WO2008121717A1 - Electrophysiological method to pharmacologically classify ligands for gs protein-coupled receptors - Google Patents

Electrophysiological method to pharmacologically classify ligands for gs protein-coupled receptors Download PDF

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WO2008121717A1
WO2008121717A1 PCT/US2008/058453 US2008058453W WO2008121717A1 WO 2008121717 A1 WO2008121717 A1 WO 2008121717A1 US 2008058453 W US2008058453 W US 2008058453W WO 2008121717 A1 WO2008121717 A1 WO 2008121717A1
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protein
kir
stimulatory
cells
inwardly rectifying
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French (fr)
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Uyenlinh Mirshahi
Trooraj Mirshahi
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Geisinger Ventures
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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • the present invention relates to electrophysiological methods to pharmacologically classify ligands for G s -protein coupled receptors as full agonists, partial agonists, neutral antagonists, or inverse agonists.
  • G s -protein coupled receptor signaling pathway Numerous in vitro methods currently used to functionally classify ligands that interact with G s -protein coupled receptors involve measurement of activity at the G protein level, most typically GTP ⁇ S binding and GTPase activity. Other methods test for downstream activity in the G s -protein coupled receptor signaling pathway by measuring, for example, cyclic adenosine monophosphate (cAMP) accumulation or protein kinase A activation, using techniques that include cystic fibrosis transmembrane conductance regulator (CFTR) channel activation, radioimmunoassay, and fluorescence measurements. Additionally, reporter gene assays such as beta galactosidase, beta lactamase, or luciferase assays have been used to functionally classify G s -protein coupled receptor ligands.
  • cAMP cyclic adenosine monophosphate
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Particular aspects of the invention relate to methods for functionally classifying potential agonists for a stimulatory G protein-coupled receptor that comprise providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein- coupled receptor, and at least one copy of an inwardly rectifying potassium channel; contacting the cells with a potential agonist for the stimulatory G protein-coupled receptor; measuring the current through the inwardly rectifying potassium channels; and determining whether the potential agonist is an agonist based upon the level of current measured through the inwardly rectifying potassium channels.
  • Other aspects of the invention relate to methods for functionally classifying potential inverse agonists for a stimulatory G protein-coupled receptor that comprise providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein- coupled receptor, and at least one copy of an inwardly rectifying potassium channel; contacting the cells with the potential inverse agonist for the stimulatory G protein-coupled receptor; measuring the current through the inwardly rectifying potassium channels; and determining whether the potential inverse agonist is an inverse agonist based upon the level of current measured through the inwardly rectifying potassium channels.
  • Still further aspects of the invention relate to methods for functionally classifying potential neutral antagonists for stimulatory G protein-coupled receptors that comprise providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein-coupled receptor, and at least one copy of an inwardly rectifying potassium channel; contacting the cells with an agonist for the stimulatory G protein-coupled receptor; measuring the current through the inwardly rectifying potassium channels; contacting the cells with the potential neutral antagonist for the stimulatory G protein-coupled receptor while contacting the cells with the agonist for the stimulatory G protein-coupled receptor; measuring the current through the inwardly rectifying potassium channels; and classifying the potential neutral antagonist as a neutral antagonist if contacting the cells with the potential neutral antagonist while contacting the cells with the agonist decreases the level of current measured through the inwardly rectifying potassium channels.
  • Additional aspects of the invention relate to methods for functionally classifying potential partial agonists for a stimulatory G protein-coupled receptor that comprise providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein- coupled receptor, and at least one copy of an inwardly rectifying potassium channel; contacting the cells with an agonist for the stimulatory G protein-coupled receptor; measuring the current through the inwardly rectifying potassium channels to obtain a first current reading; providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein- coupled receptor, and at least one copy of an inwardly rectifying potassium channel; contacting the cells with the potential partial agonist for the stimulatory G protein-coupled receptor; measuring the current through the inwardly rectifying potassium channels to obtain a second current reading; and determining whether the potential partial agonist is a partial agonist based upon a comparison of the first and second current readings.
  • Figure 1 depicts a representative tracing of current induced by 3 ⁇ M (R)-3,4- dihydroxy- ⁇ -(isopropylaminomethyl)benzyl alcohol hydrochloride ((-)ISO) through Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing ⁇ 2 adrenergic receptor ( ⁇ 2 AR) and the long splice variant of G ⁇ s (G ⁇ S L), as measured by conventional two-microelectrode voltage clamp.
  • Figure 2 A depicts a representative tracing of current induced by increasing concentrations of (-)ISO through Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing ⁇ 2 AR and G ⁇ S L, as measured by conventional two-microelectrode voltage clamp.
  • Figure 2B depicts a dose response curve showing the percentage of maximal induced current at increasing (-)ISO concentrations through Kir3.1/Kir3.2 channels generated from several tracings similar to the one depicted in figure 2A.
  • Figure 3 A depicts a representative tracing of current induced by increasing concentrations of (l-(3',4'-dihydroxyphenyl)-2-isopropylamino-ethanol hydrochloride) (( ⁇ )ISO) through Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing ⁇ 2 AR and G ⁇ S L, as measured by conventional two-microelectrode voltage clamp.
  • Figure 3B depicts a dose response curve showing the percentage of maximal induced current at increasing ( ⁇ )ISO concentrations through Kir3.1/Kir3.2 channels generated from several tracings similar to the one depicted in figure 3 A.
  • Figure 4 A depicts a representative tracing of current induced by increasing concentrations of (lR,2S)-(-)- ⁇ -(l-methylaminoethyl)benzyl alcohol (ephedrine or EPH) through Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing ⁇ 2 AR and G ⁇ SL , as measured by conventional two-microelectrode voltage clamp.
  • ephedrine or EPH ephedrine or EPH
  • Figure 4B depicts a dose response curve showing the percentage of maximal induced current at increasing EPH concentrations through Kir3.1/Kir3.2 channels generated from several tracings similar to the one depicted in figure 4A.
  • Figure 4C depicts a representative tracing of current induced by increasing concentrations of ( ⁇ )-3 ,4-dihydroxy-N- [3 -(4-hydroxyphenyl)- 1 -methylpropyl] - ⁇ -phenethylamine hydrochloride (dobutamine or DOB) through Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing ⁇ 2 AR and G ⁇ S L, as measured by conventional two-microelectrode voltage clamp.
  • Figure 4D depicts a dose response curve showing the percentage of maximal induced current at increasing DOB concentrations through Kir3.1/Kir3.2 generated from several tracings similar to the one depicted in figure 4C.
  • Figure 5 A depicts a representative tracing of current induced by 10 ⁇ M DOB through Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing ⁇ 2 AR and G ⁇ SL , as measured by conventional two-microelectrode voltage clamp.
  • the DOB was removed by washing the oocytes with high potassium solution (HK) containing 91 mK KCl, 1 niM NaCl, ImM MgCl 2 , and 5 mM KOH/HEPES (pH 7.4).
  • HK high potassium solution
  • (-)ISO at 3 ⁇ M was then applied to the oocytes to determine the maximal induction.
  • Figure 5B depicts a representative tracing of current induced by 30 ⁇ M EPH through Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing ⁇ 2 AR and G ⁇ SL , as measured by conventional two-microelectrode voltage clamp. Following superfusion of the oocytes with the EPH, the EPH was removed by washing the oocytes with HK. (-)ISO at 3 ⁇ M was then applied to the oocytes to determine the maximal induction.
  • Figure 5C depicts a representative tracing of current induced by 3 ⁇ M ( ⁇ )ISO through Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing ⁇ 2 AR and G ⁇ S L, as measured by conventional two-microelectrode voltage clamp. Following superfusion of the oocytes with the ( ⁇ )ISO, the ( ⁇ )ISO was removed by washing the oocytes with HK. (-)ISO at 3 ⁇ M was then applied to the oocytes to determine the maximal induction.
  • Figure 5D is a graphical presentation of the data shown in figures 5A, 5B, and 5D. To compare relative efficacy, the induced current values are shown as a percentage of maximal (-) ISO induced current (I max ).
  • Figure 5E depicts dose response curves showing (-)ISO-, ( ⁇ )ISO-, DOB-, and EPH-induced current through the Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing ⁇ 2 AR and G ⁇ S L, as measured by conventional two-microelectrode voltage clamp, at increasing concentrations of each agonist, fitted by nonlinear regression analysis. The absolute induced current from the concentration response curves for each agonist is shown.
  • Figure 5F depicts the relative efficacy of (-)ISO, ( ⁇ )ISO, DOB, and EPH compared to (-)ISO (the % (-)ISO I max ) calculated by normalizing the induced current values for each agonist to the maximal plateau of the (-)ISO concentration response curve.
