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WO1993014408A1 - Recepteurs de myotonine et procedes d'identification - Google Patents

Recepteurs de myotonine et procedes d'identification Download PDF

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Publication number
WO1993014408A1
WO1993014408A1 PCT/US1993/000310 US9300310W WO9314408A1 WO 1993014408 A1 WO1993014408 A1 WO 1993014408A1 US 9300310 W US9300310 W US 9300310W WO 9314408 A1 WO9314408 A1 WO 9314408A1
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WIPO (PCT)
Prior art keywords
myotonin
receptor
assay method
amylin
preparation
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PCT/US1993/000310
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English (en)
Inventor
Kevin Beaumont
Original Assignee
Amylin Pharmaceuticals, Inc.
Priority date (The priority date 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 date listed.)
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Publication date
Application filed by Amylin Pharmaceuticals, Inc. filed Critical Amylin Pharmaceuticals, Inc.
Priority to CA002105167A priority Critical patent/CA2105167A1/fr
Priority to JP5512649A priority patent/JPH06506777A/ja
Publication of WO1993014408A1 publication Critical patent/WO1993014408A1/fr

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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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • 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 present invention relates to methods for identifying physiologically active materials,' such as chemical compounds, by assessing their ability to interact with naturally occurring or isolated or cloned receptor sites.
  • Resistance to insulin may be present in several serious disorders, including Type 2 diabetes mellitus, obesity and hyper ⁇ tension. Resistance to insulin is manifest by a reduction in the effectiveness of a given dose of insulin compared to that obtained in a non-resistant state.
  • Type 2 diabetes mellitus the ability of both endogenous insulin and insulin administered exogenously to control the chronic hyper- glycemia suffered by such patients is seriously compromised. Consequently, the complications that result from uncontrolled diabetes mellitus, such as premature atherosclerosis, intercapillary glomerulosclerosis, retinopathy, neuropathy and kidney failure, are more likely to occur in insulin-resistant diabetics than in insulin-sensitive diabetics.
  • Insulin resistance is present when normal or elevated glucose concentrations persist in the face of normal or elevated concentrations of insulin in the general circulation.
  • Insulin resistance in skeletal muscle represents, in essence, inhibition of glycogen synthesis from metabolic precursors due to a reduction to subnormal levels of either basal or insulin-stimulated glycogenesis, or both.
  • hyperglycemia in Type 2 diabetes mellitus: (1) failure of glucose storage to be activated (Lillioja, J. Clin. Endocr. Metab.. 62:922-927, 1986); and (2) a defect in insulin release from the pancreas (Waingot et al. r Proc. Nat'1 Acad. Sci. USA. 79:4432-4436, 1982) .
  • Treatment of this disease has focused on attempts to reverse either or both of these defects.
  • amylin insulin associated peptide
  • DAP diabetes associated peptide
  • Native amylin is a 37 amino acid protein characterized by a disulfide bridge between the Cys residues at positions 2 and 7 and an amide group at the C- terminal tyrosine.
  • the amylin subpeptide 18-27 is amyloidogenic, that is, it possesses the tendency to form amyloid.
  • the structure of amylin shows a 43% homology to calcitonin gene related peptide-1 (CGRP-1) , a 46% homology to CGRP-2, and some similarity to insulin.
  • Amylin may be one member of a family of related peptides which include
  • amylin family of peptide hormones acts through receptors present in plasma membranes. Young et al. have shown that amylin works in skeletal muscle via a receptor-mediated mechanism that promotes glycogenolysis, by activating the rate-limiting enzyme for glycogen breakdown, phosphorylase a (Young et al. 281 FEBS Lett. 149, 1991).
  • amylin reported in vivo are: (1) a reduction in insulin action observed under "euglycemic clamp" conditions whereby infusion of amylin reduces insulin mediated glucose clearance (Molina et al. Diabetes 39:260-265, 1990, and Young et al. , Am. J. Phvsiol. 259:457-461, 1990) and partly reverses insulin- mediated suppression of hepatic glucose output (Molina et al. supra; Koopmans et al. Diabetes 39:101A, 1990); (2) In lightly anaesthetized, 18-hour fasted rats, bolus injections of amylin evoke first an increase in plasma lactate, and second a sustained increase in plasma glucose.
  • lactate production in skeletal muscle consequent upon amylin stimulation of glycogenolysis.
  • This action of amylin is observed in the fed as well as the 18-hour fasted rat, and also during somatostatin infusion given to limit secretion of pancreatic hormones including insulin and glucagon.
