Thesis

Discovery and characterisation of novel anticancer compounds acting on sphingosine kinase

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Awarding institution
  • University of Strathclyde
Date of award
  • 2011
Thesis identifier
  • T12942
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Abstract
  • There is a critical need to develop novel anticancer therapeutics. Accumulating evidence has demonstrated that sphingosine kinase (SK) is a promising target for the treatment of cancer. There are two SK isoforms, termed SK1 and SK2. SK phosphorylates sphingosine to form sphingosine-1-phosphate which drives cancer cell proliferation and migration. Therefore, the aims of this project were to discover hit compounds that can be developed into effective chemical tools and/or anticancer drug leads targeting SK. Compounds from both synthetic and natural origins were screened using SK enzymatic assays. Natural compounds were isolated using bioassay-guided fractionation and structural elucidation was achieved using nuclear magnetic resonance and mass spectrometry. Cell-based assays were employed to assess cellular activity of these inhibitors. Cell growth was determined using [³H]-thymidine incorporation assays. Immunoprecipitation was used to study SK1 oligomerisation whereas fluorescence microscopy was used to examine the effects of SK inhibitors on actin distribution. Three key findings emerged from these studies. First, SK1 inhibitor kinetic characterisation with sphingosine revealed that (S)-FTY720 vinylphosphonate is a novel uncompetitive inhibitor whereas FTY720 and SKi (2-(p-Hydroxyanilino)-4-(p-chlorophenyl)thiazole) are competitive and mixed inhibitors respectively. These inhibitors also inhibited proliferation, induced proteasomal degradation of SK1 and apoptosis of cancer cells. Over-expression of an inactive SK1 mutant inhibited the catalytic activity of wild type SK1. Together with the discovery of two activators for SK1, a model was proposed in which SK1 contains allosteric site(s) that auto-inhibits catalytic activity. Second, (R)- FTY720-OMe was discovered as a selective SK2 inhibitor, which also inhibited MCF-7 cell growth and S1P-induced actin rearrangement, demonstrating SK2 as a pro-survival and migratory protein. Third, novel SK inhibiting scaffolds including resveratrol were identified from a plant hit. Balanocarpol (a resveratrol dimer) might act like a chelator to inhibit multiple SK1 molecules, giving new insights into diversity-oriented biosynthesis of resveratrol oligomers. Together, these findings provide strong impetus for the development of inhibitors targeting SK1 and/or SK2 as effective anticancer agents.
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DOI
Date Created
  • 2011
Former identifier
  • 846475

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