  • Figure 6 depicts dose response curves showing the (-)ISO induced current through Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing ⁇ 2 AR and G ⁇ SL in the presence and absence of 500 nM ⁇ -(l-[t-butylamino]ethyl)-2,5-dimethoxy-benzyl alcohol (butoxamine or BUT) , as measured by conventional two-microelectrode voltage clamp.
  • the present invention relates to electrophysiological methods for pharmacologically classifying agonists of G s -protein coupled receptors as full agonists or partial agonists and for classifying antagonists of G s -protein coupled receptors as neutral antagonists or inverse agonists.
  • potassium current is measured through inwardly rectifying potassium channels in Xenopus laevis oocytes or transfected cell lines containing G s -protein coupled receptors and G s -proteins in the presence of ligands for the receptors, allowing the ligands to be classified as full or partial agonists, antagonists, or inverse agonists, depending upon the current measured through the inwardly rectifying potassium channels.
  • These methods can be used as secondary screening assays to pharmacologically characterize and determine the potency and efficacy of ligands that interact with G s -protein coupled receptors.
  • the present electrophysiological methods measure events that occur at the G protein level, and, as a result, minimal signal amplification occurs, allowing full agonist effects to be distinguished from partial agonist effects and neutral antagonist effects to be distinguished from inverse agonist effects.
  • the same oocyte or cell can be exposed to multiple ligands following a washout period. A single oocyte or cell can thus be exposed to an antagonist, a partial agonist, and a full agonist, allowing a pharmacological profile to be performed on the same receptor population.
  • channel activity is recorded in real time during ligand application to oocytes or to cells, eliminating the need to use extraneous agents to slow product breakdown, such as enzyme inhibitors, that may interfere with receptor-ligand binding and function.
  • each oocyte or cell serves as its own internal control, reducing the need for large replicates. Because some ligands bind reversibly, a saturating concentration of full agonist can be applied following application of a partial agonist, inverse agonist, or antagonist to determine maximal activity, and all responses can be normalized to that of the full agonist, reducing the variability often observed between samples in screening assays.
  • the inwardly rectifying potassium channels are members of the Kir3 subfamily of inwardly rectifying potassium channels.
  • the basal activity of Kir3 channels is regulated by the level of free G ⁇ Subunits. High levels of G ⁇ subunits result in large basal current, while low levels of G ⁇ subunits result in small basal current, which has been most clearly demonstrated in the Xenopus oocyte expression system, where basal current can be enhanced by co-expression of exogenous G ⁇ subunits and reduced by co-expression of proteins that bind G ⁇ ⁇ subunits.
  • Proteins that sequester free G ⁇ ⁇ subunits include G ⁇ subunits, and the level of basal channel activity can thus be controlled by expressing varying amounts of G ⁇ subunits.
  • G ⁇ subunits bound to G ⁇ subunits are released when G protein-coupled receptors are activated and enhance potassium channel activity, as manifested by increased current levels.
  • the current amplitude thus reflects the level of G ⁇ ⁇ subunits, which in turn reflects the number of receptors that were activated. Using inward current as readout, the effectiveness of different chemical agents on receptor activation can thus be determined.
  • GPCRs G protein- coupled receptors
  • PTX-sensitive G proteins which include a G ⁇ i or G ⁇ o subunit.
  • GPCRs that activate G ⁇ s can be coupled to Kir3 channel activity, however, in heterologous expression systems by introducing exogenous G ⁇ s subunits into a cell.
  • basal channel activity reflects closely the intrinsic activity of ⁇ 2 AR.
  • G ⁇ s subunits As discussed above, expression of exogenous G ⁇ s subunits will sequester G ⁇ ⁇ subunits and reduce the basal channel activity.
  • the level of expression of the G protein subunits can be adjusted to induce a basal channel current that reflects the intrinsic activity of ⁇ 2 AR.
  • Full agonists produce maximal activation of Kir 3 channels at saturating concentrations, while partial agonists produce a response that is a fraction of the response induced by a full agonist.
  • Intrinsic activity of a GPCR reflects the ability of a receptor in the APO or unoccupied state to signal to G proteins.
  • An inverse agonist binds and reduces a receptor's intrinsic activity, in contrast to a neutral antagonist that can only inhibit a receptor after it has been activated by an agonist. An inverse agonist will thus inhibit a receptor's intrinsic activity while a neutral antagonist will not.
  • agonist and “full agonist” refer to any molecule that binds to a cellular receptor, resulting in maximal activation of the receptor at a saturating concentration of the agonist, which leads to a response inside the cell.
  • the term "potential agonist” refers to any molecule that binds to a cellular receptor and may act as an agonist of the receptor, but whose physiological effect has not yet been fully characterized.
  • partial agonist refers to any molecule that binds to a cellular receptor, resulting in partial activation of the receptor as compared to the level of activation that results from the binding of a full agonist to the receptor, which leads to a response inside the cell.
  • potential partial agonist refers to any molecule that binds to a cellular receptor and may act as an partial agonist of the receptor, but whose physiological effect has not yet been fully characterized.
  • antagonist refers to any molecule that partially or fully blocks, inhibits, or neutralizes the activity of an agonist.
  • the terms “potential antagonist” and “potential neutral antagonist” refer to any molecule that binds to a cellular receptor and may act as an antagonist of the receptor, but whose physiological effect has not yet been fully characterized.
  • inverse agonist refers to any molecule that that binds to a cellular receptor and partially or fully blocks, inhibits, or neutralizes the basal, baseline, or intrinsic activity of the receptor that occurs when the receptor is in its unbound state.
  • the term "potential inverse agonist” refers to any molecule that binds to a cellular receptor and may act as an inverse agonist of the receptor, but whose physiological effect has not yet been fully characterized.
  • intrinsic current refers to the level of current measured through an inwardly rectifying potassium channel when the channel is in its resting state and has not been activated, for example, by the free G ⁇ subunits of a G protein.
  • the terms “functionally classifying” and “functionally classify” refer to characterizing a ligand for a cellular receptor as an agonist, partial agonist, neutral antagonist, or inverse agonist based upon a physiological effect produced when the ligand binds the cellular receptor.
  • contacting refers to any means that directly or indirectly cause placement together of moieties, such that the moieties come into physical contact with each other. Contacting thus includes physical acts such as placing the moieties together in a container, combining the moieties, or mixing the moieties.
  • Particular aspects of the invention relate to methods for functionally classifying ligands for stimulatory G protein-coupled receptors as agonists, partial agonists, neutral antagonists, or inverse agonists.
  • a stimulatory G protein-coupled receptor ligand is a potential agonist of the receptor
  • the methods involve, in certain aspects of the invention, providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein- coupled receptor, and at least one copy of an inwardly rectifying potassium channel.
  • the cells are then contacted with the potential agonist, and the current through the inwardly rectifying potassium channels is measured.
  • the potential agonist may or may not be classified as an agonist of the receptor.
  • the potential agonist is classified as a full or partial agonist if binding of the potential agonist to the stimulatory G protein-coupled receptor results in an increase in the level of current through the inwardly rectifying potassium channel relative to the level of instrinsic, basal, or baseline current through the channel.
  • the level of current through the inwardly rectifying potassium channels that results from binding of the potential full or partial agonist to the stimulatory G protein-coupled receptor is compared to the level of current that results from the binding of a known agonist.
  • methods for functionally classifying a potential partial agonist for a stimulatory G protein-coupled receptor involve providing cells that comprise at least one stimulatory G protein, at least one copy of a stimulatory G protein-coupled receptor, and at least one copy of an inwardly rectifying potassium channel.
  • the cells are contacted with a known agonist for the stimulatory G protein-coupled receptor, and the current through the inwardly rectifying potassium channels is measured to obtain a first current reading.
  • Cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein- coupled receptor, and at least one copy of an inwardly rectifying potassium channel are then provided, and the cells are contacted with a potential partial agonist for the stimulatory G protein-coupled receptor.
  • the current through the inwardly rectifying potassium channels is then measured to obtain a second current reading.
  • cells are first contacted with a potential partial agonist and a first current reading is obtained, and cells are then contacted with a known agonist, and a second current reading is obtained.
  • the potential partial agonist is functionally classified based upon a comparison of the first and second current readings. For example, if binding of the potential partial agonist to the stimulatory G protein-coupled receptor results in an increase in the level of current through the inwardly rectifying potassium channels that is approximately the same as the increase observed upon binding of the known agonist, the potential partial agonist is classified as a full agonist. If the increase in current through the inwardly rectifying potassium channels upon binding of the potential partial agonist to the stimulatory G protein-coupled receptor is significantly less than that observed upon binding of the known agonist, but is still greater than the level of intrinsic, basal, or baseline current through the channel, the potential partial agonist is classified as a partial agonist.
