Classifications

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  • 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
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• • A—HUMAN NECESSITIES
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  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/12—Antihypertensives

Definitions

• Neuroregulators comprise a diverse group of natural products that subserve or ' modulate communication in the nervous system. They include, but are not limited to, neuropeptides, amino acids, biogenic amines, lipids and lipid metabolites, and other metabolic byproducts. Many of these neuroregulator substances interact with specific cell surface receptors which transduce signals from the outside to the inside of the cell. G-protein coupled receptors (GPCRs) represent a major class of cell surface receptors with which many neurotransmitters interact to mediate their effects. GPCRs are predicted to have seven membrane-spanning domains and are coupled to their effectors via G-proteins linking receptor activation with intracellular biochemical sequelae such as stimulation of adenylyl cyclase.
• GPCRs G-protein coupled receptors
• MCH Melanin-concentrating hormone
• MCH has been reported to participate in a variety of processes including feeding, water balance, energy metabolism, general arousal/attention state, memory and cognitive functions, and psychiatric disorders (for reviews, see Baker, 1991; Baker, 1994; Nahon, 1994; Knigge et al., 1996). Its role in feeding or body weight regulation is supported by a recent Na ture publication (Qu et al . , 1996) demonstrating that MCH is overexpressed in the hypothalamus of ob/ob mice compared with ob/+ mice, and that fasting further increased MCH mRNA in both obese and normal mice during fasting. MCH also stimulated feeding in normal rats when injected into the lateral ventricles (Rossi et al .
• MCH may serve as an integrative neuropeptide involved in the reaction to stress, as well as in the regulation of feeding and sexual activity (Baker, 1991; Knigge et al . , 1996).
• MCH precursor The gene encoding the MCH precursor (ppMCH) has been cloned and encodes two additional peptides, neuropeptide El (13 AA) and neuropeptide GE (19AA) (Nahon et al . , 1989), which may also have biological activity.
• MCH peptide is synthesized primarily in hypothalamic neurons (the zona incerta and lateral hypothalamus) which project diffusely to many brain areas and to the pituitary (Bittencourt et al . , 1992); NEI has also been identified in medium from explanted hypothalamic neurons (Parkes and Vale, 1993) .
• MCH is also present in the periphery (testes and Gl tract; Hervieu and Nahon, 1995) but the highest concentrations are in the hypothalamus. There is also evidence for differential tissue-dependent processing of proMCH in mammals. A shorter MCH gene transcript that may result from alternate splicing was found in several brain areas and peripheral tissues, and a different peptide form was also found in the periphery (Viale et al . , 1997) .
• the gene encoding authentic MCH has been localized to chromosome 12, but two copies of a variant (truncated) gene are present on chromosome 5 (Breton et al., 1993); the functional significance, if any, of the variant is not yet known.
• the rat MCH gene may encode an additional putative peptide in a different reading frame (Toumaniantz et al . , 1996).
• the ligand retained biological activity and exhibited specific binding to a variety of cell lines including mouse melanoma (B16-F1, G4F, and G4F-7), PC12, and COS cells.
• MCH-elicited pigment aggregation in fish melanophores is associated with a reduction in basal cAMP levels, similar to that observed with norepinephrine (Svensson et al . , 1991; Morishita et al . , 1993) .
• Arguing against G-protein coupling is the general structural homology of MCH with ANF, whose receptors are not in the GPCR superfamily.
• Recently the actions of MCH were reported to be mediated via activation of a phosphatidylinositol-3-kinase pathway which is typical of tyrosine kinase and cytokine receptors (Qu et al . , 1998); however, since multiple signaling pathways (receptor cross talk) may produce this mediator no conclusions can be reached regarding MCH signal transduction pathways in mammalian systems.
• MCH methylcellulose
• lateral hypothalamus a brain area implicated in the regulation of thirst and hunger
• orexins A and B which are potent orexigenic agents, have been shown to have very similar localization to MCH in the lateral hypothalamus (Sakurai et al., 1998).
• MCH mRNA levels in this brain region are increased in rats after 24 hours of food-deprivation (Herve and Fellman, 1997); after insulin injection, a significant increase in the abundance and staining intensity of MCH immunoreactive perikarya and fibres was observed concurrent with a significant increase in the level of MCH mRNA (Bahjaoui- Bouhaddi et al . , 1994). Consistent with the ability of MCH to stimulate feeding in rats (Rossi et al . , 1997) is the observation that MCH mRNA levels are upregulated in the hypothalami of obese ob/ob mice (Qu et al .
• MCH appears to act as a functional antagonist of the melanocortin system in its effects on food intake and on hormone secretion within the HPA (hypothalamopituitary /adrenal axis) (Ludwig et al . , 1998) . Further evidence of the involvement of MCH in the regulation of feeding behavior came from studies in mice in which the gene encoding the MCH peptide has been deleted (Shimada et al . , 1998) .
• mice In these mice, the genetic deficiency of MCH led to a phenotype characterized by reduced body weight, low body fat content, and increased metabolic rate. More recently, it has been shown that the overexpression of the gene encoding MCH in different strains of mice can lead to obese phenotypes with and without secondary impairment of glucose homeostasis and insulin resistance (Tritos et al . , 2000) .
• the MCH cell group occupies a rather constant location in those areas of the lateral hypothalamus and subthalamus where they lie and may be a part of some of the so-called "extrapyramidal" motor circuits. These involve substantial striato- and pallidofugal pathways involving the thalamus and cerebral cortex, hypothalamic areas, and reciprocal connections to subthalamic nucleus, substantia nigra, and mid-brain centers (Bittencourt et al . , 1992) . In their location, the MCH cell group may offer a bridge or mechanism for expressing hypothalamic visceral activity with appropriate and coordinated motor activity. Clinically it may be of some value to consider the involvement of this MCH system in movement disorders, such as Parkinson's disease and Huntingdon's Chorea in which extrapyramidal circuits are known to be involved.
• MCH may regulate reproductive functions in male and female rats .
• MCH transcripts and MCH peptide were found within germ cells in testes of adult rats, suggesting that MCH may participate in stem cell renewal and/or differentiation of early spermatocytes (Hervieu et al . , 1996).
• MCH injected directly into the medial preoptic area (MPOA) or ventromedial nucleus (VMN) stimulated sexual activity in female rats (Gonzalez et al . , 1996) .
• MCH stimulated luteinizing hormone
• anti-MCH antiserum inhibited LH release
• the zona incerta which contains a large population of MCH cell bodies, has previously been identified as a regulatory site for the pre-ovulatory LH surge (MacKenzie et al . , 1984).
• MCH has been reported to influence release of pituitary hormones including ACTH and oxytocin.
• MCH analogues may also be useful in treating epilepsy.
• MCH has- also been observed to affect behavioral correlates of cognitive functions. MCH treatment hastened extinction of the passive avoidance response in rats (McBride et al . , 1994), raising the possibility that MCH receptor antagonists may be beneficial for memory storage and/or retention. A possible role for MCH in the modulation, or perception of pain is supported by the dense innervation of the periaqueductal grey (PAG) by MCH-positive fibers.
• PAG periaqueductal grey
• MCH may participate in the regulation of fluid intake. ICV infusion of MCH in conscious sheep produced diuretic, natriuretic, and kaliuretic changes in response to increased plasma volume (Parkes, 1996) . Together with anatomical data reporting the presence of MCH in fluid regulatory areas of the brain, the results indicate that MCH may be an important peptide involved in the central control of fluid homeostasis in mammals.
• the rat Forced Swim Test is a behavioral test that is used to screen compounds for antidepressant efficacy
• animals are placed in a cylinder of water, from which there is no escape, for an extended period of time.
• animals will display a range of behaviors such as immobility, climbing, swimming, and diving, with immobility being predominant after several minutes of immersion in the water. Consequently, many past studies have only measured or scored immobility after the administration of the test agent.
• this method does not score any other active behaviors that may be produced by potential antidepressants. Thus, if a particular class of antidepressant were to have very little effect on immobility, yet produce characteristic behaviors during the FST, these behaviors would not be scored and the conclusion would be that the compound in question does not possess antidepressant action.
• Rat Social Interaction Test There are a number of paradigms that have been used to determine whether a compound possesses anxiolytic action. A number of these tests involve food or water deprivation, punishment or measurement of consummatory behavior (see File, et al . , 1980, File, 1985, Rodgers, et al . , 1997 and Treit, 1985, for review) . In addition, in these models, prior conditioning reduces the uncertainty or anxiety. In general, these tests lack ethological validity.
• This test can detect anxiolytic agents such as the benzodiazepines (File and Hyde, 1978; File and Hyde, 1979; File, 1980) , in addition to non-benzodiazepines, including paroxetine and other SSRIs (Lightow ⁇ er, et al . , 1994) .
• the social interaction test can detect anxiogenic agents, including the inverse benzodiazepine receptor agonists (File, et al., 1982, File and Pellow, 1983; File and Pellow, 1984, File, 1985) .
• MCHl receptor antagonists are effective in animal models of obesity, depression and anxiety, which are predictive of efficacy in humans.
• MCHl receptor antagonists provide a novel method to treat obesity.
• MCHl receptor antagonists provide a novel method to treat depression and/or anxiety.
• This invention provides an isolated nucleic acid encoding a human MCHl receptor or a mutant of such human MCHl receptor which is activated by MCH or an analog or homolog thereof .
• This invention provides a nucleic acid encoding a human MCHl receptor, wherein the nucleic acid (a) hybridizes to a nucleic acid having the defined sequence shown in Figure 1 (SEQ ID NO: 1) under low stringency conditions or a sequence complementary thereto and (b) is further characterized by its ability to cause a change in the pH of a culture of CHO cells when an MCHl ligand is added to the culture and the CHO cells contain the nucleic acid which hybridized to the nucleic acid having the defined sequence or its complement.
• SEQ ID NO: 1 SEQ ID NO: 1
• This invention provides a purified human MCHl receptor protein.
• This invention provides a vector comprising a nucleic acid encoding a human MCHl receptor, particularly a vector adapted for expression of the human MCHl receptor in mammalian or non-mammalian cells.
• a vector is a plasmid designated pEXJ.
• HR-TL231 (ATCC Accession No. 203197) which comprises a nucleotide sequence encoding a human MCHl receptor.
• This invention also provides a cell comprising a vector which comprises a nucleic acid encoding a human MCHl receptor as well as a membrane preparation isolated from such cells.
• This invention further provides a nucleic acid probe comprising at least 15 nucleotides which specifically hybridizes with a nucleic acid encoding a mammalian MCHl receptor, wherein the probe has a unique sequence corresponding to a sequence present within the nucleic acid which encodes the human MCHl receptor or its complement, both of which are present in plasmid pEXJ.HR- TL231 (ATCC Accession No. 203197).
• This invention further provides a nucleic acid probe comprising at least 15 nucleotides which specifically hybridizes with a nucleic acid encoding a mammalian MCHl receptor, wherein the probe has a unique sequence corresponding to a sequence present within (a) the nucleic acid sequence shown in Figure 1 (SEQ ID NO: 1) or (b) the reverse complement thereof.
• This invention also provides an antisense oligonucleotide having a sequence capable of specifically hybridizing an RNA encoding a human MCHl receptor, so as to prevent translation of the RNA and an antisense oligonucleotide having a sequence capable of specifically hybridizing to the genomic DNA encoding a human MCHl receptor.
• This invention further provides an antibody capable of binding to a human MCHl receptor as well as an agent capable of competitively inhibiting the binding of the antibody to a human MCHl receptor.
• This invention provides a pharmaceutical composition
• a pharmaceutical composition comprising (a) an amount of the oligonucleotide described above capable of passing through a cell membrane and effective to reduce expression of a human MCHl receptor and (b) a pharmaceutically acceptable carrier capable of passing through the cell membrane.
• this invention provides a transgenic, nonhuman mammal expressing DNA encoding a human MCHl receptor.
• This invention also provides a transgenic, nonhuman mammal comprising a homologous recombination knockout of the native human MCHl receptor.
• This invention further provides a transgenic, nonhuman mammal whose genome comprises antisense DNA complementary to the DNA encoding a human MCHl receptor so placed within the genome as to be transcribed into antisense mRNA which is complementary to mRNA encoding the human MCHl receptor and which hybridizes to mRNA encoding the human MCHl receptor, thereby reducing its translation.
• this invention provides a process for identifying a chemical compound which specifically binds to a mammalian MCHl receptor which comprises contacting cells containing DNA encoding and expressing on their cell surface a mammalian MCHl receptor, wherein such cells do not normally express the mammalian MCHl receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian MCHl receptor.
• This invention provides a process for identifying a chemical compound which specifically binds to a mammalian MCHl receptor which comprises contacting a membrane preparation from cells transfected with DNA encoding and expressing on their cell surface the mammalian MCHl receptor, wherein such cells do not normally express the mammalian MCHl receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian MCHl receptor.
• This invention provides a process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian MCHl receptor which comprises separately contacting cells expressing on their cell surface the mammalian MCHl receptor, wherein such cells do not normally express the mammalian MCHl receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the mammalian MCHl receptor, a decrease in the binding of the second chemical compound to the mammalian MCHl receptor in the presence of the chemical compound indicating that the chemical compound binds to the mammalian MCHl receptor.
• This invention provides a process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian MCHl receptor which comprises separately contacting a membrane fraction from a cell extract of cells expressing on their cell surface the mammalian MCHl receptor, wherein such cells do not normally express the mammalian MCHl receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the mammalian MCHl receptor, a decrease in the binding of the second chemical compound to the mammalian MCHl receptor in the presence of the chemical compound indicating that the chemical compound binds to the mammalian MCHl receptor.
• This invention provides a method of screening a plurality of chemical compounds not known to bind to a mammalian MCHl receptor to identify a compound which specifically binds to the mammalian MCHl receptor, which comprises (a) contacting cells transfected with and expressing DNA encoding the mammalian MCHl receptor with a compound known to bind specifically to the mammalian MCHl receptor; (b) contacting the preparation of step (a) with the plurality of compounds not known to bind specifically to the mammalian MCHl receptor, under conditions permitting binding of compounds known to bind the mammalian MCHl receptor; (c) determining whether the binding of the compound known to bind to the mammalian MCHl receptor is reduced in the presence of the compounds within the plurality of compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so (d) separately determining the binding to the mammalian MCHl receptor of compounds included in the plurality of compounds, so as to thereby identify the compound which specifically
• This invention provides a method of screening a plurality of chemical compounds not known to bind to a mammalian MCHl receptor to identify a compound which specifically binds to the mammalian MCHl receptor, which comprises (a) contacting a membrane preparation from cells transfected with and expressing DNA encoding a mammalian MCHl receptor with a compound known to bind specifically to the mammalian MCHl receptor; (b) contacting the preparation of step (a) with the plurality of compounds not known to bind specifically to the mammalian MCHl receptor, under conditions permitting binding of compounds known to bind the mammalian MCHl receptor; (c) determining whether the binding of the compound known to bind to the mammalian MCHl receptor is reduced in the presence of the compounds within the plurality of compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so (d) separately determining the binding to the mammalian MCHl receptor of compounds included in the plurality of compounds, so as to
• This invention provides a method of detecting expression of a mammalian MCHl receptor by detecting the presence of mRNA coding for the mammalian MCHl receptor which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained with a nucleic acid probe under hybridizing conditions, detecting the presence of mRNA hybridizing to the probe, and thereby detecting the expression of the mammalian MCHl receptor by the cell.
• This invention provides a method of detecting the presence of a mammalian MCHl receptor on the surface of a cell which comprises contacting the cell with an antibody under conditions permitting binding of the antibody to the receptor, detecting the presence of the antibody bound to the cell, and thereby detecting the presence of the mammalian MCHl receptor on the surface of the cell .
