therefore, in this project a series of small artificial receptors were synthesised and were shown to bind PI(4,5)P2 and PI(3,4,5)P3. The aim of this was to control their effective levels and manipulate their downstream signalling pathways. PI(4,5)P2-binding receptors were developed based on existing lead compounds established within our group. Comparison of a receptor with only one binding motif with the established two motif receptor showed differences in affinity as well as specificity for the phospholipid target PI(4,5)P2 over the headgroup IP3. In cells, the effect of the receptors on the downstream signalling pathways was examined using phosphorylated Akt as an indicator of this pathway’s activation. Two fluorescent receptors were designed and one of these was used as a novel PI(4,5)P2 detection tool on immobilised phospholipid. Preliminary results show that this receptor may also be used to directly image PI(4,5)P2 in fixed cells. Novel PI(3,4,5)P3 receptors were designed with phosphate-binding motifs and a range of spacers. The specificity of each receptor was identified and binding affinities towards PI(3,4,5)P3 were established. Inhibition of protein-lipid interaction and inhibition of PIP-metabolising enzymes were also studied. The effect of these new receptors in attenuating the Akt pathway in cancer cells was also investigated, and they were found to lack the efficacy of PI(4,5)P2-binding receptors. The ability of some of these artificial receptors to bind phospholipids in the cell and affect subsequent signalling pathways indicates that phospholipids are a viable additional drug targets for many diseases.
Supervisor: Woscholski, Rudiger ; Vilar, Ramon Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
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