  • the cells contacted with the agonist are a first population of cells, and the cells contacted with the potential partial agonist are a second, separate population of cells.
  • the same population of cells is contacted with both the agonist and the potential partial agonist.
  • the cells are first contacted with the agonist and a first current reading through the inwardly rectifying potassium channels is obtained. The agonist is then substantially completely washed away from the cells, and the cells are contacted with the potential partial agonist. A second current reading through the inwardly rectifying potassium channels is then obtained.
  • the cells are first contacted with the potential partial agonist and a first current reading through the inwardly rectifying potassium channels is obtained.
  • the potential partial agonist is then substantially completely washed away from the cells, and the cells are contacted with the agonist.
  • a second current reading through the inwardly rectifying potassium channels is then obtained.
  • the level of current through inwardly rectifying potassium channels that results from the binding of a potential neutral antagonist to a stimulatory G protein-coupled receptor is measured following the binding of a known agonist to the same receptor.
  • certain embodiments of the invention relate to methods for functionally classifying potential neutral antagonists for stimulatory G protein-coupled receptors that comprise providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein-coupled receptor, and at least one copy of an inwardly rectifying potassium channel. The cells are then contacted with a known agonist for the stimulatory G protein-coupled receptor and the current through the inwardly rectifying potassium channels is measured.
  • the same cell population is then contacted with a potential neutral antagonist for the stimulatory G protein-coupled receptor and the current through the inwardly rectifying potassium channels is measured.
  • the potential neutral antagonist is classified as a neutral antagonist if contacting the cells with the potential neutral antagonist after contacting the cells with the known agonist decreases the level of current measured through the inwardly rectifying potassium channels.
  • Other aspects of the invention relate to methods for functionally classifying potential inverse agonists for a stimulatory G protein-coupled receptor that comprise providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein- coupled receptor, and at least one copy of an inwardly rectifying potassium channel.
  • the cells are contacted with a potential inverse agonist for the stimulatory G protein-coupled receptor and the current through the inwardly rectifying potassium channels is measured.
  • the potential inverse agonist may or may not be classified as an inverse agonist.
  • the potential inverse agonist is classified as an inverse agonist if binding of the potential inverse agonist decreases the level of current through the inwardly rectifying potassium channels relative to the basal, baseline, or intrinsic level of current through the cannels, in particular aspects of the invention.
  • the cells used are Xenopus laevis oocytes or Chinese Hamster ovary, HEK293, or COS cells. In particularly preferred embodiments, the cells are Xenopus laevis oocytes.
  • the stimulatory G protein-coupled receptor used in the methods of the invention can be any stimulatory G protein-coupled receptor.
  • the stimulatory G protein-coupled receptor is a ⁇ 2 adrenergic receptor or a melanocortin receptor.
  • the melanocortin receptor is preferably a MClR, MC3R, MC4R, or MC5R receptor.
  • the stimulatory G protein-coupled receptor is a ⁇ 2 adrenergic receptor.
  • the inwardly rectifying potassium channel used in the methods of the invention can be any G protein-sensitive inwardly rectifying potassium channel.
  • the inwardly rectifying potassium channel is a member of the Kir 3 subfamily of inwardly rectifying potassium channels.
  • the inwardly rectifying potassium channel is comprised of Kir 3.1 and Kir 3.2 subunits, Kir 3.1 and Kir 3.4 subunits, Kir 3.2 subunits, mutant Kir 3.4 S143T subunits, or mutant Kir 3.1 F137S subunits.
  • Mutant Kir 3.4 S143T subunits refer to Kir 3.4 subunits in which the serine residue at position 143 is replaced with a threonine residue
  • mutant Kir 3.1 F137S subunits refer to Kir 3.1 subunits in which the phenylalanine residue at position 137 is replaced with a serine residue, as described, for example, in M. Vivaudou et ah, J.Biol.Chem., 1997, 272, 31553-31560.
  • the inwardly rectifying potassium channel is comprised of Kir 3.1 and Kir 3.2 subunits.
  • the current through the inwardly rectifying potassium channels can be measured using any viable means for measuring current through ion channels.
  • the current through the inwardly rectifying potassium channels is measured in preferred embodiments of the invention using a two electrode voltage clamp or a whole cell patch clamp, as described, for example, in Hamill, O.P., et al, Pflugers Arch, 1981, 391, 85-100 and Mirshahi, T., et al., J Biol Chem, 2002, 277, 7348-7355, the disclosures of which are hereby incorporated herein by reference in their entireties.
  • Xenopus laevis oocytes were injected with 1.5 ng of Kir3.1 cRNA, 1.5 ng of Kir3.2 cRNA, 2 ng of ⁇ 2 AR cRNA, and 3 ng of the long splice variant of G ⁇ s (G s ⁇ L ) cRNA. Titration experiments were performed to optimize, with respect to the basal and induced currents, the amount of cRNAs used. Two days following injection, whole-oocyte currents were measured by conventional two-microelectrode voltage clamp (TEVC) with a GeneClamp 500 amplifier, digitized using a Digidata 1322 (Molecular Devices, CA), and collected on a hard drive on IBM-compatible PC running pClamp9.2 software (Molecular Devices, CA).
  • TEVC two-microelectrode voltage clamp
  • oocyte chamber was perfused with high-potassium solution (HK) containing (in mM): 91 KCl, 1 NaCl, 1 MgCl 2 , and 5 KOH/HEPES (pH 7.4). Drug solutions were made in HK and channels were blocked with 3mM BaCl 2 in HK.
  • oocytes were held at 0 mV (EK) and currents were constantly monitored by a command potential of from -100 to +100 mV with a 1 second voltage ramp (control current). Current amplitudes measured at -80 mV were plotted.
  • Figure 1 shows a representative tracing of (-) isoproterenol (ISO)-induced K + currents.
  • BaCl 2 (3mM) was used to measure the leftover current that was not inwardly rectifying (Ba-insensitive current).
  • the inwardly rectifying current is calculated by subtracting barium-insensitive current from control current.
  • the barium block indicates that these currents are barium-sensitive, a hallmark of currents through Kir channels.
  • the EC50 calculated from the nonlinear regression analysis of the concentration response curve was 1 l ⁇ l nM (figure 2B), which is comparable to the 2 nM EC50 previously reported in K. Wenzel-Seifert and R. Seifert, MoI. Pharmacol, 2000, 58, 954-966, as measured by GTP ⁇ S binding, the 13 nM EC50 previously reported in R. Seifert, K. Wenzel-Seifert, U. Gether, B. K. Kobilka, J. Pharmacol. Exp. Ther., 2001, 297, 1218-1226, as measured by GTPase activity, and the 10 nM EC50 previously reported in P. Samama, S. Cotecchia, T. Costa, R. J. Lefkowitz, J.Biol.Chem., 1993, 268, 4625-4636, as measured by cAMP activity.
  • Oocytes and solutions were prepared, and currents were measured, according to the procedures described in Example 1.
  • Representative tracings and concentration response curves were obtained for (l-(3',4'-dihydroxyphenyl)-2-isopropylamino-ethanol hydrochloride) (( ⁇ ) ISO), (lR,2S)-(-)- ⁇ -(l-methylaminoethyl)benzyl alcohol (ephedrine or EPH), and ( ⁇ )-3,4- dihydroxy-N-[3-(4-hydroxyphenyl)- 1 -methylpropyl]- ⁇ -phenethylamine hydrochloride (dobutamine or DOB), as shown in figures 3 and 4.
  • ( ⁇ ) ISO exhibits a robust induction of basal current (figure 3A).
  • EPH- and DOB-induced currents were significantly smaller than those of (-)ISO (figures 4A and 4C).
  • the EC 50 S generated from nonlinear regression analysis were 20 ⁇ l nM for ( ⁇ )ISO, 829 ⁇ 107 nM for EPH, and 400 ⁇ 107 nM for DOB.
  • EC50 values for ( ⁇ ) ISO, EPH, and DOB, measured using GTPase activity, have been reported as 150 nM, 560 nM, and 90 nM, respectively.
  • Oocytes and solutions were prepared, and currents were measured, according to the procedures described in Example 1.
  • Efficacy of a ligand describes the effect produced by the ligand upon binding to a receptor. In addition to receptor occupancy, efficacy also takes into consideration the ligand' s intrinsic activity. The efficacy of ligands for the ⁇ 2 AR was examined in two ways, by single doses and by a full dose response.