• This invention provides a method of determining the physiological effects of varying levels of activity of human MCHl receptors which comprises producing a transgenic, nonhuman mammal whose levels of human MCHl receptor activity are varied by use of an inducible promoter which regulates human MCHl receptor expression.
• This invention provides a method of determining the physiological effects of varying levels of activity of human MCHl receptors which comprises producing a panel of transgenic, nonhuman mammals each expressing a different amount of human MCHl receptor.
• This invention provides a method for identifying an antagonist capable of alleviating an abnormality wherein the abnormality is alleviated by decreasing the activity of a human MCHl receptor comprising administering a compound to the transgenic, nonhuman mammal and determining whether the compound alleviates the physical and behavioral abnormalities displayed by the transgenic, nonhuman mammal as a result of overactivity of a human MCHl receptor, the alleviation of the abnormality identifying the compound as an antagonist.
• This invention also provides an antagonist identified by this method.
• This invention further provides a pharmaceutical composition comprising an antagonist identified by this method and a pharmaceutically acceptable carrier.
• This invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by decreasing the activity of a human MCHl receptor which comprises administering to the subject an effective amount of this pharmaceutical composition, thereby treating the abnormality.
• This invention provides a method for identifying an agonist capable of alleviating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a human MCHl receptor comprising administering a compound to a transgenic, nonhuman mammal, and determining whether the compound alleviates the physical and behavioral abnormalities displayed by the transgenic, nonhuman mammal, the alleviation of the abnormality identifying the compound as an agonist.
• This invention also provides an agonist identified by this method.
• This invention further provides a pharmaceutical composition comprising an agonist identified by this method and a pharmaceutically acceptable carrier.
• This invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a human MCHl receptor which comprises administering to the subject an effective amount of this pharmaceutical composition, thereby treating the abnormality.
• This invention provides a method for diagnosing a predisposition to a disorder associated with the activity of a specific mammalian allele which comprises: (a) obtaining DNA of subjects suffering from the disorder; (b) performing a restriction digest of the DNA with a panel of restriction enzymes; (c) electrophoretically separating the resulting DNA fragments on a sizing gel; (d) contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human MCHl receptor and labeled with a detectable marker; (e) detecting labeled bands which have hybridized to the DNA encoding a human MCHl receptor labeled with a detectable marker to create a unique band pattern specific to the DNA of subjects suffering from the disorder; (f) preparing DNA obtained for diagnosis by steps (a) -(e); and (g) comparing the unique band pattern specific to the DNA of subjects suffering from the disorder from step (e) and the DNA
• This invention provides a method of preparing a purified human MCHl receptor which comprises: (a) inducing cells to express the human MCHl receptor; (b) recovering the human MCHl receptor from the induced cells; and (c) purifying the human MCHl receptor so recovered.
• This invention provides a method of preparing a purified human MCHl receptor which comprises: (a) inserting nucleic acid encoding the human MCHl receptor in a suitable vector; (b) introducing the resulting vector in a suitable host cell; (c) placing the resulting cell in suitable condition permitting the production of the isolated human MCHl receptor; (d) recovering the human MCHl receptor produced by the resulting cell; and (e) purifying the human MCHl receptor so recovered.
• This invention provides a process for determining whether a chemical compound is a mammalian MCHl receptor agonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian MCHl receptor with the compound under conditions permitting the activation of the mammalian MCHl receptor, and detecting an increase in mammalian MCHl receptor activity, so as to thereby determine whether the compound is a mammalian MCHl receptor agonist.
• This invention also provides a pharmaceutical composition which comprises an amount of a mammalian MCHl receptor agonist determined by this process effective to increase activity of a mammalian MCHl receptor and a pharmaceutically acceptable carrier.
• This invention provides a process for determining whether a chemical compound is a mammalian MCHl receptor antagonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian MCHl receptor with the compound in the presence of a known mammalian MCHl receptor agonist, under conditions permitting the activation of the mammalian MCHl receptor, and detecting a decrease in mammalian MCHl receptor activity, so as to thereby determine whether the compound is a mammalian MCHl receptor antagonist.
• This invention also provides a pharmaceutical composition which comprises an amount of a mammalian MCHl receptor antagonist determined by this process effective to reduce activity of a mammalian MCHl receptor and a pharmaceutically acceptable carrier.
• This invention provides a process for determining whether a chemical compound specifically binds to and activates a mammalian MCHl receptor, which comprises contacting cells producing a second messenger response and expressing on their cell surface the mammalian MCHl receptor, wherein such cells do not normally express the mammalian MCHl receptor, with the chemical compound under conditions suitable for activation of the mammalian MCHl receptor, and measuring the second messenger response in the presence and in the absence of the chemical compound, a change in the second messenger response in the presence of the chemical compound indicating that the compound activates the mammalian MCHl receptor.
• This invention also provides a compound determined by this process.
• This invention further provides a pharmaceutical composition which comprises an amount of the compound (a MCHl receptor agonist) determined by this process effective to increase activity of a mammalian MCHl receptor and a pharmaceutically acceptable carrier.
• This invention provides a process for determining whether a chemical compound specifically binds to and inhibits activation of a mammalian MCHl receptor, which comprises separately contacting cells producing a second messenger response and expressing on their cell surface the mammalian MCHl receptor, wherein such cells do not normally express the mammalian MCHl receptor, with both the chemical compound and a second chemical compound known to activate the mammalian MCHl receptor, and with only the second chemical compound, under conditions suitable for activation of the mammalian MCHl receptor, and measuring the second messenger response in the presence of only the second chemical compound and in the presence of both the second chemical compound and the chemical compound, a smaller change in the second messenger response in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound indicating that the chemical compound inhibits activation of the mammalian MCHl receptor.
• This invention also provides a compound determined by this process.
• This invention further provides a pharmaceutical composition which comprises an amount of the compound (a mammalian MCHl receptor antagonist) determined by this effective to reduce activity of a mammalian MCHl receptor and a pharmaceutically acceptable carrier.
• This invention provides a method of screening a plurality of chemical compounds not known to activate a mammalian MCHl receptor to identify a compound which activates the mammalian MCHl receptor which comprises: (a) contacting cells transfected with and expressing the mammalian MCHl receptor with the plurality of compounds not known to activate the mammalian MCHl receptor, under conditions permitting activation of the mammalian MCHl receptor; (b) determining whether the activity of the mammalian MCHl receptor is increased in the presence of the compounds; and if so (c) separately determining whether the activation of the mammalian MCHl receptor is increased by each compound included in the plurality of compounds, so as to thereby identify the compound which activates the mammalian MCHl receptor.
• This invention also provides a compound identified by this method.
• This invention further provides a pharmaceutical composition which comprises an amount of the compound (a mammalian MCHl receptor agonist) identified by this method effective to increase activity of a mammalian MCHl receptor and a pharmaceutically acceptable carrier.
• This invention provides a method of screening a plurality of chemical compounds not known to inhibit the activation of a mammalian MCHl receptor to identify a compound which inhibits the activation of the mammalian MCHl receptor, which comprises: (a) contacting cells transfected with and expressing the mammalian MCHl receptor with the plurality of compounds in the presence of a known mammalian MCHl receptor agonist, under conditions permitting activation of the mammalian MCHl receptor; (b) determining whether the activation of the mammalian MCHl receptor is reduced in the presence of the plurality of compounds, relative to the activation of the mammalian MCHl receptor in the absence of the plurality of compounds; and if so (c) separately determining the inhibition of activation of the mammalian MCHl receptor for each compound included in the plurality of compounds, so as to thereby identify the compound which inhibits the activation of the mammalian MCHl receptor.
• This invention also provides a compound identified by this method.
• This invention further provides a pharmaceutical composition which comprises an amount of the compound (a mammalian MCHl receptor antagonist) identified by this process effective to decrease activity of a mammalian MCHl receptor and a pharmaceutically acceptable carrier.
• This invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a mammalian MCHl receptor which comprises administering to the subject an amount of a compound which is a mammalian MCHl receptor agonist effective to treat the abnormality.
• This invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by decreasing the activity of a mammalian MCHl receptor which comprises administering to the subject an amount of a compound which is a mammalian MCHl receptor antagonist effective to treat the abnormality.
• This invention provides a process for making a composition of matter which specifically binds to a mammalian MCHl receptor which comprises identifying a chemical compound using any of the processes described herein for identifying a compound which binds to and/or activates or inhibits activation of a mammalian MCHl receptor and then synthesizing the chemical compound or a novel structural and functional analog or homolog thereof.
• This invention further provides a process for preparing a pharmaceutical composition which comprises administering a pharmaceutically acceptable carrier and a pharmaceutically acceptable amount of a chemical compound identified by any of the processes described herein for identifying a compound which binds to and/or activates or inhibits activation of a mammalian MCHl receptor or a novel structural and functional analog or homolog thereof.
• This invention provides a process for determining whether a chemical compound is a human MCHl receptor antagonist which comprises contacting cells transfected with and expressing DNA encoding the human MCHl receptor with the compound in the presence. of a known human MCHl receptor agonist, under conditions permitting the activation of the human MCHl receptor, and detecting a decrease in human MCHl receptor activity, so as to thereby determine whether the compound is a" human MCHl receptor antagonist, wherein the DNA encoding the human MCHl receptor comprises the sequence shown in Figure 1 (Seq. ID No. 1) or contained in plasmid pEXJ. HR-TL231 (ATCC Accession No. 203197), the known human MCHl receptor agonist is MCH or a homolog or analog of MCH, and the cells do not express the MCHl receptor prior to transfecting them.
• This invention also provides a process for determining whether a chemical compound specifically binds to and inhibits activation of a human MCHl receptor, which comprises separately contacting cells expressing on their cell surface the human MCHl receptor and producing a second messenger response upon activation of the human MCHl receptor, wherein such cells do not normally express the human MCHl receptor and the DNA encoding the human MCHl receptor comprises the sequence shown in Figure 1 (Seq. ID No. 1) or contained in plasmid pEXJ.HR-TL231 (ATCC Accession No.
• This invention further provides a method of screening a plurality of chemical compounds not known to inhibit the activation of a human MCHl. receptor to identify a compound which inhibits the activation of the human MCHl receptor, which comprises: (a) contacting cells transfected with and expressing the human MCHl receptor, wherein such cells do not normally express the human MCHl receptor and the DNA encoding the human MCHl receptor comprises the sequence shown in Figure 1 (Seq. ID No. 1) or contained in plasmid pEXJ. HR-TL231 (ATCC Accession No. 203197), with the plurality of compounds in the presence of a known human MCHl receptor agonist, under conditions permitting activation of the human MCHl receptor, wherein the known MCHl receptor agonist is MCH or a homolog or analog of MCH;
• This invention provides a process for making a composition of matter which specifically binds to a human MCHl receptor which comprises identifying a chemical compound which specifically binds to the human MCHl receptor and then synthesizing the chemical compound or a structural and functional analog or homolog thereof, wherein the chemical compound is identified as binding to the human MCHl receptor by a process involving competitive binding which comprises contacting cells expressing on their cell surface the human MCHl receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and separately with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting the extent of specific binding of the chemical compound to the human MCHl receptor, a decrease in the binding of the second chemical compound to the human MCHl receptor in the presence of the chemical compound indicating that the chemical compound binds to the human MCHl receptor, wherein the cells do not normally express the human MCHl receptor, the human MCHl receptor is encoded by nucleic acid comprising the sequence shown in Figure 1 (Seq. ID No. 1)
• This invention further provides a process for making a composition of matter which specifically binds to a human MCHl receptor which comprises identifying a chemical compound which specifically binds to the human MCHl receptor and then synthesizing the chemical compound or a structural and functional analog or homolog thereof, wherein the chemical compound is identified as binding to the human MCHl receptor by a process involving competitive binding which comprises contacting a membrane preparation from cells expressing on their cell surface the human MCHl receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and separately with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting the extent of specific binding of the chemical compound to the human MCHl receptor, a decrease in the binding of the second chemical compound to the human MCHl receptor in the presence of the chemical compound indicating that the chemical compound binds to the human MCHl receptor, wherein the cells do not normally express the human MCHl receptor, the human MCHl receptor is encoded by nucleic acid comprising the sequence shown in Figure 1 (Se
• This invention also provides a process for making a composition of matter which is a human MCHl receptor antagonist which comprises identifying a chemical compound which is a human MCHl receptor antagonist and then synthesizing the chemical compound or a structural and functional analog or homolog thereof, wherein the chemical compound is identified as a human MCHl receptor antagonist by a process which comprises contacting cells transfected with and expressing DNA encoding the human MCHl receptor with the compound in the presence of a known human MCHl receptor agonist, under conditions permitting the activation of the human MCHl receptor, and detecting a decrease in human MCHl receptor activity, so as to thereby determine whether the compound is a human MCHl receptor antagonist, wherein the cells do not normally express the human MCHl receptor, the human MCHl receptor is encoded by nucleic acid comprising the sequence shown in Figure 1 (Seq. ID No. 1) or contained in plasmid pEXJ. HR-TL231 (ATCC Accession No. 203197), and the known human MCHl receptor
• This inventions still further provides a process for making a composition of matter which specifically binds to and inhibits the activation of a human MCHl receptor which comprises identifying a chemical compound which specifically binds to and inhibits the activation of the human MCHl receptor and then synthesizing the chemical compound or a structural and functional analog or homolog thereof, wherein the chemical compound is ' identified as binding to and inhibiting the activation of the human MCHl receptor by a process which comprises separately contacting cells expressing on their cell surface the human MCHl receptor and producing a second messenger response upon activation of the human MCHl receptor, wherein such cells do not normally express the human MCHl receptor and the human MCHl receptor is encoded by nucleic acid comprising the sequence shown in Figure 1 (Seq.
• This invention provides a process for preparing a composition which comprises identifying a chemical compound which specifically binds to a human MCHl receptor, and then admixing a carrier and the chemical compound or a structural and functional analog or homolog thereof, wherein the chemical compound is identified as binding to the human MCHl receptor by a process involving competitive binding which comprises contacting cells expressing on their cell surface the human MCHl receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and separately with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting the extent of specific binding of the chemical compound to the human MCHl receptor, a decrease in the binding of the second chemical compound to the human MCHl receptor in the presence of the chemical compound indicating that the chemical compound binds to the human MCHl receptor, wherein the cells do not normally express the human MCHl receptor, the human MCHl receptor is encoded by nucleic acid comprising the sequence shown in Figure 1 (Seq. ID No. 1) or contained in plasmid
• This invention further provides a process for preparing a composition which comprises identifying a chemical compound which specifically binds to a human MCHl receptor, and then admixing a carrier and the chemical compound or a structural and functional analog or homolog thereof, wherein the chemical compound is identified as binding to the human MCHl receptor by a process involving competitive binding which comprises contacting a membrane preparation from cells expressing on their cell surface the human MCHl receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and separately with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting the extent of specific binding of the chemical compound to the human MCHl receptor, a decrease in the binding of the second chemical compound to the human MCHl receptor in the presence of the chemical compound indicating that the chemical compound binds to the human MCHl receptor, wherein the cells do not normally express the human MCHl receptor, the human MCHl receptor is encoded by nucleic acid comprising the sequence shown in Figure 1 (Seq. ID No. 1) or contained
• This invention also provides a process for preparing a composition which comprises identifying a chemical compound which is a human MCHl receptor antagonist, and then admixing a carrier and the chemical compound or a structural and functional analog or homolog thereof, wherein the chemical compound is identified as a human MCHl receptor antagonist by a process which comprises contacting cells transfected with and expressing DNA encoding the human MCHl receptor with the compound in the presence of a known human MCHl receptor agonist, under conditions permitting the activation of the human MCHl receptor, and detecting a ' decrease in human MCHl receptor activity, so as to thereby determine whether the compound is a human MCHl receptor antagonist, wherein the cells do not normally express the human MCHl receptor, the human MCHl receptor is encoded by nucleic acid comprising the sequence shown in Figure 1 (Seq. ID No. 1) or contained in plasmid pEXJ.HR-TL231 (ATCC Accession No. 203197), and the known human MCHl receptor agonist is MCH
• This invention still further provides a process for preparing a composition which comprises identifying a chemical compound which specifically binds to and inhibits the activation of a human MCHl receptor, and then admixing a carrier and the chemical compound or a structural and functional analog or homolog thereof, wherein the chemical compound is identified as binding to and inhibiting activation of the human MCHl receptor by a process which comprises separately contacting cells expressing on their cell surface the human MCHl receptor and producing a second messenger response upon activation of the human MCHl receptor, wherein such cells do not normally express the human MCHl receptor and the human MCHl receptor is encoded by nucleic acid comprising the sequence shown in Figure 1 (Seq. ID No.
