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WO2006067565A2 - Recepteur gabab tronque et procedes d'utilisation associes - Google Patents

Recepteur gabab tronque et procedes d'utilisation associes Download PDF

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Publication number
WO2006067565A2
WO2006067565A2 PCT/IB2005/003654 IB2005003654W WO2006067565A2 WO 2006067565 A2 WO2006067565 A2 WO 2006067565A2 IB 2005003654 W IB2005003654 W IB 2005003654W WO 2006067565 A2 WO2006067565 A2 WO 2006067565A2
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Prior art keywords
amino acid
polypeptide
receptor
acid sequence
seq
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PCT/IB2005/003654
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English (en)
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WO2006067565A3 (fr
Inventor
Fu-Zon Chung
Mei Cui
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Warner-Lambert Company Llc
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Publication of WO2006067565A2 publication Critical patent/WO2006067565A2/fr
Publication of WO2006067565A3 publication Critical patent/WO2006067565A3/fr

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    • 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
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • the invention relates to compositions and methods directed to truncated GABAb receptor subunits.
  • GABA Gamma amino butyric acid
  • GABAa The GABAb receptor, unlike the other two families which are ligand gated ion channels, is a seven transmembrane domain (7TM) G- protein coupled receptor with mixed effects on adenylate cyclase activity.
  • the GABAb receptor influences transmitter release by suppressing neuronal Ca ++ conductance.
  • Baclofen is a highly selective agonist of GABAb receptors.
  • GABAb(I ) and GABAB(2) Two GABAb receptor subunits, GABAb(I ) and GABAB(2), have been identified and cloned, as well as splice variants of the subunits.
  • the subunits are thought to associate, at least partly, through a coiled-coil motif.
  • This coiled-coil motif of GABAb2 is thought to mask the endoplasmic retention signal of GABAbI and allow surface expression of the receptor heterodimer.
  • Structure- function studies of the subunits have included deletion studies. See Bowery NG et al. 2002, Pharmacol. Rev. 54: 247-264; Grunewald S (2002) MoI. Pharmacol. 61 : 1070- 1080; Calver, AR (2001) J Neuroscience 21 (4): 1203-1216.
  • the invention relates to an isolated GABAbR2 receptor subunit polypeptide, wherein said polypeptide lacks a coiled-coil domain and forms a functional heterodimer with a polypeptide comprising a GABAbRI subunit polypeptide, and wherein the amino acid sequence of said polypeptide is described herein.
  • the invention also relates to an isolated GABAbR2 receptor subunit polypeptide wherein the amino acid sequence of said polypeptide is described herein.
  • the invention relates to nucleotide sequences encoding the GABAbR2 receptor subunit polypeptides of the invention, and expression vectors, and cell lines thereof.
  • the invention relates to a GABAb receptor protein comprising a heterodimer consisting of the GABAbR2 subunit polypeptides of the invention and a GABAbRI b subunit polypeptide described herein.
  • the invention in another aspect, relates to a method for identification of an agent which exhibits GABAb receptor modulating activity, comprising contacting the GABAb receptor protein of the invention with a test agent and detecting modulating activity or inactivity.
  • the invention relates to an agent identified by the inventive method for identification.
  • the invention in another aspect, relates to a method of producing a GABAb receptor comprising introducing into an appropriate cell line a suitable vector comprising a nucleotide sequence encoding the GABAbR2 subunit polypeptide of the invention.
  • the invention in another aspect, relates to a method of treating a disorder responsive to modulation of GABAb receptor activity in a mammal, said method comprising administering to said mammal an effective amount of a compound identifiable by the inventive method for identification.
  • Figure 1 depicts truncated GABAb receptor's response to GABA and dose- response to the antagonist CGP54626 in a FLIPR assay.
  • Figure 2 depicts truncated GABAb receptor's response to a positive allosteric modulator (PAM).
  • PAM positive allosteric modulator
  • FIG. 3 depicts monitoring of PAM affect by method A described herein.
  • FIG. 4 depicts monitoring of PAM affect by method B described herein.
  • the invention encompasses compositions comprising isolated GABAb subunit polypeptides; GABAb receptors comprising the polypeptides; nucleotide sequences encoding the polypeptides, and vectors and cell lines thereof; as well as methods of using the compositions.
  • the compositions are useful for production of a GABAb receptor; for identifying agents that modulate GABAb receptor activity, for treating and preventing disorders responsive to modulation of GABAb receptor activity. Methods related to such uses are encompassed by the present invention.
  • compositions that are isolated GABAb subunit polypeptides, and GABAb receptors comprising the polypeptides.
  • a composition of the present invention is an isolated GABAbR2 receptor subunit polypeptide, wherein said polypeptide lacks a coiled-coil domain and forms a functional heterodimer with a polypeptide comprising a GABAbRI subunit polypeptide, and wherein the amino acid sequence of said polypeptide comprising the GABAbRI b subunit polypeptide is selected from the group consisting of: a) an amino acid sequence set forth in SEQ ID NO: 4 and having at least one conservative amino acid substitution; b) an amino acid sequence set forth in SEQ ID NO: 4 or in a) and further comprising a tag polypeptide sequence; and c) an amino acid sequence set forth in SEQ ID NO: 4.
