Abu, Izuddin Fahmy
(2016)
Exploring block and permeation of N-methyl-D-Aspartate (NMDA) receptor channels for treatment of neurodegenerative disorders.
PhD thesis, University of Nottingham.
Abstract
N-methyl-D-aspartate receptors (NMDAR) are ionotropic glutamate receptors which can be blocked by Mg2+ in a voltage-dependent manner and are highly permeable to Ca2+, hence they represent a medically relevant target for neurodegenerative disorders caused by excitotoxicity. The two main objectives of this study were, (i) to determine the impact of Q/R/N, +1 and -8 sites modification in the M2 pore region of GluN2A NMDAR subunit on Mg2+ block and other open channel blockers; and (ii) to evaluate novel multi-target-directed ligands (MTDL) for Alzheimer’s disease therapy. The Xenopus laevis oocyte expression system was employed where NMDAR subunit cRNAs were injected into the oocytes and responses to NMDA/glycine and channel blockers were recorded using two-electrode voltage clamp (TEVC) electrophysiology.
Pore region mutations to investigate the impact of Q/R/N and adjacent sites were characterized using Mg2+, memantine, MK-801, philanthotoxin analogues and an MTDL compound, CR18. NN at the Q/R/N and +1 sites in GluN2A subunits were mutated to GR and RR, while W at the -8 position (in relation to the Q/R/N site), was mutated to N. Wild type and mutated GluN2A were co-expressed with GluN1-1a in Xenopus oocytes and antagonistic responses by channel blockers were recorded with TEVC. At -75 mV, the RR mutation significantly increased IC50s of Mg2+, memantine and MK-801 by 27-, 42- and 325-fold respectively, compared to wild-type. As for the GR mutation, IC50s were also significantly increased for memantine and MK-801 by 5- and 132-fold respectively, compared to wild type. W to N mutation at the -8 position did not significantly affect blocking potencies for all channel blockers. Blocking potency for PhTX-343 was not significantly altered by any mutations. This study provided evidence that the presence of G and R at the Q/R/N and +1 sites are likely responsible for the changes in blocking sensitivity and play important roles in ion permeability. The fact that PhTX-343 remained potent despite the mutations suggest that this compound might have a different mode of action or different binding site other than the M2 region and should be further characterized.
In the MTDL study, twenty one novel compounds were tested on GluN1-1a/GluN2A NMDAR subunits. Thirteen were memantine-derivatives (MAB) incorporated with antioxidant moieties, three were spermine-derived polyamines also incorporated with antioxidants, and five were combinatorial forms of donepezil and carvedilol. The antagonistic properties of the compounds were tested electrophysiologically at -60 mV and compared with Mg2+ and memantine. The MAB series were found to be weak NMDAR channel blockers suggesting the loss of memantine functionality due to attachment of the antioxidant structure to its amine group. Subsequently, modification of the linker point to memantine moieties to free its amine group eventually resulted in weaker NMDAR channel blockers with IC50s of more than 100 µM. The spermine-derived polyamines (CR compounds) were potent NMDAR blockers with IC50s (0.69 to 2.35 µM) comparable to memantine (2.28 µM) and significantly lower than Mg2+ (10.1 µM) and also exhibited voltage-dependence block.
Our mutation study revealed that CR18, the most potent MTDL compound was less sensitive in NMDAR containing GR or RR mutation in GluN2A subunits. This is a favourable property of an NMDAR blocker for potential Alzheimer’s disease treatment since GluN3 subunits containing GR or RR at the Q/R/N and +1 sites are less permeable to Ca2+ influx and has been shown to exert neuroprotective effects.
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