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Sigma receptors originally thought to be an opioid receptor is now categorized as a distinct class of receptor. There are two main subtypes, the sigma-1 receptor and an uncharacterised binding site, named the sigma-2 binding site. The presence of the sigma-2 binding site shows high correlation with proliferation of cells and is associated with cancer. I have categorized sigma-1 and sigma-2 binding sites in 11 human tumour cell lines. I have demonstrated that tumour cell lines from a range of tissues express both sigma-1 and sigma-2 binding sites. One exception is the MCF7 breast cancer cell line, which lacks sigma-1 receptors. I show that the quantitation of sigma-2 binding sites using the "masking" protocols are flawed, significantly overestimating levels of sigma-2 binding sites. I propose novel protocols to determine levels of sigma-1 receptors and sigma-2 binding sites in cell lines and tissue. Using radioligand binding assays in MCF7 cells, I have characterised novel sigma-2 ligands. These ligands are simple ammonium salts containing a single nitrogen atom. They are simpler than the previously recognised pharmacophore for the sigma-2 site. I have shown that these simple ammonium salts show graded affinity for the sigma-2 binding site. The highest affinity ligands were dihexylammonium (pKi 7.58) and dioctylammonium (pKi 7.9). I have used these ammonium salts and previously characterised ligands to determine sigma-2 binding site biology. I have shown that the biological activity of these drugs is related neither to their hydrophobicity nor their ability to effect calcium signalling in cells. I propose that the Hill slope of binding is inversely related to the efficacy of a ligand to inhibit metabolic activity of cancer cells. Furthermore, I offer an explanation as to why concentrations of sigma-2 ligands far higher than their determined binding affinities are required to inhibit metabolic activity.

Gamma-Amino butyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system (CNS) and inhibits the neurotransmission by targeting the ionotropic transmembrane GABAA receptor. Modulators of the GABAA receptor targets the allosteric binding sites and modifies the GABA effect and these sites acts as superior drug targets within psychopharmacology.   Gabather AB has developed the novel compound GT-002 that is known to target the receptor and cause a behavioral effect in rodents. This project characterized the binding of the lead compound GT-002 to GABAA receptor in mammalian brain tissue by development and validation of a radioligand binding assay. In the assay a comparative evaluation was performed using the benzodiazepine (BZ) antagonist Flumazenil (FLU).   All experiments were performed using GABAA receptors originating from porcine and mouse brain tissue membrane, where no significant difference between the mammals was displayed. GT-002 binds with higher affinity and associates faster than FLU to the receptor and implies a two-binding site model. GT-002 displaced FLU and no tested competitive analytes targeting various modulatory sites of the receptor displaced GT-002, implying independent binding of GT-002 and allosterically impacts the BZ binding site.

Nicotine exerts a therapeutic effect in ulcerative colitis (UC) but the mechanism underlying this effect, is not clear. However, this effect may imply that nicotine has some, as yet to be discovered, effect on the immune system. The aim of the work described in this thesis was to characterise the nicotinic acetylcholine receptors (nAChRs) on human peripheral blood lymphocytes in term of receptor subtype. To achieve this, a combination of radioligand binding assays, pharmacological and molecular biological techniques were used. The data obtained from the binding studies suggested that the presence of one binding site for (-)- nicotine on human peripheral blood lymphocytes with a Kd 15 ± 5.759 nM (1.5 ± 5.759 x 10-8 M) and Bmax 2253 ± 409 sites/cell. The competition studies showed that ligands competing with [3H]-(-)-nicotine were (-)-nicotine, epibatidine and α-bungarotoxin, while others ligands for nAChRs displaced radiolabelled nicotine in insignificant quantities. Thus, radioligand-binding experiments suggest that the binding site for nicotine on human peripheral blood lymphocytes is a nAChR containing α7 and possibly α4 or/and b2 containing nAChR subunits. No evidence was obtained to suggest the presence of a non-cholinergic nicotine receptor. Furthermore, considerable subject to subject variation in the specific binding of radiolabelled nicotine was observed. Because of this only tentative conclusions could be drawn from radioligand binding data. Polymerase chain reaction (RT-PCR) was then used to demonstrate mRNA for the subunits of nAChRs suggested by radioligand binding studies. Data obtained show that the human peripheral blood lymphocytes tested, expressed mRNAs for α4, α5, α7, β2 neuronal nAChRs subunits and β1 muscle nAChR subunit. Expression of the α5 mRNA subunit of nAChR was observed in the lymphocytes in each sample of lymphocytes tested. In contrast, the expression pattern of mRNAs for α4, α7, β1, and β2 mRNAs subunits of nAChRs, varied between individuals. Finally, Western blot analysis was used to confirm that mRNA expression resulted in the expression of protein for nAChR subunits in human peripheral lymphocytes using monoclonal antibodies against α4, α5, α7, and β2 nAChR subunits, which had been detected by RT-PCR. The results obtained from the Western blot analysis show that protein for α4, α5, and α7 nAChR subunits was expressed in most, but not all of the human peripheral blood lymphocyte samples tested and some of the bands obtained were faint. In contrast, protein for the β2 nAChR subunit was observed in a few samples tested and the bands were faint. From the results obtained in this study, it is possible to conclude that human peripheral blood lymphocytes may contain nAChRs with subunit compositions of α4β2, α4β2α5, and/or α7. However, further studies are necessary to show whether or not the single binding site for nicotine demonstrated by radioligand binding experiments is due to one or all of these nAChRs. Thus, the findings of the present study suggest the presence of nAChR on human peripheral blood lymphocytes. Nicotine and its effect may occur through these non- neuronal nAChRs mechanisms. Such a mechanism of action could account for the beneficial of nicotine in ulcerative colitis. Furthermore, a compound that acts on these receptors, but not on nAChRs found on other cells may have therapeutic utility in the treatment of inflammation.

Modulation of the endocannabinoid system (ECS) is of great interest for its therapeutic relevance in several pathophysiological processes. The CB2 subtype is largely localized to immune effectors, including microglia within the central nervous system, where it promotes anti-inflammation. Recently, a rational drug design toward precise modulation of the CB2 active site revealed the novelty of Pyrrolo[2,1-c][1,4]benzodiazepines tricyclic chemotype with a high conformational similarity in comparison to the existing leads. These compounds are structurally unique, confirming their chemotype novelty. In our continuing search for new chemotypes as selective CB2 regulatory molecules, following SAR approaches, a total of 17 selected (S,E)-11-[2-(arylmethylene)hydrazono]-PBD analogs were synthesized and tested for their ability to bind to the CB1 and CB2 receptor orthosteric sites. A competitive [H]CP-55,940 binding screen revealed five compounds that exhibited >60% displacement at 10 μM concentration. Further concentration-response analysis revealed two compounds, and , as potent and selective CB2 ligands with sub-micromolar activities ( = 146 nM and 137 nM, respectively). In order to support the potential efficacy and safety of the analogs, the oral and intravenous pharmacokinetic properties of compound were sought. Compound was orally bioavailable, reaching maximum brain concentrations of 602 ± 162 ng/g (p.o.) with an elimination half-life of 22.9 ± 3.73 h. Whether administered via the oral or intravenous route, the elimination half-lives ranged between 9.3 and 16.7 h in the liver and kidneys. These compounds represent novel chemotypes, which can be further optimized for improved affinity and selectivity toward the CB2 receptor.