• This invention provides a method of treating an eating disorder or obesity in a subject which comprises administering to the subject a therapeutically effective amount of an MCHl antagonist which inhibits the activation of the MCHl receptor.
• This invention provides a method of reducing the body mass of a subject which comprises administering to the subject an amount of an MCHl antagonist effective to reduce the body mass of the subject.
• This invention further provides a method of treating an eating disorder in a subject which comprises administering to the subject a therapeutically effective amount of an MCHl agonist which activates the MCHl receptor.
• This invention also provides a method of treating depression and/or anxiety in a subject which comprises administering to the subject a composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a MCHl receptor antagonist, wherein:
• the MCHl receptor antagonist does not inhibit the activity of central monoamine oxidase A greater than 50 percent, at a concentration of lOmM; and (2) the MCHl receptor antagonist does not inhibit the activity of central monoamine oxidase B greater than 50 percent, at a concentration of lOmM; and
• the MCHl receptor antagonist binds to the human MCHl receptor with a binding affinity at least ten-fold higher than the binding affinity with which it binds to each of the following transporters: serotonin transporter, norepinephrine transporter, and dopamine transporter.
• Nucleotide sequence encoding a human MCHl receptor (MCHl) (SEQ ID NO: 1) . Three potential start (ATG) codons and the stop (TGA) codon are underlined.
• SEQ ID NO: 2 Deduced amino acid sequence (SEQ ID NO: 2) of the human MCHl receptor (MCHl) encoded by the nucleotide sequence shown Figure 1 (SEQ ID NO-: 1) .
• Nucleotide sequence of rat MCHl (SEQ ID NO: 3) .
• One start (ATG) codon and the stop codon (TGA) are underlined.
• FIG. 17 Saturation equilibrium binding of [3H] Compound 10 to the human MCHl receptor.
• Membrane preparations from Cos-7 cells transfected with MCHl were incubated with varying concentrations of [3H] Compound 10 (SA: 56 Ci/mmol) at room temperature for 90 min, in a volume of 0.250 ml.
• the reaction was terminated by filtration in GF/C filters, and the radioactivity determined by scintillation counting. Non-specific binding was defined as the amount of radioactivity retained in the filter after incubating the reaction mixture in the presence of unlabeled Compound 10 (10 mM) .
• CA1,2,3 fields CA1, 2, 3 of hippocampus
• Ml primary motor cortex Me medial amygdaloid nucleus
• MPO medial preoptic area OC occipital cortex
• RSG retrosplenial granular cortex
• V2 secondary visual cortex VL ventrolateral thalamic nucleus
• Figure 21 Effect of Compound 10 on MCH-induced stimulation of food intake in rats.
• MCH (3 nmol) or vehicle was administered into the third venticle, and food intake measured 30, 60 and 120 minutes later.
• Some rats were pretreated with vehicle or Compound 10 (1 or 10 mg/kg) i.p. 20 minutes prior to i.e. v. injection.
• Figure 22 Effect of Compound 10 on body weight gain in young growing rats.
• Compound 10 (10 mg/kg/day) , fenfluramine (6 mg/kg/day) or vehicle were administered to rats for 14 days via subcutaneously implanted osmotic minipumps .
• Significant differences from vehicle are denoted by **P ⁇ 0.001, *P ⁇ 0.01, xP ⁇ 0.05, as determined by ANOVA and Newman-Keuls test.
• Compound 10 Effect of Compound 10 on body weight gain in young growing rats.
• Compound 10 (1, 3 or 10 mg/kg) or vehicle (dashed line) was administered to rats twice daily by i.p. injection.
• Significant differences from vehicle are denoted by **P ⁇ 0.001, *P ⁇ 0.01, as determined by ANOVA and
• Compound 94 Effect of Compound 94 on body weight gain in young growing rats.
• Compound 94 (3, 10 or 30 mg/kg) or vehicle was administered to rats twice daily by i.p. injection. Significant differences from vehicle are denoted by
• Figure 25 Effect of Compound 95 on body weight gain in young growing rats.
• Compound 67173 (3, 10 or 30 mg/kg) or vehicle was administered to rats twice daily by i.p. injection.
• Significant differences from vehicle are denoted by *P ⁇ 0.001, as determined by ANOVA and Newman-Keuls test.
• M adenine or cytosine
• R adenine or guanine
• W adenine, thymine, or uracil
• S cytosine or guanine
• Y cytosine, thymine, or uracil
• D adenine, guanine, thymine, or uracil (not cytosine)
• B cytosine, guanine, thymine, or uracil (not adenine)
• N adenine, cytosine, guanine, thymine, or uracil
• agonist is used throughout this application to indicate any peptide or non-peptidyl compound which increases the activity of any of the polypeptides of the subject invention.
• antagonist is used throughout this application to indicate any peptide or non-peptidyl compound which decreases the activity of any of the polypeptides of the subject invention.
• mimmalian is used throughout this invention to include mutant forms of the human MCHl receptor.
• the activity of a G-protein coupled receptor such as the polypeptides disclosed herein may be measured using any of a variety of functional assays in which activation of the receptor in question results in an observable change in the level of some second messenger system, including, but not limited to, adenylate cyclase, calcium mobilization, arachidonic acid release, ion channel activity, inositol phospholipid hydrolysis or guanylyl cyclase.
• Heterologous expression systems utilizing appropriate host cells to express the nucleic acid of the subject invention are used to obtain the desired second messenger coupling. Receptor activity may also be assayed in an oocyte expression system.
• the antagonist may act as an inverse agonist or an allosteric modulator, as opposed to a neutral antagonist, and suppress receptor signaling independent of the agonist (Lutz and Kenakin, 1999) .
• the categories of "antagonist compounds” are therefore seen to include 1) neutral antagonists (which block agonist actions but do not affect constitutive activity); 2) inverse agonists (which block agonist actions as well as constitutive activity by stabilizing an inactive receptor conformation); 3) and allosteric modulators (which block agonist actions to a limited extent and which may also block constitutive activity through allosteric regulation) .
• the human MCHl receptor gene contains introns and furthermore, the possibility exists that additional introns could exist in coding or non-coding regions.
• spliced form(s) of mRNA may encode additional amino acids either upstream of the currently defined starting methionine or within the coding region.
• the existence and use of alternative exons is possible, whereby the mRNA may encode different amino acids within the region comprising the exon.
• single amino acid substitutions may arise via the mechanism of RNA editing such that the amino acid sequence of the expressed protein is different than that encoded by the original gene. (Burns et al . , 1996; Chu et al . , 1996) . Such variants may exhibit pharmacologic properties differing from the polypeptide encoded by the original gene .
• This invention provides splice variants of the human MCHl receptor disclosed herein. This invention further provides for alternate translation initiation sites and alternately spliced or edited variants of nucleic acids encoding the human MCHl receptor of this invention.
• the nucleic acid of the subject invention also includes nucleic acid analogs of the human MCHl receptor gene,
• the human MCHl receptor gene comprises the nucleic acid sequence shown in Fig. 1 or contained in plasmid pEXJ.HR-TL231 (ATCC Accession No. 203197) .
• Nucleic acid analogs of the human MCHl receptor genes differ from the human MCHl receptor gene described herein in terms of the identity or location of one or more nucleic acid bases (deletion analogs containing less than all of the nucleic acid bases shown in Fig. 1 or contained in plasmid pEXJ. HR-TL231, substitution analogs wherein one or more nucleic acid bases shown in Fig.
• nucleic acid analog encodes a protein which comprises an amino acid sequence as shown in Fig. 2 or encoded by the nucleic acid sequence contained in plasmid pEXJ.HR-TL231.
• the nucleic acid analog encodes a protein comprising an amino acid sequence which differs from the amino acid sequences shown in Fig. 2 or encoded by the nucleic acid contained in plasmids pEXJ. HR-TL231.
• the protein encoded by the nucleic acid analog has a function which is the same as the function of the receptor protein comprising the amino acid sequence shown in Fig. 2.
• the function of the protein encoded by the nucleic acid analog differs from the function of the receptor protein comprising the amino acid sequence shown in Fig. 2.
• the variation in the nucleic acid sequence occurs within the transmembrane (TM) region of the protein.
• the variation in the nucleic acid sequence occurs outside of the TM region.
• nucleic acid is DNA.
• the DNA is cDNA.
• the DNA is genomic DNA.
• nucleic acid is RNA. Methods for production and manipulation of nucleic acid molecules are well known in the art .
• nucleic acid which is degenerate with respect to the DNA encoding the polypeptides described herein.
• the nucleic acid comprises a nucleotide sequence which is degenerate with respect to the nucleotides sequence shown in Figure 1 (SEQ ID NO: 2) or the nucleotide sequence contained in the plasmid pEXJ.
• HR-TL231 that is, a nucleotide sequence which is translated into the same amino acid sequence.
• This invention also encompasses DNAs and cDNAs which encode amino acid sequences which differ from those of the polypeptides of this invention, but which should not produce phenotypic changes .
• this invention also encompasses DNAs, cDNAs, and RNAs which hybridize to the DNA, cDNA, and RNA of the subject invention. Hybridization methods are well known to those of skill in the art.
• nucleic acids of the subject invention also include nucleic acid molecules coding for polypeptide analogs, fragments or derivatives of antigenic polypeptides which differ from naturally-occurring forms in terms of the identity or location of one or more amino acid residues (deletion analogs containing less than all of the residues specified for the protein, substitution analogs wherein one or more residues specified are replaced by other residues and addition analogs wherein one or more amino acid residues is added to a terminal or medial portion of the polypeptides) and which share some or all properties of naturally-occurring forms.
• These molecules include: the incorporation of codons "preferred" for expression by selected non-mammalian hosts; the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate DNA sequences that facilitate construction of readily expressed vectors.
• the creation of polypeptide analogs is well known to those of skill in the art (R.F. Spurney et al. (1997); Fong, T.M. et al . (1995); Underwood, D.J. et al. (1994); Graziano, M.P. et al . (1996); Guan X.M. et al . (1995) ) .
• modified polypeptides of this invention may be transfected into cells either transiently or stably using methods well-known in the art, examples of which are disclosed herein.
• This invention also provides for binding assays using the modified polypeptides, in which the polypeptide is expressed either transiently or in stable cell lines.
• This invention further provides a compound identified using a modified polypeptide in a binding assay such as the binding assays described herein.
• nucleic acids described and claimed herein are useful for the information which they provide concerning the amino acid sequence of the polypeptide and as products for the large scale synthesis of the polypeptides by a variety of recombinant techniques .
• the nucleic acid molecule is useful for generating new cloning and expression vectors, transformed and transfected prokaryotic and eukaryotic host cells, and new and useful methods for cultured growth of such host cells capable of expression of the polypeptide and related products.
• nucleic acid encoding a human MCHl receptor or a mutant of such human MCHl receptor which is activated by MCH or an analog or homolog thereof.
• the nucleic acid is DNA.
• the DNA is cDNA.
• the DNA is genomic DNA.
• the nucleic acid is RNA.
• This invention also provides methods of using an isolated nucleic acid encoding species homologs of the MCHl receptor encoded by the nucleic acid sequence shown in Fig. 1 (SEQ ID NO: 1) or encoded by the plasmid pEXJ.HR- TL231.
• the nucleic acid encodes a mammalian MCHl receptor homolog which has substantially the same amino acid sequence as does the MCHl receptor encoded by the plasmid pEXJ. HR-TL231.
• the nucleic acid encodes a mammalian MCHl receptor homolog which has above 65% amino acid identity to the MCHl receptor encoded by the plasmid pEXJ.
• the mammalian MCHl receptor homolog has above 70% nucleic acid identity to the MCHl receptor gene contained in plasmid pEXJ. HR-TL231; preferably above 80% nucleic acid identity to the MCHl receptor gene contained in the plasmid pEXJ.
• HR-TL231 more preferably above 90% nucleic acid identity to the MCHl receptor gene contained in the plasmid pEXJ. HR-TL231. Examples of methods for isolating and purifying species homologs are described elsewhere (e.g., U.S. Patent No. 5,602,024, W094/14957, W097/26853, WO98/15570) .
• the nucleic acid encodes a MCHl receptor which has an amino acid sequence identical to that encoded by the plasmid pEXJ.HR-TL231.
• the MCHl receptor comprises a sequence substantially the same as the amino acid sequence shown in Figure 2 (SEQ ID NO: 2) .
• the MCHl receptor comprises an amino acid sequence as shown in Figure 2 (SEQ ID NO: 2) .
• the mutant human MCHl receptor comprises an amino acid sequence as shown in Figure 13 (SEQ ID NO: 26) . In another embodiment, the mutant human MCHl receptor comprises an amino acid sequence as shown in Figure 14 (SEQ ID NO: 27) . In still another embodiment, the mutant human MCHl receptor comprises an amino acid sequence as shown in Figure 15 (SEQ ID NO: 28) .
• the human MCHl receptor is encoded by the nucleic acid sequence shown in Figure 1 beginning with any of the three indicated start (ATG) codons .
• This invention provides an isolated nucleic acid encoding a modified human MCHl receptor, which differs from a human MCHl receptor by having an amino acid(s) deletion, replacement, or addition in the third intracellular domain .
• This invention provides a nucleic acid encoding a human MCHl receptor, wherein the nucleic acid (a) hybridizes to a nucleic acid having the defined sequence shown in Figure 1 (SEQ ID NO: 1) under low stringency conditions or a sequence complementary thereto and (b) is further characterized by its ability to cause a change in the pH of a culture of CHO cells when a MCHl ligand is added to the culture and the CHO cells contain the nucleic acid which hybridized to the nucleic acid having the defined sequence or its complement.
• SEQ ID NO: 1 SEQ ID NO: 1
• Hybridization at low stringency is performed at 40"C in a hybridization buffer containing 25% formamide, 5X SCC, 7mM Tris, IX Denhardt's, 25 ⁇ l/ml salmon sperm DNA. Wash at 40°C in 0. IX SCC, 0.1% SDS. Changes in pH are measured through microphysiometric measurement of receptor mediated extracellular acidification rates. Because cellular metabolism is intricately involved in a broad range of cellular events
• Receptors and/or control vectors are transiently expressed in CHO-K1 cells, by liposome mediated transfection according to the manufacturers recommendations (LipofectAMINE, GibcoBRL, Gaithersburg, MD) , and maintained in Ham' s F-12 complete (10% serum) .
• a total of lO ⁇ g of DNA is used to transfect each 75cm 2 flask which had been split 24 hours prior to the transfection and judged to be 70-80% confluent at the time of transfection.
• 24 hours post transfection the cells are harvested and 3 x 10 5 cells seeded into microphysiometer capsules. Cells are allowed to attach to the capsule membrane for an additional 24 hours; during the last 16 hours, the cells are switched to serum-free F- 12 complete to minimize ill-defined metabolic stimulation caused by assorted serum factors.
• the cell capsules are transferred to the microphysiometer and allowed to equilibrate in recording media (low buffer RPMI 1640, no bicarbonate, no serum
• fatty acid free BSA 0.1% fatty acid free BSA
• a standard recording protocol specifies a lOO ⁇ l/min flow rate, with a 2 min total pump cycle which includes a 30 sec flow interruption during which the acidification rate measurement is taken.
