EP1423700A2 - Affinity-assay for the human erg potassium channel - Google Patents
Affinity-assay for the human erg potassium channelInfo
- Publication number
- EP1423700A2 EP1423700A2 EP02762684A EP02762684A EP1423700A2 EP 1423700 A2 EP1423700 A2 EP 1423700A2 EP 02762684 A EP02762684 A EP 02762684A EP 02762684 A EP02762684 A EP 02762684A EP 1423700 A2 EP1423700 A2 EP 1423700A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- assay
- tris
- kci
- dofetilide
- erg
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Definitions
- the invention relates to an assay to establish the affinity of compounds at the "ether-a- go-go" (ERG) potassium (K + ) channel, in particular the human ERG (hERG) potassium channel, using a labelled rapid delayed rectifying potassium channel (IKR) blocker, for example [ 3 H]-dofetilide or [ 3 H]-MK-499.
- ERG ether-a- go-go
- IKR rapid delayed rectifying potassium channel
- the QT interval is the portion of an ECG that represents the time from the beginning of ventricular depolarization to the end of ventricular repolarisation. Because the QT interval can be affected by heart rate lengthening with a decrease in heart rate and shortening with an increase in heart rate, the QT is often "corrected" for heart rate, resulting in the QT C interval. In rare cases the administration of some drug molecules results in a prolongation of the QT interval of the ECG in man.
- ECGs of these patients resemble those of individuals suffering from an inherited disorder known as long QT syndrome. Drug-induced ventricular fibrillation, in these cases, can eventually lead to sudden death (Morganroth J et al. (1993) Am J Cardioi. 72, 26B-31 B; De Ponti F. et al., (2000) Eur J. Clin. Pharmacol. 56, 1-18).
- a number of drug molecules, including, E-4031 , cisapride and terfenadine, are all known to prolong the QT interval of the electrocardiogram in man (Fuliki A, et al. (1994), Cardiovascular Pharmacol. 23: 374-378; Van Haarst AD et al., (1998) Clin Pharmacol. Ther. 64: 542-546; Honig P.K. et al. (1993) J.A.M.A. 269; 1513- 1518).
- an assay that comprises, or consists of, the following steps: a) incubation of cells expressing ERG or membranes derived from cells expressing ERG or membranes derived from tissue expressing ERG with labelled IKR blocker in assay buffer in the presence or absence of different amounts of a test compound or a mixture of test compounds; b) determination of specifically bound labelled IKR blocker; c) calculation of the inhibition of labelled IKR blocker binding by the test compound or mixture of test compounds.
- the assay is useful as a preclinical predictive indicator for identification of compounds with a propensity to prolong the QT interval in man.
- the assay is a competitive binding assay that measures the ability of a test compound or mixture of compounds to displace labelled IKR blocker from the ERG K + channel (ether-a-go-go K + channel, herein called ERG).
- ERG ether-a-go-go K + channel
- the assay can be performed in a high throughput test system.
- ligand binding assays using labelled IKR blockers can be used to assist in the design of new drugs devoid of, or with reduced affinity to ERG, in particular human ERG (hERG).
- the assay buffer used is particularly important for optimising binding of the IKR blocker or test compound(s) to ERG. It has been found that optimal assay performance is achieved using a Tris based buffer (pH 7.2 to 7.6, preferably pH 7.4 at room temperature) containing potassium (K + ) ions. Potassium ions in the assay buffer may be provided, for example as potassium choride (KCI). The concentration of potassium ions in the assay buffer determines the predictive value of the assay. Assays performed in assay buffer containing from 7.5 to 12.5mM KCI, preferably from 8.5 to 11.5mM KCI, most preferably 10mM KCI are particularly useful to provide an IC 2 o value predictive of onset of QT prolongation.
- the assay buffer of the invention preferably comprises or consists of Tris.CI and KCI.
- MgCI 2 may be included in the assay buffer.
- the concentration of Tris.CI in the assay buffer is preferably from 30mM to 10OmM Tris.CI, more preferably from 30mM to 70mM Tris.CI, yet more preferably from 40mM to 60mM Tris.CI, further preferably from 45mM to 55 mM Tris.CI, most preferably 50mM Tris.CI.
