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WO1994021665A1 - Recepteurs clones et methodes de criblage associees - Google Patents

Recepteurs clones et methodes de criblage associees Download PDF

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Publication number
WO1994021665A1
WO1994021665A1 PCT/US1994/003248 US9403248W WO9421665A1 WO 1994021665 A1 WO1994021665 A1 WO 1994021665A1 US 9403248 W US9403248 W US 9403248W WO 9421665 A1 WO9421665 A1 WO 9421665A1
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Prior art keywords
receptor
cla
clb
binding
calcitonin
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PCT/US1994/003248
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English (en)
Inventor
Keith Albrandt
Kevin Beaumont
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Amylin Pharmaceuticals, Inc.
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Application filed by Amylin Pharmaceuticals, Inc. filed Critical Amylin Pharmaceuticals, Inc.
Priority to AU65247/94A priority Critical patent/AU6524794A/en
Publication of WO1994021665A1 publication Critical patent/WO1994021665A1/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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors

Definitions

  • the present invention relates to cloned receptors and to methods for screening for, identifying, isolating, characterizing, or quantitating physiologically active materials, such as chemical compounds, by assessing their ability to interact with receptor sites.
  • Calcitonin is known to be a regulator of calcium homeostasis, acting principally on bone. It also has a direct action on the kidneys and on gastrointestinal secretory activity, as well as having both direct and indirect effects on the central nervous system.
  • Azria, M The Calcitonins: Physiology and Pharmacology (S. Karger AG, Basel 1989) . Effects reported following intraventricular injection of salmon calcitonin include anorexia (Freed et al.. Science 206:850-852, 1979), analgesia (Bragor et al.
  • CGRP Tissue-specific RNA processing of calcitonin gene transcripts leads to mRNAs encoding different peptide products, including precursors of calcitonin and the calcitonin gene-related peptides ( ⁇ -CGRP and S-CGRP) .
  • ⁇ -CGRP and S-CGRP precursors of calcitonin and the calcitonin gene-related peptides
  • CGRP is distributed throughout the central nervous system.
  • CGRP is also present in high concentrations in perivascular nerves throughout the body, including in the coronary and cerebral vessels.
  • the most striking effect of CGRP is vasodilation, and in healthy volunteers infusion of CGRP causes hypotension and reflex tachycardia.
  • CGRP is the principal circulating product of the human calcitonin gene, suggesting that it has an important physiological role in the control of blood flow and vascular tone.
  • CGRP Like calcitonin, CGRP also inhibits bone resorption, gastric acid secretion and the perception of pain, albeit less potently.
  • Amylin is a recently discovered peptide hormone that is co-secreted with insulin from the pancreas (see, e.g. , Cooper et al. , Diabetalogia 32:104, 1989; Cooper et al. , Diabetes 88:493-496, 1989; Cooper et al. , Biochim. Biophvs. Acta 1014:247-258, 1989).
  • Major metabolic effects of amylin reported in vivo include (1) a reduction in insulin-mediated glucose clearance (Molina et al.
  • Amylin is 100- to 1000-fold less potent as a vasodilator than the related peptide CGRP. Amylin is also reported to lower plasma calcium in rabbits and rats (Datta et al.. Biochem. Biophvs. Res. Commun. 162:876-881. 1989), although human calcitonin is reported to be more effective than amylin in inducing hypocalcemia.
  • amylin Principal therapeutic uses for amylin are those proposed for the treatment of diabetes. It has been determined that type-1 (insulin-dependent) diabetics, in addition to their life-threatening lack of insulin, have a marked amylin deficiency. The treatment of diabetes with amylin or agonists of amylin is described in Cooper, U.S. Patent No. 5,175,145, issued December 29, 1992, for "Treatment of Diabetes Mellitus With Amylin Agonists.” It has also been proposed that excess amylin action contributes to the disordered metabolism in type 2 diabetes, glucose intolerance, insulin resistance and obesity, and the blockade of amylin with amylin antagonists has been identified as an appropriate therapeutic strategy for those conditions. Cooper et al. , International Application No. PCT/US89/00049, "Treatment of Type 2
  • Calcitonin and ⁇ -CGRP share common parentage in the calcitonin gene where alternative processing of the primary mRNA transcript leads to the generation of the two distinct peptides, which share only limited sequence homology (about 30% (Amara et al.. Science. 229:1094-1097, 1985).
  • The. structure of amylin shows a 43% homology to ⁇ -CGRP, a 46% homology to jS-CGRP, and some similarity to insulin.
  • Amylin may be one member of a family of related peptides which include CGRP, insulin, insulin-like growth factors, and the relaxins and which share common genetic heritage (Cooper et al. , Prog. Growth Factor Research 1:99-105, 1989).
  • calcitonin and the related peptides CGRP and amylin act via membrane receptors at least some of which serve to activate adenylate cyclase and generate cyclic AMP as an intracellular second messenger.
  • 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 receptors and receptor preparations are described in Beaumont et al., International Application No.
  • calcitonin was 500- to 1000-fold less potent than either rat or human CGRP in competition for CGRP binding sites.
  • CGRP was 500- to 1000-fold less potent in competing for calcitonin sites in both brain and renal membranes (Goltzman and Mitchell, Science 227:1343-1345. 1985) , as well as in whole kidney sections (Sexton et al.. Kidnev Int. 32:862-868, 1987).