• Ligand challenges involve a 1 min 20 sec exposure to the sample just prior to the first post challenge rate measurement being taken, followed by two additional pump cycles for a total of 5 min 20 sec sample exposure.
• drugs in a primary screen are presented to the cells at lO ⁇ M final concentration.
• Ligand samples are then washed out and the acidification rates reported are expressed as a percentage increase of the peak response over the baseline rate observed just prior to challenge.
• An examples of a MCH ligand includes, but is not limited to, the endogenous MCH peptide.
• This invention provides a purified human MCHl receptor protein .
• This invention provides a vector comprising nucleic acid encoding a human MCHl receptor.
• the vector is adapted for expression in a cell which comprises the regulatory elements necessary for expression of the nucleic acid in the cell operatively linked to the nucleic acid encoding the human MCHl receptor as to permit expression thereof.
• the cell is a bacterial cell, an amphibian cell, a yeast cell, an insect cell or a mammalian cell.
• the vector is a baculovirus.
• the vector is a plasmid.
• This invention provides a plasmid designated pEXJ.HR-TL231
• This plasmid comprises the regulatory elements necessary for expression of DNA in a mammalian cell operatively linked to DNA encoding the human MCHl receptor so as to permit expression thereof.
• This plasmid (pEXJ. HR-TL231) was deposited on September 17, 1998, with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and was accorded ATCC Accession No. 203197.
• This invention further provides for any vector or plasmid which comprises modified untranslated sequences, which are beneficial for expression in desired host cells or for use in binding or functional assays.
• a vector or plasmid with untranslated sequences of varying lengths may express differing amounts of the polypeptide depending upon the host cell used.
• the vector or plasmid comprises the coding sequence of the polypeptide and the regulatory elements necessary for expression in the host cell.
• This invention provides a cell comprising a vector comprising a nucleic acid encoding the human MCHl receptor.
• the cell is a non-mammalian cell.
• the non-mammalian cell is a Xenopus oocyte cell or a Xenopus melanophore cell.
• the cell is a mammalian cell.
• the mammalian cell is a COS-7 cell, a 293 human embryonic kidney cell, a NIH-3T3 cell, a LM(tk-) cell, a mouse Yl cell, or a CHO cell.
• This invention provides an insect cell comprising a vector adapted for expression in an insect cell which comprises a nucleic acid encoding a human MCHl receptor.
• the insect cell is an Sf9 cell, an Sf21 cell or a Trichoplusia ni 5B1-4 (HighFive) cell.
• This invention provides a membrane preparation isolated from any one of the cells described above.
• This invention provides a nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with a nucleic acid encoding a human MCHl receptor, wherein the probe has a unique sequence corresponding to a sequence present within one of the two strands of the nucleic acid encoding a human MCHl receptor present in plasmid pEXJ. HR-TL231.
• This invention also provides a nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with a nucleic acid encoding a human MCHl receptor, wherein the probe has a unique sequence corresponding to a sequence present within (a) the nucleic acid sequence shown in Figure 1 (SEQ ID NO: 1) or (b) the reverse complement thereto.
• the nucleic acid is DNA.
• the nucleic acid is RNA.
• the phrase "specifically hybridizing” means the ability of a nucleic acid molecule to recognize a nucleic acid sequence complementary to its own and to form double-helicalsegments through hydrogen bonding between complementary base pairs.
• Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary greatly in length and may be labeled with a detectable label, such as a radioisotope or flourescent dye, to facilitate detection of the probe.
• DNA probe molecules may be produced by insertion of a DNA molecule which encodes the polypeptides of this invention into suitable vectors, such as plasmids or bacteriophages, followed by transforming into suitable bacterial host cells, replication in the transformed bacterial host cells and harvesting of the DNA probes, using methods well known in the art. Alternatively, probes may be generated chemically from DNA synthesizers.
• RNA probes may be generated by inserting the DNA molecule which encodes the polypeptides of this invention downstream of a bacteriophage promoter such as T3, T7, or SP6. Large amounts of RNA probe may be produced by incubating the labeled nucleotides with the linearized fragment where it contains an upstream promoter in the presence of the appropriate RNA polymerase.
• This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to RNA encoding a human MCHl receptor, so as to prevent translation of the RNA.
• This invention also provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to genomic DNA encoding a human MCHl receptor.
• the oligonucleotide comprises chemically modified nucleotides or nucleotide analogues .
• This invention provides an antibody capable of binding to a human MCHl receptor encoded by a nucleic acid encoding a human MCHl receptor. This invention also provides an agent capable of competitively inhibiting the binding of the antibody to a human MCHl receptor.
• the antibody is a monoclonal antibody or antisera.
• This invention provides a pharmaceutical composition
• a pharmaceutical composition comprising (a) an amount of the oligonucleotide capable of passing through a cell membrane and effective to reduce expression of a human MCHl receptor and (b) a pharmaceutically acceptable carrier capable of passing through the cell membrane.
• the oligonucleotide is coupled to a substance which inactivates mRNA.
• the substance which inactivates mRNA is a ribozyme.
• the pharmaceutically acceptable carrier comprises a structure which binds to a human MCHl receptor on a cell capable of being taken up by the cells after binding to the structure.
• the pharmaceutically acceptable carrier is capable of binding to a human MCHl receptor which is specific for a selected cell type .
• This invention provides a pharmaceutical composition which comprises an amount of an antibody effective to block binding of a ligand to a human MCHl receptor and a pharmaceutically acceptable carrier.
• pharmaceutically acceptable carrier means any of the standard pharmaceutically acceptable carriers and is any pharmaceutical carrier known to those of ordinary skill in the art as useful in formulating pharmaceutical compositions. Examples include, but are not limited to, phosphate buffered saline, physiological saline, water, and emulsions, such as oil/water emulsions.
• the guidance characterized the following solvents as Class 2 Solvents: acetonitrile, chlorobenzene , chloroform, cyclohexane, 1,2- dichloroethene, dichloromethane, 1, 2-dimethoxyethane, N,N- dimethylacetamide, N, N-dimethylformamide, 1,4-dioxane, 2- ethoxyethanol, ethyleneglycol, formamide, hexane, methanol, 2-methoxyethanol, methylbutyl ketone, methylcyclohexane, N-methylpyrrolidone, nitromethane, pyridine, sulfolane, tetralin, toluene, 1,1,2- trichloroethene and xylene.
• pharmaceutically acceptable carrier shall not include Class 1 or Class 2 Solvents.
• the pharmaceutical carrier may be a liquid and the pharmaceutical composition would be in the form of a solution.
• the pharmaceutically acceptable carrier is a solid and the composition is in the form of a powder or tablet.
• the pharmaceutical carrier is a gel and the composition is in the form of a suppository or cream.
• the compound may be formulated as a part of a pharmaceutically acceptable transdermal patch.
• the compound may be delivered to the subject by means of a spray or inhalant.
• a solid carrier can include one or more substances which may also act as endogenous carriers (e.g. nutrient or micronutrient carriers), flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents; it can also be an encapsulating material.
• the carrier is a finely divided solid which is in admixture with the finely divided active ingredient.
• the active ingredient is mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active ingredient.
• Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidme, low melting waxes and ion exchange resins .
• Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
• the active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
• the liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmoregulators .
• suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives as above, e.g.
• cellulose derivatives preferably sodium carboxymethyl cellulose solution
• alcohols including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil)
• oils e.g. fractionated coconut oil and arachis oil
• the carrier can also be an oily ester such as ethyl oleate or isopropyl myristate.
• Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration.
• the liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellent.
• Liquid pharmaceutical compositions which are sterile solutions or suspensions can be utilized by for example, intramuscular, intratheca ' l, epidural, intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously.
• the compounds may be prepared as a sterile solid composition which may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.
• Carriers are intended to include necessary and inert binders, suspending agents, lubricants, flavorants, sweeteners, preservatives, dyes, and coatings.
• the MCHl antagonist can be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic) , bile salts, acacia, gelatin, sorbitan onoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
• solutes or suspending agents for example, enough saline or glucose to make the solution isotonic
• bile salts for example, enough saline or glucose to make the solution isotonic
• acacia gelatin
• sorbitan onoleate sorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
• the MCHl antagonist can also be administered orally either in liquid or solid composition form.
• Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions.
• forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
• Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular compound in use, the strength of the preparation, the mode of administration, and the advancement of the disease condition. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.
• This invention provides a transgenic, nonhuman mammal expressing DNA encoding a human MCHl receptor.
• This invention also provides a transgenic, nonhuman mammal comprising a homologous recombination knockout of the native human MCHl receptor.
• This invention further provides a transgenic, nonhuman mammal whose genome comprises antisense DNA complementary to the DNA encoding a human MCHl receptor so placed within the genome as to be transcribed into antisense mRNA which is complementary to mRNA encoding the human MCHl receptor and which hybridizes to mRNA encoding the human MCHl receptor, thereby reducing its translation.
• the DNA encoding the human MCHl receptor additionally comprises an inducible promoter.
• the DNA encoding the human MCHl receptor additionally comprises tissue specific regulatory elements.
• the transgenic, nonhuman mammal is a mouse.
• Animal model systems which elucidate the physiological and behavioral roles of the polypeptides of this invention are produced by creating transgenic animals in which the activity of the polypeptide is either increased or decreased, or the amino acid sequence of the expressed polypeptide is altered, by a variety of techniques.
• these techniques include, but are not limited to: 1) Insertion of normal or mutant versions of DNA encoding the polypeptide, by microinjection, electroporation, retrovir.al transfection or other means well known to those in the art, into appropriate fertilized embryos in order to produce a transgenic animal or 2) Homologous recombination of mutant or normal, human or animal versions of these genes with the native gene locus in transgenic animals to alter the regulation of expression or the structure of these polypeptide sequences.
• homologous recombination is well known in the art. It replaces the native gene with the inserted gene and so is useful for producing an animal that cannot express native polypeptides but does express, for example, an inserted mutant polypeptide, which has replaced the native polypeptide in the animal's genome by recombination, resulting in underexpression of the transporter. Microinjection adds genes to the genome, but does not remove them, and so is useful for producing an animal which expresses its own and added polypeptides, resulting in overexpression of the polypeptides.
• transgenic animal One means available for producing a transgenic animal, with a mouse as an example, is as follows: Female mice are mated, and the resulting fertilized eggs are dissected out of their oviducts. The eggs are stored in an appropriate medium such as M2 medium. DNA or cDNA encoding a polypeptide of this invention is purified from a vector by methods well known in the art. Inducible promoters may be fused with the coding region of the DNA to provide an experimental means to regulate expression of the transgene. Alternatively, or in addition, tissue specific regulatory elements may be fused with the coding region to permit tissue-specific expression of the trans-gene.
• microinjection needle which may be made from capillary tubing using a pipette puller
• the egg to be injected is put in a depression slide.
• the needle is inserted into the pronucleus of the egg, and the DNA solution is injected.
• the injected egg is then transferred into the oviduct of a pseudopregnant mouse ( a mouse stimulated by the appropriate hormones to maintain pregnancy but which is not actually pregnant ) , where it proceeds to the uterus, implants, and develops to term.
• pseudopregnant mouse a mouse stimulated by the appropriate hormones to maintain pregnancy but which is not actually pregnant
• microinjection is not the only method for inserting DNA into the egg cell, and is used here only for exemplary purposes .
• This invention provides a process for identifying a chemical compound which specifically binds to a mammalian MCHl receptor which comprises contacting cells comprising DNA encoding, and expressing on their cell surface, the mammalian MCHl receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian MCHl receptor, wherein the cells do not normally express the mammalian MCHl receptor and the DNA encoding the mammalian MCHl receptor (a) hybridizes to a nucleic acid having the defined sequence shown in Figure 1 (SEQ ID NO: 1) under low stringency conditions or a sequence complementary thereto and (b) is further characterized by its ability to cause a change in the pH of a culture of CHO cells when a MCHl ligand is added to the culture and the CHO cells contain the nucleic acid which hybridized to the nucleic acid having the defined sequence or its complement.
• This invention also provides a process for identifying a chemical compound which specifically binds to a mammalian MCHl receptor which comprises contacting a membrane preparation from cells comprising DNA encoding, and expressing on their cell surface, the mammalian MCHl receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian MCHl receptor, wherein the cells do not normally express the mammalian MCHl receptor and the DNA encoding the mammalian MCHl receptor (a) hybridizes to a nucleic acid having the defined sequence shown in Figure 1 (SEQ ID NO: 1) under low stringency conditions or a sequence complementary thereto and (b) is further characterized by its ability to cause a change in the pH of a culture of CHO cells when a MCHl ligand is added to the culture and the CHO cells contain the nucleic acid which hybridized to the nucleic acid having the defined sequence or its complement.
• a hybridizes to a nucleic acid having the defined sequence shown
• the MCHl receptor is a human MCHl receptor. In another embodiment, the MCHl receptor is a rat MCHl receptor. In another embodiment, the mammalian MCHl receptor comprises substantially the same amino acid sequence as the sequence of the human MCHl receptor encoded by plasmid pEXJ.HR- TL231. In a further embodiment, the mammalian MCHl receptor comprises substantially the same amino acid sequence as that shown in Figure 2 (SEQ ID NO: 2 ) . In another embodiment, the mammalian MCHl receptor comprises the amino acid sequence shown in Figure 2 (SEQ ID NO: 2) . In a different embodiment, the mammalian MCHl receptor comprises the amino acid sequence shown in Figure 13 (SEQ ID NO: 26) .
• the mammalian MCHl receptor comprises the amino acid sequence shown in Figure 14 (SEQ ID NO: 27) .
• the mammalian MCHl receptor comprises the amino acid sequence shown in Figure 15 (SEQ ID NO: 28) .
• the compound is not previously known to bind to a mammalian MCHl receptor. This invention further provides a compound identified by the above-described processes.
• the cell is an insect cell.
• the cell is a mammalian cell.
• the cell is nonneuronal in origin.
• the nonneuronal cell is a COS-7 cell, 293 human embryonic kidney cell, a CHO cell, a NIH-3T3 cell, a mouse Yl cell, or a LM(tk-) cell.
• This invention provides a process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian MCHl receptor which comprises contacting cells expressing on their cell surface the mammalian MCHl receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and separately with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the mammalian MCHl receptor, a decrease in the binding of the second chemical compound to the mammalian MCHl receptor in the presence of the chemical compound indicating that the chemical compound binds to the mammalian MCHl receptor, wherein the cells do not normally express the mammalian MCHl receptor and the DNA encoding the mammalian MCHl receptor (a) hybridizes to a nucleic acid having the defined sequence shown in Figure 1 (SEQ ID NO: 1) under low stringency conditions or a sequence complementary thereto and (b) is further characterized by its ability to cause a change in the pH of a culture
• This invention also provides a process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian MCHl receptor which comprises contacting a membrane preparation from cells expressing on their cell surface the mammalian MCHl receptor, with both the chemical compound and a second chemical compound known -to bind to the receptor, and separately with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the mammalian MCHl receptor, a decrease in the binding of the second chemical compound to the mammalian MCHl receptor in the presence of the chemical compound indicating that the chemical compound binds to the mammalian MCHl receptor, wherein the cells do not normally express the mammalian MCHl receptor and the DNA encoding the mammalian MCHl receptor (a) hybridizes to a nucleic acid having the defined sequence shown in Figure 1 (SEQ ID NO: 1) under low stringency conditions or a sequence complementary thereto and (b) is further characterized by its ability to cause a
• the mammalian MCHl receptor is a human MCHl receptor or a mutant of such human MCHl receptor which is activated by MCH or an analog or homolog thereof.
• the mammalian MCHl receptor is a rat MCHl receptor.
• the mammalian MCHl receptor comprises substantially the same amino acid sequence as the human MCHl receptor encoded by plasmid pEXJ. HR-TL231.