- the concentration of KCI in the assay buffer is preferably from 5 to 20mM KCI, more preferably from 6 to 15mM KCI, yet more preferably from 7.5 to 12.5mM KCI, further preferably from 8.5 to 11.5mM KCI, most preferably 10mM KCI.
- the assay buffer comprises or consists of from 30 to 100mM Tris.CI and from 5 to 20mM KCI, preferably from 30 to 70mM or from 30 to 100mM Tris.CI and from 6 to 15mM KCI ; yet more preferably from 40 to 60mM Tris.CI and from 7.5 to 12.5mM KCI ; further preferably from 45 to 55mM Tris.CI and from 8.5 to 11.5mM KCI.
- the assay buffer comprise or consist of 50mM Tris.CI and lOmM KCI.
- the concentration of MgCl 2 is preferably from 0.6mM to 2.0mM MgCI 2 , more preferably from 0.6mM to 1.6mM MgCI 2 , yet more preferably from 0.8mM to 1.4mM MgCI 2 , further preferably from 0.9mM to 1.3mM MgCI 2 , yet further preferably from 1.OmM to 1.2mM MgCI 2 , most preferably 1.OmM or 1.2mM MgCI 2 .
- the assay buffer used comprises or consists of from 30 to 100mM Tris.CI, from 5 to 20mM KCI, and from 0.6 to 2.0mM MgCI 2; preferably from 30 to 100mM Tris.CI or from 30 to 70mM Tris.CI, from 6 to 15mM KCI, and from 0.6 to 1.6mM MgCI 2 , yet more preferably from 40 to 60mM Tris.CI, from 7.5 to 12.5mM KCI and from 0.8 to 1.4mM MgCI 2 , further preferably from 45 to 55mM Tris.CI, from 8.5 to 11.5mM KCI and from 0.9 to 1.3mM MgCI 2 or from 1.0 to 1.2mM MgCI 2
- the assay buffer may comprise or consist of 50mM Tris.CI, 10mM KCI and LOmM MgCI 2 ; or 50mM Tris.CI, 10mM KCI and 1.2mM MgCI 2 It is preferred that the assay buffer be at a pH between 7.2 and 7.6 at room temperature; it is particularly preferred that the assay buffer be at pH 7.4 at room temperature.
- the ERG gene can be from a vertebrate or invertebrate source; for vertebrates the ERG gene may be from a mammalian source (e.g. human, simian, bovine, porcine, canine, rabbit, guinea pig, rat, or mouse) or an invertebrate source such as an insect source (e.g. drosophila).
- a mammalian source e.g. human, simian, bovine, porcine, canine, rabbit, guinea pig, rat, or mouse
- an invertebrate source such as an insect source (e.g. drosophila).
- a prokaryotic homologue of mammalian ERG may be used. It is preferred that the ERG gene be mammalian ERG, in particular human ERG (hERG) or canine ERG (cERG).
- the ERG gene may be expressed in a mammalian cell line e.g. HEK-293 (Human embryonic kidney) cells, CHO (Chinese hamster ovary) cells; CHL (Chinese hamster lung) cells, COS (monkey) cells; or in an insect cell line e.g. SF9.
- HEK-293 Human embryonic kidney
- CHL Choinese hamster ovary
- COS monkey
- a baculovirus vector system can be used for expression of ERG in a compatible insect cell line.
- ERG may be expressed in yeast or bacterial cells. It is preferred that the ERG gene is hERG or cERG and is expressed in either HEK-293, CHO or CHL cells.
- the assay may be performed using whole cells expressing ERG or membrane preparations derived from cells expressing ERG, or membrane preparations derived from tissue expressing ERG.
- Dofetilide is an IKR blocker (selective inhibitor of the rapid component of the delayed rectifier potassium current), which prolongs the action potential duration and the effective refractory period in a concentration-dependent manner. Clinical studies have demonstrated that dofetilide is effective in treating patients with atrial as well as ventricular arrhythmias. Dofetilide has formula I below.