  • Cla receptor is meant a receptor of the amino acid sequence shown as Cla in Fig. 1, i.e.. the deduced 479 amino acid sequence of cDNA clone L2175-D20, and any functional homolog thereof.
  • Clb receptor is meant a receptor of the amino acid sequence shown as Clb in Fig. 1, i.e. , the deduced 516 amino acid sequence of the cDNA clone U3237-A2, and any functional homolog thereof.
  • functional homolog is meant a receptor having variation( ⁇ ) in amino acid sequence while retaining Cla or Clb binding activity, respectively.
  • the deduced amino acid sequence of the Cla receptor is 78% and 66% homologous with the human and pig calcitonin receptors, respectively.
  • the amino acid sequence of the Clb receptor is identical to the Cla receptor except for a 37 amino acid insert in the second extracellular domain between transmembrane domains 2 and 3.
  • the Cla and Clb receptors are useful in methods for identifying calcitonin agonist or antagonist compounds useful in the treatment of various disease states or conditions, such as obesity, anorexia or pain. These receptors are also useful in methods for identifying receptor selectivity characteristics of amylin agonist or antagonist compounds useful in the treatment of various disease states or conditions, such as diabetes mellitus, impaired glucose tolerance, and insulin resistance.
  • the Cla and Clb receptors are useful in the screening procedures detailed below, as well as those described (with respect to amylin receptors) in Beaumont, U.S. Serial No. 07/670,231, and those described (with respect to myotonin receptors) in Beaumont, U.S. Serial No. 07/821,731 the disclosures of which are hereby incorporated by reference.
  • the invention features rapid, inexpensive and physiological methods for identifying, screening and characterizing potential compounds useful for treatment of diseases or conditions characterized by an elevated or undesired level of amylin activity (in the case of antagonists) and conditions which are benefitted by amylin (in the case of agonists) .
  • 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 Cla or Clb receptor binding sites.
  • the Cla and Clb binding sites may be present in transfected cells, or in membranes prepared or isolated from said cells.
  • the invention provides an assay method for identifying or screening for calcitonin agonist or antagonist compounds.
  • the method includes bringing together a test sample and a Cla or Clb receptor preparation.
  • the test sample contains one or more test compounds, and the Cla or Clb receptor preparation contains a Cla or Clb receptor protein capable of binding to a Cla or Clb receptor-binding compound.
  • the test sample is incubated with the receptor preparation under conditions that allow binding to the Cla or Clb receptor protein. Those test samples containing one or more test compounds which detectably bind to the Cla or Clb receptor protein are then identified.
  • this method further includes the steps of screening test samples which detectably bind to a Cla or Clb receptor for in vitro or in vivo stimulation or inhibition of calcitonin receptor-mediated activity, and identifying those test samples which act as receptor agonists or antagonists at either receptor.
  • test samples which detectably bind to a Cla or Clb 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, rat amylin, and rat CGRP.
  • Useful receptor preparations include transfected cells bearing a Cla or Clb receptor, membrane preparations bearing a Cla or Clb receptor, and isolated Cla or Clb receptor protein.
  • 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 an amylin, a calcitonin, an ⁇ -CGRP, and a j8-CGRP (e.g..
  • test sample comprises one or more known or unknown test compounds.
  • One or more non-specific Cla or Clb binding sites which may be present on cells that do not comprise a desired or target receptor may optionally be blocked.
  • the invention provides an assay method for evaluating one or more receptor binding characteristics sought to be determined for a known or a candidate Cla or Clb 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 Cla or Clb receptor preparation, as described above; assessing or measuring the ability of the compound to compete against the labeled ligand for binding to a CGRP or amylin receptor preparation, or assessing or measuring the ability of the compound to compete against the labeled ligand for binding to more than one or all of said receptor preparations; 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 primary cell cultures or established cell lines e.g.. the SK-N-MC cell line and the L6 cell line.
  • Amylin receptor preparations include cell or membrane preparations, e.g. , such as those described in Beaumont et al., supra.
  • Cla and Clb receptor preparations may be used in selectivity assays or screens in which, for example, an amylin agonist or antagonist compound is tested for Cla or Clb receptor binding.
  • amylin agonists or antagonists having a preferred receptor binding profile may be identified and selected.
  • molecules that bind to the Cla and Clb receptor, or which have certain receptor binding potency can be selected or rejected as candidate therapeutics based on such assay results.
  • the invention provides an assay method for determining the presence or amount of a
  • the method includes the steps of bringing together the test sample and a Cla or Clb receptor preparation, as described above; measuring the ability of the test sample to compete against a labeled ligand for binding to the Cla or Clb receptor preparation; and, optionally, relating the amount of Cla or Clb receptor binding compound in the test sample with the amount of Cla or Clb receptor binding compound measured for a negative control sample, the negative control sample being known to be free of any Cla or Clb receptor binding compound, and/or relating the amount of Cla or Clb receptor binding compound in the test sample with the amounts of Cla or Clb receptor binding compound measured for positive control samples_ which contain known amounts of Cla or Clb receptor binding compound, in order to determine the presence or amount of Cla or Clb receptor binding compound present in the test sample.
  • This assay method can be utilized to evaluate the stability, potency or solubility characteristics of Cla or Clb binding ligand preparation, such as a calcitonin preparation.