• the mammalian MCHl receptor comprises substantially the same amino acid sequence as that shown in Figure 2 (SEQ ID NO: 2) .
• the mammalian MCHl receptor comprises the amino acid sequence shown in Figure 2 (SEQ ID NC : 2) .
• the cell is an insect cell. In another embodiment, the cell is a mammalian cell. In a further embodiment, the cell is nonneuronal in origin. In another embodiment, the nonneuronal cell is a COS-7 cell, 293 human embryonic kidney cell, a CHO cell, a NIH-3T3 cell, a mouse Yl cell, or a LM(tk-) cell. In one embodiment, the compound is not previously known to bind to a mammalian MCHl receptor. This invention provides a compound identified by the above-described processes.
• This invention provides a method of screening a plurality of chemical compounds not known to bind to a mammalian MCHl receptor to identify a compound which specifically binds to the mammalian MCHl receptor, which comprises (a) contacting cells transfected with and expressing DNA encoding the mammalian MCHl receptor with the plurality of compounds not known to bind specifically to the mammalian MCHl receptor, under conditions permitting binding of compounds known to bind the mammalian MCHl receptor; (b) determining whether the binding of a compound known to bind to the mammalian MCHl receptor is reduced in the presence of the compounds within the plurality of compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so (c) separately determining the binding to the mammalian MCHl receptor of compounds included in the plurality of compounds, so as to thereby identify the compound which specifically binds to the mammalian MCHl receptor.
• This invention provides a method of screening a plurality of chemical compounds not known to bind to a mammalian MCHl receptor to identify a compound which specifically binds to the mammalian MCHl receptor, which comprises (a) contacting a membrane preparation from cells transfected with and expressing the mammalian MCHl receptor with the plurality of compounds not known to bind specifically to the mammalian MCHl receptor, under conditions permitting binding of compounds known to bind the mammalian MCHl receptor; (b) determining whether the binding of a compound known to bind to the mammalian MCHl receptor is reduced in the presence of the compounds within the plurality of compounds, relative to the binding of the co pound in the absence of the plurality of compounds; and if so (c) separately determining the binding to the mammalian MCHl receptor of compounds included in the plurality of compounds, so as to thereby identify the compound which specifically binds to the mammalian MCHl receptor .
• the mammalian MCHl receptor is a human MCHl receptor or a mutant of such human MCHl receptor which is activated by MCH or an analog or homolog thereof.
• the mammalian MCHl receptor is a rat MCHl receptor.
• the cell is a mammalian cell.
• the mammalian cell is non- neuronal in origin.
• the nonneuronal cell is a COS-7 cell, a 293 human embryonic kidney cell, a LM(tk-) cell, a CHO cell, a mouse Yl cell, or an NIH-3T3 cell.
• This invention also provides a method of detecting expression of a mammalian MCHl receptor by detecting the presence of mRNA coding for the mammalian MCHl receptor which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained from a nucleic acid probe under hybridizing conditions, detecting the presence of mRNA hybridizing to the probe, and thereby detecting the expression of the mammalian MCHl receptor by the cell.
• This invention further provides a method of detecting the presence of a mammalian MCHl receptor on the surface of a cell which comprises contacting the cell with an antibody under conditions permitting binding of the antibody to the receptor, detecting the presence of the antibody bound to the cell, and thereby detecting the presence of the mammalian MCHl receptor on the surface of the cell.
• This invention provides a method of determining the physiological effects of- varying levels of activity of human MCHl receptors which comprises producing a transgenic, nonhuman mammal whose levels of human MCHl receptor activity are varied by use of an inducible promoter which regulates human MCHl receptor expression.
• This invention also provides a method of determining the physiological effects of varying levels of activity of human MCHl receptors which comprises producing a panel of transgenic, nonhuman mammals each expressing a different amount of human MCHl receptor.
• This invention provides a method for identifying an antagonist capable of alleviating an abnormality wherein the abnormality is alleviated by decreasing the activity of a human MCHl receptor comprising administering a compound to a transgenic, nonhuman mammal, and determining whether the compound alleviates the physical and behavioral abnormalities displayed by the transgenic, nonhuman mammal as a result of overactivity of a human MCHl receptor, the alleviation of the abnormality identifying the compound as an antagonist.
• This invention also provides an antagonist identified by the above- described method.
• This invention further provides a pharmaceutical composition comprising an antagonist identified by the above-described method and a pharmaceutically acceptable carrier.
• This invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by decreasing the activity of a human MCHl receptor which comprises administering to the subject an effective amount of this pharmaceutical composition, thereby treating the abnormality.
• This invention provides a method for identifying an agonist capable of alleviating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a human MCHl receptor comprising administering a compound to transgenic, nonhuman mammal, and determining whether the compound alleviates the physical and behavioral abnormalities displayed by the transgenic, nonhuman mammal, the alleviation of the abnormality identifying the compound as an agonist.
• This invention also provides an agonist identified by the above-described method.
• This invention further provides a pharmaceutical composition comprising an agonist identified by the above- described method and a pharmaceutically acceptable carrier.
• This invention further provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a human MCHl receptor which comprises administering to the subject an effective amount of this pharmaceutical composition, thereby treating the abnormality.
• This invention provides a method for diagnosing a predisposition to a disorder associated with the activity of a specific mammalian ' allele which comprises: (a) obtaining DNA of subjects suffering from the disorder; (b) performing a restriction digest of the DNA with a panel of restriction enzymes; (c) electrophoretically separating the resulting DNA fragments on a sizing gel; (d) contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human MCHl receptor and labeled with a detectable marker; (e) detecting labeled bands which have hybridized to the DNA encoding a human MCHl receptor labeled with a detectable marker to create a unique band pattern specific to the DNA of subjects suffering from the disorder; (f) preparing DNA obtained for diagnosis by steps (a) -(e); and (g) comparing the unique band pattern specific to the DNA of subjects suffering from the disorder from step (e) and
• This invention provides a method of preparing the purified human MCHl receptor which comprises: (a) inducing cells to express the human MCHl receptor; (b) recovering the human MCHl receptor from the induced cells; and (c) purifying the human MCHl receptor so recovered.
• This invention provides a method of preparing the purified human MCHl receptor which comprises: (a) inserting nucleic acid encoding the human MCHl receptor in a suitable vector; (b) introducing the resulting vector in a suitable host cell; (c) placing the resulting cell in suitable condition permitting the production of the isolated human MCHl receptor; (d) recovering the human MCHl receptor produced by the resulting cell; and (e) purifying the human MCHl receptor so recovered.
• This invention provides a process for determining whether a chemical compound is a mammalian MCHl receptor agonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian MCHl receptor with the compound under conditions permitting the activation of the mammalian MCHl receptor, and detecting an increase in mammalian MCHl receptor activity, so as to thereby determine whether the compound is a mammalian MCHl receptor agonist.
• This invention also provides a process for determining whether a chemical compound is a mammalian MCHl receptor antagonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian MCHl receptor with the compound in the presence of a known mammalian MCHl receptor agonist, under conditions permitting the activation of the mammalian MCHl receptor, and detecting a decrease in mammalian MCHl receptor activity, so as to thereby determine whether the compound is a mammalian MCHl receptor antagonist.
• the mammalian MCHl receptor is a human MCHl receptor or a mutant of such human MCHl receptor which is activated by MCH or an analog or homolog thereof.
• This invention further provides a pharmaceutical composition which comprises an amount of a mammalian MCHl receptor agonist determined by the above-described process effective to increase activity of a mammalian MCHl receptor and a pharmaceutically acceptable carrier.
• a mammalian MCHl receptor agonist is not previously known.
• This invention provides a pharmaceutical composition which comprises an amount of a mammalian MCHl receptor antagonist determined by the above-described process effective to reduce activity of a mammalian MCHl receptor and a pharmaceutically acceptable carrier.
• the mammalian MCHl receptor antagonist is not previously known.
• This invention provides a process for determining whether a chemical compound specifically binds to and activates a mammalian MCHl receptor, which comprises contacting cells producing a second messenger response and expressing on their cell surface the mammalian MCHl receptor, wherein such cells do not normally express the mammalian MCHl receptor, with the chemical compound under conditions suitable for activation of the mammalian MCHl receptor, and measuring the second messenger response in the presence and in the absence of the chemical compound, a change in the second messenger response in the presence of the chemical compound indicating that the compound activates the mammalian MCHl receptor.
• the second messenger response comprises chloride channel activation and the change in second messenger is an increase in the level of inward chloride current.
• This invention also provides a process for determining whether a chemical compound specifically binds to and inhibits activation of a mammalian MCHl receptor, which comprises separately contacting cells producing a second messenger response and expressing on their cell surface the mammalian MCHl receptor, wherein such cells do not normally express the mammalian MCHl receptor, with both the chemical compound and a second chemical compound known to activate the mammalian MCHl receptor, and with only the second chemical compound, under conditions suitable for activation of the mammalian MCHl receptor, and measuring the second messenger response in the presence of only the second chemical compound and in the presence of both the second chemical compound and the chemical compound, a smaller change in the second messenger response in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound indicating that the chemical compound inhibits activation of the mammalian MCHl receptor.
• the second messenger response comprises chloride channel activation and the change in second messenger response is a smaller increase in the level of inward chloride current in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound.
• This invention also provides the above-described processes performed with membrane preparations from cells producing a second messenger response and transfected with and expressing the mammalian MCHl receptor.
• the mammalian MCHl receptor is a human MCHl receptor or a mutant of such human MCHl receptor which is activated by MCH or an analog or homolog thereof.
• the mammalian MCHl receptor is a rat MCHl receptor.
• the mammalian MCHl receptor comprises substantially the same amino acid sequence as encoded by the plasmid pEXJ. HR-TL231.
• the mammalian MCHl receptor comprises substantially the same amino acid sequence as that shown in Figure 2 (SEQ ID NO: 2) .
• the mammalian MCHl receptor comprises an amino acid sequence as shown in Figure 2 (SEQ ID NO: 2) .
• the cell is an insect cell.
• the cell is a mammalian cell.
• the mammalian cell is nonneuronal in origin.
• the nonneuronal cell is a COS-7 cell, CHO cell, 293 human embryonic kidney cell, NIH-3T3 cell or LM(tk-) cell.
• the compound is not previously known to bind to a mammalian MCHl receptor. This invention also provides a compound determined by the above-described processes.
• This invention also provides a pharmaceutical composition which comprises an amount of a mammalian MCHl receptor agonist determined by the above-described processes effective to increase activity of a mammalian MCHl receptor and a pharmaceutically acceptable carrier.
• a mammalian MCHl receptor agonist is not previously known.
• This invention further provides a pharmaceutical composition which comprises an amount of a mammalian MCHl receptor antagonist determined by the above-described processes effective to reduce activity of a mammalian MCHl receptor and a pharmaceutically acceptable carrier.
• a mammalian MCHl receptor antagonist determined by the above-described processes effective to reduce activity of a mammalian MCHl receptor and a pharmaceutically acceptable carrier.
• the mammalian MCHl receptor antagonist is not previously known.
• This invention provides a method of screening a plurality of chemical compounds not known to activate a mammalian MCHl receptor to identify a compound which activates the mammalian MCHl receptor which comprises: (a) contacting cells transfected with and expressing the mammalian MCHl receptor with the plurality of compounds not known to activate the mammalian MCHl receptor, under conditions permitting activation of the mammalian MCHl receptor; (b) determining whether the activity of the mammalian MCHl receptor is increased in the presence of the compounds; and if so (c) separately determining whether the activation of the mammalian MCHl receptor is increased by each compound included in the plurality of compounds, so as to thereby identify the compound which activates the mammalian MCHl receptor.
• the mammalian MCHl receptor is a human MCHl receptor or a mutant of such human MCHl receptor which is activated by MCH or an analog or homolog thereof. In another embodiment, the mammalian MCHl receptor is a rat MCHl receptor .
• This invention provides a method of screening a plurality of chemical compounds not known to inhibit the activation of a mammalian MCHl receptor to identify a compound which inhibits the activation of the mammalian MCHl receptor, which comprises: (a) contacting cells transfected with and expressing the mammalian MCHl receptor with the plurality of compounds in the presence of a known mammalian MCHl receptor agonist, under conditions permitting activation of the mammalian MCHl receptor; (b) determining whether the activation of the mammalian MCHl receptor is reduced in the presence of the plurality of compounds, relative to the activation of the mammalian MCHl receptor in the absence of the plurality of compounds; and if so (c) separately determining the inhibition of activation of the mammalian MCHl receptor for each compound included in the plurality of compounds, so as to thereby identify the compound which inhibits the activation o ' f the mammalian MCHl receptor.
• the mammalian MCHl receptor is a human MCHl receptor or a mutant of such human MCHl receptor which is activated by MCH or an analog or homolog thereof. In another embodiment, the mammalian MCHl receptor is a rat MCHl receptor.
• the cell is a mammalian cell.
• the mammalian cell is non-neuronal in origin.
• the non-neuronal cell is a COS-7 cell, a 293 human embryonic kidney cell, a LM(tk-) cell or an NIH-3T3 cell.
• This invention provides a pharmaceutical composition
• a pharmaceutical composition comprising a compound identified by the above-described methods effective to increase mammalian MCHl receptor activity and a pharmaceutically acceptable carrier.
• This invention also provides a pharmaceutical composition comprising a compound identified by the above-described methods effective to decrease mammalian MCHl receptor activity and a pharmaceutically acceptable carrier.
• This invention further provides a method of measuring receptor activation in an oocyte expression system such as a Xenopus oocyte expression system or melanophore.
• receptor activation is determined by measurement of ion channel activity.
• receptor activation is measured by aequorin luminescence .
• genes in Xenopus oocytes is well known in the art (Coleman, A., 1984; Masu, Y.,et al . , 1994) and is performed using microinjection of native mRNA or in vitro synthesized mRNA into frog oocytes.
• the preparation of in vitro synthesized mRNA can be performed by various standard techniques (Sambrook, et al . 1989) including using T7 polymerase with the mCAP RNA mapping kit (Stratagene) .
• This invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a mammalian MCHl receptor which comprises administering to the subject an amount of a compound which is a mammalian MCHl receptor agonist effective to treat the abnormality.
• the abnormality is a regulation of a steroid or pituitary hormone disorder, an epinephrine release disorder, a gastrointestinal disorder, a cardiovascular disorder, an electrolyte balance disorder, hypertension, diabetes, a respiratory disorder, asthma, a reproductive function disorder, an immune disorder, an endocrine disorder, a musculoskeletal disorder, a neuroendocrine disorder, a cognitive disorder, a memory disorder such as Alzheimer's disease, a sensory modulation and transmission disorder, a motor coordination disorder, a sensory integration disorder, a motor integration disorder, a dopaminergic function disorder such as Parkinson's disease, a sensory transmission disorder, an olfaction disorder, a sympathetic innervation disorder, an affective disorder such as depression, a stress-related disorder, a fluid-balance disorder, a urinary disorder such as urinary incontinence, a seizure disorder, pain, psychotic behavior such as schizophrenia, morphine tolerance, opiate addiction or migraine.
• This invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by decreasing the activity of a mammalian MCHl receptor which comprises administering to the subject an amount of a compound which is a mammalian MCHl receptor antagonist effective to treat the abnormality.
• the abnormality is a regulation of a steroid or pituitary hormone disorder, an epinephrine release disorder, a gastrointestinal disorder, a cardiovascular disorder, an electrolyte balance disorder, hypertension, diabetes, a respiratory disorder, asthma, a reproductive function disorder, an immune disorder, an endocrine disorder, a musculoskeletal disorder, a neuroendocrine disorder, a cognitive disorder, a memory disorder such as Alzheimer's disease, a sensory modulation and transmission disorder, a motor coordination disorder, a sensory integration disorder, a motor integration disorder, a dopaminergic function disorder such as Parkinson's disease, a sensory transmission disorder, an olfaction disorder, a sympathetic innervation disorder, an affective disorder such as depression, a stress-related disorder, a fluid-balance disorder, a urinary disorder such as urinary incontinence, a seizure disorder, pain, psychotic behavior such as schizophrenia, morphine tolerance, opiate addiction or migraine.