  • a coiled-coil domain is intended a polypeptide segment having the motif EEKSRLLEKENRELEKIIAEKEERVSELRHQLQS or the motif TSRLEGLQSENHRLRMKITELDKDLEEVTMQLQD.
  • a “functional heterodimer” is intended a GABAb receptor heterodimer that modulates the level of cytoplasmic calcium in response to an agonist or an antagonist.
  • a GABAbR2 receptor subunit polypeptide of the invention can form a functional heterodimer with a polypeptide comprising a GABAbRI subunit polypeptide, the subunits can be expressed in a cell line, and compared to a cell line expressing wildtype GABAb heterodimers, which wildtype heterodimers can measurably modulate cytoplasmic calcium levels in the FLIPR assay in the presence of chimeric G proteins such as Gqi3(5).
  • “Modulate” means an increase or a decrease.
  • a composition of the present invention is an isolated GABAbR2 receptor subunit polypeptide wherein the amino acid sequence of said polypeptide is selected from the group consisting of: a) an amino acid sequence set forth in SEQ ID NO: 2 and having at least one conservative amino acid substitution; b) an amino acid sequence set forth in SEQ ID NO: 2 or in a) and further comprising a tag polypeptide sequence; and c) an amino acid sequence set forth in SEQ ID NO: 2.
  • Conservative amino acid substitutions are well known in the art.
  • conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, VaI, Leu, and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and GIu, substitution between the amide residues Asn and GIn, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, and Tyr.
  • Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306- 1310 (1990).
  • Conservative amino acid substitutions involve exchanging a member within one group for another member within the same group, including the aromatic, hydrophobic, polar, basic, acidic, and small groups as follows:
  • Aromatic Phenylalanine, Tryptophan, Tyrosine
  • “Phenotypically silent” means that the conservative amino acid substitution does not significantly alter the biological function of the polypeptide comprising one or more of the substitutions in comparison to the corresponding unsubstituted polypeptide; wherein the unsubstituted polypeptide has an identical amino acid sequence to the substituted polypeptide, but for the conservative amino acid substitutions.
  • a conservative amino acid substitution of a GABAbR2 polypeptide of the invention is phenotypically silent, is tested by providing a first cell line that expresses the substituted GABAbR2 polypeptide and a GABAbRI b polypeptide having the amino acid sequence set forth in SEQ ID NO:4, and a second cell line that expresses the substituted GABAbR2 polypeptide and a GABAbRIb polypeptide having the amino acid sequence set forth in SEQ ID NO:4; contacting said cell lines separately with known GABAb receptor agonists and antagonists; and determining whether the order of potency of the agonists and antagonists for heterodimers comprising the said polypeptides in said cell lines is altered by the amino acid substitutions.
  • Typical agonists and antagonists include SKF97541 , R-baclofen, (R,S)-baclofen, CGP54626, CGP55845, CGP52432, and SCH50911.
  • a composition of the present invention is a GABAb receptor protein comprising a heterodimer consisting of GABAbR2- and a GABAbRI b subunit described above.
  • the receptor protein of the invention can modulate the level of cytosolic calcium in response to a GABAb receptor agonist or antagonist.
  • the receptor of the invention exhibits a pharmacology that is similar to that of the wild type.
  • the agonist SKF97541 has higher potency for a receptor of the invention than does R-baclofen, that R-baclofen has higher potency for a receptor of the invention than does (R,S)-baclofen, that the antagonist CGP54626 or CGP55845 has higher potency for a receptor of the invention than does CGP52432, and that the antagonist CGP52432 has higher potency for a receptor of the invention than does SCH50911.
  • isolated means that the subunit polypeptide is not in its native state, in that it has been purified at least to some extent, or it has been synthetically produced, for example by recombinant methods.
  • isolated therefore includes the possibility of the receptor protein being in combination with other biological or non-biological material, such as cells, suspensions of cells or cell fragments, proteins, peptides, organic or inorganic solvents, or other materials where appropriate, but excludes the situation where the receptor protein is in a state as found in nature.
  • the term “isolated” is applicable to the polypeptides and receptor proteins of the invention.
  • the present invention encompasses GABAb receptor polypeptide sequences set forth herein recited, and variants that have at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity with the sequences. These variants may include the deletion, modification or addition of single amino acids or groups of amino acids within the protein sequence, as long as the biological functionality of the peptide is not adversely affected.
  • Whether a particular deletion, modification or addition affects the biological function can be tested in a manner similar to testing whether a conservative amino acid substitution affects the biological function as described above, except for providing cell lines expressing receptors comprising the variant polypeptide, and cell lines expressing receptors comprising polypeptides identical to the variant polypeptide but for the variation.
  • the invention also provides nucleotide sequences which encode the GABAbR2 polypeptides of the invention or variants thereof, and nucleotide sequences complementary thereto.
  • the nucleotide sequences are DNA, RNA, or cDNA sequences.
  • the invention is a nucleotide sequence encoding a GABAbR2 receptor subunit polypeptide wherein the amino acid sequence of said polypeptide is selected from the group consisting of: a) an amino acid sequence set forth in SEQ ID NO: 2 and having at least one conservative amino acid substitution; b) an amino acid sequence set forth in SEQ ID NO: 2 or in a) and further comprising a tag polypeptide sequence; and c) an amino acid sequence set forth in SEQ ID NO:.2.