- MK-499 (Merck) is methylsulphonamide antiarrhythmic drug that acts as an IKR blocker. MK-499 has formula II shown below.
- the IKR blocker used in the assay is labelled with a detectable label, for example a radiolabel or fluorescent tag.
- the labelled IKR blocker used in the assay is labelled dofetilide, preferably radiolabelled dofetilide, most preferably tritiated dofetilide ([ 3 H]-dofetilide).
- the labelled IKR blocker used in the assay is labelled MK-499, preferably radiolabelled MK-499, most preferably tritiated MK-499 ([ 3 H]-MK-499).
- Preferred assay formats include the filter binding technique, whereby bound and unbound labelled IKR blocker e.g. labelled dofetilide or labelled MK-499; preferably radiolabelled dofetilide or radiolabelled MK-499; most preferably [ 3 H]-dofetilide or [ 3 H]- MK-499, are separated by filtration.
- the assay can be performed utilising the scintillation proximity assay (SPA) technique, using radiolabelled IKR blocker e.g. radiolabelled dofetilide or radiolabelled MK-499, preferably [ 3 H]-dofetilide or [ 3 H]-MK- 499.
- SPA scintillation proximity assay
- cells expressing ERG or membranes derived from cells expressing ERG or membranes derived from tissue expressing ERG are incubated in assay buffer with labelled IKR blocker e.g. [ 3 H]-dofetilide or [ 3 H]-MK-499, in the presence (test) or absence (control) of the test compound or mixture of test compounds. Incubations are preferably carried out at room temperature for from 60 to 120 minutes, preferably for 90 minutes. Non-specific binding is determined in the presence of unlabelled IKR blocker, e.g. 10 ⁇ M dofetilide or 10 ⁇ M MK-499.
- IKR blocker e.g. 10 ⁇ M dofetilide or 10 ⁇ M MK-499.
- Bound labelled IKR blocker is separated from unbound IKR blocker by filtration through filter mats, or onto multiwell filter plates. Filter mats or plates are washed to remove unbound labelled IKR blocker, bound labelled IKR blocker is quantified e.g. for tritiated IKR blocker such as [ 3 H]-dofetilide or [ 3 H]-MK-499 by scintillation spectroscopy using an appropriate counter for radioactivity.
- tritiated IKR blocker such as [ 3 H]-dofetilide or [ 3 H]-MK-499 by scintillation spectroscopy using an appropriate counter for radioactivity.
- beads are used to bind cells expressing ERG or membranes derived from cells expressing ERG or membranes derived from tissue expressing ERG.
- a variety of bead types are suitable for use in a SPA assay according to the invention, these include PVT wheat germ agglutinin, yttrium oxide polylysine beads, or yttrium silicate beads (YSi) (Amersham Biosciences) such as YSi polylysine or YSi wheat germ agglutinin.
- the optimum bead type for , use in a SPA assay of the invention depends on the cells or cell membranes used; bead to cell or bead to membrane binding may be assessed to identify the optimum bead type for the cell or cell membrane used.
- Beads bound to ERG material whole cell, cell membrane preparation or tissue membrane preparation
- IKR blocker e.g. [ 3 H]-dofetilide or [ 3 H]-MK-499 in the presence (test) or absence (control) of the test compound or mixture of test compounds.
- the ability of the test compound or mixture of test compounds to displace bound radiolabelled IKR blocker is determined by detecting light emissions, for example using standard counters that can be used with SPA technology.
- the assay may also include one or more of the steps of: calculation of the concentration of the test compound(s) that gives 20% inhibition of dofetilide binding (IC 2 o), calculation of the concentration of the test compound(s) that gives 50% inhibition of dofetilide binding (IC 50 ), calculation of the compound affinity as Ki or calculation of the compound affinity as pKi.
- the IC 20 values generated from competitive displacement of IKR blocker binding, e.g. [ 3 H]-dofetilide binding, using the assay of the invention are comparable to the free drug concentration associated with QT prolongation in man.