  • a Cla or Clb receptor binding compound and/or labelled ligand is an calcitonin agonist or a calcitonin antagonist; and the test sample is a biological fluid, selected from the group consisting of blood, plasma, urine, cerebrospinal fluid, lymph fluid, or a calcitonin preparation; and the assay method includes evaluation of the stability, potency or the solubility of a calcitonin preparation.
  • the receptor preparations of the invention can be utilized to prepare anti-Cla or anti-Clb receptor antibodies, including polyclonal antisera and monoclonal antibodies, utilizing art-known methods.
  • the invention is used to screen cell lines, cells from tissue, and cells from human or animal blood in order to identify those which carry Cla or Clb receptors.
  • the Cla or Clb 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 a calcitonin, or the evaluation of samples known or suspected to contain calcitonin or calcitonin agonists or antagonists.
  • the invention features purified Cla or Clb receptor; and purified nucleic acids encoding such calcitonin receptors, e.g.. by standard techniques.
  • purified is meant that the Cla or Clb receptor or nucleic acid encoding it 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 depiction of the complete amino acid sequences of a Cla receptor and a Clb calcitonin receptor, and putative receptor domains have been assigned.
  • TM refers to the transmembrane portions of the receptors, I to the intracellular portions, and E to the extracellular portions.
  • the receptors appear to have 7 transmembrane regions, 4 intracellular domains, and 4 extracellular domains.
  • the amino-terminus domain is extracellular (El) and the carboxy- erminus domain is intracellular (14) .
  • Dashes indicate gaps in the sequences introduced to optimize homology.
  • A alanine
  • C cysteine
  • D aspartic acid
  • E glutamic acid
  • F phenylalanine
  • G glycine
  • H histidine
  • I isoleucine
  • K lysine
  • L leucine
  • M methionine
  • N asparagine
  • P proline
  • Q glutamine
  • R arginine
  • S serine
  • T threonine
  • V valine
  • W tryptophan
  • Y tyrosine.
  • Figure 2 is a graphical representation of the saturation isotherm of 125 I-salmon calcitonin binding to Cla receptor membranes. Amount of bound 125 I-salmon calcitonin (in pmol/mg) is plotted against l25 I-salmon calcitonin receptor concentration (pM) .
  • Figure 3 depicts a Scatchard plot of Cla receptor binding.
  • Figure 4 is a graphical representation of the saturation isotherm of 125 I-salmon calcitonin binding to Clb receptor membranes. Amount of bound 125 I-salmon calcitonin (in pmol/mg) is plotted against 125 I-salmon calcitonin receptor concentration (pM) .
  • Figure 5 depicts a Scatchard plot of Clb receptor binding.
  • Figure 6 is a graphical representation of competition studies for the Cla receptor. Data were fit to a 4- parameter logistic equation, using the Cheng-Prusoff relationship to derive apparent inhibition constants (Ki) from IC JO values. Values shown represent means ⁇ standard deviations from duplicate experiments. Percent bound 125 I- salmon calcitonin is plotted against concentrations of the unlabeled ligands salmon calcitonin (open circle) , rat amylin (open square) , rat and CGRP (open triangle) , rat ⁇ - CGRP (filled triangle) , rat calcitonin (open diamond) and human calcitonin (filled diamond) .
  • Figure 7 is a graphical representation of competition studies for the Clb receptor. Data were fit to a 4- parameter logistic equation, using the Cheng-Prusoff relationship to derive apparent inhibition constants (Ki) from ICj o values. Values shown represent means + standard deviations from duplicate experiments. Percent bound X2S I- salmon calcitonin is plotted against concentrations of the unlabeled ligands salmon calcitonin (open circle) , rat amylin (open square) , rat and CGRP (open triangle) , rat ⁇ - CGRP (filled triangle) , rat calcitonin (open diamond) and human calcitonin (filled diamond) .
  • Figure 8 shows cyclic AMP production in COS cells transiently transfected with the Cla or Clb receptor.
  • the Cla and Clb receptors of the present invention were cloned using the technique of mixed oligonucleotide- primed amplification of cDNA (MOPAC) .
  • MOPAC mixed oligonucleotide- primed amplification of cDNA
  • degenerate oligonucleotide primers corresponding to conserved regions of potentially related sequences are used to amplify homologous sequences by the polymerase chain reaction (PCR) from cDNA prepared from RNA of tissues or cell lines.
  • PCR polymerase chain reaction
  • oligonucleotides A312 and A313 corresponding to conserved regions in TM3 and TM7 of the pig calcitonin, opossum PTH/PTHrP, and rat secretin G-protein coupled receptors were used as primers in an attempt to amplify amylin receptor sequences from rat nucleus accumbens poly(A) + RNA.
  • PCR amplified products were analyzed by Southern blotting with an oligonucleotide probe (A315) corresponding to a third conserved region of the calcitonin and PTH/PTHrP receptors. The PCR product was gel purified and subjected to a second round of amplification.
  • the secondary PCR product was subcloned into pCRlOOO and sequenced.
  • An oligo(dT) -primed rat nucleus accumbens cDNA library was subsequently screened for a full-length cDNA sequence corresponding to the PCR product.
  • the cDNA library was non-directionally cloned in eukaryotic expression vector pcDNAI and subsequently transformed into E. col i MC1061/P3. Plasmid DNA was prepared from 72 pools each representing -5000 individual library transformants.