  • the increase in plasma glucose is associated with a dilution of infused trace glucose, indicating increased hepatic glucose output. It is not presently known whether the actions of amylin infusion in "euglycemic clamped" rats, and amylin injections, to increase hepatic glucose output is a result of direct actions of amylin on liver, or solely an indirect effect of the amylin actions, such as the release of lactate from muscle. For instance, it was reported that amylin increases gluconeogenesis and glycogenolysis in cultured Hep G2 cells (a cell line derived from a human liver tumor) . Ciaraldi et al. , Diabetes 39: Supp. 1, 145A, 1990. On the other hand, it was reported that amylin had no observable effects on glucose metabolism in isolated rat hepatoyctyes or in perfused rat liver (Stephens et al. Diabetes in press, 1991) .
  • amylin can exert certain other actions in vivo, including vasodilatation. Brain et al. Am. J. Pathol. 136:487-490, 1990. Amylin was 100 to 1000 fold less potent as vasodilator than the related peptide CGRP. This could reflect a weak action of amylin on CGRP receptors although no evidence was provided in support of this. Amylin is also reported to lower plasma calcium in rabbits and rats (Datta et al. Biochem. Biophys. Res. Commun. 162 :876-881, 1989) . This action resembles that of calcitonin. Human calcitonin was more effective than amylin in adducing hypocalcaemia and it is possible, though also unproven, that amylin acts less potently than calcitonin at calcitonin receptors on bone cells.
  • CGRP and calcitonin act via membrane receptors at least some of which serve to activate adenylate cyclase and generate cyclic AMP as an intracellular second messenger.
  • high affinity binding sites (receptors) for CGRP on liver membrane were reported; at these sites CGRP is said to potently activate adenylate cyclase.
  • Amylin was reported to displace labelled CGRP from these binding sites but with much lower affinity, approximately 200 nM, than CGRP, approximately 20 pM.
  • amylin agonist activity is assessed by measuring the inhibition of insulin-stimulated glycogen synthesis in intact rat soleus muscle. Leighton et al.
  • the soleus assay is effective in quantitating relative potencies of agonists but the affinity of a ligand for its receptor cannot accurately be determined from agonist dose-response relations in whole tissues or organs. For instance, different molecular sizes, or solubility, or propensity to bind to tissue components can influence the defined potencies.
  • the soleus assay furthermore, has little or no value as an effective, high-throughput primary screening assay for compounds active at amylin receptors.
  • myotonin receptor a novel peptide receptor, herein termed the myotonin receptor.
  • This receptor is useful in methods for identifying compounds useful in treatment of hypoglycemic conditions, or insulin resistance. It is also useful for other screening procedures as detailed below, and described (with respect to amylin receptors) in Beaumont et al., supra.
  • the invention features rapid, inexpensive and physiological methods for identifying, screening and characterizing potential compounds useful for treatment of a hypoglycemic condition, or a disease characterized by insulin resistance.
  • the methods include assessing the ability of candidate molecules to compete against tracer concentrations of certain labeled peptides, including certain labeled peptide hormones and fragments and analogs thereof, for binding to specific myotonin receptor binding sites in cells or membranes prepared or isolated from said cells or from tissues containing cells with membrane receptors for myotonin.
  • Myotonin receptors can be identified by the binding characteristics described below for one example of a myotonin receptor.
  • the invention provides an assay method for identifying or screening compounds useful for treatment of a hypoglycemic condition or a disease characterized by insulin resistance.
  • the method includes bringing together a test sample and a myotonin receptor preparation.
  • the test sample contains one or more test compounds, and the myotonin receptor preparation contains a myotonin receptor protein capable of binding to calcitonin.
  • the test sample is incubated with the receptor preparation under conditions that allow the binding of calcitonin or related radioligand to the myotonin receptor protein.
  • Those test samples containing one or more test compounds which detectably bind to the myotonin receptor protein are then identified.
  • this method further includes the steps of screening test samples which detectably bind to the myotonin receptor for in vitro or in vivo stimulation or inhibition of myotonin receptor- mediated activity, and identifying those test samples which act as receptor agonists or antagonists at this receptor.
  • the test samples which detectably bind to the myotonin receptor protein are identified by measuring the displacement of a labeled first ligand from the receptor preparation by the test sample, and comparing the measured displacement of the first labeled ligand from the receptor preparation by the test sample with the measured displacement of the labeled first ligand from the receptor preparation by one or more known second ligands.
