56 research outputs found
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
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