  • FIGS. 5A, 5B, and 5C show representative tracings of the efficacy studies. Setting the maximal induction response of (-)ISO at 100%, the relative induction of other compounds was quantified and compared to that of (-)ISO in the same oocyte. For example, a saturating concentration of 10 ⁇ M DOB induced a mean increase ⁇ SEM of 20 ⁇ 3% as compared to that of (-) ISO (figure 5A). Similarly, a saturating concentration of 30 ⁇ M EPH increased K + current by 9 ⁇ 4% as compared to (-) ISO (figure 5B).

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Abstract

The invention provides methods for functionally classifying ligands for stimulatory G protein-coupled receptors as full or partial agonists, inverse agonists, or antagonists that comprise providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein-coupled receptor, and at least one copy of an inwardly rectifying potassium channel; contacting the cells with a ligand for the stimulatory G protein-coupled receptor; measuring the current through the inwardly rectifying potassium channels; and determining whether ligand is full or partial agonist, inverse agonist, or antagonist based upon the level of current measured through the inwardly rectifying potassium channels.

Description

ELECTROPHYSIOLOGICAL METHOD TO PHARMACOLOGICALLY CLASSIFY LIGANDS FOR Gs PROTEIN-COUPLED RECEPTORS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. application number 60/908,835, filed March 29, 2007, which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to electrophysiological methods to pharmacologically classify ligands for Gs-protein coupled receptors as full agonists, partial agonists, neutral antagonists, or inverse agonists.
BACKGROUND OF THE INVENTION
[0003] Numerous in vitro methods currently used to functionally classify ligands that interact with Gs-protein coupled receptors involve measurement of activity at the G protein level, most typically GTPγS binding and GTPase activity. Other methods test for downstream activity in the Gs-protein coupled receptor signaling pathway by measuring, for example, cyclic adenosine monophosphate (cAMP) accumulation or protein kinase A activation, using techniques that include cystic fibrosis transmembrane conductance regulator (CFTR) channel activation, radioimmunoassay, and fluorescence measurements. Additionally, reporter gene assays such as beta galactosidase, beta lactamase, or luciferase assays have been used to functionally classify Gs-protein coupled receptor ligands.
[0004] These methods have numerous drawbacks and disadvantages, however. Some of the methods require the use of radioisotopes and most of the methods indirectly measure receptor-ligand interactions because they examine events that occur several steps downstream of the signal transduction cascade. As a result, the responses measured are sometimes artificially amplified, resulting in a higher incidence of false positives. Artificially amplified responses also make it difficult to distinguish full agonists from partial agonists and neutral antagonists from inverse agonists. W. Thomsen, J. Frazer, D. Unett, Curr .Opin. Biotechnol, 2005, 16, 655-665.
[0005] A need therefore exists in the art for methods to classify ligands for Gs-protein coupled receptors as full agonists, partial agonists, neutral antagonists, or inverse agonists that do not have the disadvantages of the methods currently used to classify Gs-protein coupled receptor ligands.
SUMMARY OF THE INVENTION
[0006] Particular aspects of the invention relate to methods for functionally classifying potential agonists for a stimulatory G protein-coupled receptor that comprise providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein- coupled receptor, and at least one copy of an inwardly rectifying potassium channel; contacting the cells with a potential agonist for the stimulatory G protein-coupled receptor; measuring the current through the inwardly rectifying potassium channels; and determining whether the potential agonist is an agonist based upon the level of current measured through the inwardly rectifying potassium channels.
[0007] Other aspects of the invention relate to methods for functionally classifying potential inverse agonists for a stimulatory G protein-coupled receptor that comprise providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein- coupled receptor, and at least one copy of an inwardly rectifying potassium channel; contacting the cells with the potential inverse agonist for the stimulatory G protein-coupled receptor; measuring the current through the inwardly rectifying potassium channels; and determining whether the potential inverse agonist is an inverse agonist based upon the level of current measured through the inwardly rectifying potassium channels. [0008] Still further aspects of the invention relate to methods for functionally classifying potential neutral antagonists for stimulatory G protein-coupled receptors that comprise providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein-coupled receptor, and at least one copy of an inwardly rectifying potassium channel; contacting the cells with an agonist for the stimulatory G protein-coupled receptor; measuring the current through the inwardly rectifying potassium channels; contacting the cells with the potential neutral antagonist for the stimulatory G protein-coupled receptor while contacting the cells with the agonist for the stimulatory G protein-coupled receptor; measuring the current through the inwardly rectifying potassium channels; and classifying the potential neutral antagonist as a neutral antagonist if contacting the cells with the potential neutral antagonist while contacting the cells with the agonist decreases the level of current measured through the inwardly rectifying potassium channels.
[0009] Additional aspects of the invention relate to methods for functionally classifying potential partial agonists for a stimulatory G protein-coupled receptor that comprise providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein- coupled receptor, and at least one copy of an inwardly rectifying potassium channel; contacting the cells with an agonist for the stimulatory G protein-coupled receptor; measuring the current through the inwardly rectifying potassium channels to obtain a first current reading; providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein- coupled receptor, and at least one copy of an inwardly rectifying potassium channel; contacting the cells with the potential partial agonist for the stimulatory G protein-coupled receptor; measuring the current through the inwardly rectifying potassium channels to obtain a second current reading; and determining whether the potential partial agonist is a partial agonist based upon a comparison of the first and second current readings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Figure 1 depicts a representative tracing of current induced by 3 μM (R)-3,4- dihydroxy-α-(isopropylaminomethyl)benzyl alcohol hydrochloride ((-)ISO) through Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing β2 adrenergic receptor (β2AR) and the long splice variant of Gαs (GαSL), as measured by conventional two-microelectrode voltage clamp. [0011] Figure 2 A depicts a representative tracing of current induced by increasing concentrations of (-)ISO through Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing β2AR and GαSL, as measured by conventional two-microelectrode voltage clamp.
[0012] Figure 2B depicts a dose response curve showing the percentage of maximal induced current at increasing (-)ISO concentrations through Kir3.1/Kir3.2 channels generated from several tracings similar to the one depicted in figure 2A.
[0013] Figure 3 A depicts a representative tracing of current induced by increasing concentrations of (l-(3',4'-dihydroxyphenyl)-2-isopropylamino-ethanol hydrochloride) ((±)ISO) through Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing β2AR and GαSL, as measured by conventional two-microelectrode voltage clamp.
[0014] Figure 3B depicts a dose response curve showing the percentage of maximal induced current at increasing (±)ISO concentrations through Kir3.1/Kir3.2 channels generated from several tracings similar to the one depicted in figure 3 A.
[0015] Figure 4 A depicts a representative tracing of current induced by increasing concentrations of (lR,2S)-(-)-α-(l-methylaminoethyl)benzyl alcohol (ephedrine or EPH) through Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing β2AR and GαSL, as measured by conventional two-microelectrode voltage clamp.
[0016] Figure 4B depicts a dose response curve showing the percentage of maximal induced current at increasing EPH concentrations through Kir3.1/Kir3.2 channels generated from several tracings similar to the one depicted in figure 4A.
[0017] Figure 4C depicts a representative tracing of current induced by increasing concentrations of (±)-3 ,4-dihydroxy-N- [3 -(4-hydroxyphenyl)- 1 -methylpropyl] -β-phenethylamine hydrochloride (dobutamine or DOB) through Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing β2AR and GαSL, as measured by conventional two-microelectrode voltage clamp.
[0018] Figure 4D depicts a dose response curve showing the percentage of maximal induced current at increasing DOB concentrations through Kir3.1/Kir3.2 generated from several tracings similar to the one depicted in figure 4C.
[0019] Figure 5 A depicts a representative tracing of current induced by 10 μM DOB through Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing β2AR and GαSL, as measured by conventional two-microelectrode voltage clamp. Following superfusion of the oocytes with the DOB, the DOB was removed by washing the oocytes with high potassium solution (HK) containing 91 mK KCl, 1 niM NaCl, ImM MgCl2, and 5 mM KOH/HEPES (pH 7.4). (-)ISO at 3 μM was then applied to the oocytes to determine the maximal induction.
[0020] Figure 5B depicts a representative tracing of current induced by 30 μM EPH through Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing β2AR and GαSL, as measured by conventional two-microelectrode voltage clamp. Following superfusion of the oocytes with the EPH, the EPH was removed by washing the oocytes with HK. (-)ISO at 3 μM was then applied to the oocytes to determine the maximal induction.
[0021] Figure 5C depicts a representative tracing of current induced by 3 μM (±)ISO through Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing β2AR and GαSL, as measured by conventional two-microelectrode voltage clamp. Following superfusion of the oocytes with the (±)ISO, the (±)ISO was removed by washing the oocytes with HK. (-)ISO at 3 μM was then applied to the oocytes to determine the maximal induction.