  • Labeled first ligands and second ligands include salmon calcitonin, an amylin agonist, or an amylin antagonist.
  • Useful receptor preparations include isolated cells bearing the myotonin receptor, isolated membrane preparations bearing the myotonin receptor and isolated myotonin receptor protein. Test samples used in any of the above methods that contain more than one test compound and which yield positive results can then be divided and retested as many times as necessary, and as appropriate, to identify the compound or compounds in the test sample which are responsible for yielding the positive result.
  • the first ligand is labelled with a member selected from the group consisting of radioactive isotopes, nonradioactive isotopes, fluorescent molecules, chemiluminescent molecules, and biotinylated molecules;
  • the known second ligand or ligands are selected from the group consisting of amylin, calcitonin, cu-CGRP, and ⁇ -CGRP, e.g. , human amylin, dog amylin, rat amylin, human calcitonin, rat calcitonin, eel calcitonin, salmon calcitonin, human or-
  • the test sample comprises one or more known or unknown test compounds; and one or more non-specific myotonin binding sites on cells which do not comprise a myotonin receptor are blocked, e.g. , one binding site includes a calcitonin or CGRP receptor, and the assay method includes the step of providing an antagonist for the calcitonin or CGRP receptor in the assay method to block the calcitonin or CGRP receptor.
  • the cells bearing the myotonin receptor are smooth muscle cells, e.g. , from a vas deferens, or the cell line DDT-MF-2 leiomyosarcoma.
  • the invention provides an assay method for evaluating one or more receptor binding characteristics sought to be determined for a known or a candidate amylin agonist or antagonist compound.
  • the method includes the steps of assessing or measuring the ability of the compound to compete with a labeled ligand for binding to a myotonin receptor preparation described above; assessing or measuring the ability of the compound to compete against the labeled ligand for binding to a
  • CGRP receptor preparation or assessing or measuring the ability of the compound to compete against the labeled ligand for binding to a calcitonin receptor, or assessing or measuring the ability of the compound to compete against the labeled ligand for binding to a CGRP receptor preparation and to a calcitonin receptor preparation; and, determining the receptor binding characteristic sought to be determined for the compound.
  • Receptor binding characteristics which may be determined include binding affinity and binding specificity.
  • CGRP receptor preparations include hepatocyte preparations, including primary cell cultures or established cell lines e.g. , the Hep G2 cell line.
  • Calcitonin receptor preparations include cell or membrane preparations, e.g. , from osteoclasts or equivalent calcitonin receptor-bearing cell lines, such as the LLC-PK1 cell line.
  • the invention provides an assay method for determining the presence or amount of a myotonin receptor binding compound in a test sample to be assayed.
  • the method includes the steps of bringing together the test sample and a myotonin receptor preparation described above; measuring the ability of the test sample to compete against a labeled ligand for binding to the myotonin receptor preparation; and, optionally, relating the amount of myotonin receptor binding compound in the test sample with the amount of myotonin receptor binding compound measured for a negative control sample, the negative control sample being known to be free of any myotonin receptor binding compound, and/or relating the amount of myotonin receptor binding compound in the test sample with the amounts of myotonin receptor binding compound measured for positive control samples which contain known amounts of myotonin receptor binding compound, in order to determine the presence or amount of myotonin receptor binding compound present in the test sample.
  • This assay method in still further embodiments, can be utilized to evaluate the stability of an amylin preparation, to evaluate the potency of an amylin preparation, and to evaluate the solubility characteristics of an amylin preparation.
  • the myotonin receptor binding compound and/or labelled ligand is an amylin agonist such as salmon calcitonin, or an amylin antagonist; and the test sample is a biological fluid, selected from the group consisting of blood, plasma, urine, cerebrospinal fluid, lymph fluid, or an amylin preparation; and the assay method includes evaluation of the stability, potency or the solubility of an amylin preparation.
  • the receptor preparations of the invention can be utilized to prepare anti-myotonin receptor antibodies, including polyclonal antisera and monoclonal antibodies, utilizing art-known methods.
  • the invention is used to screen cell lines, cells desegregated from tissue, and cells from human or animal blood in order to identify those which carry myotonin receptors.
  • the myotonin receptor preparations of the invention may also be bound to a solid phase and used in various affinity chromatography methods and used, for example, for the purification of peptides such as amylin, or the evaluation of samples known or suspected to contain amylin, amylin agonists or amylin antagonists.