  • the nucleic acid molecules of the invention further comprise sequences that encode a tag polypeptide.
  • the tag polypeptide facilitates purification of the expressed protein comprising said tag polypeptide.
  • the tag is a histidine tag. It is recognized that the tag polypeptide can be fused to the N-terminal side, or to the C-terminal side of a GABAb polypeptide of the invention. It is also recognized that proteolytic cleavage sites can readily and optionally be engineered between the tag and the GABAb polypeptide of the invention, in order to optionally cleave the tag polypeptide.
  • the invention includes expression vectors which are modified to comprise nucleotide sequences encoding the polypeptides of the invention or variants thereof.
  • the expression vectors are modified to comprise nucleotide sequences encoding for a GABAbR2 polypeptide of the invention, or a variant thereof.
  • the expression vectors are modified to additionally comprise nucleotide sequences encoding for a GABAbRIb polypeptide set forth herein; for coexpression in a single vector.
  • the invention includes cell lines modified to express the polypeptides and receptors of the invention.
  • Such cell lines include transiently transfected cells; stably transfected cell lines; higher eukaryotic cell lines, such as mammalian cells or insect cells; or lower eukaryotic cells, such as yeast; or prokaryotic cells such as bacterial cells.
  • Particular examples of cells modified by insertion of vectors encoding for the polypeptides and receptors of the invention include Chinese Hamster Ovary (CHO) cells.
  • the selected cell line allows for mature glycosylation and cell surface expression of the inventive polypeptides and receptors.
  • the cell line may include a single vector which allows for expression of both of the receptor subtypes, or alternatively separate vectors for each subunit.
  • the subunits are co-expressed in order to optimize the dimerization, glycosylation, and transport of the glycosylated dimer to the cell surface.
  • polypeptides and receptors of the invention are transiently expressed in a cell line (e.g CHO-Gqi3(5)-NFAT- ⁇ -Iactamase ) or in an in vitro membrane translation system.
  • a cell line e.g CHO-Gqi3(5)-NFAT- ⁇ -Iactamase
  • in vitro membrane translation system e.g CHO-Gqi3(5)-NFAT- ⁇ -Iactamase
  • the heterodimer which forms a functional GABAb receptor protein of the invention comprises a first polypeptide that is a GABAbR2 polypeptide of the invention, or a variant thereof; and further comprises a second polypeptide that is any GABAbRI receptor subtype or splice variant, or variants thereof, that forms a functional GABAb receptor heterodimer with the GABAbR2 polypeptide of the invention, or a variant thereof.
  • the second polypeptide is a GABAbRI b polypeptide as set forth herein.
  • Other examples of this second polypeptide include known GABAbRI splice variants including those having a deletion in the C-terminal intercellular domain. In one embodiment, this deletion comprises the amino acids constituting a coiied-coil motif.
  • Another aspect of the invention is the use of the receptor proteins according to the invention, in screening methods designed to identify agents which act as receptor ligands and which may be useful to modulate receptor activity.
  • screening methods will involve contacting the protein concerned, the heterodimeric GABAb receptor, with a test agent; and detecting whether or not there is modulation in the receptor activity.
  • the present invention also includes within its scope those agents which are identified as possessing useful GABAb receptor modulation activity, by the screening methods described to herein.
  • Agent means any chemical or biochemical that can be subjected to one or more of the methods of the invention that recite use of an agent.
  • Agent includes a small molecule or compound, amino acid, protein, polypeptide, peptide, antibody, and nucleic acid.
  • Another aspect of the invention is the use of agents which have been identified by screening techniques referred to above in the treatment or disorders which are responsive to modulation of a GABAb receptor activity, in a mammal in need thereof.
  • Modulation means that there is agonism or antagonism or allosteric modulation at the receptor site, which results from ligand binding of the agent at the receptor.
  • GABAb receptors have been implicated in disorders of the central nervous system (CNS), gastrointestinal (Gl) tract, lungs and bladder and therefore modulation of GABAb receptor activity in these tissues is envisioned to result in a positive therapeutic outcome in relation to such disorders.
  • the agents identified using the screening techniques according to the invention are useful for treatment and/or prophylaxis of disorders such as cognitive disorders, spasticity, epilepsy, Alzheimer's disease, pain, anxiety, depression, addiction, fibromyalgia as well as affective disorders and feeding disorders.
  • disorders such as cognitive disorders, spasticity, epilepsy, Alzheimer's disease, pain, anxiety, depression, addiction, fibromyalgia as well as affective disorders and feeding disorders.
  • the agents identified using the screening techniques according to the invention are useful for treatment and/or prophylaxis of disorders such as those described below.