  • plasmid DNA pools were used as template in a PCR amplification with the A312 and A313 degenerate primer mixtures and products were analyzed by Southern blotting and probing with the A315 oligonucleotide.
  • a PCR product -470 bp that hybridized with A315 was amplified in 4 of the 72 pools examined.
  • the bacteria corresponding to these pools were screened by colony hybridization using A315 as a probe .and an isolated bacterial transformant was isolated from 2 of the 4 pools. Restriction endonuclease analysis indicated that both rat nucleus accumbens cDNA library clones (pcDNAI-175 and pcDNAI-237) were about 2.5 kb in length.
  • Oligonucleotide A333 was used to prime 1st strand cDNA synthesis from rat nucleus accumbens poly(A) + RNA.
  • Oligonucleotide A361, from the 3rd extracellular loop was used as the second gene-specific primer for PCR amplification. Product bands were isolated, subcloned and partially sequenced.
  • Full-length cDNA for the Cla receptor was constructed by ligating corresponding 5' RACE (L2) and cDNA library (175) sequences into eukaryotic expression vector pcDNAI.
  • Full-length cDNA construct pcDNAI-L2175-D20 contains a -3.4 kb insert in the correct orientation for expression.
  • Full-length cDNA for the Clb receptor was similarly constructed by ligating corresponding 5'RACE (U3) and cDNA library (237) sequences into eukaryotic expression vector pcDNAI.
  • Full-length cDNA construct preDNAI-U3237-A2 contains a -3.5 kb insert in the correct orientation for expression.
  • the unique StuI site in the 3rd intracellular domain was restored in both constructs indicating regeneration of the correct reading frame at the only site within the protein coding region used for construction of the full-length molecules from the 5' RACE and cDNA products.
  • the present invention provides novel inexpensive, rapid and physiological methods for screening, identifying, and characterizing potential agonists and antagonists at two novel receptors, the Cla receptor and the Clb receptor, as well as the use of these receptors to identify calcitonin agonists and antagonists, and in agonist and antagonist receptor selectivity screening assays. This includes assessing the relative abilities of candidate agonists and antagonists to compete against relevant peptides for binding to specific Cla or Clb 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, transfected cells expressing said receptor sites, membrane preparations derived from said prepared or transfected cells, or isolated receptor protein preparations, including cloned receptor preparations using recombinant DNA techniques.
  • a Cla or Clb receptor assay can be used to determine the concentration of Cla receptor- or Clb receptor- ctive compounds in unknown solutions or mixtures.
  • Cla and Clb receptors are assayed as described below.
  • a membrane or cell preparation containing a high density of Cla or Clb receptors is incubated with, for example, radiolabeled calcitonin and unlabelled calcitonin.
  • a competition curve is generated relating the amount of calcitonin in the assay tube to the inhibition of radiolabelled calcitonin binding produced.
  • unlabelled peptide is replaced by a solution containing an unknown amount of calcitonin 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 effects 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 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 unlabelled calcitonin in order to determine the content of calcitonin or calcitonin 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.J. Kuhar (Raven Press, New York, 1991).
  • This method is used to quantitate the amount of Cla or Clb receptor active compounds in a known or an unknown sample, and may be used to quantitate Cla or Clb receptor active compounds in plasma or other body fluids and tissues, for use in identifying active metabolites, pharmacokinetics, stability, solubility, or distribution of Cla or Clb receptor agonists and antagonists, calcitonin agonists and antagonists, and amylin agonists and amylin antagonists. It may also be used to identify, isolate and purify peptides having a high affinity for the Cla or Clb receptor.
  • a Cla or Clb 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, for example, calcitonin from either receptor and, thus, for example, for identifying candidate Cla, Clb, and calcitonin agonists or antagonists.
  • the assay can be used to screen, for example, libraries of synthetic compounds, extracts of plants, extracts of animal tissue, extracts of marine organisms, or bacterial or fungal fermentation broths.
  • an initial step brings together a Cla or Clb 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 calcitonin. 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 a Cla or Clb 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 potency of interaction with a Cla or Clb receptor and, if relevant, the amylin, CGRP and/or myotonin 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 a Cla or Clb, an amylin, a CGRP, myotonin or other 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 calcitonin agonist or antagonist activity.
  • calcitonin agonist may be determined through inhibition of insulin-stimulated incorporation of labelled glucose into glycogen in rat soleus muscle.
  • the material can also be tested for calcitonin 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.
  • Calcitonin agonist activity can also be assessed by measuring hypocalcemia analgesia, or anorexia following ij vivo administration (e.g.. Zaidi, M.
  • Antagonist activity is measured by assessing the ability of the test compound to block these actions of calcitonin. Also, by applying different concentrations of the test material in these assays, the potency of Cla, Clb, or calcitonin agonist or antagonist action can be determined.
  • the test materials are brought together with Cla or Clb responsive membrane or cell systems in which calcitonin changes rates of synthesis of cyclic AMP (cAMP) .
  • cAMP cyclic AMP
  • Such preparations include membranes prepared from cultured or transfected cell lines with abundant Cla or Clb or amylin receptors, or the cells themselves. Changes in cAMP levels are measured by radioim-munoassay following exposure of the membrane or cell preparations, incubated according to art-known methods. Materials testing positive in displacing calcitonin from Cla or Clb receptors and having no effect on cAMP production can be expected to be antagonists.