  • the invention features purified myotonin receptor, e.g. , isolated from DDT-MF-2 leiomyosarcoma cell line cells; a method for isolating a myotonin receptor by purifying cell membrane from DDT-MF-2 cell line cells, and separating components of the cell membrane from the myotonin receptor; purified nucleic acid encoding a myotonin receptor; and a method for purifying nucleic acid encoding a myotonin receptor, e.g.
  • purified is meant that the myotonin, myotonin receptor or nucleic acid encoding either of these is separated from its natural environment, preferably as a homogeneous preparation having at least 60-90% by weight of the desired product.
  • Figure 1 is a graphical representation of a saturation isotherm of 125 I-salmon-calcitonin binding to rat hindlimb muscle membranes
  • Figure 3 is a graphical representation of competition of salmon-calcitonin with amylin and CGRP-1 in DDT-MF-2 cells.
  • Figure 4 is a graphical representation of AC66 Antagonism of sCT-Induced cAMP production by DDT-MF-2 cells.
  • Figure 5 is a Schild Plot of AC66 Antagonism of sCT- Induced cAMP Production in DDT-MF-2 cells.
  • Figure 6 is a graphical representation of amylin, calcitonin, and CGRP-induced cAMP production in DDT-MF-2 cells.
  • Figure 7 is a graphical representation of the effects of heterologous calcitonins on cAMP production in DDT-MF2 cells.
  • the present invention provides novel inexpensive, rapid and physiological methods for screening, identifying, and characterizing potential agonists and antagonists at a novel adenylate-cyclase-coupled receptor, the myotonin receptor, as well as the use of this receptor to identify agonists and antagonists at the related amylin receptor. This includes assessing the relative abilities of candidate agonists and antagonists to compete against relevant peptides for binding to specific myotonin receptor sites.
  • the receptor sites used for these and other purposes may be present as isolated receptor-bearing tissues, cells prepared from said receptor bearing tissues, membrane preparations derived from said cells, and isolated receptor protein preparations including cloned receptor preparations using recombinant DNA techniques.
  • tissue, cells, membrane, or receptor preparations used in the competition-based screening methods of the invention exhibit binding characteristics identical to or equivalent to a natural receptor.
  • Tissue preparations or cell or membrane preparations derived from tissues bearing the myotonin receptor can be identified for use in methods for screening myotonin receptor antagonists and agonists as described below.
  • cells or membranes bearing the myotonin receptor, or myotonin receptor protein preparations are preferably isolated from rat skeletal muscle or cells such as DDT-MF-2 cells.
  • the myotonin receptor assay can be used to determine the concentration of myotonin receptor- or amylin receptor-active compounds in unknown solutions or mixtures.
  • Myotonin receptors are assayed as described below.
  • a membrane or cell preparation containing a high density of myotonin receptors is incubated with, for example, radiolabelled calcitonin and unlabelled amylin.
  • a competition curve is generated relating the amount of amylin in the assay tube to the inhibition of radiolabelled calcitonin binding produced.
  • unlabelled peptide is replaced by a solution containing an unknown amount of amylin to be quantified.
  • This solution may be plasma, serum or other fluid, or solid mixture dissolved in assay buffers.
  • the unknown solution is preferably added in a volume of less than or equal to about 10% of the final assay volume, so as not to significantly alter the ionic content of the solution. If larger volumes of unknown are used, a solution containing an equivalent salt content is included as a control for effect's of altered ionic content on binding.
  • Nonspecific binding i.e.. binding of radiolabelled calcitonin in the presence of a high concentration (10"°M) of unlabelled amylin or salmon calcitonin, is subtracted from total binding for each sample to yield specific binding.
  • the amount of inhibition of specific binding of radiolabelled calcitonin produced by the unknown is compared to the inhibition curve produced by amylin in order to determine the content of amylin or amylin receptor-active substances in the unknown sample.
  • Methods for performing these calculations are described in several sources, such as in Neurotransmitter Receptor Binding, eds H. Yamamura, S.J. Enna, and M. . Kuhar (Raven Press, New York, 1991) .
  • This method is used to quantitate the amount of myotonin-receptor active compounds in a known or an unknown sample, and may be used to quantitate myotonin- receptor active compounds in plasma or other body fluids and tissues, for use in identifying active metabolites, pharmacokinetics, stability, solubility, or distribution of myotonin receptor agonists and antagonists, amylin agonists and amylin antagonists. It may also be used to identify, isolate and purify peptides having a high affinity for the myotonin receptor.
  • the quantity of CGRP in the unknown sample can be determined through a radio- receptor assay for CGRP.