- the assay can be used to predict the concentration of a compound liable to cause undesirable cardiac side effects.
- steps are carried out: a) An assay is carried out according to the invention.
- IC 20 value is obtained; this indicates the real or predicted free drug concentration at which QT prolongation will occur in man; c) The IC 2 o value is compared with the free drug concentration required for the desired therapeutic effect of the compound or mixture of compounds in vivo.
- the free drug concentration required for the desired therapeutic effect of the compound or mixture of compounds is within 10 to 30 fold of the IC 20 of the compound or mixture of compounds in the assay, the compound or mixture of compounds is likely to show QT interval prolongation in man.
- the assay of the invention is a better predictor of in vivo QT prolongation effect of drug molecules than existing assays such as the HERG patch clamp assay.
- Figure 1 Representative saturation curve data for [ 3 H]-dofetilide binding to HERG in (a) filter binding, (b) SPA 96 well format and (c) SPA 384 well format.
- Figure 2 Correlation plots comparing p i values obtained from filter binding and SPA binding assays: (a) correlation between 96 well hERG [ 3 H] dofetilide SPA assay and radioligand binding assay, (b) correlation between 96 well and 384 well hERG [ 3 H] dofetilide SPA assay.
- Figure 3 Comparison of inhibition of [ 3 H]-dofetilide binding to hERG, hERG patch clamp, and free drug concentration known to induce QT interval prolongation in man, for (a) E-4031 , (b) dofetilide, (c) terfenadine and (d) cisapride.
- Figure 4 Comparison of the dofetilide IC 50 in the dofetilide binding assay carried out in cell membranes from HEK-293 cells transfected with human ERG (hERG (A)) or with canine ERG (cERG ( ⁇ )) . .y .c. ⁇ ii.pai .son of terfenadine IC 50 in the dofetilide binding assay in cERG or hERG transfected HEK-293 cell membranes
- Example 1 Preparation of membranes from HEK-293 cells expressing human or canine ERG
- HEK-293 cell line expressing human ERG (Zhou, Z et al (1998) Biophys. J. 74, 230-241) was provided by Dr. Craig January, University of Wisconsin, USA; this cell line was designated the "January” cell line.
- Full length cDNA for human ERG was inserted downstream of the CMV promoter in pcDNA3.1 (Invitrogen), the vector also has a SV40 promoter that drives expression of a neomycin resistance gene.
- the construct was transfected into human embryonic kidney 293S (HEK-293) cells. Stable transformants were selected using G418 (Gibco). Although cell line 15 has slightly lower expression of hERG than the January cell line, it has improved growth characteristics.
- Cell line 15 (293S-HERG (Clone 15) was deposited on 26 June 2002 with the ECACC (CAMR Salisbury, Wiltshire, SP4 OJG, UK) in accordance with the terms of the Budapest Treaty 1977 under deposit accession number 02062678.
- Adherent HEK-293 cells expressing human ERG were grown in MEM Earles medium (Life Technologies) supplemented with 10% foetal calf serum (PAA Laboratories), 2 mM L-glutamine (Sigma), 1 mM sodium pyruvate (Sigma), 0.4 mg/ml G418 (Life Technologies) and an addition of 1x non-essential amino acids (Life Technologies). The cells were grown at 37°C in a humidified atmosphere with 5% C0 2 in T225 cm 3 flasks.
- the cells were split 1 :3 to 1 :5 after reaching 80% confluence using cell dissociation solution (Sigma, cat no: C5914 in 2001) and later seeded into 850 cm 2 CO2 gassed roller bottles (Corning, cat no: 430849 in 2001) in the absence of G418.
- a HEK-293 cell line expressing canine ERG was produced by transient transfection of HEK-293 cells.
- the complete coding sequence of cERG cDNA (Zehelein et al (2001). Pflugers Archiv. European Journal of Physiology. 442(2): 188 - 191) was provided in the pBluescript® vector (Stratagene) by Professor Zehelein University of Heidelberg, Germany.
- the cERG cDNA was flanked by SamHI Sites.
- an indirect cloning method was devised using the cloning vector pSP73 (Promega).