  • Antagonist action can be further evaluated by incubating various concentrations of the material with calcitonin or a calcitonin agonist and measuring the degree of inhibition of the changes in cAMP evoked by the calcitonin or calcitonin agonist.
  • the invention can also be used to screen cell lines, cells disaggregated from tissue, and cells from human or animal blood for Cla. Clb, or calcitonin receptors. These cells will be used as a readily available source for additional Cla or Clb receptor preparations for development of agonists and antagonists of calcitonin.
  • Membranes from cells are obtained by homogenization of cells with an instrument such as Polytron (Brinkman Instruments) followed by centrifugation. 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 salmon calcitonin.
  • Cells in which there is a significant difference between total binding (triplicate tubes) and nonspecific binding (triplicate tubes) at the P ⁇ 0.05 level will be used for further study of Cla or Clb receptor function.
  • Subcellular membrane fractions obtained by differential or density gradient centrifugation are assayed for specific binding of radiolabelled calcitonin in order to identify the membrane fraction containing the highest density of Cla or Clb 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 radiolabelled 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.
  • solubilized receptors After determining conditions suitable for solubilizing Cla or Clb receptors and for assaying solubilized receptors, these solubilized receptors are purified away from other solubilized membrane proteins by chromatographic procedures, such as affinity chromatography on supports to which calcitonin has been coupled, ion exchange chromatography, leetin agarose chromatography, gel filtration, and hydrophobic interaction chromatography. Chromatography column eluates are tested for specific Cla or Clb 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 radiolabelled calcitonin or amylin binding per milligram protein. Desired chromatography steps are combined sequentially, using large quantities of starting material, in order to obtain partially or completely purified receptors, as desired.
  • Receptors for example, those which have been partially or completely purified by this method may be used to generate Cla or Clb receptor-specific antibodies for use in diagnosis (disease states with altered receptor density, distribution, or antigenicity) and for use in screening, for example, tissues or recombinant libraries for Cla or Clb receptor expression.
  • the Cla or Clb receptor sequences can also be used to probe for other Cla or Clb receptor- encoding gene sequences by art-known methods.
  • a pair of degenerate oligonucleotides corresponding to conserved regions of the calcitonin, PTH/PTHrP, and secretin receptors were synthesized for use in MOPAC.
  • A312 is a mixture of 16 18-mers corresponding to the transmembrane (TM) region 3 sense strand:
  • A313 is a mixture of 64 17-mers corresponding to the TM7 antisense Strand: 5'-(T/C) (C/G) (A/G)TTG(C/A) (A/G)GAA(G/A)CAGTA-3' .
  • oligonucleotides specific to the novel receptors described herein were also synthesized: A333 (TM7 antisense strand; positions 1372-1392 of clone L2175-D20) 5'- CCCTGGAAATGAATCAGAGAG-3' ; and, A361 (4th extracellular domain antisense strand; positions 1342-1363) 5'- (CAU) 4 ATAATCATAGATCTTCCCAAGC-3' .
  • Oligonucleotides were synthesized on an Applied Bio ⁇ ystem (Foster City, CA) model 381A DNA synthesizer using standard 3-cyanoethyl phosphoramidite chemistry. Following synthesis they were cleaved from the support, deprotected and eluted. After evaporation to dryness they were dissolved in water.
  • RNA samples were obtained from rats and immediately frozen in liquid N 2 and then stored at -80 " c. Frozen tissue was pulverized to a fine powder using a porcelain mortar and pestle immersed in a bath of liquid N 2 . Poly(A) + RNA was isolated from powdered tissue samples by a guanidinium isothiocyanate procedure and oligo (dT) cellulose affinity chromatography (Fast Track; Invitrogen, San Diego, CA) according to the manufacturer' ⁇ instructions.
  • Poly(A) + RNA from rat ovary, pancreas, skeletal muscle, smooth muscle, and kidney was obtained from Clonetech, Palo Alto, CA.
  • RNA Reverse transcription of poly(A) + RNA to cDNA and subsequent complification by the PCR was accomplished using reagents from the GeneAmp RNA PCR kit (Perkin Elmer Cetus; Norwalk, CT) .
  • rat nucleus accumbens poly(A) + RNA was reverse transcribed in a final 20 ⁇ l volume containing 10 mM Tris-HCl, pH 8.3/50 mM KC1/5 mM MgCl 2 / 1 mM each dNTP/2.5 ⁇ M random hexamer primer ⁇ /1 unit ⁇ l' 1 RNase inhibitor/2.5 units ⁇ l "1 Moloney urine leukemia virus reverse transcriptase for 10 min at 22°C followed by 45 min at 42°C. The reaction was terminated by heating for 5 min at 99°C followed by chilling to 4°C.
  • the above 20 ⁇ l reaction was adjusted to a final 100 ⁇ l volume containing 10 mM Tri ⁇ -HCl, pH 8.3/50 mM KC1/2 mM MgCl 2 /2 ⁇ M each upstream (A312) and downstream (A313) degenerate ⁇ oligonucleotide primers/0.2 mM each dNTP (contributed from the reverse transcription reaction) containing 2.5 units AmpliTaq DNA Polymerase (Perkin Elmer Cetus; Norwalk, CT) .
  • Blots were subsequently hybridized with 32 P-labeled A315 oligonucleotide in 6X SSPE/1% SDS/2X Denhardt's at 42°C overnight. Blots were washed in IX SSPE/1% SDS at 37°C and exposed to Kodak XAR film with an inten ⁇ ifying screen at - 80°C.