  • a radio-receptor assay can be performed using 125 I-hCGRP and rat liver membranes according to the methods described for the myotonin radio- receptor assay. With this assay, the CGRP content of the unknown sample can be determined. Since the myotonin radio-receptor assay can identify all compounds active on myotonin receptors, including CGRP, it is useful to subtract CGRP content, as determined by radio-receptor or other assay (e.g..).
  • radioimmunoassay from total content of myotonin-receptor active compounds to yield the amount of amylin in samples (e.g. , serum) which may contain both amylin and CGRP.
  • samples e.g. , serum
  • the myotonin receptor can also be used in a high throughput screen, optionally utilizing robotic systems such as those known in the art, for identifying compounds which displace myotonin or amylin from this receptor and, thus, identifying candidate myotonin or amylin agonists or antagonists.
  • the assay can be used to screen, for example, libraries of synthetic compounds, extracts of plants, extracts of marine organisms, or bacterial or fungal fermentation broths. In one embodiment, .
  • an initial step brings together the myotonin receptor preparation, pre-incubated with radiolabelled calcitonin and a solution of test compound.
  • the final concentration of solvent should generally not exceed that which displaces the standard displacement curve of labelled calcitonin by cold calcitonin by 25%, i.e. , shifts the measured IC 50 by less than 25%. This can be evaluated for each selected solvent.
  • the test concentration will be about lOOnM, l ⁇ M, or lO ⁇ M depending on the frequency with which positive tests occur.
  • a positive will typically be represented by at least about a 20% reduction of specific binding of labelled amylin. With broths and extracts, a positive test will be denoted by at least about 20%, 50% or 80% reduction in specific calcitonin binding, according to the frequency of positive tests.
  • a suitable assay for evaluating non-specific effects will be a radiolabelled standard reagent for determination of binding to a standard receptor in the vas deferens or tissue being used. Those receptors which are relatively abundant in the tissue and readily assayed should be chosen. Any compound, broth, or extract that tests positive in the myotonin receptor screen, and which also tests positive by the same quantitative criteria in the standard receptor screen is rejected as non-selectively interfering with ligand binding to membrane receptors.
  • the invention also includes determination of the interaction with CGRP receptors of compounds, broths or extracts which selectively reduce amylin binding to myotonin receptors.
  • steps similar to those described above are undertaken with a binding assay consisting of cells or membranes having myotonin receptors pre-incubated with I25 I-his 10 _human ⁇ -CGRP.
  • compounds, broths or extracts are identified which selectively act at the myotonin receptor but not at the CGRP receptor.
  • the potency of interaction with the myotonin receptor and, if relevant, the amylin, CGRP or calcitonin receptors are determined by measuring the displacement of ligand from the membrane preparations by a range of concentrations of the test compound.
  • the desired activity is isolated and purified by art-known methods including HPLC, followed by testing the separated materials to determine which retain the desired activity.
  • HPLC HPLC
  • its potency at the myotonin, amylin, and CGRP receptor can also be determined.
  • Art- known methods including NMR, mass spectroscopy, and elemental analysis may be used to make a chemical identification of any isolated material having the desired receptor binding activities.
  • a positive testing material can be assessed in a functional assay to assess amylin agonist activity through, for example, inhibition of insulin-stimulated incorporation of labelled glucose into glycogen in rat soleus muscle.
  • the material can also be tested for antagonist activity in this assay by assessing its ability to restore insulin-stimulated incorporation of labelled glucose into glycogen in rat soleus muscle incubated with 10, 20, 50 or 100 nM rat amylin. Also, by applying different concentrations of the test material in these assays, the potency of myotonin or amylin agonist or antagonist action can be determined.
  • Another test of myotonin receptor agonist action uses the measurement of elevation of plasma lactate and/or glucose in, for example, halothane-anaesthetized, 18-hour fasted rats following intravenous bolus injections of the test material.
  • the potency of the material as an amylin agonist can be determined.
  • the test material is infused intravenously into 18-hour fasted, anaesthetized rats.
  • the reduction (compared to control conditions) of the hyperlactemic and/or hyperglycemic response to intravenous injections of a known amount of amylin agonist is then measured or otherwise evaluated.
  • the antagonist potency of such materials can be determined by repeating the test at different infusion rates of the test material.
  • the test materials are brought together with myotonin-responsive membrane or cell systems in which amylin changes rates of synthesis of cyclic AMP (cAMP) .