- the cERG/pBluescript construct and pSP73 vector were subjected to SamHI digestion, to reduce interference by the presence of pBluescript SamHI fragments in the ligation reaction, the cERG/pBluescript SamHI digested material was also subjected to Seal digestion to cleave pBluescript and ensure more effective separation of the cERG SamHI fragment on agarose gel.
- the restriction mixtures were subjected to agarose gel electrophoresis, bands containing the cERG and pSP73 SamHI fragments were visualized following staining with ethidium bromide and UV illumination.
- the cERG and pSP73 bands were excised and eluted from the gel using a QIAgen MinELute Gel extraction kit according to the manufacturers instructions.
- the plasmid DNA fragments were subjected to CIP treatment using a standard protocol.
- the cERG SamHI fragments were ligated into the pSP73 SamHI fragments using a standard ligation protocol.
- the ligation mixture was transformed into cJM109 competent E. coli cells using a standard transformation protocol. Transformants were selected by plating on LB agar (Millers) containing ampicillin (50 ⁇ g/ml) and incubated overnight at 37°C.
- the cERG cDNA was excised from cERG/pSP73 as an Xho ⁇ (5') EcoRI (3') fragment, this fragment was ligated into an XhoMEctiP ⁇ fragment of the reverse poly linker form of pcDNA3.1 , pcDNA3.1 (-) XhoUEcoRl.
- the reverse polylinker form was used because the cERG/pSP73 clone selected contained the reverse orientation of cERG.
- the 5' end of cERG was located adjacent to the enhancer-promoter sequence from human cytomegalovirus (CMV).
- CMV human cytomegalovirus
- transformants were selected via plating onto LB agar (Millers) containing ampicillin (50 ⁇ g/ml) and incubating overnight at 37°C along with required controls. Colonies picked at random from the cERG/pcDNA3.1 (-) plates were inoculated into 5ml of LB media containing ampicillin (50 ⁇ g/ml) and incubated at 37°C, 200rpm overnight. These overnight cultures were subsequently used to produce mini- preps of DNA using a QIAgen Miniprep Kit. The resulting DNA was subjected to a Xho ⁇ and EcoRI double digestion and analysis on 1% agarose gel.
- the colony PCR protocol permitted rapid detection of cERG/pcDNA3.1(-) clones.
- Three primers were designed and made for use in the PCR protocol:
- Primer 1 'CERG01 ' (SEQ ID NO: 1) which hybridises to cERG at nucleotide positions 601-620 of the coding sequence:
- Primer 2 5'-ACCACATCCACCAGGCACAG-3' Primer 2: 'NHE PCDNA3' (SEQ ID NO: 2) which hybridises to pcDNA3.1 (-) at nucleotide positions 886-910 (within the multicloning site flanking the Nhe ⁇ cloning site):
- T7 SP73' (SEQ ID NO: 3) which was used as a control and used against a colony known to produce cERG/pSP73. This hybridised to pSP73 at nucleotide positions 98-121 , within the T7 polymerase promoter sequence:
- Taqman Gold buffer (X10) 10.O ⁇ l 10 ⁇ l dNTPs (X10, 2mM/dNTP) 2.0 ⁇ l 2 ⁇ l Taqman Gold Polymerase (5u/ ⁇ l) 0.5 ⁇ l 0.5 ⁇ l
- the bacterial pellet was resuspended in the PCR reaction mixture.
- the PCR reaction was performed as specified by the manufacturers protocol for the Taqman Gold PCR kit (Applied Biosystems, 1999 edition) thus:
- Step 2 denaturation 95°C 1 minute
- Step 3 - annealing 60°C 1 minute
- Step 4 extension 72°C 1 minute To step 2 for 35 cycles, then step 5.
- biep t> - ⁇ ena ⁇ ura ⁇ on 95°C 45 sees
- Step 6 - annealing 60°C 45 sees
- PCR products for each well were then separated by electrophoresis on a 1.5% agarose gel using a 100bp DNA ladder marker at 100V for 25 minutes in 1X TAE buffer and visualised using UV light. Putative positive clones were identified and samples from these PCR reaction mixtures were run on a second separate 1.5% agarose gel at 100V for one hour to examine the sizes of the PCR products.