  • This invention relates to a method of treating a disorder or condition selected from faintness attacks, epilepsy, asphyxia, general anoxia, hypoxia, spinal cord trauma, traumatic brain injury, head trauma, cerebral ischemia, stroke (including thromboembolic stroke, focal ischemia, global ischemia, transient cerebral ishemia attacks and other cerebral vascular problems accompanied by cerebral ischemia such as in patients undergoing carotid endarterectomy or other vascular surgical procedures in general or diagnostic vascular surgical procedures such as angiography), cramp caused by thiosemicarbazide, cardiazole cramp, and cerebral vascular disorders due to acute or chronic cerebrovascular damage such as cerebral infarction, subarachnoid haemorrhage or cerebral edema in a mammal, comprising administering to a mammal in need of such treatment a therapeutically effective amount of an agent or a pharmaceutically acceptable salt thereof wherein said agent is identified by a method of the invention for identifying agents that exhibit GABAb receptor modulating activity.
  • This invention also relates to a method of treating a disorder or condition selected from the group consisting of neurocardiac disorders such as neurocardiac syncope, neurogenic syncope, hypersensitive Carotid sinus, neurovascular syndrome and arrythmias including arrythmias secondary to gastrointestinal disturbances in a mammal, comprising administering to a mammal in need of such treatment a therapeutically effective amount of an agent or a pharmaceutically acceptable salt thereof wherein said agent is identified by a method of the invention for identifying agents that exhibit GABAb receptor modulating activity.
  • a disorder or condition selected from the group consisting of neurocardiac disorders such as neurocardiac syncope, neurogenic syncope, hypersensitive Carotid sinus, neurovascular syndrome and arrythmias including arrythmias secondary to gastrointestinal disturbances in a mammal
  • This invention also relates to a method of treating a disorder or condition selected from acute pain, chronic pain, pain resulting from soft tissue and peripheral damage such as acute trauma; postherpetic neuralgia, occipital neuralgia, trigeminal neuralgia, segmental or intercostal neuralgia and other neuralgias; pain associated with osteoarthritis and rheumatoid arthritis; musculoskeletal pain such as pain associated with strains, sprains and trauma such as broken bones; spinal pain, central nervous system pain such as pain due to spinal cord or brain stem damage; lower back pain, sciatica, dental pain, myofascial pain syndromes, episiotomy pain, gout pain, and pain resulting from burns; deep and visceral pain, such as heart pain; muscle pain, eye pain, inflammatory pain, orofacial pain, for example, odontalgia; abdominal pain, and gynecological pain, for example, dysmenorrhoea, labour pain
  • This invention also relates to a method of treating a disorder or condition selected from mood disorders, such as depression, or more particularly, depressive disorders, for example, major depressive disorder, severe unipolar recurrent major depressive episodes, dysthymic disorder, depressive neurosis and neurotic depression, melancholic depression including anorexia, weight loss, insomnia, early morning waking or psychomotor retardation, atypical depression (or reactive depression) including increased appetite, hypersomnia, psychomotor agitation or irritability; treatment resistant depression; seasonal affective disorder and pediatric depression; premenstrual syndrome, premenstrual dysphoric disorder, hot flashes, bipolar disorders or manic depression, for example, bipolar I disorder, bipolar II disorder and cyclothymic disorder; seasonal affective disorder, conduct disorder and disruptive behavior disorder; stress related somatic disorders and anxiety disorders, such as childhood anxiety disorder, panic disorder with or without agoraphobia, agoraphobia without history of panic disorder, specific phobias (e.g., specific animal phobias), social anxiety disorder, social
  • This invention also relates to a method of treating a disorder or condition selected from the group consisting of borderline personality disorder; schizophrenia and other psychotic disorders, for example, schizophreniform disorders, schizoaffective disorders, delusional disorders, brief psychotic disorders, shared psychotic disorders, psychotic disorders due to a general medical condition, psychotic disorders with delusions or hallucinations, substance induced psychotic disorder, psychotic episodes of anxiety, anxiety associated with psychosis, psychotic mood disorders such as severe major depressive disorder; mood disorders associated with psychotic disorders such as acute mania and depression associated with bipolar disorder, mood disorders associated with schizophrenia; and behavioral disturbances associated with mental retardation in a mammal, comprising administering to a mammal in need of such treatment a therapeutically effective amount of an agent or a pharmaceutically acceptable salt thereof wherein said agent is identified by a method of the invention for identifying agents that exhibit GABAb receptor modulating activity.
  • a disorder or condition selected from the group consisting of borderline personality disorder; schizophrenia and other psychotic disorders, for
  • This invention also relates to a method of treating a disorder or condition selected from the group consisting of sleep disorders such as insomnia (e.g., primary insomnia including psychophysiological and idiopathic insomnia, secondary insomnia including insomnia secondary to restless legs syndrome, Parkinson's disease or another chronic disorder, and transient insomnia), somnambulism, sleep deprivation, REM sleep disorders, sleep apnea, hypersomnia, parasomnias, sleep-wake cycle disorders, jet lag, narcolepsy, sleep disorders associated with shift work or irregular work schedules, deficient sleep quality due to a decrease in slow wave sleep caused by medications or other sources, and other sleep disorders in a mammal, comprising administering to a mammal in need of such treatment a therapeutically effective amount of an agent or a pharmaceutically acceptable salt thereof wherein said agent is identified by a method of the invention for identifying agents that exhibit GABAb receptor modulating activity.
  • insomnia e.g., primary insomnia including psychophysiological and idiopathic insomnia, secondary insomnia including
  • This invention also relates to a method of increasing slow wave sleep and increasing growth hormone secretion in a human subject comprising administering to a human subject in need of such treatment a therapeutically effective amount of an agent or a pharmaceutically acceptable salt thereof wherein said agent is identified by a method of the invention for identifying agents that exhibit GABAb receptor modulating activity.