  • Such preparations include membranes prepared from cultured cell lines with abundant myotonin or amylin receptors, or the cells themselves. Changes in cAMP levels are measured by radioimmunoassay following exposure of the membrane or cell preparations, incubated according to art-known methods. Materials testing positive in displacing amylin from its receptors or myotonin receptors and having no effect on cAMP production can be expected to be amylin receptor antagonists. Antagonist action can be further evaluated by incubating various concentrations of the material analog with amylin or an amylin agonist and measuring the degree of inhibition of the changes in cAMP evoked by amylin or an amylin agonist.
  • the invention can also be used to screen cell lines, cells disaggregated from tissue, and cells from human or animal blood for amylin receptors. These cells will be used as a readily available source for additional myotonin receptor preparations for development of agonists and antagonists of amylin. Membranes from cells are obtained by homogenization of cells with an instrument such as
  • Membranes so obtained are combined with, e.g. , 125 I-salmon calcitonin in a buffer system such as that described in the examples below, and are incubated and collected as described in those examples.
  • Specific binding of 125 I-salmon calcitonin to the cell membrane is identified by measuring the decrease in binding obtained in the presence of, for example, 10" 7 M rat amylin or
  • Subcellular membrane fractions obtained by differential or density gradient centrifugation are assayed for specific binding of radiolabeled calcitonin in order to identify the membrane fraction containing the highest density of myotonin receptors per milligram protein (as assayed by Bradford or Lowry protein assays) .
  • the membrane fraction with highest receptor density is preferably used for further purification.
  • This membrane fraction is collected and treated in a buffered solution with several membrane solubilizing agents, including triton, digitonin, octyl glucoside, deoxycholate, and cholate, at concentrations of from 0.001% to 1% detergent at reduced temperature (4°C) for about 1 hour.
  • Protease inhibitors including phenylmethylsulfonyl fluoride, EDTA, aprotinin are included in the buffer system to prevent receptor degradation during or after solubilization.
  • solubilized membranes After treatment of membranes with detergents, unsolubilized membranes are sedimented by centrifugation at high speed (100,000 x g for 1 hour) and resulting supernatants containing solubilized receptors are assayed for binding of radiolabeled calcitonin as described above.
  • Solubilized receptors can be collected by filtration on polyethyleneimine-coated filters (Bruns et al. Anal. Biochem. 132:74-81. 1983. Alternatively, solubilized receptors are collected by methods such as precipitation with polyethyleneglycol, gel filtration, or equilibrium dialysis.
  • Binding characteristics (such as affinity for amylin, CGRP and calcitonins) of solubilized receptors are assessed and should match the characteristics of membrane- localized receptors. After determining conditions suitable for solu ⁇ bilizing myotonin receptors and for assaying solubilized receptors, these solubilized receptors are purified away from other solubilized membrane proteins by chromato- graphic procedures, such as affinity chromatography on supports to which myotonin, amylin or calcitonin has been coupled, ion exchange chromatography, lectin agarose chromatography, gel filtration, and hydrophobic inter ⁇ action chromatography.
  • Chromatography column eluates are tested for specific myotonin receptor binding to protein content, in order to identify peaks containing receptors and the extent of purification.
  • each chromatographic step is tested to determine the extent to which it contributes to receptor purification, as measured by an increase in specific radiolabeled myotonin, amylin, or calcitonin binding per milligram protein. Desired chromatography steps are combined sequentially, using large quantities of starting material, in order to obtain partially or com ⁇ pletely purified amylin receptors, as desired.
  • Receptors which have been partially or completely purified by this method are used to generate myotonin receptor-specific antibodies for use in diagnosis (disease states with altered receptor density, distribution, or antigenicity) and for use in screening recombinant libraries for myotonin receptor expression.
  • Purified receptor preparations can also be used to obtain partial sequence information, which is useful in preparing oligonucleotide probes for screening recombinant libraries for myotonin receptor-encoding gene sequences.
  • Example 1 Identification of Myotonin Receptor It was discovered that salmon calcitonin has a high affinity for amylin receptors in rat nucleus accumbens, and that salmon calcitonin is more potent than amylin at inhibiting insulin-stimulated glycogen synthesis in isolated soleus muscle. To determine whether radioiodinated salmon calcitonin might be a useful ligand for labeling amylin receptors in rat soleus, salmon calcitonin was radioiodinated using chloramine-T procedures. Binding experiments were done to test the ability of 125 I-salmon calcitonin to bind to membranes prepared from skeletal muscle and other tissues according to the following procedure:
  • Sprague-Dawley rats (200-250 gm) were sacrificed by decapitation, and skeletal muscles (hindlimb or soleus) removed to cold phosphate-buffered saline (PBS) and weighed. Muscles were transferred to approximately 10 volumes cold HEPES buffer (20 mM HEPES, pH brought to 7.4 with NaOH) , and homogenized with a Polytron. Homogenates were centrifuged 10 min at 200 x g and the pellet discarded. The supernatant was centrifuged 15 minutes at 48,000 x g to obtain muscle membranes.