[0022] Figure 5D is a graphical presentation of the data shown in figures 5A, 5B, and 5D. To compare relative efficacy, the induced current values are shown as a percentage of maximal (-) ISO induced current (Imax).
[0023] Figure 5E depicts dose response curves showing (-)ISO-, (±)ISO-, DOB-, and EPH-induced current through the Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing β2AR and GαSL, as measured by conventional two-microelectrode voltage clamp, at increasing concentrations of each agonist, fitted by nonlinear regression analysis. The absolute induced current from the concentration response curves for each agonist is shown.
[0024] Figure 5F depicts the relative efficacy of (-)ISO, (±)ISO, DOB, and EPH compared to (-)ISO (the % (-)ISO Imax) calculated by normalizing the induced current values for each agonist to the maximal plateau of the (-)ISO concentration response curve.
[0025] Figure 6 depicts dose response curves showing the (-)ISO induced current through Kir3.1/Kir3.2 channels in Xenopus laevis oocytes containing β2AR and GαSL in the presence and absence of 500 nM α-(l-[t-butylamino]ethyl)-2,5-dimethoxy-benzyl alcohol (butoxamine or BUT) , as measured by conventional two-microelectrode voltage clamp.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0026] The present invention relates to electrophysiological methods for pharmacologically classifying agonists of Gs-protein coupled receptors as full agonists or partial agonists and for classifying antagonists of Gs-protein coupled receptors as neutral antagonists or inverse agonists. Specifically, potassium current is measured through inwardly rectifying potassium channels in Xenopus laevis oocytes or transfected cell lines containing Gs-protein coupled receptors and Gs-proteins in the presence of ligands for the receptors, allowing the ligands to be classified as full or partial agonists, antagonists, or inverse agonists, depending upon the current measured through the inwardly rectifying potassium channels. These methods can be used as secondary screening assays to pharmacologically characterize and determine the potency and efficacy of ligands that interact with Gs-protein coupled receptors.
[0027] The present electrophysiological methods measure events that occur at the G protein level, and, as a result, minimal signal amplification occurs, allowing full agonist effects to be distinguished from partial agonist effects and neutral antagonist effects to be distinguished from inverse agonist effects. In addition, because some ligands bind to receptors in a reversible manner, the same oocyte or cell can be exposed to multiple ligands following a washout period. A single oocyte or cell can thus be exposed to an antagonist, a partial agonist, and a full agonist, allowing a pharmacological profile to be performed on the same receptor population. Moreover, channel activity is recorded in real time during ligand application to oocytes or to cells, eliminating the need to use extraneous agents to slow product breakdown, such as enzyme inhibitors, that may interfere with receptor-ligand binding and function. Furthermore, each oocyte or cell serves as its own internal control, reducing the need for large replicates. Because some ligands bind reversibly, a saturating concentration of full agonist can be applied following application of a partial agonist, inverse agonist, or antagonist to determine maximal activity, and all responses can be normalized to that of the full agonist, reducing the variability often observed between samples in screening assays.
[0028] In certain embodiments of the invention, the inwardly rectifying potassium channels are members of the Kir3 subfamily of inwardly rectifying potassium channels. In the absence of receptor activation, the basal activity of Kir3 channels is regulated by the level of free GβγSubunits. High levels of Gβγ subunits result in large basal current, while low levels of Gβγ subunits result in small basal current, which has been most clearly demonstrated in the Xenopus oocyte expression system, where basal current can be enhanced by co-expression of exogenous Gβγ subunits and reduced by co-expression of proteins that bind Gβγ subunits. Proteins that sequester free Gβγ subunits include Gα subunits, and the level of basal channel activity can thus be controlled by expressing varying amounts of Gα subunits. [0029] Gβγ subunits bound to Gα subunits are released when G protein-coupled receptors are activated and enhance potassium channel activity, as manifested by increased current levels. The current amplitude thus reflects the level of Gβγ subunits, which in turn reflects the number of receptors that were activated. Using inward current as readout, the effectiveness of different chemical agents on receptor activation can thus be determined.
[0030] Activation of Kir 3 channels in native tissue is mediated through G protein- coupled receptors (GPCRs) that couple to the pertussis toxin-sensitive (PTX-sensitive) G proteins, which include a Gαi or Gαo subunit. GPCRs that activate Gαs (a PTX-insensitive G protein) can be coupled to Kir3 channel activity, however, in heterologous expression systems by introducing exogenous Gαs subunits into a cell. In oocytes expressing β2AR, Gαs, Kir3.1 and Kir3.2, basal channel activity reflects closely the intrinsic activity of β2AR. As discussed above, expression of exogenous Gαs subunits will sequester Gβγ subunits and reduce the basal channel activity. The level of expression of the G protein subunits can be adjusted to induce a basal channel current that reflects the intrinsic activity of β2AR.
[0031] Full agonists produce maximal activation of Kir 3 channels at saturating concentrations, while partial agonists produce a response that is a fraction of the response induced by a full agonist. Intrinsic activity of a GPCR reflects the ability of a receptor in the APO or unoccupied state to signal to G proteins. An inverse agonist binds and reduces a receptor's intrinsic activity, in contrast to a neutral antagonist that can only inhibit a receptor after it has been activated by an agonist. An inverse agonist will thus inhibit a receptor's intrinsic activity while a neutral antagonist will not.
[0032] As used herein, the terms "agonist" and "full agonist" refer to any molecule that binds to a cellular receptor, resulting in maximal activation of the receptor at a saturating concentration of the agonist, which leads to a response inside the cell.
[0033] As used herein, the term "potential agonist" refers to any molecule that binds to a cellular receptor and may act as an agonist of the receptor, but whose physiological effect has not yet been fully characterized.
[0034] As used herein, the term "partial agonist" refers to any molecule that binds to a cellular receptor, resulting in partial activation of the receptor as compared to the level of activation that results from the binding of a full agonist to the receptor, which leads to a response inside the cell. [0035] As used herein, the term "potential partial agonist" refers to any molecule that binds to a cellular receptor and may act as an partial agonist of the receptor, but whose physiological effect has not yet been fully characterized.
[0036] As used herein, the terms "antagonist" and "neutral antagonist" refer to any molecule that partially or fully blocks, inhibits, or neutralizes the activity of an agonist.
[0037] As used herein, the terms "potential antagonist" and "potential neutral antagonist" refer to any molecule that binds to a cellular receptor and may act as an antagonist of the receptor, but whose physiological effect has not yet been fully characterized.
[0038] As used herein, the term "inverse agonist" refers to any molecule that that binds to a cellular receptor and partially or fully blocks, inhibits, or neutralizes the basal, baseline, or intrinsic activity of the receptor that occurs when the receptor is in its unbound state.
[0039] As used herein, the term "potential inverse agonist" refers to any molecule that binds to a cellular receptor and may act as an inverse agonist of the receptor, but whose physiological effect has not yet been fully characterized.
[0040] As used herein, the term "intrinsic current" refers to the level of current measured through an inwardly rectifying potassium channel when the channel is in its resting state and has not been activated, for example, by the free Gβγ subunits of a G protein.
[0041] As used herein, the terms "functionally classifying" and "functionally classify" refer to characterizing a ligand for a cellular receptor as an agonist, partial agonist, neutral antagonist, or inverse agonist based upon a physiological effect produced when the ligand binds the cellular receptor.
[0042] As used herein, the terms "contacting," "contact," and all variations thereof, refer to any means that directly or indirectly cause placement together of moieties, such that the moieties come into physical contact with each other. Contacting thus includes physical acts such as placing the moieties together in a container, combining the moieties, or mixing the moieties.
[0043] Particular aspects of the invention relate to methods for functionally classifying ligands for stimulatory G protein-coupled receptors as agonists, partial agonists, neutral antagonists, or inverse agonists. If a stimulatory G protein-coupled receptor ligand is a potential agonist of the receptor, the methods involve, in certain aspects of the invention, providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein- coupled receptor, and at least one copy of an inwardly rectifying potassium channel. The cells are then contacted with the potential agonist, and the current through the inwardly rectifying potassium channels is measured. Depending upon the level of current measured, the potential agonist may or may not be classified as an agonist of the receptor. For example, if binding of the potential agonist to the stimulatory G protein-coupled receptor results in an increase in the level of current through the inwardly rectifying potassium channel relative to the level of instrinsic, basal, or baseline current through the channel, the potential agonist is classified as a full or partial agonist.