• This invention provides a process for making a composition of matter which specifically binds to a mammalian MCHl receptor which comprises identifying a chemical compound using any of the processes described herein for identifying a compound which binds to and/or activates or inhibits activation of a mammalian MCHl receptor and then synthesizing the chemical compound or a novel structural and functional analog or homolog thereof.
• the mammalian MCHl receptor is a human MCHl receptor or a mutant of such human MCHl receptor which is activated by MCH or an analog or homolog thereof.
• the mammalian MCHl receptor is a rat MCHl receptor.
• This invention further provides a process for preparing a composition which comprises admixing a pharmaceutically acceptable carrier and a therapeutically effective amount of a chemical compound identified by any of the processes described herein for identifying a compound which binds to and/or activates or inhibits activation of a mammalian MCHl receptor or a novel structural and functional analog or homolog thereof.
• the mammalian MCHl receptor is a human MCHl receptor or a mutant of such human MCHl receptor which is activated by MCH or an analog or homolog thereof.
• the mammalian MCHl receptor is a rat MCHl receptor.
• This invention provides a process for determining whether a chemical compound is a human MCHl receptor antagonist which comprises contacting cells transfected with and expressing DNA encoding the human MCHl receptor with the compound in the presence of a known human MCHl receptor agonist, under conditions permitting the activation of the human MCHl receptor, and detecting a decrease in human MCHl receptor activity, so as to thereby determine whether the compound is a human MCHl receptor antagonist, wherein the DNA encoding the human MCHl receptor comprises the sequence shown in Figure 1 (Seq. ID No. 1) or contained in plasmid pEXJ.HR-TL231 (ATCC Accession No. 203197), the known human MCHl receptor agonist is MCH or a homolog or analog of MCH, and the cells do not express the MCHl receptor prior to transfecting them.
• This invention also provides a process for determining whether a chemical compound specifically binds to and inhibits activation of a human MCHl receptor, which comprises separately contacting cells expressing on their cell surface the human MCHl receptor and producing a second messenger response upon activation of the human MCHl receptor, wherein such cells do not normally express the human MCHl receptor and the DNA encoding the human MCHl receptor comprises the sequence shown in Figure 1 (Seq. ID No. 1) or contained in plasmid pEXJ. HR-TL231 (ATCC Accession No.
• the second messenger response comprises chloride channel activation and the change in second messenger response is a smaller increase in the level of inward chloride current in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound.
• This invention further provides a method of screening a plurality of chemical compounds not known to inhibit the activation of a human MCHl receptor to identify a compound which inhibits the activation of the human MCHl receptor, which comprises:
• the cell is an insect cell.
• the cell is a mammalian cell.
• the cell is a mammalian cell which is nonneuronal in origin.
• the cell is a COS-7 cell, a CHO cell, a 293 human embryonic kidney cell, a NIH-3T3 cell, a mouse Yl cell, or a LM(tk-) cell.
• This invention provides a process for making a composition of matter which specifically binds to a human MCHl receptor which comprises identifying a chemical compound which specifically binds -to the human MCHl receptor and then synthesizing the chemical compound or a structural and functional analog or homolog thereof, wherein the chemical compound is identified as binding to the human MCHl receptor by a process involving competitive binding which comprises contacting cells expressing on their cell surface the human MCHl receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and separately with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting the extent of specific binding of the chemical compound to the human MCHl receptor, a decrease in the binding of the second chemical compound to the human MCHl receptor in the presence of the chemical compound indicating that • the chemical compound binds to the human MCHl receptor, wherein the cells do not normally express the human MCHl receptor, the human MCHl receptor is encoded by nucleic acid comprising the sequence shown in Figure 1 (Seq. ID
• This invention further provides a process for making a composition of matter which specifically binds to a human MCH1 receptor which comprises identifying a chemical compound which specifically binds to the human MCHl receptor and then synthesizing the chemical compound or a structural and functional analog or homolog thereof, wherein the chemical compound is identified as binding to the human MCHl receptor by a process involving competitive binding which comprises contacting a membrane preparation from cells expressing on their cell surface the human MCHl receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and separately with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting the extent of specific binding of the chemical compound to the human MCHl receptor, a decrease in the binding of the second chemical compound to the human MCHl receptor in the presence of the chemical compound indicating that the chemical compound binds to the human MCHl receptor, wherein the cells do not normally express the human MCHl receptor, the human MCHl receptor is encoded by nucleic acid comprising the sequence shown in Figure 1 (Se
• This invention also provides a process for making a composition of matter which is a human MCHl receptor antagonist which comprises identifying a chemical compound which is a human MCHl receptor "antagonist and then synthesizing the chemical compound or a structural and functional analog or homolog thereof, wherein the chemical compound is identified as a human MCHl receptor antagonist by a process which comprises contacting cells transfected with and expressing DNA encoding the human MCHl receptor with the compound in the presence of a known human MCHl receptor agonist, under conditions permitting the activation of the human MCHl receptor, and detecting a decrease in human MCHl receptor activity, so as to thereby determine whether the compound is a human MCHl receptor antagonist, wherein the cells do not normally express the human MCHl receptor, the human MCHl receptor is encoded by nucleic acid comprising the sequence shown in Figure 1 (Seq. ID No. 1) or contained in plasmid pEXJ. HR-TL231 (ATCC Accession No. 203197), and the known human MCHl
• This invention still further provides a process for making a composition of matter which specifically binds to and inhibits the activation of a human MCHl receptor which comprises identifying a chemical compound which specifically binds to and inhibits the activation of the human MCHl receptor and then synthesizing the chemical compound or a structural and functional analog or homolog thereof, wherein the chemical compound is identified as binding to and inhibiting the activation of the human MCHl receptor by a process which comprises separately contacting cells expressing on their cell surface the human MCHl receptor and producing a second messenger response upon activation of the human MCHl receptor, wherein such cells do not normally express the human MCHl receptor and the human MCHl receptor is encoded by nucleic acid comprising the sequence shown in Figure 1 (Seq. ID No.
• the second messenger response comprises chloride channel activation and the change in second messenger response is a smaller increase in the level of inward chloride current in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound.
• This invention provides a process for preparing a composition which comprises identifying a chemical compound which specifically binds to a human MCHl receptor, and then admixing a carrier and the chemical compound or a structural and functional analog or homolog thereof, wherein the chemical compound is identified as binding to the human MCHl receptor by a process involving competitive binding which comprises contacting cells expressing on their cell surface the human MCHl receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and separately with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting the extent of specific binding of the chemical compound to the human MCHl receptor, a decrease in the binding of the second chemical compound to the human MCHl receptor in the presence of the chemical compound indicating that the chemical compound binds to the human MCHl receptor, wherein the cells do not normally express the human MCHl receptor, the human MCHl receptor is encoded by nucleic acid comprising the sequence shown in Figure 1 (Seq. ID No. 1) or contained in plasmid
• This invention further provides a process for preparing a composition which comprises identifying a chemical compound which specifically binds to a human MCHl receptor, and then admixing a carrier and the chemical compound or a structural and functional analog or homolog thereof, wherein the chemical compound is identified as binding to the human MCHl receptor by a process involving competitive binding which comprises contacting a membrane preparation from cells expressing on their cell surface the human MCHl receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and separately with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting the extent of specific binding of the chemical compound to the human MCHl receptor, a decrease in the binding of the second chemical compound to the human MCHl receptor in the presence of the chemical compound indicating that the chemical compound binds to the human MCHl receptor, wherein the cells do not normally express the human MCHl receptor, the human MCHl receptor is encoded by nucleic acid comprising the sequence shown in Figure 1 (Seq. ID No. 1) or contained
• This invention also provides a process for preparing a composition which comprises identifying a chemical compound which is a human MCHl receptor antagonist, and then admixing a carrier and the chemical compound or a structural and functional analog or homolog thereof, wherein the chemical compound is identified as a human MCHl receptor antagonist by a process which comprises contacting cells transfected with and expressing DNA encoding the human MCHl receptor with the compound in the presence of a known human MCHl receptor agonist, under conditions permitting the activation of the human MCHl receptor, and detecting a decrease in human MCHl receptor activity, so as to thereby determine whether the compound is a human MCHl receptor antagonist, wherein the cells do not normally express the human MCHl receptor, the human MCHl receptor is encoded by nucleic acid comprising the sequence shown in Figure 1 (Seq. ID No. 1) or contained in plasmid pEXJ.HR-TL231 (ATCC Accession No. 203197), and the known human MCHl receptor agonist is MCH or
• This invention still further provides a process for preparing a composition which comprises identifying a chemical compound which specifically binds to and inhibits the activation of a human MCHl receptor, and then admixing a carrier and the chemical compound or a structural and functional analog or homolog thereof, wherein the chemical compound is identified as binding to and inhibiting activation of the human MCHl receptor by a process which comprises separately contacting cells expressing on their cell surface the human MCHl receptor and producing a second messenger response upon activation of the human MCHl receptor, wherein such cells do not normally express the human MCHl receptor and the human MCHl receptor is encoded by nucleic acid comprising the sequence shown in Figure 1 (Seq. ID No.
• the second messenger response comprises chloride channel activation and the change in second messenger response is a smaller increase in the level of inward chloride current in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound.
• the cell is an insect cell.
• the cell is a mammalian cell.
• the mammalian cell is nonneuronal in origin.
• the nonneuronal cell is a COS-7 cell, a 293 human embryonic kidney cell, a CHO cell, a NIH-3T3 cell, a mouse Yl cell, or a LM(tk-) cell.
• antagonist potency is measured as K B which is defined as the equilibrium dissociation constant for the antagonist-receptor complex.
• agonist potency is measured as EC50 which is defined as the concentration that is required to elicit 50% of the maximum response in a functional assay.
• binding affinity describes the concentration of a compound required to occupy one-half of the binding sites in a receptor population, as detectable by radioligand binding. Binding affinity concentration can be represented as K l f inhibition constant, or K D , dissociation constant.
• selectivity of binding affinity refers to the ability of a chemical compound to discriminate one receptor from another. For example, a compound showing selectivity for receptor A versus receptor B will bind receptor A at lower concentrations than those required to bind receptor B.
• the statements of the form "binds to the MCHl receptor with a binding affinity at least ten-fold higher than" a named receptor, indicates that the binding affinity at the MCHl receptor is at least ten-fold greater than that for a named receptor, and binding affinity measurements (i.e. K x or K) for the compound are at least ten-fold lower in numerical value.
• This invention provides a method of treating an eating disorder or obesity in a subject which comprises administering to the subject a therapeutically effective amount of an MCHl antagonist which inhibits the activation of the MCHl receptor.
• the MCHl antagonist additionally inhibits the activation of the MCHl receptor with an antagonist potency which is at least 30-fold greater than the antagonist potency with which the MCHl antagonist inhibits the activation of each of the 5- HT2C and MC-4 receptors.
• the MCHl antagonist additionally inhibits the activation of the MCHl receptor with an antagonist potency which is at least 10-fold greater than the antagonist potency with which the MCHl antagonist inhibits the activation of each of the NPY1, NPY5, GALRl, GALR2, and GALR3 receptors.
• the MCHl antagonist additionally inhibits the activation of the MCHl receptor with an antagonist potency which is at least 100-fold greater than the antagonist potency with which the MCHl antagonist inhibits the activation of each of the 5-HT2C and MC-4 receptors.
• the MCHl antagonist additionally inhibits the activation of the MCHl receptor with an antagonist potency which is at least 100-fold greater than the antagonist potency with which the MCHl antagonist inhibits the activation of each of the NPY1, NPY5, GALR1, GALR2 , and GALR3 receptors. In an embodiment, the MCHl antagonist additionally inhibits the activation of the MCHl receptor with an antagonist potency which - is at least 30-fold greater than the binding affinity with which the MCHl antagonist binds to each of the 5-HT2C and MC-4 receptors.
• the MCHl antagonist additionally inhibits the activation of the MCHl receptor with an antagonist potency which is at least 10-fold greater than the binding affinity with which the MCHl antagonist binds to each of the NPY1, NPY5, GALR1, GALR2, and GALR3 receptors.
• the MCHl antagonist additionally inhibits the ' activation of the MCHl receptor with an antagonist potency which is at least 100-fold greater than the binding affinity with which the MCHl antagonist binds to each of the 5-HT2C and MC-4 receptors.
• the MCHl antagonist additionally inhibits the activation of the MCHl receptor with an antagonist potency which is at least 100-fold greater than the binding affinity with which the MCHl antagonist binds to each of the NPYl, NPY5, GALRl, GALR2 , and GALR3 receptors.
• the MCHl antagonist additionally binds to the MCHl receptor with a binding affinity which is at least 30-fold greater than the binding affinity with which the MCHl antagonist binds to each of the 5-HT2C and MC-4 receptors.
• the MCHl antagonist additionally binds to the MCHl receptor with a binding affinity which is at least 10-fold greater than the binding affinity with which the MCHl antagonist binds to each of the NPY1, NPY5, GALR1, GALR2, and GALR3 receptors.
• the MCHl antagonist additionally binds to the MCHl receptor with a binding affinity which is at least 100-fold greater than the binding affinity with which the MCHl antagonist binds to each of the 5-HT2C and MC-4 receptors. In an additional embodiment, the MCHl antagonist additionally binds to the MCHl receptor with a binding affinity which is at least 100-fold greater than the binding affinity with which the MCHl antagonist binds to each of the NPY1, NPY5, GALR1, GALR2 , and GALR3 receptors .
• the MCHl antagonist additionally binds to the MCHl receptor with a binding affinity which is at least 30-fold greater than the binding affinity with which the MCHl antagonist binds to the dopamine D2 receptor. In another embodiment, the MCHl antagonist additionally binds to the MCHl receptor with a binding affinity which is at least 30-fold greater than the binding affinity with which the MCHl antagonist binds to the histamine HI receptor.
• the MCHl antagonist additionally binds to the MCHl receptor with a binding affinity which is at least 100-fold greater than the -go- binding affinity with which the MCHl antagonist binds the dopamine D2 receptor. In another embodiment, the MCHl antagonist additionally binds to the MCHl receptor with a binding affinity which is at least 100-fold greater than the binding affinity with which the MCHl antagonist binds to the HI histamine receptor.
• the MCHl antagonist additionally binds to the MCHl receptor with a binding affinity which is at least 200-fold greater than the binding affinity with which the MCHl antagonist binds the dopamine D2 receptor. In still another embodiment, the MCHl antagonist additionally binds to the MCHl receptor with a binding affinity which is at least 200-fold greater than the binding affinity with which the MCHl antagonist binds to the HI histamine receptor.
• the MCHl antagonist additionally binds to the MCHl receptor with a binding affinity which is at least 10-fold greater than the binding affinity with which the MCHl antagonist binds to the ⁇ 1A adrenoceptor . In another embodiment, the MCHl antagonist additionally binds to the MCHl receptor with a binding affinity which is at least 100-fold greater than the binding affinity with which the MCHl antagonist binds to the ⁇ 1A adrenoceptor.
• the MCHl antagonist additionally binds to the ⁇ 1A adrenoceptor with a binding affinity which is no more than 10-fold greater than the binding affinity with which the MCHl antagonist binds to the MCHl receptor. In still other embodiments, the MCHl antagonist additionally binds to the ⁇ 1A adrenoceptor with a binding affinity which is no more than 100-fold greater than the binding affinity with which the MCHl antagonist binds to the MCHl receptor.
• the eating or feeding disorder is bulimia, obesity or bulimia nervosa.
• the subject is a vertebrate, a mammal, a human or a canine. In another embodiment, the MCHl antagonist is administered in combination with food.