  • This invention also relates to a method of treating a disorder or condition selected from the group consisting of respiratory diseases, particularly those associated with excess mucus secretion, such as chronic obstructive airways disease, bronchopneumonia, chronic bronchitis, cystic fibrosis, adult respiratory distress syndrome, and bronchospasm; cough, whooping cough, angiotensin converting enzyme (ACE) induced cough, pulmonary tuberculosis, allergies such as eczema and rhinitis; contact dermatitis, atopic dermatitis, urticaria, and other eczematoid dermatitis; itching, hemodialysis associated itching; inflammatory diseases such as inflammatory bowel disease, psoriasis, osteoarthritis, cartilage damage (e.g., cartilage damage resulting from physical activity or osteoarthritis), rheumatoid arthritis, psoriatic arthritis, asthma, pruritis and sunburn; and hypersensitivity
  • This invention also relates to a method of treating a disorder or condition selected from the group consisting of neurodegenerative disorders, such as Parkinson's disease (PD), Huntington's disease (HD) and Alzheimer's disease (AD); delerium, dementias ⁇ e.g., senile dementia of the Alzheimer's type, senile dementia, vascular dementia, HIV-1 associated dementia, AIDS dementia complex (ADC), dementias due to head trauma, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, or due to multiple etiologies), amnestic disorders, other cognitive or memory disorders, and behavioral symptoms of dementia in a mammal, comprising administering to a mammal in need of such treatment a therapeutically effective amount of an agent or a pharmaceutically acceptable salt thereof wherein said agent is identified by a method of the invention for identifying agents that exhibit GABAb receptor modulating activity.
  • a disorder or condition selected from the group consisting of neurodegenerative disorders, such as Parkinson's disease (
  • This invention also relates to a method of treating a disorder or condition selected from the group consisting of Down's syndrome; Sjogren's syndrome, hypertension, hematopoiesis, postoperative neuroma, benign prostatic hypertrophy, periodontal disease, hemorrhoids and anal fissures, infertility, reflex sympathetic dystrophy, hepatitis, tenalgia attendant to hyperlipidemia, vasodilation, fibrosing and collagen diseases such as scleroderma and eosinophilic fascioliasis; and vasospastic diseases such as angina, migraine and Reynaud's disease in a mammal, comprising administering to a mammal in need of such treatment a therapeutically effective amount of an agent or a pharmaceutically acceptable salt thereof wherein said agent is identified by a method of the invention for identifying agents that exhibit GABAb receptor modulating activity.
  • a disorder or condition selected from the group consisting of Down's syndrome; Sjogren'
  • This invention also relates to a method of treating a disorder or condition selected from the group consisting of ophthalmic diseases such as dry eye syndrome, conjunctivitis, vernal conjunctivitis, and the like; and ophthalmic conditions associated with cell proliferation such as proliferative vitreoretinopathy in a mammal, comprising administering to a mammal in need of such treatment a therapeutically effective amount of an agent or a pharmaceutically acceptable salt thereof wherein said agent is identified by a method of the invention for identifying agents that exhibit GABAb receptor modulating activity.
  • ophthalmic diseases such as dry eye syndrome, conjunctivitis, vernal conjunctivitis, and the like
  • ophthalmic conditions associated with cell proliferation such as proliferative vitreoretinopathy in a mammal
  • This invention also relates to a method of treating a disorder or condition selected from the group consisting of autism, attention deficit hyperactivity disorder (ADHD), angiogenesis (i.e., use for the inhibition of angiogenesis), Reiter's syndrome and arthropathies in a mammal, comprising administering to a mammal in need of such treatment a therapeutically effective amount of an agent or a pharmaceutically acceptable salt thereof wherein said agent is identified by a method of the invention for identifying agents that exhibit GABAb receptor modulating activity.
  • ADHD attention deficit hyperactivity disorder
  • angiogenesis i.e., use for the inhibition of angiogenesis
  • Reiter's syndrome and arthropathies in a mammal
  • This invention also relates to a method of treating a disorder or condition selected from the group consisting of substance-related disorders arising from the use of alcohol, amphetamines (or amphetamine-like substances) caffeine, cannabis, cocaine, hallucinogens, inhalants and aerosol propellants, nicotine, opioids, phenylgiycidine derivatives, sedatives, hypnotics, and anxiolytics, which substance-related disorders include dependence and abuse, intoxication, withdrawal, intoxication delerium and withdrawal delerium; and addiction disorders involving addictions to be ⁇ aviura ⁇ .y., addictions to gambling and other addictive behaviors) in a mammal, comprising administering to a mammal in need of such treatment a therapeutically effective amount of an agent or a pharmaceutically acceptable salt thereof wherein said agent is identified by a method of the invention for identifying agents that exhibit GABAb receptor modulating activity.