  • cold HEPES buffer (20 mM HEPES, pH brought to 7.4 with NaOH
  • Membranes were washed three times in fresh buffer by centrifugation for 15 min at 48,000 x g, followed each time by rehomogeni- zation of the membrane pellet in 40 ml fresh buffer. The final membrane pellet was resuspended in 20 mM HEPES buffer containing 0.2 mM phenylmethylsulfonyl fluoride (PMSF) at a concentration of 50 mg original wet weight/ml.
  • PMSF phenylmethylsulfonyl fluoride
  • HBBP buffer (20 mM HEPES; 1 mg/ml bacitracin, 2.5 mg/ml BSA, 0.2 mM PMSF); 0.05 ml 125 I- salmon calcitonin (specific activity 1000 - 2000 Ci/mmol, final concentration - 2-10 pM) 0.05 ml HBBP or test peptide dissolved in HBBP; and 0.1 ml muscle membrane in HEPES/PMSF buffer. The muscle membranes were added last to start the incubation, which was carried out for 90 minutes at ambient temperature.
  • Incubations were terminated by filtration with a har ⁇ vesting apparatus (Brandel cell harvester) through glass fiber filters (Whatman, GF/B filters) which were presoaked for at least 4 hours in 0.3% polyethyleneimine. Filters were washed with 15 ml cold phosphate-buffered saline (PBS) and radioactivity on individual filters determined with a gamma counter.
  • a har ⁇ vesting apparatus Brandel cell harvester
  • Glass fiber filters Whatman, GF/B filters
  • Competition curves were generated by measuring binding of 125 I-salmon calcitonin in the presence of 10" 12 to 10 "6 M unlabeled peptide and were analyzed by non linear regression using a 4-parameter logistic equation (Inplot program; GraphPAD Software, San Diego) .
  • Fig. 1 to determine binding constants, saturation curves were generated and analyzed according to modifications of the method of Scatchard, as described in detail by Bylund & Yamamura.
  • binding of 12S I-salm ⁇ n calcitonin was measured at concentrations varying from 1 - 100 pM to obtain total binding, and again in the presence of 100 nM unlabeled salmon calcitonin to obtain nonspecific binding.
  • Scatchard plots of saturation binding curves indicate that a high affinity salmon calcitonin binding site is present in rat hindlimb muscle membranes with a dissociation constant (Kd) of approximately 11 pM.
  • Example 2 Cell lines with myotonin receptor While binding assays demonstrate the affinity of compounds for receptors, functional assays are needed to determine whether compounds active in binding assays are agonists or antagonists, or indeed whether they interfere with binding through nonspecific actions (e.g. , membrane effects) . For this reason, and also in order to investigate post-receptor actions of amylin, binding assays were used to screen several cell lines for the presence of 125 I-rat amylin, 125 l-human CGRP-1, and 125 l-salmon calcitonin binding sites. Cell lines were obtained from ATCC, grown in tissue culture flasks in the recommended media, and harvested by scraping with a cell scraper into cold PBS.
  • Binding was measured as in Example 1 with the following modifications: final volume was 0.2 ml containing 0.1 ml HBBP, 0.02 ml HBBP or competing peptide in HBBP, 0.03 ml radiolabeled ligand, and 0.05 ml cell membrane. Nonspecific binding was determined in the presence of 10 "6 M unlabeled peptide (rat amylin, hCGRP, or salmon calcitonin for their respective binding sites) .
  • Numerous cell lines were found not to contain specific 125 I- salmon calcitonin binding sites, including L6 myoblast, HepG2, RBA mammary carcinoma, GH 3 pituitary, L8 skeletal muscle, BC 3 H1 smooth muscle, C 2 C 12 myoblast, Hs729T Rhabdomyosarcoma, SK-N-MC neuroblastoma, C6 glioma, G7 myoblast, G8 myoblast and Nor 10 muscle cells lines.