• This invention also provides a method of treating an eating disorder in a subject which comprises administering to the subject a therapeutically effective amount of an MCHl agonist which activates the MCHl receptor.
• the MCHl agonist additionally activates the MCHl receptor with an agonist potency which is at least 30-fold greater than the agonist potency with which the MCHl agonist activates each of the 5-HT2C and MC-4 receptors.
• the MCHl agonist additionally activates the MCHl receptor with an agonist potency which is at least 10-fold greater than the agonist potency with which the MCHl agonist activates each of the NPY1, NPY5, GALR1, GALR2, and GALR3 receptors. In a further embodiment, the MCHl agonist additionally activates the MCHl receptor with an agonist potency which is at least 100-fold greater than the agonist potency with which the MCHl agonist activates each of the 5-HT2C and MC-4 receptors .
• the MCHl agonist additionally activates the MCHl receptor with an agonist potency which is at least 100-fold greater than the agonist potency with which the MCHl agonist activates each of the NPY1, NPY5, GALR1, GALR2, and GALR3 receptors.
• the eating disorder is anorexia nervosa.
• the subject is a vertebrate, a mammal, a human or a canine.
• the MCHl agonist is administered in combination with food.
• a "therapeutically effective amount” is any amount of a compound which, when administered to a subject suffering from a disease against which the compounds are effective, causes reduction, remission, or regression of the disease.
• a "subject” is a vertebrate, a mammal, a human or a canine .
• This invention further provides a method of modifying feeding behavior of a subject which comprises administering to the subject an amount of a compound of the present invention effective to decrease the consumption of food by the subject and/or decrease the body mass of the subject.
• the subject is a vertebrate, a mammal, a human or a canine.
• the MCHl antagonist is administered in combination with food.
• the present invention includes within its scope prodrugs of the compounds of the invention.
• prodrugs will be functional derivatives of the compounds of the invention which are readily convertible in vivo into the required compound.
• the term "administering" shall encompass the treatment of the various conditions described with the MCHl antagonist specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified MCHl antagonist in vivo after administration to the patient.
• Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in Design of Prodrugs, ed. H. Bundgaard, Elsevier, 1985.
• the present invention provides a method of treating depression and/or anxiety in a subject which comprises administering to the subject a composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a MCHl antagonist, wherein:
• the MCHl antagonist does not inhibit the activity of central monoamine oxidase A greater than 50 percent, at a concentration of lOmM; and (2) the MCHl antagonist does not inhibit the activity of central monoamine oxidase B greater than 50 percent, at a concentration of lOmM; and
• the MCHl antagonist binds to the MCHl receptor with a binding affinity at least ten-fold higher than the binding affinity with which it binds to each of the following transporters: serotonin transporter, norepinephrine transporter, and dopamine transporter.
• the MCHl antagonist also binds to the MCHl receptor with a binding affinity at least ten- fold higher than the binding binding affinity with which it binds to each of the human 5HT 1A , human 5HT 1B , human 5HT 1D , human 5HT 1E , human 5HT 1F , human 5HT 2A , rat 5HT 2C , human 5HT 4 , human 5HT 6 and human 5HT 7 receptors.
• the MCHl antagonist also binds to the MCHl receptor with a binding affinity at least ten-fold higher than the binding affinity with which it binds to the human histamine H : and H 2 receptors.
• the MCHl antagonist also binds to the MCHl receptor with a binding affinity at least ten-fold higher than the binding affinity with which it binds to the human dopamine D l r D 2 , D 3 , D 4 and D 5 receptors .
• the MCHl antagonist also binds to the MCHl receptor with a ' binding affinity at least tenfold higher than the binding affinity with which it binds to the human ⁇ 1A adrenoceptor, the human ⁇ 1B adrenoceptor and the human ⁇ 1D adrenoceptor.
• the MCHl antagonist also binds to the MCHl receptor with a binding affinity at least tenfold higher than the binding affinity with which it binds to the human ⁇ 2A adrenoceptor, the human ⁇ 2B adrenoceptor and the human ⁇ 2c adrenoceptor.
• the MCHl antagonist does not inhibit the activity of central monoamine oxidase A greater than 60 percent. In further embodiments the MCHl antagonist does not inhibit the activity of central monoamine oxidase B greater than 60 percent. In other embodiments the MCHl antagonist does not inhibit the activity of central monoamine oxidase A greater than 70 percent. In still other embodiments the MCHl antagonist does not inhibit the activity of central monoamine oxidase B greater than 70 percent .
• the binding properties of compounds at different receptors were determined using cultured cell lines that selectively express the receptor of interest.
• Cell lines were prepared by transfecting the cloned cDNA or cloned genomic DNA or constructs containing both genomic DNA and cDNA encoding the receptors as further, described in the Experimental Details herein below.
• the binding interactions of compounds at different transporters and enzymes can be determined using tissue preparations and specific assays well known in the art.
• GenBank DNA database which is managed by the National Center for Biotechnology (Bethesda, MD) .
• the "human D l ⁇ ” receptor was renamed the "human D ⁇ receptor.
• the gene for a targeted receptor subtype is cloned, it is placed into a recipient cell which then expresses the targeted receptor subtype on its surface.
• This cell which expresses a single population of the targeted human receptor subtype, is then propagated resulting in the establishment of a cell line.
• This cell line which constitutes a drug discovery system, is used in two different types of assays: binding assays and functional assays. In binding assays, the affinity of a compound for both the receptor subtype that is the target of a particular drug discovery program and other receptor subtypes that could be associated with side effects are measured.
• the data obtained from binding assays also enable chemists to design compounds toward or away from one or more of the relevant subtypes, as appropriate, for optimal therapeutic efficacy.
• functional assays the nature of the response of the receptor subtype to the compound is determined. Data from the functional assays show whether the compound is acting to inhibit or enhance the activity of the receptor subtype, thus enabling pharmacologists to evaluate compounds rapidly at their ultimate human receptor subtypes targets permitting chemists to rationally design drugs that will be more effective and have fewer or substantially less severe side effects than existing drugs.
• Combinatorial chemistry involves automated synthesis of a variety of novel compounds by assembling them using different combinations of chemical building blocks .
• the use of combinatorial chemistry greatly accelerates the process of generating compounds.
• the resulting arrays of compounds are called libraries and are used to screen for compounds (“lead compounds”) that demonstrate a sufficient level of activity at receptors of interest.
• Using combinatorial chemistry it is possible to synthesize "focused" libraries of compounds anticipated to be highly biased toward the receptor target of interest.
• a BLAST search of GENEMBL was performed with the GCG sequence analysis package (Genetics Computer Group, Madison, WI) using a Synaptic Pharmaceutical Corporation proprietary sequence, FB41a, as a query. This resulted in the identification of an EST (accession number F07228) with a high degree of homology to FB41a and somatostatin, opiate and galanin receptors .
• Poly A+ RNA was purified from human hippocampal RNA
• DS- cDNA was synthesized from poly A+ RNA according to Gubler and Hoffman (1983) with minor modifications. The resulting cDNA was ligated to BstXI adaptors (Invitrogen, Corp.) and the excess adaptors removed by exclusion column chromatography. High molecular weight fractions of size- selected ds-cDNA were ligated in pEXJ.BS, an Okayama and Berg expression vector modified from pcEXV (Miller and Germain, 1986) to contain BstXI and other additional restriction sites.
• a total of 2.2 xl0 ⁇ independent clones with a mean insert size of 3.0 kb were generated.
• the library was plated on agar plates (ampicillin selection) and glycerol stocks for 450 pools of 5000 independent clones were prepared. Primary glycerol stocks were also grouped together in groups of approximately 10 to create superpools .
• Cloning of the full-length sequence of MCHl Glycerol stocks of the superpools and primary pools from the human hippocampal cDNA library were screened by PCR with F07228 specific primers T579 and T580 using Taq DNA Polymerase (Boehringer-Mannheim, Indianapolis, IN) and the following PCR protocol: 94°C hold for 5 minutes; 40 cycles of 94°C for 2 minute, 68°C for 4 minutes; 7 minute hold at 68 J C; 4°C hold until the samples are run on a gel.
• One positive primary pool 490 was successively divided into subpools, amplified in LB medium overnight and screened by PCR using primers T579 and T580.
• One positive subpool, 490-4-10-23 was plated on agar plates (ampicillin selection) , and colonies were transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH) . Filters were hybridized for two days under high stringency conditions with 10 b cpm/ml of a 32 P-labeled cDNA probe, T581, designed against the F07228 EST sequence. Filters were washed and apposed to Biomax MS film (Kodak) . Seven positive colonies were picked, streaked on LB-AMP plates, and grown overnight. Two individual colonies from each of the original seven were picked and subjected to vector-anchored PCR using the following primer pairs: T95, T580 and T94, T579.
• TL230 One positive colony, Gl, was amplified overnight in TB and processed for plasmid purification. This plasmid was designated TL230 and sequenced on both strands with a Sequenase kit (US Biochemical, Cleveland, Ohio) . Nucleotide and peptide sequence analysis were performed with GCG programs (Genetics Computer Group, ' Madison, WI). A Hin lll- Kpnl fragment of TL230 was subcloned into the mammalian expression vector pEXJ, and named TL231.
• TL579 5 ' -GGGAACTCCACGGTCATCTTCGCGGT-3 ' (SEQ ID NO: 5)
• TL580 5'-TAGCGGTCAATGGCCATGGCGGTCAG-3' (SEQ ID NO: 6)
• TL581 5'-TAGCGGTCAATGGCCATGGCGGTCAG-3'
• the species genomic DNA (Clontech) may be amplified with a forward PCR primer corresponding to one of the TM regions of TL231 and a reverse primer corresponding to another TM region of TL231.
• PCR may be performed with the Expand Long Template PCR System (Boeringer Mannheim) , for example, under the following conditions: 30 sec at 94°C, 1.5 min at 50°C, 1.5 min at 68 n C for 40 cycles, with a pre- and post-incubation of 5 min at 94°C and 7 min at 68°C, respectively.
• a band is isolated, subcloned using the TA cloning kit (Invitrogen), and sequenced.
• the sequence is run and analyzed on an ABI PRISM 377 BigDye Terminator Cycle Sequencing Kit Sequencer. Forward and reverse PCR primers are designed against this sequence and used to amplify a band from genomic DNA using, for example, the following conditions: 30 sec at 94°C, 1.5 min at 68 n C for 35 cycles, with a pre- and post-incubation of 5 min at 94 n C and 5 min at 68 n C, respectively.
• the PCR product is subcloned using the TA cloning kit (Invitrogen). Miniprep cultures of transformants are prepared and sequenced as above .
• a nucleic acid sequence encoding an MCHl receptor may be isolated using standard molecular biology techniques and approaches such as those briefly described below:
• the full-length sequence may be obtained by sequencing this cosmid clone with additional sequencing primers. Since one intron is present in this gene the full-length intronless gene may be obtained from cDNA using standard molecular biology techniques. For example, a forward PCR primer designed in the 5 ' UT and a reverse PCR primer designed in the 3 ' UT may be used to amplify a full-length, intronless gene from cDNA. Standard molecular biology techniques could be used to subclone this gene into a mammalian expression vector.
• Approach #3 As yet another alternative method, one could utilize 3' and 5' RACE to generate PCR products from cDNA expressing MCHl which contain the additional sequences of MCHl. These RACE PCR products could then be sequenced to determine the missing sequence. This new sequence could then be used to design a forward PCR primer in the 5 ' UT and a reverse primer in the 3 ' UT . These primers could then be used to amplify a full-length MCHl clone from cDNA.
• the plasmid TL231 encodes three in frame methionine residues, any of which could potentially initiate translation of the MCHl receptor.
• the ability of these residues to function in a heterologous expression system was examined by constructing mutants of TL231 in which one or more of the downstream methionine residues was mutated to alanine. Mutagenesis was performed using the QuickChange site-directed mutagenesis kit (Stratagene) .
• Each 50 ul PCR reaction contained 10 mM KC1, 10 M (NH . SO., 20 mM Tris-HCl (pH 8.8), 2 mM MgS0 4 , 0.1% Triton X-100, 0.
• Img/ml nuclease-free BSA 114 ng each of two mutagenesis primers (see below), 50 ng of plasmid DNA template (see below), 2.5 units of PfuTurbo DNA polymerase, and 1 ul of the proprietary dNTP mix provided in the kit.
• Thermocycling was performed with an Applied Biosystems 9700 machine using the following cycling parameters: one cycle of 95° for 30 seconds; eighteen cycles of 95° for 30 seconds, 55° for 1 minute, 68° for 2.5 minutes; a final hold at 4°. Next, 1 ul (10 units) of Dpnl restriction enzyme was added to the mutagenesis reaction followed by incubation at 37° for 1 hour.
• the template DNA was TL231 and the mutagenesis primers were RP192 and RP193.
• This clone is designated R106 (SEQ ID NO: 16) and encodes only the first two potential start codons
• the template DNA was RIO 6 and the mutagenesis primers were RP190 and RP191.
• the resulting clone is designated R114 (SEQ ID NO: 17) and encodes only first start codon (See Figure 12) .
• the same mutagenesis technology can be employed to construct additional MCHl mutants that encode other combinations of the available methionine residues.
• the mutation MIA could be constructed using primers XI and X2. Such a change would eliminate the first methionine but retain the two downstream residues.
• the double mutation MIA, M70A could be constructed by sequentially using primer pairs Xl/ X2 and RP192/RP193. This would create a gene in which only the second methionine was left intact.
• TL231 was amplified with BB1122 (a forward primer beginning 10 nucleotides upstream of the third methionine in TL231, and also incorporating a Hindlll site) and BB1123 (a reverse primer in the second transmembrane domain) and the resulting product digested with Hindlll and B ⁇ rlllA.
• PCR was performed with the Expand Long Template PCR System (Roche Molecular Biochemicals, Indianapolis, IN) under the following conditions: 20 seconds at 94°C, 1 minute at 68°C for 40 cycles, with a pre- and post-incubation of 5 minutes at 94°C and 7 minutes at 68°C respectively.
• the 270 bp product was gel purified and ligated to a 4 kb Hindlll /Bglll restriction fragment from TL231.
• the resulting construct was named BO120.
• a broad variety of host cells can be used to study heterologously expressed proteins. These cells include but are not restricted to assorted mammalian lines such as; Cos-7, CHO, LM(tk-), HEK293, etc.; insect cell lines such as; Sf9, Sf21, etc.; amphibian cells such as xenopus oocytes; and others.
• COS-7 cells are grown on 150 mm plates in DMEM with supplements (Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 ⁇ g/ml streptomycin) at 37°C, 5% C0 2 . Stock plates of COS-7 cells are trypsinized and split 1:6 every 3-4 days.
• supplements Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 ⁇ g/ml streptomycin
• Human embryonic kidney 293 cells are grown on 150 mm plates in DMEM with supplements (10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 ⁇ g/ml streptomycin) at 37°C, 5% C0 2 . Stock plates of 293 cells are trypsinized and split 1 : 6 every 3-4 days .
• Mouse fibroblast LM(tk-) cells are grown on 150 mm plates in D-MEM with supplements (Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 ⁇ g/ml streptomycin) at 37°C, 5% CO_. Stock plates of LM(tk-) cells are trypsinized and split 1:10 every 3-4 days.
• CHO cells Chinese hamster ovary (CHO) cells were grown on 150 mm plates in HAM' s F-12 medium with supplements (10% bovine calf serum, 4 mM L-glutamine and 100 units/ml penicillin/ 100 ⁇ g/ml streptomycin) at 37°C, 5% C0 2 . Stock plates of CHO cells are trypsinized and split 1:8 every 3-4 days.
• Mouse embryonic fibroblast NIH-3T3 cells are grown on 150 mm plates in Dulbecco's Modified Eagle Medium (DMEM) with supplements (10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 ⁇ g/ml streptomycin) at 37°C, 5% C0_. Stock plates of NIH-3T3 cells are trypsinized and split 1:15 every 3-4 days.