- mini-cultures which gave an amplified a PCR product were each seeded from the original deep-well 96-well plate into sterile tubes with 5ml LB broth containing 50 ⁇ g/ml ampicillin and incubated at 37°C overnight at 200rpm. The overnight cultures were then used to produce mini-preps of DNA using a QIAgen Miniprep Kit. The resulting DNA was subjected to an Xho ⁇ and EcoRI double digestion to check for the presence of cERG/pcDNA3.1 (-).
- restriction digest was analysed via a 1% agarose gel run for 1 hour at 100V with 1 kb DNA ladder markers (20 ⁇ l sample loading with 2 ⁇ l gel loading solution) ⁇ Further restriction digestion analysis was performed to confirm that the purified plasmids from the transformants were indeed cERG/pcDNA3.1 (-).
- MEM Mesarcoma
- FCS foetal calf serum
- 2mM L- glutamine 1 mM sodium pyruvate
- 1 mM non-essential amino acids 1 mM non-essential amino acids.
- Cells were seeded into 225cm 2 ventilated cap flasks and were maintained in a humidified atmosphere containing 5% CO 2 .
- the HEK-293 cells used in this study were between passage numbers 39-48. Cells were passaged typically every three days in a ratio of
- the cERG/pcDNA3.1 (-) construct was transfected into HEK-293 cells grown to 80-95% confluency in 225cm 2 ventilated flasks using the following method. Endotoxin free cERG/pcDNA3.1(-) DNA (94 ⁇ g) and Lipofectamine2000 (Gibco BRL) (94 ⁇ g) were added to 2.25ml of OPTIMEM-I media (Gibco BRL) in sterile 10ml centrifuge tubes; mixing was carried out after incubation at room temperature for five minutes.
- the Lipo ⁇ ec ⁇ am ⁇ nek.uu/DNA/OPTIMEM-1 mix was then incubated at room temperature for twenty minutes before the addition of a further 10.5ml OPTIMEM-I.
- HEK-293 cells were washed with 10ml PBS and the Lipofectamine2000/DNA/OPTIMEM-I mixture added and incubated for 3.5 hours at 37°C in a humidified atmosphere containing 5% C0 2 . After incubation, 50ml of MEM (supplemented with 10% (v/v) FCS, 2mM L-glutamine,
- HEK-293 cells were incubated for 24 hours at 37°C.
- Transfected cells were harvested after 24 hours by washing with PBS, scraping the cells into 10ml PBS and centrifuging at lOOOrpm for 5 minutes at room temperature.
- the resulting cERG/pcDNA3.1 (-) transfected HEK-293 cell pellet was stored at -80°C until required.
- Cell membrane fractions were prepared from frozen aliquots of cells. All procedures were carried out at 4°C unless otherwise stated. Frozen aliquots of cells were thawed at room temperature and resuspended in assay buffer (e.g. 50mM Tris.CI, 10mM KCI, 1 to ;! .2mM MgCI 2 , pH7.4, or 50mM Tris.CI, 10mM KCI, pH7.4). The cells were then disrupted by homogenisation in an Omni LabTek homogeniser at 20,000 rpm for 30 seconds. The homogenate was centrifuged for 20 minutes at 48,000xg (4°C, Sorvall RC5B centrifuge) and the supernatant removed. The resulting pellets were resuspended in assay buffer and homogenised as above for 10 seconds. The pellets were collected by centrifugation and the final pellet resuspended in assay buffer.
- assay buffer e.g. 50mM Tris.
- Protein content was determined using a Coomassie Blue based protein assay kit.
- [ 3 H]-dofetilide (80-83 Ci/mmol) was synthesized by catalytic tritiation (a custom service provided, for example, by Amersham Life Science).
- catalytic tritiation a custom service provided, for example, by Amersham Life Science.
- detectable labels known to the skilled person can be used instead of 3 H, e.g. fluorescent tags, other radiolabels, antibodies etc.
