Ion channels in drug discovery : Focus on biological assays

Ion channels are well characterised drug targets. However, the techniques used to study ion channel pharmacology have not been particularly applicable to modern drug discovery. The aim of this thesis was to examine the usefulness of both established and novel methods used in the discovery and pharmacological characterisation of drugs interacting with ion channels. Three different classes of ion channels have been studied; the GABAA receptor representing the classical ligand-gated ion channel, the voltage-gated sodium channel (NaV-1.7) and the newly discovered non-selective cation channel, Transient Receptor Potential Melastatin-8 (TRPM8). Due to the difficulty in expression and purification of membrane proteins, no crystal structures of GABAA receptors are currently available and structure-based drug design is not directly applicable. Using pharmacophore modelling and site-directed mutagenesis, ligand interactions were studied at a structural level. A pharmacophore model for the benzodiazepine binding site (BzR) on the GABAA receptor, based on structure-activity relationship studies for 136 different ligands from 10 structurally different classes was used to model flavonoids binding to the GABAA receptor. By synthesising a series of flavonoids, the structure activity relationship (SAR) was investigated using 3H-flumazenil binding to rat cortical membranes in-vitro. The results demonstrate that flavonoids with high affinity for the BzR spanning the whole efficacy range from agonists to inverse agonists can be synthesised and the receptor binding properties of the flavonoids can successfully be fitted in a comprehensive pharmacophore model. In order to investigate which arginine residues potentially contribute to the formation of the GABA binding pocket, six arginines conserved in all human GABAA receptor alpha subunits as well as two non-conserved arginines in the extracellular, N-terminal segment of the alpha5 subunit, were substituted by lysines. The individual alpha5 subunit mutants were co-expressed with human beta2 and gamma2s GABAA receptor subunits in CHO cells using transient transfection. Electrophysiological whole-cell patch-clamp recordings showed that, of the eight arginine residues tested, only the two arginines, at position 70 and 123, appear to be essential for GABA-gated chloride current, since the EC50 values of the two mutant constructs increase more than 100-fold as compared to wild-type alpha5beta2gamma2s GABAA receptors. Ion channels are challenging targets particularly in the early phases of the drug discovery process, due to the lack of technologies available to screen large numbers of compounds in functionally relevant assays. The human sodium channel NaV-1.7 was stably expressed in HEK293 cells and 3 high throughput screening (HTS) assays were compared. 1) A Li+ flux atomic absorption spectroscopy (AAS) assay, 2) a Fluorometric Imaging Plate Reader (FLIPR) membrane potential assay 3) a Fluorescent Energy Transfer (FRET) based membrane potential assay. These 3 assays were then compared to an automated electrophysiological assay (the Ionworks-HT platform) to characterize eleven known sodium channel inhibitors. The results demonstrated that all 3 HTS assays are suitable for the identification of NaV-1.7 inhibitors, but for an HTS assay the Li+-flux assay was more robust than the FLIPR and FRET based membrane potential assays. Furthermore, there was a better correlation between the Ionworks assay and the Li+-flux assay regarding the IC50 values of the sodium channel inhibitors investigated. Human TRPM8 channels stably expressed in HEK293 were used to develop a FLIPR based HTS assay using the calcium sensitive dye Fluo-4. The two known TRPM8 agonists menthol and icilin induced [Ca2+]i increases with EC50 values comparable to electrophysiological measurements. Screening of a compound library identified 15 novel antagonists, which were characterized using both the FLIPR assay and twoelectrode voltage clamp electrophysiology in Xenopus oocytes expressing TRPM8. The antagonists were additionally tested in a FLIPR assay using cold as the agonist, which additionally demonstrated concentration dependent antagonism. These results show that menthol, icilin and cold can all be used as agonists when searching for antagonists and demonstrate that a cellular HTS assay for investigating the pharmacological activity of TRPM8 ligands can be developed. The work in this thesis illustrates the validity of multiple methods and technologies when studying ion channel pharmacology. Traditional ligand binding assays in combination with molecular modelling are important tools for structure activity relationship studies, but functional assays are needed to determine ligand efficacy. The functional cellular assays that have become available recently to study ion channels have shown great potential in terms of screening throughput and delivering quality data. These assays are capable of determine both the pharmacological properties of ion channel ligands and perform SAR studies. With these developments ion channels may become more tractable targets for the pharmaceutical industry, leading to new improved drugs for the patients

Corporate Social Responsibility - Lost in Translation? : Hur CSR som idé tas emot i offentlig sektor

År 2007 uppmärksammades de oegentligheter som förekom i produktionen av de varor som landstingen köpte in till den svenska sjukvården vilket medförde att landstingen startade ett samarbete runt socialt ansvar i upphandling. Då CSR främst kopplas ihop med företag i privat sektor, avser denna kvalitativa studie undersöka hur CSR som idé har tagits emot i offentlig sektor och hur den har gjorts om för att passa i sin nya kontext. Det empiriska materialet har samlats in genom semistrukturerade intervjuer och har sedan analyserats med skandinavisk nyinstitutionell översättningsteori samt teorin om inramning. Undersökningen visar att det tidigare var svårt att veta vilka sociala krav man som enskild förvaltning kunde ställa i upphandlingar och att när CSR som idé togs emot av aktörer i landstingen startade dessa ett samarbete där idén aktivt gjordes om för att passa i offentlig sektor. Det innebar att idén materialiserades i en uppförandekod som tog hänsyn till det unika med den nya kontexten, vilket främst var lagen om offentlig upphandling. Studien visar att CSR inte har förlorat sin innebörd i översättningen, utan den har enbart anpassats till de nya förutsättningarna.