  • EXAMPLE 7 CDNA LIBRARY SCREENING An oligo(dT)-primed and size-selected (>800 bp) Wistar rat nucleus accumbens cDNA library was bidirectionally cloned in eukaryotic expression vector pcDNAI (Invitrogen; San Diego, CA) and transformed into E . col i MC1061/P3.
  • Plasmid DNA wa ⁇ prepared from 72 sublibrary pools of -5000 clones each and subjected to PCR amplification in a final 50 ⁇ l volume of 10 mM Tris-HCl, pH 8.3/50 mM KCl/2 mM MgCl 2 /0.2 mM each dNTP/2 ⁇ M each A312 and A313 primers. Reactions were heated to 94°C for 5 min to en ⁇ ure denaturation of the template DNA and then held at the 40°C annealing temperature.
  • rat nucleus accumbens poly(A) + RNA wa ⁇ converted to first strand cDNA using A333 gene-specific primer.
  • homopolymeric dC tails were added with terminal deoxynucleotidyl transfera ⁇ e.
  • Amplification products were purified from 1% low melting point agarose gel slices, subcloned into plasmid vector pAMPl and transformed into E . col i DH5 ⁇ (CloneAmp System; Gibco BRL, Gaithersburg, MD) .
  • EXAMPLE 9 CONSTRUCTION OF FULL-LENGTH RECEPTOR cDNA ⁇
  • the first full-length receptor cDNA was constructed as follows. Two receptor DNA fragments were prepared by restriction endonuclea ⁇ e dige ⁇ tion and purification from 1% low melting point agarose gels: i) ⁇ 1.2kb Pstl-StuI fragment from 5' RACE subclone pAMPl-L2; and ii) ⁇ 2.2kb Stul-Xjal fragment from cDNA library clone pcDNAI-275.
  • the second full-length receptor cDNA was constructed in a similar manner.
  • a -1.3 kB Pstl-StuI fragment from 5'RACE clone pAMPl-U3 and a -2.2 kb Stul- Spel fragment from cDNA library clone pcDNAI-237 were isolated from 1% low melting point agarose gels. They were introduced into Ps l-Xbal linearized and phosphata ⁇ ed eukaryotic expre ⁇ ion pla ⁇ mid pcDNAI in a three part ligation.
  • TRANSIENT TRANSFECTION COS-7 cells were seeded at 1 x 10 7 cells per T-150 tis ⁇ ue culture fla ⁇ k and grown overnight. The monolayer (-75% confluent) was then transfected with 14 ⁇ g pla ⁇ mid DNA and 120 ⁇ g Lipofectin reagent (Gibco BRL) in 7.5 ml Optimem media (Gibco BRL) containing 5.5 ⁇ M 2- mercaptoethanol.
  • DMEM low gluco ⁇ e
  • Cells were harvested 60 hr ⁇ po ⁇ t-tran ⁇ fection by ⁇ craping into ice cold PBS and pelleted by centrifugation at -160 x g for 4 min at 4°C.
  • the cell pellet was resu ⁇ pended in ice cold 20 mM HEPES and disrupted twice for 15 sec using a Brinkmann polytron homogenizer on setting number 3.
  • the homogenate was then centrifuged at 48,400 x g for 20 min at 4°C and the pellet resuspended in ice cold 20 mM HEPES by brief homogenization. Aliquot ⁇ were fla ⁇ h frozen in a dry ice/ethanol bath and then ⁇ tored at -80°C.
  • DNA was sequenced by the dideoxy chain-termination method (Sanger et al.. Proc. Natl. Acad. Sci. USA 74:5463-5467, 1977) with modified phage T7 DNA polymerase (Sequenase; United States Biochemical ⁇ , Cleveland, OH) .
  • RADIOLIGAND BINDING ASSAYS Radioligand studies were carried out in 20 mM N-2- Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, Sigma, St. Louis, MO), pH 7.4, containing 1.0 mg/ml BSA (Protease free, Fraction V, Sigma), 1.0 mg/ml bacitracin (Sigma) , 5 ⁇ g/ml bestatin-HCl (Sigma) , 1 ⁇ g/ml phosphoramidon (Sigma) .
  • Membranes from COS-7 cells transfected with Cla receptor were thawed and diluted to a concentration of approximately 1.3 ⁇ g protein/ml while membranes from COS-7 cells transfected with Clb receptor (Clb membranes) were thawed and diluted to 13 ⁇ g protein/ml for all binding studies.
  • Transfected COS cells from Example 9 were u ⁇ ed to prepare the membrane ⁇ . Protein determinations were made using the Bradford assay (Biorad, Richmond, CA) with BSA as a control.
  • Table 1 below indicates the concentration of ligand which produces half-maximal inhibition (IC50) of 12S I-salmon calcitonin binding to membranes from COS cells expres ⁇ ing the indicated receptor. Results are means of IC50s measured in 2-3 separate experiments.
  • EXAMPLE 13 ADENYLATE CYCLASE COUPLING Transfected COS cells were cultured in Dulbecco's Minimum Es ⁇ ential medium (Irvine Scientific, Santa Ana, CA) containing 10% fetal bovine serum (Gemini Bioproducts, Calabasa ⁇ , CA) , 1 mg gluco ⁇ e/ml, and 2 mM L-glutamine. Twenty-four hour ⁇ after transfection (described in Example 10), cells were ⁇ ubcultured at 1 x 10 5 cells/0.2 ml medium/well in 96-well tis ⁇ ue culture plate ⁇ (Corning Glass Works, Corning NY). Cells were maintained at 37°C and 5% C0 2 /95% humidified air.