- test compounds were dissolved at 1 mM in 50% DMSO or 100% DMSO, and then diluted to the desired concentrations (e.g. up to 100 ⁇ M, or up to the boundaries of solubility for the compound) in assay buffer.
- the final DMSO concentration in assay incubations is preferably 1.0 to 1.5% or less for optimal assay conditions.
- Incubations included membrane homogenate at 50 ⁇ g/ml in assay buffer (50 mM Tris.CI, 10mM KCI, LOmM to 1.2mM MgCI 2 , pH7.4) unless otherwise indicated, [ 3 H]- dofetilide (4 to 7nM) and test compound or mixture of test compounds or control vehicle. Filtration assays were incubated at room temperature for 90 minutes. Nonspecific binding was determined in the presence of 10 ⁇ M dofetilide and was usually less than 15 % of total binding.
- Bound ligand was separated from free ligand by rapid filtration through GF/B glass fibre filter mats using, for example, a Brandel cell harvester, or onto GF/B Unifilter 96-well filter plates (Packard) using a Packard Filterr ate 96 harvester. Filter mats and plates were pre-soaked in 5% PEI (w/v) for 60 minutes and washed after harvesting with 3 x 1 ml washes of ice-cold assay buffer. Unifilter plates were air dried for a minimum of 1.5 hours at 37°C prior to the addition of Microscint-0 (Packard).
- Bound [ 3 H]-dofetilide was determined by liquid scintillation spectroscopy using an appropriate counter, for example in a Packard TopCount Scintillation Counter (NXT Counter) or Wallac Counter (Trilux) for Unifilter plates and in a Wallac Big Spot Counter when filter mats were used.
- NXT Counter Packard TopCount Scintillation Counter
- Triplelux Wallac Counter
- the scintillation proximity assay was carried out in assay buffer consisting of 50mM Tris.CI, 10mM KCI, LOmM to 1.2mM MgCI 2 , pH7.4, or using assay buffer consisting of 50mM Tris base, 10mM KCI, pH7.4.
- Bead to membrane binding was assessed to determine the optimum bead type for the cell line used.
- YSi wheatgerm agglutinin beads were used with cell membranes derived from the January HEK-293 hERG expressing cell line; YSi polylysine beads were used in studies using membranes derived from Cell Line 15 (HEK-293 hERG expressing cell line).
- test compound or mixture of test compounds.
- the plates were incubated at room temperature and shaken for approximately 1 hour. Beads were allowed to settle for a minimum of 30 minutes before plates were counted for retained radioactivity on a TopCount NXT scintillation counter.
- Nonspecific binding i.e. background count, was determined by the addition of 10 ⁇ M dofetilide. Background counts were usually less than 15% of the total binding.
- specific binding of [ 3 H]-dofetilide was determined over a range of concentrations (5 to 500nM) in the absence or presence of cold (i.e. unlabelled) 10 ⁇ M dofetilide.
- Buffer 25mM HEPES free acid 50mM Tris 135mM NaCI, 5mM KCI lOmM KCI and 1 mM MgS0 4 , 50 ⁇ M CaCI 2 1.0 or 1.2mM MgCI 2 pH 7.4 at room temp pH 7.4 at room temp
- the assay buffer used in Examples 1 to 8 was the Tris-based incubation buffer (50mM Tris.CI, 10mM KCI, 1 mM MgCI 2 ). Additionally, experiments were performed to optimise the cell membrane protein concentration and bead concentration for filter and SPA binding assays.
- the IC 2 o values generated from competitive displacement of [ 3 H]-dofetilide binding using the assay of the invention are comparable to the free drug concentration associated with QT prolongation in man as is shown in Figure 3 for a range of compounds, including E-4031 (Figure 3a), dofetilide (Figure 3b), terfenadine ( Figure 3c) and cisapride ( Figure 3d).
- E-4031 Figure 3a
- dofetilide Figure 3b
- terfenadine Figure 3c
- cisapride Figure 3d
- the inhibition of dofetilide binding in the binding assay filter binding technique
- a hERG patch clamp assay is compared with the concentration of free drug associated with QT interval prolongation in man
- the ERG patch clamp assay provides a measure of the current through the ERG channel and indicates the number of ion channels present in a cell.