  • DDT-MF-2 Hamster leiomyosarcoma cells
  • DDT-MF-2 cells were grown to confluence in 24-well tissue culture clusters, media was removed and replaced with Dulbecco's PBS containing 0.1 mg/ml BSA + 0.1 mg/ml isobutylmethy- Ixanthine. After 15 minutes incubation at 37°C, cells were stimulated with indicated peptide for 15 minutes. Reaction was stopped by addition of trichloroacetic acid, and cycli AMP was measured in neutralized supernatants using a commercially available radioimmunoassay (RIA) kit. Referring to Fig. 4, Salmon calcitonin (1 nM) strongly stimulated cyclic AMP accumulation (greater than 3Ox baseline) and this effect was blocked by coincubation with AC66 (4 ⁇ t ⁇ ) .
  • RIA radioimmunoassay
  • DDT-MF-2 cells are plated at 10 4 cells per well.
  • cells are washed once with Delbecco's Phosphate Buffered Saline (DBPS, Sigma Chemicals) containing 0.1 mg/ml isobu- tylmethylxanthine and test compound at 37°C.
  • DBPS Delbecco's Phosphate Buffered Saline
  • the reaction is stopped by addition of trichloroacetic acid, followed by neutra ⁇ lization with TRIS base.
  • Example 5 Antagonist Assay The ability of antagonists to block salmon calcitonin-mediated effects on glucose metabolism in soleus muscle was measured to determine whether myotonin receptors contributed to these effects. For these assays, insulin-stimulated glycogen synthesis was measured in isolated soleus muscle in the presence of 1 nM salmon calcitonin, which suppresses the effect of insulin. The IC 50 s for reversing the effects of salmon calcitonin were: AC66 (0.151 ⁇ ) and hCGRP 8 . 37 (48 ⁇ M) . This is a 320-fold difference in potency, which compares favorably with the 230-fold difference in potency at the myotonin receptor.

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Abstract

L'invention concerne des récepteurs de myotonine purifiée, un acide nucléique codant de tels récepteurs, et des procédés d'identification ou de caractérisation ou de dosage ou d'isolement de composés connus ou candidats utiles pour le traitement d'un état d'hypoglycémie ou d'une maladie caractérisée par une résistance à l'insuline, y compris des dosages de liaison utilisant des préparations contenant des récepteurs spécifiques de myotonine. Des membranes de cellules de leiomyosarcome qui contiennent des récepteurs de myotonine sont particulièrement utiles pour les procédés de cette invention et également comme source de protéines de récepteurs de myotonine.
PCT/US1993/000310 1992-01-14 1993-01-14 Recepteurs de myotonine et procedes d'identification WO1993014408A1 (fr)

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CA002105167A CA2105167A1 (fr) 1992-01-14 1993-01-14 Recepteurs a myotonine et methodes de criblage
JP5512649A JPH06506777A (ja) 1992-01-14 1993-01-14 ミオトニン受容体およびスクリーニング方法

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995005601A1 (fr) * 1993-08-18 1995-02-23 Zeneca Limited Procede de detection d'interactions biologiques, notamment dans des titrages de liaison de recepteurs
WO1999040928A1 (fr) 1998-02-13 1999-08-19 Amylin Pharmaceuticals, Inc. Nouveaux composes a activite mixte par rapport a l'amyline
US6936584B1 (en) 1998-02-13 2005-08-30 Amylin Pharmaceuticals, Inc. Mixed amylin activity compounds
US8168592B2 (en) 2005-10-21 2012-05-01 Amgen Inc. CGRP peptide antagonists and conjugates

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
No relevant documents disclosed *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995005601A1 (fr) * 1993-08-18 1995-02-23 Zeneca Limited Procede de detection d'interactions biologiques, notamment dans des titrages de liaison de recepteurs
US6054282A (en) * 1993-08-18 2000-04-25 Zeneca Limited Method for detecting biological interactions especially in receptor binding assays
WO1999040928A1 (fr) 1998-02-13 1999-08-19 Amylin Pharmaceuticals, Inc. Nouveaux composes a activite mixte par rapport a l'amyline
US6936584B1 (en) 1998-02-13 2005-08-30 Amylin Pharmaceuticals, Inc. Mixed amylin activity compounds
EP1053001A4 (fr) * 1998-02-13 2006-01-04 Amylin Pharmaceuticals Inc Nouveaux composes a activite mixte par rapport a l'amyline
US8168592B2 (en) 2005-10-21 2012-05-01 Amgen Inc. CGRP peptide antagonists and conjugates

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