Novel Inhibitors of Glutamate and Nucleoside Transporters

• Author / Creator

  Wu, Cody G

• Membrane transporters play integral roles in the functioning of cells by controlling movement of molecules across the phospholipid bilayer. In this thesis, utilization of radioisotope uptake assays permitted examination of intentional and unintentional inhibition of three membrane transporter families: the excitatory amino acid transporter family (hEAATs), the concentrative nucleoside transporter family (hCNTs), and the equilibrative nucleoside transporter family (hENTs). hEAATs, while being the topic of intensive neurophysiological research as well as attractive pharmacological targets for treatment of glutamate related-diseases, suffer from lack of availability of isoform-specific inhibitors. In collaboration with Dr. S.A. Baldwin and colleagues at the University of Leeds (UK), a series of glutamate analogues were synthesized as potential glutamate transport inhibitors. By varying the concentration of inhibitors, we were able to determine the concentration of each compound required to cause 50% inhibition of L-glutamate uptake (IC50 value) for each of the four human EAAT family members produced in Xenopus laevis oocytes. Of eight compounds tested, seven showed high specificity for the hEAAT2 isoform. Of these seven hEAAT2-specific inhibitors, two showed high-potency (IC50 ~ 5 µM). With further testing, these compounds may prove useful in hEAAT research and pharmaceutical development. The design of new and novel classes of inhibitors for research and pharmaceutical purposes requires care and awareness to prevent, or at least minimize, spillover inhibition of other proteins. This was not the case with mitogen-activated protein kinase kinase (MEK) inhibitors, a class of small-molecule kinase inhibitors designed to inhibit proteins within the mitogen activated protein kinase (MAPK) pathway. This pathway plays an integral role in the transduction of cell growth and proliferation signals from receptors on the cell surface to the nucleus through a complex multi-protein intracellular phosphorylation cascade. Deregulation of this pathway and its protein mediators can result in abnormal, uncontrolled cell growth and the development of cancer. Due to the high prevalence of MAPK pathway deregulation in cancer patients (roughly 30%), development of inhibitory compounds that target its various protein mediators has received intense attention in recent years. Inhibitors such as binimetinib, trametinib, and selumetinib targeting MEK1/2 have demonstrated unexpectedly poor clinical performance when used in combined therapy with nucleoside analogue anticancer drugs. Since these nucleoside analogues require human nucleoside transporter (hNT) proteins to enter the cell at pharmacologically effective concentrations, we hypothesized that the purine and pyridopyrimidine-like structures found within these inhibitors allowed for binding to and inhibition of hNTs. Radioisotope uptake assays using radiolabelled uridine and adenosine in Xenopus laevis oocytes producing individual hNTs from both the hCNT and hENT transporter families (hCNT1-3 and hENT1-4, respectively) have revealed high-potency binimetinib, trametinib, and selumetinib inhibition of hENT3 (average IC50 values in the range 2.7 - 5.1 µM), more moderate inhibition of hENT4 (average IC50 values in the range 57.1 - 274 µM), and low-potency inhibition of other hNTs. Some adverse effects of MEK administration may be attributable to spillover inhibition of hENT3/4.

• Subjects / Keywords

  • MEK
  • Nucleoside
  • Glutamate
  • HENT
  • Inhibitors
  • EAAT
  • Transporters
  • HCNT

• Graduation date

  Fall 2016

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3J09WC4V

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.