  • substance-related disorders include dependence and abuse, intoxication, withdrawal, intoxication delerium and withdrawal
  • This invention also relates to a method of treating a disorder or condition selected from the group consisting of Down's syndrome; demyelinating diseases such as multiple sclerosis (MS) and amylolateral sclerosis (ALS) in a mammal, comprising administering to a mammal in need of such treatment a therapeutically effective amount of an agent or a pharmaceutically acceptable salt thereof wherein said agent is identified by a method of the invention for identifying agents that exhibit GABAb receptor modulating activity.
  • a disorder or condition selected from the group consisting of Down's syndrome; demyelinating diseases such as multiple sclerosis (MS) and amylolateral sclerosis (ALS) in a mammal, comprising administering to a mammal in need of such treatment a therapeutically effective amount of an agent or a pharmaceutically acceptable salt thereof wherein said agent is identified by a method of the invention for identifying agents that exhibit GABAb receptor modulating activity.
  • MS multiple sclerosis
  • ALS amylolateral sclerosis
  • This invention also relates to a method of treating a disorder or condition selected from the group consisting of pervasive development disorder, fibromyalgia, human immunodeficiency virus (HIV) infections; HIV encephalopathy; dissociative disorders such as body dysmorphic disorders; eating disorder such as anorexia and bulimia; ulcerative colitis; Crohn's disease; irritable bowel syndrome; chronic pancreatitis, chronic fatigue syndrome; sudden infant death syndrome (SIDS); overactive bladder; lower urinary tract symptoms of overactive bladder; chronic cystitis; chemotherapy induced cystitis; cough, angiotensin converting enzyme (ACE) induced cough, itch, hiccups, premenstrual syndrome, premenstrual dysphoric disorder, amenorrheic disorders such as desmenorrhea; reflex sympathetic dystrophy such as shoulder/hand syndrome; plasma extravasation resulting from cytokine chemotherapy; disorders of bladder function such as chronic cystitis, bladder detrusor hyper-reflexia, inflammation of the
  • This invention also relates to a method of treating a disorder or condition selected from the group consisting of movement disorders such as primary movement disorders, akinesias, dyskinesias (e.g., familial paroxysmal dyskinesia, tardive dyskinesia, tremor, chorea, myoclonus, tics and other dyskinesias) spasticities, Tourette's syndrome, Scott syndrome, palsys ⁇ e.g., Bell's palsy, cerebral palsy, birth palsy, brachial palsy, wasting palsy, ischemic palsy, progressive bulbar palsy and other palsys), akinetic-rigid syndrome; extrapyramidal movement disorders such as medication-induced movement disorders, for example, neuroleptic-induced Parkinsonism, neuroleptic malignant syndrome, neuroleptic-induced acute dystonia, neuroleptic-induced acute akathisia, neuroleptic- induced tardive dyskinesia and medication-induced postural
  • This invention also relates to a method of treating a disorder or condition selected from the group consisting of mastalgia syndromes, motion sickness, systemic lupus erythematosis and immune dysfunctions (e.g., stress induced immune dysfunctions such as idiopathic immune dysfunctions, post infection immune dysfunctions, post lumpectomy immune dysfunctions, porcine stress syndrome, bovine shipping fever, equine paroxysmal fibrillation, confinement dysfunction in chicken, sheering stress in sheep, and human-animal interaction stress in dogs) in a mammal, comprising administering to a mammal in need of such treatment a therapeutically effective amount of an agent or a pharmaceutically acceptable salt thereof wherein said agent is identified by a method of the invention for identifying agents that exhibit GABAb receptor modulating activity.
  • a disorder or condition selected from the group consisting of mastalgia syndromes, motion sickness, systemic lupus erythematosis and immune dysfunctions (e.g., stress induced immune dysfunctions such as
  • This invention also relates to a method of treating a disorder or condition selected from the group consisting of gastrointestinal (Gl) disorders, including inflammatory gastrointestinal disorders such as inflammation bowel disease, disorders caused by Helicobacter pylori and diseases of the Gl tract such as gastro-esophageal reflux disease (GERD), gastritis, proctitis, gastroduodenal ulcers, peptic ulcers, dyspepsia, disorders associated with the neuronal control of viscera, ulcerative colitis, Crohn's disease, irritable bowel syndrome and emesis, including post operative nausea and post operative vomiting, and including acute, delayed or anticipatory emesis (emesis includes emesis induced by chemotherapy, radiation, toxins, viral or bacterial infections, pregnancy, vestibular disorders, for example, motion sickness, vertigo, dizziness and Meniere's disease, surgery, migraine, variations in intercraniai pressure, gastro-oesophageal reflux disease, acid indigestion, over indulgence in food
  • This invention also relates to a method of treating a disorder or condition selected from the group consisting of neoplasms, including breast tumours, gastric carcinomas, gastric lymphomas, neuroganglioblastomas and small cell carcinomas such as small cell lung cancer in a mammal, comprising administering to a mammal in need of such treatment a therapeutically effective amount of an agent or a pharmaceutically acceptable salt thereof wherein said agent is identified by a method of the invention for identifying agents that exhibit GABAb receptor modulating activity.
  • a disorder or condition selected from the group consisting of neoplasms, including breast tumours, gastric carcinomas, gastric lymphomas, neuroganglioblastomas and small cell carcinomas such as small cell lung cancer in a mammal
  • the compounds which are identified according to the screening methods outlined above may be formulated with standard pharmaceutically acceptable carriers and/or excipients as is routine in the pharmaceutical art, and as fully described in Remmington's Pharmaceutical Sciences, Mack Publishing Company, Eastern Pennsylvania, 17th Ed, 1985, the disclosure of which is included herein in its entirety by way of reference.