[0044] To distinguish full from partial agonists, in particular embodiments of the invention, the level of current through the inwardly rectifying potassium channels that results from binding of the potential full or partial agonist to the stimulatory G protein-coupled receptor is compared to the level of current that results from the binding of a known agonist. For example, in certain embodiments of the invention, methods for functionally classifying a potential partial agonist for a stimulatory G protein-coupled receptor involve providing cells that comprise at least one stimulatory G protein, at least one copy of a stimulatory G protein-coupled receptor, and at least one copy of an inwardly rectifying potassium channel. The cells are contacted with a known agonist for the stimulatory G protein-coupled receptor, and the current through the inwardly rectifying potassium channels is measured to obtain a first current reading. Cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein- coupled receptor, and at least one copy of an inwardly rectifying potassium channel are then provided, and the cells are contacted with a potential partial agonist for the stimulatory G protein-coupled receptor. The current through the inwardly rectifying potassium channels is then measured to obtain a second current reading. Alternatively, cells are first contacted with a potential partial agonist and a first current reading is obtained, and cells are then contacted with a known agonist, and a second current reading is obtained.
[0045] The potential partial agonist is functionally classified based upon a comparison of the first and second current readings. For example, if binding of the potential partial agonist to the stimulatory G protein-coupled receptor results in an increase in the level of current through the inwardly rectifying potassium channels that is approximately the same as the increase observed upon binding of the known agonist, the potential partial agonist is classified as a full agonist. If the increase in current through the inwardly rectifying potassium channels upon binding of the potential partial agonist to the stimulatory G protein-coupled receptor is significantly less than that observed upon binding of the known agonist, but is still greater than the level of intrinsic, basal, or baseline current through the channel, the potential partial agonist is classified as a partial agonist.
[0046] In particular aspects of the invention, the cells contacted with the agonist are a first population of cells, and the cells contacted with the potential partial agonist are a second, separate population of cells. In other embodiments of the invention, if either the agonist or the potential partial agonist binds reversibly to the G protein-coupled receptor, the same population of cells is contacted with both the agonist and the potential partial agonist. In certain aspects of such embodiments, the cells are first contacted with the agonist and a first current reading through the inwardly rectifying potassium channels is obtained. The agonist is then substantially completely washed away from the cells, and the cells are contacted with the potential partial agonist. A second current reading through the inwardly rectifying potassium channels is then obtained. Alternatively, the cells are first contacted with the potential partial agonist and a first current reading through the inwardly rectifying potassium channels is obtained. The potential partial agonist is then substantially completely washed away from the cells, and the cells are contacted with the agonist. A second current reading through the inwardly rectifying potassium channels is then obtained.
[0047] To functionally classify potential neutral antagonists, in particular embodiments of the invention, the level of current through inwardly rectifying potassium channels that results from the binding of a potential neutral antagonist to a stimulatory G protein-coupled receptor is measured following the binding of a known agonist to the same receptor. Accordingly, certain embodiments of the invention relate to methods for functionally classifying potential neutral antagonists for stimulatory G protein-coupled receptors that comprise providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein-coupled receptor, and at least one copy of an inwardly rectifying potassium channel. The cells are then contacted with a known agonist for the stimulatory G protein-coupled receptor and the current through the inwardly rectifying potassium channels is measured. The same cell population is then contacted with a potential neutral antagonist for the stimulatory G protein-coupled receptor and the current through the inwardly rectifying potassium channels is measured. The potential neutral antagonist is classified as a neutral antagonist if contacting the cells with the potential neutral antagonist after contacting the cells with the known agonist decreases the level of current measured through the inwardly rectifying potassium channels. [0048] Other aspects of the invention relate to methods for functionally classifying potential inverse agonists for a stimulatory G protein-coupled receptor that comprise providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein- coupled receptor, and at least one copy of an inwardly rectifying potassium channel. The cells are contacted with a potential inverse agonist for the stimulatory G protein-coupled receptor and the current through the inwardly rectifying potassium channels is measured. Depending upon the level of current measured through the inwardly rectifying potassium channels, the potential inverse agonist may or may not be classified as an inverse agonist. For example, if binding of the potential inverse agonist decreases the level of current through the inwardly rectifying potassium channels relative to the basal, baseline, or intrinsic level of current through the cannels, in particular aspects of the invention, the potential inverse agonist is classified as an inverse agonist.
[0049] Any cell type can be used in the methods of the invention. In preferred embodiments, the cells used are Xenopus laevis oocytes or Chinese Hamster ovary, HEK293, or COS cells. In particularly preferred embodiments, the cells are Xenopus laevis oocytes.
[0050] The stimulatory G protein-coupled receptor used in the methods of the invention can be any stimulatory G protein-coupled receptor. In preferred embodiments of the invention, the stimulatory G protein-coupled receptor is a β2 adrenergic receptor or a melanocortin receptor. The melanocortin receptor is preferably a MClR, MC3R, MC4R, or MC5R receptor. In particularly preferred embodiments of the invention, the stimulatory G protein-coupled receptor is a β2 adrenergic receptor.
[0051] The inwardly rectifying potassium channel used in the methods of the invention can be any G protein-sensitive inwardly rectifying potassium channel. In preferred embodiments, the inwardly rectifying potassium channel is a member of the Kir 3 subfamily of inwardly rectifying potassium channels. In certain aspects of the invention, the inwardly rectifying potassium channel is comprised of Kir 3.1 and Kir 3.2 subunits, Kir 3.1 and Kir 3.4 subunits, Kir 3.2 subunits, mutant Kir 3.4 S143T subunits, or mutant Kir 3.1 F137S subunits. Mutant Kir 3.4 S143T subunits refer to Kir 3.4 subunits in which the serine residue at position 143 is replaced with a threonine residue, and mutant Kir 3.1 F137S subunits refer to Kir 3.1 subunits in which the phenylalanine residue at position 137 is replaced with a serine residue, as described, for example, in M. Vivaudou et ah, J.Biol.Chem., 1997, 272, 31553-31560. In particularly preferred embodiments of the invention, the inwardly rectifying potassium channel is comprised of Kir 3.1 and Kir 3.2 subunits.
[0052] The current through the inwardly rectifying potassium channels can be measured using any viable means for measuring current through ion channels. The current through the inwardly rectifying potassium channels is measured in preferred embodiments of the invention using a two electrode voltage clamp or a whole cell patch clamp, as described, for example, in Hamill, O.P., et al, Pflugers Arch, 1981, 391, 85-100 and Mirshahi, T., et al., J Biol Chem, 2002, 277, 7348-7355, the disclosures of which are hereby incorporated herein by reference in their entireties.
[0053] The following examples are illustrative of certain embodiments of the invention and should not be considered to limit the scope of the invention.
Example 1: Measurement of (-) Isoproterenol-Induced Potassium Currents Through Kir3.1/3.2 Channels
[0054] Xenopus laevis oocytes were injected with 1.5 ng of Kir3.1 cRNA, 1.5 ng of Kir3.2 cRNA, 2 ng of β2AR cRNA, and 3 ng of the long splice variant of Gαs (GsαL) cRNA. Titration experiments were performed to optimize, with respect to the basal and induced currents, the amount of cRNAs used. Two days following injection, whole-oocyte currents were measured by conventional two-microelectrode voltage clamp (TEVC) with a GeneClamp 500 amplifier, digitized using a Digidata 1322 (Molecular Devices, CA), and collected on a hard drive on IBM-compatible PC running pClamp9.2 software (Molecular Devices, CA). Data were analyzed using Clampfit9 and Origin 7.5 (OriginLab Corp., MA). Agarose-cushioned microelectrodes were used with resistances between 0.1 to 1.0 MΩ W. Schreibmayer, H. A. Lester, N. Dascal, Pflugers Arch., 1994, 426, 453-458. Oocytes were constantly superfused with a low-potassium solution (LK) containing (in mM): 91 NaCl, 1 KCl, 1 MgCl2, and 5 NaOH/HEPES (pH 7.4). To measure inward currents, the oocyte chamber was perfused with high-potassium solution (HK) containing (in mM): 91 KCl, 1 NaCl, 1 MgCl2, and 5 KOH/HEPES (pH 7.4). Drug solutions were made in HK and channels were blocked with 3mM BaCl2 in HK. Typically, oocytes were held at 0 mV (EK) and currents were constantly monitored by a command potential of from -100 to +100 mV with a 1 second voltage ramp (control current). Current amplitudes measured at -80 mV were plotted. Figure 1 shows a representative tracing of (-) isoproterenol (ISO)-induced K+ currents. As shown, typically approximately 5 to 8 μA basal currents were obtained, depending on the efficiency of expression. Application of 3μM (-) ISO to oocytes typically caused a 6 to 8 fold increase in basal current, followed by a complete washout in HK solution.
[0055] BaCl2 (3mM) was used to measure the leftover current that was not inwardly rectifying (Ba-insensitive current). The inwardly rectifying current is calculated by subtracting barium-insensitive current from control current. The barium block indicates that these currents are barium-sensitive, a hallmark of currents through Kir channels.