  • cyclic AMP cyclic AMP
  • medium was replaced with 100 ⁇ l Dulbecco's pho ⁇ phate buffered ⁇ aline (DPBS; Sigma) containing 0.1 mg BSA/ml and 0.05 mg i ⁇ obutylmethyl xanthine/ml, pH 7.3.
  • DPBS Dulbecco's pho ⁇ phate buffered ⁇ aline
  • DPBS Dulbecco's pho ⁇ phate buffered ⁇ aline
  • test substance( ⁇ ) at 2x final concentration ⁇ were added and plates were incubated an additional 25 minutes. The response was halted by acidification with 25 ⁇ l 10% trichloracetic acid, followed by neutralization with 25 ⁇ l 0.8 M Tris (hydroxymethyl) aminomethane.
  • Immunoreactive cAMP in supernatants was acetylated and measured using a ⁇ cintillation proximity a ⁇ ay (Amer ⁇ ham, Arlington Heights, IL) . Prior to as ⁇ ay, supernatants were diluted 1:25 in assay buffer and cAMP was acetylated following a protocol provided by the manufacturer.
  • Results shown in Figure 8 demonstrate that salmon calcitonin at 10"*M strongly stimulates cAMP synthesis in COS cells expressing either Cla or Clb receptors, but not in vector-transfected control cells. Transfection did not alter /S-adrenegic receptor coupling to adenylate cyclase in COS cell ⁇ , a ⁇ seen by the equal respon ⁇ ivene ⁇ s to isoproterenol in all three cell populations. These result ⁇ indicate that Cla and Clb transcripts encode functional receptors that are positively coupled to adenylate cyclase. Stimulation of adenylate cyclase activity or inhibition of agonist- ⁇ timulated activity in Cla- or Clb-expressing cells can be taken to indicate receptor agoni ⁇ t or antagonist activity, respectively.

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Abstract

L'invention porte sur récepteurs C1a et C1b clonés de rats, sur l'acide nucléique codant pour lesdits récepteurs, et sur des méthodes d'identification, de criblage, de caractérisation, d'essai ou d'isolement d'agonistes ou d'antagonistes de la calcitanine, connus ou candidats, y compris sur des essais de liaison et de sélection recourant à des préparations contenant des récepteurs C1a ou C1b.
PCT/US1994/003248 1993-03-24 1994-03-24 Recepteurs clones et methodes de criblage associees WO1994021665A1 (fr)

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WO1996031526A1 (fr) * 1995-04-06 1996-10-10 Amylin Pharmaceuticals, Inc. Agents antiobesite
US5622839A (en) * 1992-09-30 1997-04-22 Zymogenetics, Inc. Recombinant production of human calcitonin receptor polypeptides
WO2003045424A1 (fr) * 2001-11-26 2003-06-05 Protemix Corporation Limited Procedes et compositions pour normaliser les niveaux lipidiques dans les tissus mammaliens
WO2002057792A3 (fr) * 2000-12-29 2003-11-06 Neogenesis Pharmaceuticals Inc Criblage de proteines hydrophobes par selection d'affinites
US6964660B2 (en) 1997-07-08 2005-11-15 Atrionix, Inc. Tissue ablation device assembly and method of electrically isolating a pulmonary vein ostium from an atrial wall
US6966908B2 (en) 1997-07-08 2005-11-22 Atrionix, Inc. Tissue ablation device assembly and method for electrically isolating a pulmonary vein ostium from an atrial wall
US6997925B2 (en) 1997-07-08 2006-02-14 Atrionx, Inc. Tissue ablation device assembly and method for electrically isolating a pulmonary vein ostium from an atrial wall
US8932208B2 (en) 2005-05-26 2015-01-13 Maquet Cardiovascular Llc Apparatus and methods for performing minimally-invasive surgical procedures
US20150361173A1 (en) 2005-11-14 2015-12-17 Labrys Biologics, Inc. Antagonist antibodies directed against calcitonin gene-related peptide and methods using same
US9328167B2 (en) 2008-03-04 2016-05-03 Labrys Biologics, Inc. Methods of treating chronic pain
US20160207975A1 (en) * 2013-07-30 2016-07-21 Christopher J. Soares Cgrp agonist peptides
US9896502B2 (en) 2014-03-21 2018-02-20 Teva Pharmaceuticals International Gmbh Antagonist antibodies directed against calcitonin gene-related peptide and methods using same
US10058380B2 (en) 2007-10-05 2018-08-28 Maquet Cordiovascular Llc Devices and methods for minimally-invasive surgical procedures
US10370425B2 (en) 2012-01-26 2019-08-06 Christopher Joseph Soares Peptide antagonists of the calcitonin CGRP family of peptide hormones and their use
US10392434B2 (en) 2016-09-23 2019-08-27 Teva Pharmaceuticals International Gmbh Treating refractory migraine
US10556945B2 (en) 2014-03-21 2020-02-11 Teva Pharmaceuticals International Gmbh Antagonist antibodies directed against calcitonin gene-related peptide and methods using same
US10597448B2 (en) 2009-08-28 2020-03-24 Teva Pharmaceuticals International Gmbh Methods for treating visceral pain associated with interstitial cystitis by administering antagonist antibodies directed against calcitonin gene-related peptide