  • the compounds may be administered via enteral or parenteral routes such as via oral, buccal, anal, pulmonary, intravenous, intraarterial, intramuscular, intraperitoneal, topical or other appropriate administration routes.
  • Truncated human GABAb R1 b (SEQ ID NO: 3 encoding SEQ ID NO:4) and truncated human GABAb R2 (SEQ ID NO:1 encoding SEQ ID NO:2) were cloned from human whole brain cDNA library (BD Bioscience) using Polymerase Chain Reaction (PCR) and following the vendors's protocol. The PCR products were then cloned into the pcDNA3.1 vector using the TOPO cloning kit (Invitrogen) and following the vendor's protocol. The resulting plasmid DNA was checked by restriction enzyme digestion and the corrected plasmids were confirmed by sequencing. The DNA primers used in the PCR reactions were:
  • GABAbRI b Primer 1 ⁇ '-ACCACCATGGGGCCCGGGGCCCCTTTTGCC-S' Primer 2 ⁇ '-TTACCCTCGGGTGATCAGCCTGCGCAT-S' GABAbR2:
  • Primer 3 ⁇ '-ACCACCATGGCTTCCCCGCGAAGCTCCGGG-S'
  • the GABAb R1 b truncated receptor subunit contains 747 amino acids ("aa”), which is the N-terminus to the 747th aa of the wild type receptor out of the 844 full length aa sequence. It encompasses the N-terminal extracellular domain and the seven- transmembrane (“7TM”) domain. The truncation eliminated the entire intracellular C- terminal (including the coiled-coiled domain) except 4 amino acids downstream of the putative 7TM domain.
  • the GABAb R2 truncated receptor contains 777 amino acids, which is the N- terminus to the 777 aa of the wild type receptor out of the 941 aa full length sequence. It encompasses the N-terminal extracellular domain, the seven-transmembrane (7TM) domain and the intracellular domain downstream of the 7TM, before the coiled-coiled domain. The truncation eliminated the majority of the intracellular C terminal, including the coiled-coiled domain.
  • Plasmid DNA encoding truncated GABAbRI b and plasmid DNA encoding truncated GABAbR2 were then co-transfected into CHO-Gqi3(5)-NFAT- ⁇ -lactamase cells (Aurora) using electroporation.
  • Cells were grown in T75 flask to confluence in DMEM medium (Invitrogen, Cat#10569) supplemented with 25mM HEPE, 1x NEAA, 10% dFBS, 100 ⁇ g/ml Zeocin and 2.5 ⁇ g/ml blasticindin (all reagents were from Invitrogen).
  • the cells were then detached, and 20 ⁇ g of each DNA were added to 10 7 cells in 0.8 ml of serum free medium in an electroporation chamber. After electroporation, cells were diluted and grown in the above medium with added selection antibiotic, geneticin (Invitrogen) at 1mg/ml. After about 10 days, most cells died and the remaining resistant cells were sorted using flow cytometry based on the activation of ⁇ -lactamase under GABA stimulation. Positively responding cells were collected. Once these collected cells grew to sufficient amount, they were evaluated using FLIPR assay in response to GABA. The cells that responded well to GABA were then used as the established stable cell line expressing functional truncated GABAbRIb and truncated GABAbR2. These are the cells used in the following EXAMPLES.
  • Cells were seeded in a 96-well back-well clear-bottom plate in growth medium until confluent. Cells were washed with FLIPR Assay Buffer (HBSS, with 20 mM HEPES and 2.5 mM probenecid), and then loaded with Fluo4-AM dye (Molecular Probes) in Assay Buffer for 60 to 90 minutes in the cell incubator. Cells were washed a second time with the assay buffer and then a certain amount of the assay buffer was left in the plate. Compounds such as the agonists GABA and baclofen, or the antagonists such as CGP54626 (Tocris), were diluted in assay buffer and prepared into a compound plate for the FLIPR run. The cell plate was then run on the FLIPR machine for measurement of Ca 2+ signal when stimulated with agonist in the presence or absence of antagonist. The maximal signal of the FLIPR response was exported and calculated for agonist or antagonist activity.
  • FLIPR Assay Buffer HBSS, with 20 mM HEPES and
  • the above cloned truncated GABAb receptors form functional heterodimer that has similar pharmacology to the wild type receptors.
  • GABAb R1 and GABAb R2 were co-expressed in CHO cells that have Gqi3(5) proteins, the functional response of the GABAb receptors can be monitored by measuring the intracellular Ca 2+ signal using FLIPR.
  • the truncated GABAb receptor heterodimer composed of the GABAb R1 b and GABAb R2 polypeptides described above, responded dose-dependently to a variety of agonists and antagonists, as shown in Table 1 and Figure 1 , in a manner similar to the wild type GABAb receptors.
  • the SKF97541 showed about ten times more potency than baclofen (Froestl et al (1995) J.Med.Chem. 38: 3297).