Corporate Social Responsibility - Lost in Translation? : Hur CSR som idé tas emot i offentlig sektor

År 2007 uppmärksammades de oegentligheter som förekom i produktionen av de varor som landstingen köpte in till den svenska sjukvården vilket medförde att landstingen startade ett samarbete runt socialt ansvar i upphandling. Då CSR främst kopplas ihop med företag i privat sektor, avser denna kvalitativa studie undersöka hur CSR som idé har tagits emot i offentlig sektor och hur den har gjorts om för att passa i sin nya kontext. Det empiriska materialet har samlats in genom semistrukturerade intervjuer och har sedan analyserats med skandinavisk nyinstitutionell översättningsteori samt teorin om inramning. Undersökningen visar att det tidigare var svårt att veta vilka sociala krav man som enskild förvaltning kunde ställa i upphandlingar och att när CSR som idé togs emot av aktörer i landstingen startade dessa ett samarbete där idén aktivt gjordes om för att passa i offentlig sektor. Det innebar att idén materialiserades i en uppförandekod som tog hänsyn till det unika med den nya kontexten, vilket främst var lagen om offentlig upphandling. Studien visar att CSR inte har förlorat sin innebörd i översättningen, utan den har enbart anpassats till de nya förutsättningarna.

Corporate Social Responsibility - Lost in Translation? : Hur CSR som idé tas emot i offentlig sektor

År 2007 uppmärksammades de oegentligheter som förekom i produktionen av de varor som landstingen köpte in till den svenska sjukvården vilket medförde att landstingen startade ett samarbete runt socialt ansvar i upphandling. Då CSR främst kopplas ihop med företag i privat sektor, avser denna kvalitativa studie undersöka hur CSR som idé har tagits emot i offentlig sektor och hur den har gjorts om för att passa i sin nya kontext. Det empiriska materialet har samlats in genom semistrukturerade intervjuer och har sedan analyserats med skandinavisk nyinstitutionell översättningsteori samt teorin om inramning. Undersökningen visar att det tidigare var svårt att veta vilka sociala krav man som enskild förvaltning kunde ställa i upphandlingar och att när CSR som idé togs emot av aktörer i landstingen startade dessa ett samarbete där idén aktivt gjordes om för att passa i offentlig sektor. Det innebar att idén materialiserades i en uppförandekod som tog hänsyn till det unika med den nya kontexten, vilket främst var lagen om offentlig upphandling. Studien visar att CSR inte har förlorat sin innebörd i översättningen, utan den har enbart anpassats till de nya förutsättningarna.

The heteroyohimbine mayumbine binds with high affinity to rat brain benzodiazepine receptors in vitro

Mayumbine, a naturally occurring heteroyohimbine, was isolated from Rauwolfia vomitoria extracts and shown to potently (IC = 76 ± 3.5 nM) inhibit the in vitro binding of H-diazepam to the benzodiazepine sites within the rat GABA(A) receptor complex. 50

6-Methylflavone, a benzodiazepine receptor ligand with antagonistic properties on rat brain and human recombinant GABA(A) receptors in vitro

Seventeen flavonoids were found to have inhibitory activity on the central nervous system GABA(A)/benzodiazepine (BZD) receptors with IC values ranging from 0.12 to 8 μM. 6-Methylflavone, the most potent, was pharmacologically characterized by radioligand binding assays on rat brain membranes in vitro and human recombinant GABA(A)/BZD receptors expressed in Sf-9 insect cells, as well as electrophysiologically by the whole-cell patch clamp technique. Scatchard plot analysis showed that 6-methylflavone was a competitive inhibitor of [ H]-Ro 15-1 788, binding to rat brain cortical membranes. The GABA ratio of 1.06 for [ H]-diazepam binding to cortex and 1.23 for cerebellum indicated an antagonistic or a weak partial agonistic profile of 6-methylflavone on the rat BZD receptors, while the GABA ratio of 0.76 on hippocampus indicated an antagonistic or partial-inverse agonistic profile on the BZD receptors. In Sf-9 insects cells, the GABA ratios showed a weak partial agonistic profile on the α β γ(2S) (GABA ratio 1.29) subtype combination, an antagonistic profile on the α β γ(2S) (1.13) and α β γ(2S) (1.03), and a partial inverse agonistic profile on the α β γ(2S) (0.79) subtype combination. The modulation of GABA-induced chloride currents by 6-methylflavone suggests that the compound is an antagonist at human GABA(A) receptor subtypes. Based on our data of GABA(A)/BZD receptor active as well as inactive flavonoids, some general structure-activity relationships are discussed. 50 1 2 1 2 2 2 3 2 5 2 3