US11390654B2 (en) 2016-09-02 2022-07-19 Christopher Joseph Soares Use of CGRP receptor antagonists in neuroprotection and neurological disorders

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Cited By (41)

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US5622839A (en) * 1992-09-30 1997-04-22 Zymogenetics, Inc. Recombinant production of human calcitonin receptor polypeptides
US5674689A (en) * 1992-09-30 1997-10-07 Zymogenetics, Inc. Human calcitonin receptor polypeptides and methods of use
US5674981A (en) * 1992-09-30 1997-10-07 Zymogenetics, Inc. Human calcitonin receptor polypeptides
US5683884A (en) * 1992-09-30 1997-11-04 Zymogenetics, Inc. Methods for identifying modulators of human calcitonin mediated metabolism
WO1996031526A1 (fr) * 1995-04-06 1996-10-10 Amylin Pharmaceuticals, Inc. Agents antiobesite
US6964660B2 (en) 1997-07-08 2005-11-15 Atrionix, Inc. Tissue ablation device assembly and method of electrically isolating a pulmonary vein ostium from an atrial wall
US6966908B2 (en) 1997-07-08 2005-11-22 Atrionix, Inc. Tissue ablation device assembly and method for electrically isolating a pulmonary vein ostium from an atrial wall
US6997925B2 (en) 1997-07-08 2006-02-14 Atrionx, Inc. Tissue ablation device assembly and method for electrically isolating a pulmonary vein ostium from an atrial wall
US7340307B2 (en) 1997-07-08 2008-03-04 Atrionix, Inc. Tissue ablation device assembly and method for electrically isolating a pulmonary vein ostium from an atrial wall
WO2002057792A3 (fr) * 2000-12-29 2003-11-06 Neogenesis Pharmaceuticals Inc Criblage de proteines hydrophobes par selection d'affinites
WO2003045424A1 (fr) * 2001-11-26 2003-06-05 Protemix Corporation Limited Procedes et compositions pour normaliser les niveaux lipidiques dans les tissus mammaliens
US8932208B2 (en) 2005-05-26 2015-01-13 Maquet Cardiovascular Llc Apparatus and methods for performing minimally-invasive surgical procedures
US9884908B2 (en) 2005-11-14 2018-02-06 Teva Pharmaceuticals International Gmbh Methods for treating headache using antagonist antibodies directed against calcitonin gene-related peptide
US9890210B2 (en) 2005-11-14 2018-02-13 Teva Pharmaceuticals International Gmbh Antagonist antibodies directed against calcitonin gene-related peptide
US9266951B2 (en) 2005-11-14 2016-02-23 Labrys Biologics, Inc. Antagonist antibodies directed against calcitonin gene-related peptide and methods using same
US9328168B2 (en) 2005-11-14 2016-05-03 Labrys Biologics, Inc. Methods of using anti-CGRP antagonist antibodies
US9340614B2 (en) 2005-11-14 2016-05-17 Labrys Biologics, Inc. Antagonist antibodies directed against calcitonin gene-related peptide and methods using same
US9346881B2 (en) 2005-11-14 2016-05-24 Labrys Biologics, Inc. Antagonist antibodies directed against calcitonin gene-related peptide and methods using same
US9365648B1 (en) 2005-11-14 2016-06-14 Labrys Biologics, Inc. Methods of using anti-CGRP antagonist antibodies
US10329343B2 (en) 2005-11-14 2019-06-25 Teva Pharmaceuticals International Gmbh Methods for treating headache using antagonist antibodies directed against calcitonin gene-related peptide
US20150361173A1 (en) 2005-11-14 2015-12-17 Labrys Biologics, Inc. Antagonist antibodies directed against calcitonin gene-related peptide and methods using same
US9884907B2 (en) 2005-11-14 2018-02-06 Teva Pharmaceuticals International Gmbh Methods for treating headache using antagonist antibodies directed against calcitonin gene-related peptide
US9890211B2 (en) 2005-11-14 2018-02-13 Teva Pharmaceuticals International Gmbh Antagonist antibodies directed against calcitonin gene-related peptide
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US10370425B2 (en) 2012-01-26 2019-08-06 Christopher Joseph Soares Peptide antagonists of the calcitonin CGRP family of peptide hormones and their use
US9951115B2 (en) * 2013-07-30 2018-04-24 Christopher J. Soares CGRP agonist peptides
US20160207975A1 (en) * 2013-07-30 2016-07-21 Christopher J. Soares Cgrp agonist peptides
US10556945B2 (en) 2014-03-21 2020-02-11 Teva Pharmaceuticals International Gmbh Antagonist antibodies directed against calcitonin gene-related peptide and methods using same
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US10519224B2 (en) 2014-03-21 2019-12-31 Teva Pharmaceuticals International Gmbh Treating headache comprising administering an antibody to calcitonin gene-related peptide
US11555064B2 (en) 2014-03-21 2023-01-17 Teva Pharmaceuticals International Gmbh Treating headache comprising administering an antibody to calcitonin gene-related peptide
US11390654B2 (en) 2016-09-02 2022-07-19 Christopher Joseph Soares Use of CGRP receptor antagonists in neuroprotection and neurological disorders
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