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Philanthotoxin-433 (PhTX-433) is an active component of the Egyptian solitary digger wasp, Philanthus triangulum, venom which non-selectively inhibits several excitatory ion channels. With the aim of improving potency and selectivity, several synthetic analogues were developed based on the single or multiple modifications in the hydrophilic polyamine tail and hydrophobic aromatic head group of the PhTX. In the first part of this study, we investigated the pharmacological actions of two synthetic analogues, Philanthotoxin-343 (PhTX-343) and Philanthotoxin-12 (PhTX-12), on mammalian hetero- and homooligomeric nicotinic acetylcholine receptor (nAChR) subunit combinations expressed in Xenopus oocytes. Whole-cell currents in response to application of acetylcholine alone or co-applied with PhTX-analogue were studied electrophysiologically using two-electrode voltage-clamp at three different membrane holding potentials (VH = -60 mV, -80 mV and -100 mV). Concentration-inhibition curves were constructed and IC50 values estimated for each holding potential. The IC50 (95% CI, n=oocytes) values for PhTX-343 inhibition of α3β4, α3β2, α4β2, α4β4, α7, α3β4(F255V), α4β2(V253F) and α1β1δγ peak currents at -100 mV were 0.07 µМ (0.05-0.10 µМ, n=9), 3.20 µМ (1.69-6.26 µМ, n=8), 0.14 µМ (0.09-0.21 µМ, n=7), 0.28 µМ (0.17-0.46 µМ, n=6), 8.7 µМ (6.9-11.0 µМ, n =9), 0.01 µМ (0.01-0.02 µМ, n=8), 0.10 µМ (0.06-0.16 µМ, n=8) and 3.2 µМ (2.5-4.1 µМ, n=9) respectively; for PhTX-12 they were 2.03 µМ (1.22-3.26 µМ, n=8), 74.0 µМ (21-259 µМ, n=10), 1.6 µМ (0.7-3.6 µМ, n=11), 1.82 µМ (0.81-4.11 µМ, n=9), 12.1 µМ (9.0-16.0 µМ, n =10), 1.0 µМ (0.5-2.0 µМ, n=5), 3.4 µМ (1.3-8.7 µМ, n=9) and 5.0 µМ (2.8-8.8 µМ, n=8) respectively; i.e. in contrast to M-nAChR, PhTX-343 was more potent than PhTX-12 in all cases. All IC50s were lower when inhibition was measured after 1 minute of application indicating use dependence. For inhibition of heteromeric nAChRs, the peak potency of PhTX-343 was strongly augmented by holding the cell at more negative VH while this was not the case for PhTX-12 where only weak voltage-dependence was observed. The inhibition of homomeric α7 receptors by PhTX-343 was voltage-independent, whereas the block by PhTX-12 was voltage-dependent. In addition, these two synthetic analogues were different in their recovery rates (PhTX-12 was generally faster than PhTX-343) but they were similar in their mechanism of action, non-competitive inhibition. In the second part of the project, I explored the structure-activity relations of twenty one synthetic analogues, with a view to improving their activity and selectivity on rat neuronal α4β2 and α3β4 nAChRs at VH = -100 mV. We showed that the presence of positive charge in the polyamine tail of PhTX compounds is essential for nAChR subtype selectivity and its removal made the molecule lose its selectivity. Also, it appears that adding a bulky group to the terminal ammonium drastically reduced activity whereas a similar addition to the head region it increased their potency. In addition, we identified the key regions and substitutions responsible for increasing PhTX activity, cyclohexylalanine in place of tyrosine, and selectivity, phenyl group. Analogues having cyclohexylalanine and a phenolic group in the head region showed IC50 values in the low nano-molar and pico-molar (160-400 pM) range. These data suggest that PhTXs could serve as lead compounds for highly potent and selective inhibitors of N-nAChRs.

Calcium ions play an important role in many signalling pathways involved in normal cell metabolism. Pertrebations of normal Ca++ signalling may also play a pivotal role in the initiation of cell death. In these studies I have examined the influx of 45Ca++ into the extensor tibiae muscle of the locust (Schistocerca gregaria ). 45Ca++ entry could be stimulated by the addition of glutamate receptor-agonists or by activation of voltage activated calcium channels. L-glutamate, L-quisqualate and NMDA stimulated the influx of 45Ca++ while L-aspartate had only a small effect. DL-ibotenate, kainate, AMPA and glycine had no effect on 45Ca++ uptake (all agonists were tested at concentrations up to (100μM). Glycine (1μM) enhanced the 45Ca++ entry induced by NMDA and L-glutamate. Only the glycine potentiation of L-glu-stimulated responses was abolished in the presence of Mg++ (2mM) or AP5 (10μM) whereas the NMDA-stimulated response was completely abolished by these agents. These finding suggests that in the presence of glycine, L-glutamate may activate NMDA receptors and that in the absence of glycine L-glu-stimulated 45Ca++ entry occurs via activation of the qGluR. Depolarisation of the extensor tibiae muscles (50mM KCl) stimulated 45Ca++ influx by activation of voltage-sensitive calcium channels. Philanthotoxin-343 (0.1μM) had no effect on depolarisation activated calcium entry, however, nifedipine (1μM) an L-type calcium channel antagonist inhibited this Ca++ influx. Nifedipine did not inhibit L-glu-stimulated Ca++ entry suggesting that in these muscles L-type Ca++ channels are not involved in the Ca++ influx pathway following G1uR activation. Philanthotoxin-433 (PhTX-433) and many of its synthetic analogues are potent inhibitors of locust GluR. In the future these analogues may prove as useful potential neuroprotective agents or as novel pesticides. Over 100 analogues of PhTX-433 have been synthesized with changes made in the four regions of the structure, the thermospermine moiety, the tyrosyl moiety, the butyryl moiety and the terminal amino moiety. The effects of different concentrations (10-4M to 10-14M) of synthetic analogues of PhTX-433 (PhTX-343, PhTX-343-Arg, PhTX-4) were investigated in the 45Ca++ influx assay using locust extensor tibiae muscle. PhTX-343-Arg was more potent (IC50= - 7x10-9) than PhTX- 343 (IC50= - 10-8M) or PhTX-4 in blocking 45Ca H uptake. These findings were further supported by electrophysiological studies. The interaction of these synthetic analogues of philanthotoxin with GluR of locust muscle were further investigated by examining the effect of these compounds on evoked excitatory post synaptic potentials. In recent years control of ticks have been very important issue because of the social and economical damage they cause. Neuromuscular transmission is a main target site for the chemical control of many pests. Philanthotoxin and its analogues block the glutamate receptors which are involved in arthropod neuromuscular transmission and thus may prove useful as novel pesticides. The action of synthetic analogues of philanthotoxin (C7PhTX-343, DNP12-, PhTX-343 and PhTX-343) were examined on evoked excitatory postsynaptic potential in tick coxal muscle. These compounds all antagonized the evoked EPSP. C7PhTX-343 and DNP12-PhTX-343 exhibited same potency (IC50 = 10-8M) and both were more potent than PhTX-343 (IC50 ='2X10-5M). In recent years Xenopus oocyte has taken over a new role as a test tube for the study of the biogenesis, functional architecture and modulation of plasma membrane protein. Attempts were made to express mRNA from embryonic tissue of tick and locust leg muscle in to Xenopus oocyte for pharmacological studies. Xenopus oocytes failed to translate RNA faithfully and efficiently from these sources. Rat brain RNA injected oocytes used as control, expressed routinely.

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.