Example 2: Determination of the Concentration Response of Kir3.1/3.2 Channels to (-) Isoproterenol
[0056] Oocytes and solutions were prepared, and currents were measured, according to the procedures described in Example 1. To determine the EC50 of (-) ISO, increasing concentrations of (-) ISO were applied to a single oocyte following perfusion in HK solution. Recordings were taken with application of (-) ISO at increasing concentration followed by Ba2+ block, on oocytes from two to four different frogs. As shown by the representative TEVC tracing in figure 2A, (-) ISO-induced currents are dose-responsive and saturable in the recording system used. A dose response curve was generated using nonlinear regression analysis by GraphPad Prism 4 software (GraphPad, CA). The EC50 calculated from the nonlinear regression analysis of the concentration response curve was 1 l±l nM (figure 2B), which is comparable to the 2 nM EC50 previously reported in K. Wenzel-Seifert and R. Seifert, MoI. Pharmacol, 2000, 58, 954-966, as measured by GTPγS binding, the 13 nM EC50 previously reported in R. Seifert, K. Wenzel-Seifert, U. Gether, B. K. Kobilka, J. Pharmacol. Exp. Ther., 2001, 297, 1218-1226, as measured by GTPase activity, and the 10 nM EC50 previously reported in P. Samama, S. Cotecchia, T. Costa, R. J. Lefkowitz, J.Biol.Chem., 1993, 268, 4625-4636, as measured by cAMP activity.
Example 3: Determination of the Concentration Response of Kir3.1/3.2 Channels to (±) Isoproterenol, Ephedrine, and Dobutamine
[0057] Oocytes and solutions were prepared, and currents were measured, according to the procedures described in Example 1. Representative tracings and concentration response curves were obtained for (l-(3',4'-dihydroxyphenyl)-2-isopropylamino-ethanol hydrochloride) ((±) ISO), (lR,2S)-(-)-α-(l-methylaminoethyl)benzyl alcohol (ephedrine or EPH), and (±)-3,4- dihydroxy-N-[3-(4-hydroxyphenyl)- 1 -methylpropyl]-β-phenethylamine hydrochloride (dobutamine or DOB), as shown in figures 3 and 4. Similar to (-) ISO, (±) ISO exhibits a robust induction of basal current (figure 3A). In contrast, EPH- and DOB-induced currents were significantly smaller than those of (-)ISO (figures 4A and 4C). The EC50S generated from nonlinear regression analysis were 20±l nM for (±)ISO, 829±107 nM for EPH, and 400±107 nM for DOB. EC50 values for (±) ISO, EPH, and DOB, measured using GTPase activity, have been reported as 150 nM, 560 nM, and 90 nM, respectively. Furthermore, EC50S of for EPH and DOB, measured by GTPγS binding in Sf9 membranes expressing P2AR-G8L fusion proteins, have been reported as 923 nM and 90 nM, respectively. K. Wenzel-Seifert and R. Seifert, Mol.Pharmacol, 2000, 58, 954-966.
Example 4: Determination of the Efficacy of β∑AR Agonists
[0058] Oocytes and solutions were prepared, and currents were measured, according to the procedures described in Example 1. Efficacy of a ligand describes the effect produced by the ligand upon binding to a receptor. In addition to receptor occupancy, efficacy also takes into consideration the ligand' s intrinsic activity. The efficacy of ligands for the β2AR was examined in two ways, by single doses and by a full dose response.
[0059] For the single dose analysis, saturating but non-desensitizing concentrations of the ligands were applied to each oocyte, with application of (-) ISO serving as an internal control. From the dose response curve, 3μM (-) ISO was selected to be the reference point for determining maximal activity. At this concentration, (-) ISO increases K+ current by as much as 6 fold from basal level, depending mainly on the expression levels of channels, β2AR, and GαSL in oocytes. All recordings were performed by superfusion of oocytes with test compounds at the concentrations shown in figures 5 A to 5 C followed by complete wash out of the test compounds with HK, and then application of the reference compound (3μM (-) ISO). When a test compound could not be completely washed out, (-) ISO was applied first. Figures 5A, 5B, and 5C show representative tracings of the efficacy studies. Setting the maximal induction response of (-)ISO at 100%, the relative induction of other compounds was quantified and compared to that of (-)ISO in the same oocyte. For example, a saturating concentration of 10 μM DOB induced a mean increase ±SEM of 20±3% as compared to that of (-) ISO (figure 5A). Similarly, a saturating concentration of 30 μM EPH increased K+ current by 9±4% as compared to (-) ISO (figure 5B). At equiconcentration, the racemic form of (-) ISO, (±) ISO, showed a similar increase in induced current (figure 5C). Figure 5D summarizes the data from the tracings. To compare relative efficacy, the induced current values are shown as a percentage of maximal (-)ISO-induced current (IM3X)-
[0060] Ligand efficacy was then examined using the full concentration response curve. The absolute induced current from the concentration response curve of each agonist fitted by nonlinear regression analysis is depicted in figure 5E. The relative efficacy of the agonists compared to (-)ISO (% (-)ISO IM3X) was calculated by dividing each data point by the maximal plateau value of (-)ISO generated from the nonlinear regression analysis (figure 5F). The relative efficacies of (±)ISO, DOB, and EPH were 87±7%, 25±3%, and 23±3%, respectively. Within experimental variability, results for (±) ISO and DOB from the full concentration response experiments were similar to those obtained from the single dose analysis. However, the relative efficacies determined for EPH were significantly different using the two techniques. The differences can be explained by the small sample size in the single dose analysis (n=4). Nonetheless, the data indicate that the two isoforms of ISO are full agonists, and EPH and DOB are partial agonists at β2AR. The rank order of efficacies for the agonists at β2AR according to these results was (-)ISO « (±)ISO > DOB > EPH. The efficacies of DOB and EPH for stimulating GTPγS binding to β2AR-GsαL fusion proteins expressed in Sf9 membranes have been reported as 98% and 87%, respectively, as compared to (-)ISO by K. Wenzel-Seifert and R. Seifert, MoI. Pharmacol, 2000, 58, 954-966.
[0061] Furthermore, the efficacies of (±)ISO, DOB, and EPH for inducing GTPase activity in the same cell membrane have been reported as 91%, 76%, and 66%, respectively, compared to (-)ISO by R. Seifert, K. Wenzel-Seifert, U. Gether, B. K. Kobilka, J. Pharmacol. Exp. Ther., 2001, 297, 1218-1226. Although the rank order of these efficacies is similar to the rank order of the efficacies determined from the experiments described above, the reported studies used fusion proteins between β2AR and Gαs that had been previously shown to possess distinct pharmacological profiles, so it is plausible that the reported values are higher than those generated from the above experiments because of differences in the assays employed. The efficacies generated from the above experiments were determined using internal controls for receptor population, G protein levels, and effector expression. Example 5: Determination of the Effectiveness of a β∑AR Antagonist
[0062] Oocytes and solutions were prepared, and currents were measured, according to the procedures described in Example 1. The potency of the β2AR specific antagonist α-(l-[t- butylamino]ethyl)-2,5-dimethoxy-benzyl alcohol (butoxamine or BUT) was determined. Butoxamine (lμM) caused no change in basal K+ current (data not shown). However, 500 nM BUT shifted rightward the concentration response curve of (-)ISO (figure 6). The EC50 values determined from nonlinear regression analysis were 1 l±l nM (n = 4) for (-)ISO alone, and 28±4 nM (n = 8) for (-)ISO + BUT. These data indicate that BUT acts as a neutral antagonist of β2AR.
[0063] The entire disclosure of each patent, patent application, and publication cited or described in this document is hereby incorporated herein by reference.

Claims

We Claim:
1. A method for functionally classifying a potential agonist for a stimulatory G protein- coupled receptor comprising: providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein-coupled receptor, and at least one copy of an inwardly rectifying potassium channel; contacting the cells with the potential agonist for the stimulatory G protein-coupled receptor; measuring the current through the inwardly rectifying potassium channels; and determining whether the potential agonist is an agonist based upon the level of current measured through the inwardly rectifying potassium channels.
2. The method of claim 1 wherein the potential agonist is determined to be an agonist if the level of current measured through the inwardly rectifying potassium channels when the cells are contacted with the potential agonist is greater than the intrinsic current measured through the inwardly rectifying potassium channels when the stimulatory G protein-coupled receptors are unoccupied.
3. The method of claim 1 wherein the cells are Xenopus laevis oocytes or Chinese Hamster ovary, HEK293, or COS cells.
4. The method of claim 3 wherein the cells are Xenopus laevis oocytes.
5. The method of claim 1 wherein the stimulatory G protein-coupled receptor is a β2 adrenergic receptor or a melanocortin receptor.