  • the functional response of the truncated GABAb receptors is robust and reliable, and amenable to high thoroughput (HTS) format to search for novel antagonists, and/or agonists.
  • HTS high thoroughput
  • PAM Positive allosteric modulator
  • PAM Fluo4-AM dye as above.
  • the PAM assay was carried out with protocol A and protocol B.
  • protocol A after the second wash, the cells were pretreated with PAM, such as CGP7930 (Tocris) at the indicated concentrations for 15 to 30 minutes. Then on the FLIPR, certain concentration of GABA was added and the Ca 2+ signal was measured.
  • protocol B after the second wash, the cells were pretreated with certain concentration of GABA for 15-30 minutes. Then on the FLIPR, certain concentration of CGP7930 was added and Ca 2+ signal was measured.
  • the truncated GABAb receptors' response to positive allosteric modulator (PAM) is in a similar manner to that of the wild type receptors.
  • PAM positive allosteric modulator
  • GABAb PAM CGP7930 enhanced the response of the truncated GABAb receptor to GABA.
  • the maximal response was enhanced by about 30%, and the dose response curve of GABA were left shifted.
  • the PAM effect on GABAb receptors can be monitored by two methods. As shown in Figure 3, in method A, the truncated GABAb receptor expressing cells were either pre-treated with PAM CGP7930 or were without treatment. After 15 minutes, all cells were added with 10 nM GABA on FLIPR and the Ca 2+ signal was measured. Without PAM, only minimal activity was observed. In contrast, in the presence of PAM, a dramatic enhanced signal was observed. Alternatively, the PAM activity can be monitored using method B as shown in Figure 4. In this method, the truncated GABAb receptor expressing cells were pre-treated with 100 nM GABA. After 15 minutes, cells were either treated with PAM CGP7930, or with buffer without PAM; and the Ca2+ signal was measured.
  • High throughput screening (HTS) method uses High throughput screening (HTS) to determine whether HTS is High throughput screening (HTS).
  • the above selected cell line expressing the truncated GABAbR2 and GABAbRI b, as well as the Gqi3(5) protein can be used in the functional FLIPR assay for HTS to search for GABAb agonists, antagonists, or allosteric modulators.
  • the following method was developed, optimized and validated with the use of robotic system for cell wash, and reagent transfer and addition.
  • the cells were grown in T300 flasks in Growth Media (DMEM, 5% dialyzed FBS, 25mM HEPES, 1x NEAA, 100 ⁇ g/ml Zeocin, 2.5 ⁇ g/ml blastocidin, 725 ⁇ g/ml g418), and seeded in Seeding Media (DMEM, 5% dialyzed FBS, 25mM HEPES, 1x NEAA) in 384- well black-well clear bottom plates at 15k cells/well. After 20-24 hrs, the cells are confluent and ready for assay. For every 20 cell plates, 1 mg Flou-4 AM dye was dissolved and prepared into assay buffer as dye solution.
  • Growth Media DMEM, 5% dialyzed FBS, 25mM HEPES, 1x NEAA
  • the cell plates were washed with assay buffer and loaded with dye solution at 3OuI /well for 60 minutes at 37 0 C in the incubator. Then the cell plates were washed again and incubated at room temperature for 60 minutes in assay buffer. The cells were then read on FLIPR. First, testing compounds at desired concentration were added to the cell plate and the Ca 2+ signal was measured. After 5 minutes, GABA was added at about EC 2O and Ca 2+ signal was measured continuously on FLIPR. In this way, a variety of compounds that modulate GABAb activity can be identified, such as agonists, antagonists and allosteric modulators.
  • SEQ ID NO:1 GABAbR2 cDNA ;
  • SEQ ID NO:2 Polypeptide sequence encoded by above GABAbR2 cDNA:
  • SEQ ID NO:3 GABAbRI b cDNA
  • SEQ ID NO:4 Polypeptide sequence encoded by above GABAbRIb cDNA ;
  • SEQ ID NQ's: 5-8 Primers.

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Abstract

L'invention porte sur des polypeptides de sous-unité du récepteur de GABAbR2 isolés, sur des séquences nucléotidiques codant les polypeptides de l'invention et des vecteurs d'expression, et sur des lignes cellulaires associées. L'invention se rapporte aussi à une protéine du récepteur GABAb comprenant ces polypeptides. Elle concerne également un procédé d'identification d'un agent qui possède une activité de modulation du récepteur de GABAb, un agent identifié par le procédé d'identification de l'invention, un procédé de fabrication d'un récepteur de GABAb, ainsi qu'un procédé de traitement d'un trouble en réponse à la modulation d'une activité du récepteur de GABAb.
PCT/IB2005/003654 2004-12-22 2005-12-16 Recepteur gabab tronque et procedes d'utilisation associes WO2006067565A2 (fr)

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CN115137829A (zh) * 2022-08-08 2022-10-04 山东大学 用于减轻放疗诱导的肠道损伤的药物

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WO1999040114A1 (fr) * 1998-02-05 1999-08-12 Merck & Co., Inc. Nouvelles sequences d'adn codant le recepteur gaba¿b?

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* Cited by examiner, † Cited by third party
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CN115137829A (zh) * 2022-08-08 2022-10-04 山东大学 用于减轻放疗诱导的肠道损伤的药物

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