- the ligand binding assay provides a better predictor of in vivo QT prolongation effect of a drug than the hERG patch clamp technique ( Figure 3d).
- a binding assay is carried out according to the invention, for example as as described in Example 2 or Example 3, to test the affinity of the compound or mixture of compounds for ERG, preferably hERG or cERG; b) The IC-2 0 is obtained, e.g. as described at the end of Example 2; the IC 2 o being the real or predicted free drug concentration at which QT prolongation occurs in man; c) The IC 2 o value is compared with the free drug concentration required for the desired therapeutic effect of the compound in vivo. If the free drug concentration required for the desired therapeutic effect of the compound is within 10 to 30 fold of the IC 20 of the compound in the assay of the invention, the compound is highly likely to cause QT interval prolongation in man.
- Example 6 Comparison of dofetilide binding assay carried out HEK-293 cells transfected with cERG or hERG.
- the dofetilide binding assay was carried out as described Example 2 using HEK 293 cells transfected with either human ERG or canine ERG. The results are shown in figure 4, from which it can be seen that the IC 5 o for dofetilide is similar for canine and human ERG, being 13.9nM and 15.6nM respectively. IC 2 o values for dofetilide were 1.92nM and 2.15nM for canine and human ERG, respectively.
- Example 7 Comparison of terfenadine competition assay using HEK-293 cells transfected with cERG or hERG
- the dofetilide binding assay was carried out using terfenadine as the test compound.
- Transiently transfected cERG HEK-293 cell membranes (200 ⁇ g/well), or stable hERG HEK-293 cell membranes (100 ⁇ g/well) were incubated with twelve different concentrations of terfenadine and 5nM [ 3 H]-dofetilide for 90 minutes at room temperature.
- Total and non-specific binding were measured by incubating with 10% DMSO and 10 ⁇ M unlabelled dofetilide to a total assay volume of 200 ⁇ l.
- the membranes were harvested by filtration with a Packard Unifilter cell harvester and radioactivity (cpm) was measured.
- FIG. 5 shows the mean values of the experiments for each cell type (cERG or hERG transfected) and indicates that the IC 50 for terfenadine is similar for cERG and hERG, being 77.2nM and 88.9nM respectively.
- IC20 values for terfenadine were 10.7nM and 12.3nM for canine and human ERG, respectively.
- the dofetilide binding assay was carried out using E4031 as the test compound.
- Transiently transfected cERG HEK-293 cell membranes (200 ⁇ g/well) or stable hERG HEK-293 cell membranes (100 ⁇ g/well) were incubated with twelve different concentrations of E4031 and 5nM [ 3 H]-dofetilide for 90 minutes at room temperature.
- Total and non-specific binding values were measured by incubation with 10% DMSO and 10 ⁇ M unlabelled dofetilide in a total assay volume of 200 ⁇ l.
- the membranes were harvested by filtration with a Packard Unifilter cell harvester and radioactivity (cpm) was measured. Two saturation experiments were carried out each for cERG and hERG expressing cell membrane samples.
- Figure 6 shows the mean values of the experiments for each cell membrane type (cERG or hERG transfected) and indicates that the IC 5 o for E4031 is similar for cERG and hERG, being 27.3 nM and 35.4 nM respectively.
- IC2 0 values for E4031 were 3.8 nM and 4.9 nM for canine and human ERG, respectively.
- IC- 50 or IC20 values are compared for the compounds tested, they were found to be very similar for cERG and hERG. This indicates that either hERG or cERG can be used in the assay of the invention to predict the onset of QT prolongation in man.
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US7927820B2 (en) | 2004-07-20 | 2011-04-19 | Janssen Pharmaceutica Nv | Assay systems and methods for detecting molecules that interact with membrane channels |
US7326772B2 (en) * | 2005-05-12 | 2008-02-05 | Penta Biotech, Inc. | Peptide for assaying hERG channel binding |
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