6. The method of claim 5 wherein the melanocortin receptor is MClR, MC3R, MC4R, or MC5R.
7. The method of claim 5 wherein the stimulatory G protein-coupled receptor is a β2 adrenergic receptor.
8. The method of claim 1 wherein the inwardly rectifying potassium channel is a member of the Kir 3 subfamily of inwardly rectifying potassium channels.
9. The method of claim 8 wherein the inwardly rectifying potassium channel is comprised of Kir 3.1 and Kir 3.2 subunits, Kir 3.1 and Kir 3.4 subunits, Kir 3.2 subunits, mutant Kir 3.4 S143T subunits, or mutant Kir 3.1 F137S subunits.
10. The method of claim 9 wherein the inwardly rectifying potassium channel is comprised of Kir 3.1 and Kir 3.2 subunits.
11. The method of claim 1 wherein the current through the inwardly rectifying potassium channels is measured using a two electrode voltage clamp or a whole cell patch clamp.
12. A method for functionally classifying a potential inverse agonist for a stimulatory G- protein coupled receptor comprising: providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein-coupled receptor, and at least one copy of an inwardly rectifying potassium channel; contacting the cells with the potential inverse agonist for the stimulatory G protein- coupled receptor; measuring the current through the inwardly rectifying potassium channels; and determining whether the potential inverse agonist is an inverse agonist based upon the level of current measured through the inwardly rectifying potassium channels.
13. The method of claim 1 wherein the potential inverse agonist is determined to be an inverse agonist if the level of current measured through the inwardly rectifying potassium channels when the cells are contacted with the potential inverse agonist is less than the intrinsic current measured through the inwardly rectifying potassium channels when the stimulatory G protein-coupled receptors are unoccupied.
14. The method of claim 12 wherein the cells are Xenopus laevis oocytes or Chinese Hamster ovary, HEK293, or COS cells.
15. The method of claim 14 wherein the cells are Xenopus laevis oocytes.
16. The method of claim 12 wherein the stimulatory G protein-coupled receptor is a β2 adrenergic receptor or a melanocortin receptor.
17. The method of claim 16 wherein the melanocortin receptor is MClR, MC3R, MC4R, or MC5R.
18. The method of claim 16 wherein the stimulatory G protein-coupled receptor is a β2 adrenergic receptor.
19. The method of claim 12 wherein the inwardly rectifying potassium channel is a member of the Kir 3 subfamily of inwardly rectifying potassium channels.
20. The method of claim 19 wherein the inwardly rectifying potassium channel is comprised of Kir 3.1 and Kir 3.2 subunits, Kir 3.1 and Kir 3.4 subunits, Kir 3.2 subunits, mutant Kir 3.4 S143T subunits, or mutant Kir 3.1 F137S subunits.
21. The method of claim 20 wherein the inwardly rectifying potassium channel is comprised of Kir 3.1 and Kir 3.2 subunits.
22. The method of claim 11 wherein the current through the inwardly rectifying potassium channels is measured using a two electrode voltage clamp or a whole cell patch clamp.
23. A method for functionally classifying a potential neutral antagonist for a stimulatory G- protein coupled receptor comprising: providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein-coupled receptor, and at least one copy of an inwardly rectifying potassium channel; contacting the cells with an agonist for the stimulatory G protein-coupled receptor; measuring the current through the inwardly rectifying potassium channels; contacting the cells with the potential neutral antagonist for the stimulatory G protein- coupled receptor while contacting the cells with the agonist for the stimulatory G protein- coupled receptor; measuring the current through the inwardly rectifying potassium channels; and classifying the potential neutral antagonist as a neutral antagonist if contacting the cells with the potential neutral antagonist after contacting the cells with the agonist decreases the level of current measured through the inwardly rectifying potassium channels.
24. The method of claim 23 wherein the cells are Xenopus laevis oocytes or Chinese Hamster ovary, HEK293, or COS cells.
25. The method of claim 24 wherein the cells are Xenopus laevis oocytes.
26. The method of claim 23 wherein the stimulatory G protein-coupled receptor is a β2 adrenergic receptor or a melanocortin receptor.
27. The method of claim 26 wherein the melanocortin receptor is MClR, MC3R, MC4R, or MC5R.
28. The method of claim 26 wherein the stimulatory G protein-coupled receptor is a β2 adrenergic receptor.
29. The method of claim 23 wherein the inwardly rectifying potassium channel is a member of the Kir 3 subfamily of inwardly rectifying potassium channels.
30. The method of claim 29 wherein the inwardly rectifying potassium channel is comprised of Kir 3.1 and Kir 3.2 subunits, Kir 3.1 and Kir 3.4 subunits, Kir 3.2 subunits, mutant Kir 3.4 S143T subunits, or mutant Kir 3.1 F137S subunits.
31. The method of claim 30 wherein the inwardly rectifying potassium channel is comprised of Kir 3.1 and Kir 3.2 subunits.
32. The method of claim 23 wherein the current through the inwardly rectifying potassium channels is measured using a two electrode voltage clamp or a whole cell patch clamp.
33. A method for functionally classifying a potential partial agonist for a stimulatory G- protein coupled receptor comprising: providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein-coupled receptor, and at least one copy of an inwardly rectifying potassium channel; contacting the cells with an agonist for the stimulatory G protein-coupled receptor; measuring the current through the inwardly rectifying potassium channels to obtain a first current reading; providing cells comprising at least one stimulatory G protein, at least one copy of a stimulatory G protein-coupled receptor, and at least one copy of an inwardly rectifying potassium channel; contacting the cells with the potential partial agonist for the stimulatory G protein- coupled receptor; measuring the current through the inwardly rectifying potassium channels to obtain a second current reading; and determining whether the potential partial agonist is a partial agonist based upon a comparison of the first and second current readings.
34. The method of claim 33 wherein the potential partial agonist is determined to be a partial agonist if the second current reading is less than the first current reading but is greater than the intrinsic current measured through the inwardly rectifying potassium channels when the stimulatory G protein-coupled receptors are unoccupied.
35. The method of claim 33 wherein the cells contacted with the agonist are a first population of cells and the cells contacted with the potential partial agonist are a second, separate population of cells.
36. The method of claim 33 wherein the population of cells contacted with the agonist is the same population of cells contacted with the potential partial agonist.
37. The method of claim 33 wherein the cells are Xenopus laevis oocytes or Chinese Hamster ovary, HEK293, or COS cells.
38. The method of claim 37 wherein the cells are Xenopus laevis oocytes.
39. The method of claim 33 wherein the stimulatory G protein-coupled receptor is a β2 adrenergic receptor or a melanocortin receptor.
40. The method of claim 39 wherein the melanocortin receptor is MClR, MC3R, MC4R, or MC5R.
41. The method of claim 39 wherein the stimulatory G protein-coupled receptor is a β2 adrenergic receptor.
42. The method of claim 33 wherein the inwardly rectifying potassium channel is a member of the Kir 3 subfamily of inwardly rectifying potassium channels.
43. The method of claim 42 wherein the inwardly rectifying potassium channel is comprised of Kir 3.1 and Kir 3.2 subunits, Kir 3.1 and Kir 3.4 subunits, Kir 3.2 subunits, mutant Kir 3.4 S143T subunits, or mutant Kir 3.1 F137S subunits.
44. The method of claim 43 wherein the inwardly rectifying potassium channel is comprised of Kir 3.1 and Kir 3.2 subunits.
45. The method of claim 33 wherein the current through the inwardly rectifying potassium channels is measured using a two electrode voltage clamp or a whole cell patch clamp.
PCT/US2008/058453 2007-03-29 2008-03-27 Electrophysiological method to pharmacologically classify ligands for gs protein-coupled receptors WO2008121717A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5882944A (en) * 1993-06-23 1999-03-16 The Regents Of The University Of California Methods for G protein coupled receptor activity screening
US6203987B1 (en) * 1998-10-27 2001-03-20 Rosetta Inpharmatics, Inc. Methods for using co-regulated genesets to enhance detection and classification of gene expression patterns
US6436642B1 (en) * 1999-04-20 2002-08-20 Curagen Corporation Method of classifying a thyroid carcinoma using differential gene expression

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5882944A (en) * 1993-06-23 1999-03-16 The Regents Of The University Of California Methods for G protein coupled receptor activity screening
US6203987B1 (en) * 1998-10-27 2001-03-20 Rosetta Inpharmatics, Inc. Methods for using co-regulated genesets to enhance detection and classification of gene expression patterns
US6436642B1 (en) * 1999-04-20 2002-08-20 Curagen Corporation Method of classifying a thyroid carcinoma using differential gene expression

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