• DMEM Dulbecco's Modified Eagle Medium
• Sf9 and Sf21 cells are grown in monolayers on 150 mm tissue culture dishes in TMN-FH media supplemented with 10% fetal calf serum, at 27°C, no C0 2 .
• High Five insect cells are grown on 150 mm tissue culture dishes in Ex-Cell 400TM medium supplemented with L-Glutamine, also at 27 °C, no CO..
• cell lines that grow as adherent monolayers can be converted to suspension culture to increase cell yield and provide large batches of uniform assay material for routine receptor screening projects.
• Xenopus oocytes can also be used as a host system for transient expression of heterologous proteins. Their maintenance and usage is described in the electrophysiological methods section that follows. Transient expression
• DNA encoding proteins to be studied can be transiently expressed in a variety of mammalian, insect, amphibian and other cell lines by several methods including but not restricted to; calcium phosphate-mediated, DEAE-dextran mediated, Liposomal-mediated, viral-mediated, electroporation-mediated and microinjection delivery. Each of these methods may require optimization of assorted experimental parameters depending on the DNA, cell line, and the type of assay to be subsequently employed.
• a typical protocol for the calcium phosphate method as applied to LM(tk-) cells is described as follows; Adherent cells are harvested approximately twenty-four hours before transfection and replated at a density of 1-2 x 10 5 cells/cm 2 in a 100 mm tissue culture dish and allowed to incubate over night at 37°C at 5% C0 2 . 250 ⁇ l of a mixture of CaCl 2 and DNA (20 ⁇ g DNA in 250 M CaCl,) is added to a 5 ml plastic tube and 250 ul of 2X HBS (250 mM NaCl, 10 mM KCl, 1.5 mM Na 2 HP0 4 , 12 mM dextrose, 50 mM HEPES) is slowly added with gentle mixing.
• 2X HBS 250 mM NaCl, 10 mM KCl, 1.5 mM Na 2 HP0 4 , 12 mM dextrose, 50 mM HEPES
• the mixture is allowed to incubate for 20 minutes at room temperature to allow a DNA precipitate to form.
• the cells are then washed with complete medium, 10 ml of culture medium is added to each plate, followed by addition of the DNA precipitate.
• the cells are then incubated for 24 to 48 hours at 37°C at 5% CO,.
• a typical protocol for the DEAE-dextran method as applied to Cos-7 cells is described as follows; Cells to be used for transfection are split 24 hours prior to the transfection to provide flasks which are 70-80% confluent at the time of transfection. Briefly, 8 ⁇ g of receptor DNA plus 8 ⁇ g of any additional DNA needed (e.g. G ⁇ protein expression vector, reporter construct, antibiotic -Ill- resistance marker, mock vector, etc.) are added to 9 ml of complete DMEM plus DEAE-dextran mixture (10 mg/ml in PBS) . Cos-7 cells plated into a T225 flask (sub-confluent) are washed once with PBS and the DNA mixture is added to each flask.
• any additional DNA needed e.g. G ⁇ protein expression vector, reporter construct, antibiotic -Ill- resistance marker, mock vector, etc.
• the cells are allowed to incubate for 30 minutes at 37°C, 5% C0 2 . Following the incubation, 36 ml of complete DMEM with 80 ⁇ M chloroquine is added to each flask and allowed to incubate an additional 3 hours. The medium is then aspirated and 24 ml of complete medium containing 10% DMSO for exactly 2 minutes and then aspirated. The cells are then washed 2 times with PBS and 30 ml of complete DMEM added to each flask. The cells are then allowed to incubate over night. The next day the cells are harvested by trypsinization and reseeded as needed depending upon the type of assay to be performed.
• a typical protocol for liposomal-mediated transfection as applied to CHO cells is described as follows; Cells to be used for transfection are split 24 hours prior to the transfection to provide flasks which are 70-80% confluent at the time of transfection. A total of lO ⁇ g of DNA which may include varying ratios of receptor DNA plus any additional DNA needed (e.g. G protein expression vector, reporter construct, antibiotic resistance marker, mock vector, etc.) is used to transfect each 75 cm 2 flask of cells. Liposomal mediated transfection is carried out according to the manufacturer's recommendations (LipofectAMINE, GibcoBRL, Bethesda, MD) . Transfected cells are harvested 24 h post transfection and used or reseeded according the requirements of the assay to be employed.
• Liposomal mediated transfection is carried out according to the manufacturer's recommendations (LipofectAMINE, GibcoBRL, Bethesda, MD) . Transfected cells are harvested 24 h post transfection and
• a typical protocol for the electroporation method as applied to Cos-7 cells is described as follows; Cells to be used for transfection are split 24 hours prior to the transfection to provide flasks which are subconfluent at the time of transfection. The cells are harvested by trypsinization resuspended in their growth media and counted. 4 x 10 b cells are suspended in 300 ⁇ l of DMEM and placed into an electroporation cuvette. 8 ⁇ g of receptor DNA plus 8 ⁇ g of any additional DNA needed (e.g.
• G ⁇ protein expression vector, reporter construct, antibiotic resistance marker, mock vector, etc. is added to the cell suspension, the cuvette is placed into a BioRad Gene Pulser and subjected to an electrical pulse (Gene Pulser settings: 0.25 kV voltage, 950 ⁇ F capacitance) . Following the pulse, 800 ⁇ l of complete DMEM is added to each cuvette and the suspension transferred to a sterile tube. Complete medium is added to each tube to bring the final cell concentration to 1 x 10 " cells/100 ⁇ l . The cells are then plated as needed depending upon the type of assay to be performed.
• a typical protocol for viral mediated expression of heterolgous proteins is described as follows for baculovirus infection of insect Sf9 cells.
• the coding region of DNA encoding the receptor disclosed herein may be subcloned into pBlueBacIII into existing restriction sites or sites engineered into sequences 5' and 3' to the coding region of the polypeptides.
• 0.5 ⁇ g of viral DNA (BaculoGold) and 3 ⁇ g of DNA construct encoding a polypeptide may be co-transfected into 2 x 10 6 Spodoptera frugiperda insect Sf9 cells by the calcium phosphate co-precipitation method, as outlined in by Phar ingen (in "Baculovirus Expression Vector System: Procedures and Methods Manual") .
• the cells then are incubated for 5 days at 27°C.
• the supernatant of the co- transfection plate may be collected by centrifugation and the recombinant virus plaque purified.
• the procedure to infect cells with virus, to prepare stocks of virus and to titer the virus stocks are as described in Pharmingen' s manual. Similar principals would in general apply to mammalian cell expression via retro-viruses, Simliki forest virus and double stranded DNA viruses such as adeno-, herpes-, and vacinia-viruses, and the like.
• Microinjection of cRNA encoding for proteins of interest is useful for the study of protein function in xenopus oocytes as well as cultured mammalian cells.
• a typical protocol for the preparation of cRNA and injection into xenopus oocytes can be found in the following electrophysiology section.
• Stable expression Heterologous DNA can be stably incorporated into host cells, causing the cell to perpetually express a foreign protein.
• Methods for the delivery of the DNA into the cell are similar to those described above for transient expression but require the co-transfection of an ancillary gene to confer drug resistance on the targeted host cell. The ensuing drug resistance can be exploited to select and maintain cells that have taken up the heterologous DNA.
• An assortment of resistance genes are available including but not restricted to Neomycin, Kanamycin, and Hygromycin.
• stable expression of a heterologous receptor protein is carried out in, but not necessarily restricted to, mammalian cells including, CHO, HEK293, LM(tk-) , etc.
• pellets of transfected cells are suspended in ice-cold buffer (20 mM Tris. HCl, 5 mM EDTA, pH 7.4) and homogenized by sonication for 7 sec.
• the cell lysates are centrifuged at 200 x g for 5 min at 4°C.
• the supernatants are then centrifuged at 40,000 x g for 20 min at 4°C.
• the resulting pellets are washed once in the homogenization buffer and suspended in binding buffer (see methods for radioligand binding) . Protein concentrations are determined by the method of Bradford (1976) using bovine serum albumin as the standard. Binding assays are usually performed immediately, however it is possible to prepare membranes in batch and store frozen in liquid nitrogen for future use.
• Cells may be screened for the presence of endogenous human receptor by radioligand binding (described in detail below) . Cells with either no or a low level of the endogenous human receptor disclosed herein may be transfected with the exogenous receptor.
• MCHl binding experiments with membranes (20-40 ⁇ g membrane protein) from transfected cells are performed with 0.1 nM [ : - " l] Phe 13 -Tyr 19 -MCH (Custom labeled by NEN) using incubation buffer consisting of 50mM Tris pH 7.4, lOmM MgCl_, 2 ⁇ g/ml aprotonin, 0.5mM PMSF and 50 ⁇ g/ml bacitracin. Binding 'is performed at 25°C for 1 hr . Incubations are terminated by rapid vacuum filtration over GF/C glass fiber filters, presoaked in 5% PEI using 50 mM Tris pH 7.4 containing 0.01% triton X-100 as wash buffer. In all experiments nonspecific binding is defined using 10 ⁇ M unlabeled MCH.
• Cells may be screened for the presence of endogenous mammalian receptor using functional assays (described in detail below) .
• Functional assays described in detail below
• Cells with no or a low level of endogenous receptor present may be -transfected with the exogenous receptor for use in the following functional assays.
• a wide spectrum of assays can be employed to screen for receptor activation.
• the receptor-mediated stimulation or inhibition of cyclic AMP (cAMP) formation may be assayed in cells expressing the mammalian receptors.
• Cells are plated in 96-well plates and incubated in Dulbecco's phosphate buffered saline (PBS) supplemented with 10 M HEPES, ImM isobutylmethylxanthine for 20 min at 37 °C, in 5% C0 2 .
• Test compounds are added with ⁇ or without 10 ⁇ M forskolin and incubated for an additional 10 min at 37 °C.
• the medium is then aspirated and the reaction stopped by the addition of 100 mM HCl.
• the plates are stored at 4°C for 15 min, and the cAMP content in the stopping solution measured by radioimmunoassay . Radioactivity may be quantified using a gamma counter equipped with data reduction software.
• Cells expressing the mammalian receptor are seeded into 96 well plates and grown for 3 days in HAM' s F-12 with supplements.
• the labeled cells are washed three times with 200 ⁇ L HAM' s F-12.
• the wells are then filled with medium (200 ⁇ L) and the assay is initiated with the addition of peptides or buffer (22 ⁇ L) .
• Cells are incubated for 30 min at 37°C, 5% CO_ .
• the intracellular free calcium concentration may be measured by microspectroflourometry using the fluorescent indicator dye Fura-2/AM (Bush et al, 1991) .
• Cells are seeded onto a 35 mm culture dish containing a glass coverslip insert, washed with HBS and loaded with 100 ⁇ L of Fura-2/AM (10 ⁇ M) for 20 to 40 min. After washing with HBS to remove the Fura-2/AM solution, cells are equilibrated in HBS for 10 to 20 min. Cells are then visualized under the 40X objective of a Leitz Fluovert FS microscope and fluorescence emission is determined at 510 nM with excitation wavelengths alternating between 340 nM and 380 nM. Raw fluorescence data are converted to calcium concentrations using standard calcium concentration curves and software analysis techniques.
• IP second messenger inositol phosphate
• the transfectants are challenged with agonist (10 ⁇ l/well; 10X concentration) for 30 minutes at 37°C, 5% C0 2 .
• the challenge is terminated and the cells lysed by the addition of 100 ⁇ l cold 5% v/v trichloroacetic acid (TCA) , followed by an incubation at 4°C for greater than 30 minutes.
• TCA cold 5% v/v trichloroacetic acid
• Total IPs are isolated from the lysate by ion exchange chromatography. Briefly, the lysed contents of the wells are transferred to a Multiscreen HV filter plate (Millipore) containing 100 ⁇ l Dowex AG1-X8 suspension (50% v/v, water : resin) (200-400 mesh, formate form).
• the filter plates are placed on a vacuum manifold to wash and elute the resin bed. Each well is first washed 2 times with 200 ⁇ l 5 mM myoinositol . Total [ 3 H] IPs are eluted with 75 ⁇ l of 1.2 M ammonium formate/0.1 M formic acid into Wallac 96-well plates. 200 ⁇ l of SuperMix scintillation cocktail is added to each well, mixed well, allowed to equilibrate and counted on a Micro Beta Trilux scintillation counter. (Note: The assay may be scaled to a 24 well format by simple adjustment of reagent volumes and employing individual chromatographic columns.)
• GTPyS assays are well-known in the art, and it is expected that variations on the method described above, such as are described by e.g., Tian et al . (1994) or Lazareno and Birdsall (1993), may be used by one of ordinary skill in the art.
• the c-fos- ⁇ -gal promoter/reporter construct used for these studies consists of the cfos promoter region (-384 to +19) (Schilling et al 1991, Yalkinoglu et al, 1995) inserted upstream of ⁇ - galactosidase cDNA containing expression vector pNASS ⁇ (Clontech) . Transcription activity is measured by assay of ⁇ -galactosidase enzyme activity as detected in a colorimetric assay.
• the medium is removed and replaced with medium containing drug (e.g. MCH) typically at a concentration of 10 ⁇ M.
• MCH medium containing drug
• the cells are allowed to incubate at 37°C, 5% C0 2 for at least 18 hours, after which the medium is aspirated and the cells washed with 200 ⁇ l PBS/well.
• the cells are then lysed with 100 ⁇ l AB buffer (100 mM Sodium Phosphate buffer, pH 8.0, 2 mM MgSOschreib, 0.1 mM MnCl 2 ) for 10 minutes at room temperature.
• AB/Tx/ ⁇ -mercaptoethanol AB buffer with 0.5% Triton X-100, 40 mM ⁇ -mercaptoethanol
• ONPG/AB 4 mg/ml O-nitrophenyl-b-D-galactopyranoside in AB buffer
• the reaction is allowed to proceed for 30 minutes or until yellow color becomes evident.
• Measurement of optical density is taken at 405 nm using a Dynatech microplate reader .
• MAP kinase mitogen activated kinase
• mitogen activated kinase may be monitored to evaluate receptor activation.
• MAP kinase is activated by multiple pathways in the cell. A primary mode of activation involves the ras/raf/MEK/MAP kinase pathway. Growth factor (tyrosine kinase) receptors feed into this pathway via SHC/Grb-2/SOS/ras . Gi coupled receptors are also known to activate ras and subsequently produce an activation of MAP kinase.
• Receptors that activate phospholipase C (Gq and Gil) produce diacylglycerol (DAG) as a consequence of phosphatidyl inositol hydrolysis. DAG activates protein kinase C which in turn phosphorylates MAP kinase.
• DAG diacylglycerol
• MAP kinase activation can be detected by several approaches.
• One approach is based on an evaluation of the phosphorylation state, either unphosphorylated (inactive) or phosphorylated (active) .
• the phosphorylated protein has a slower mobility in SDS-PAGE and can therefore be compared with the unstimulated protein using Western blotting.
• antibodies specific for the phosphorylated protein are available (New England Biolabs) which can be used to detect an increase in the phosphorylated kinase.
• cells are stimulated with the mitogen and then extracted with Laemmli buffer. The soluble fraction is applied to an SDS-PAGE gel and proteins are transferred electrophoretically to nitrocellulose or Immobilon.
• Immunoreactive bands are detected by standard Western blotting technique. Visible or chemiluminescent signals are recorded on film and may be quantified by densitometry .
• Another approach is based on evaluation of the MAP kinase activity via a phosphorylation assay.
• Cells are stimulated with the mitogen and a soluble extract is prepared.
• the extract is incubated at 30°C for 10 min with gamma-32-ATP, an ATP regenerating system, and a specific substrate for MAP kinase such as phosphorylated heat and acid stable protein regulated by insulin, or PHAS-I .
• the reaction is terminated by the addition of H 3 P0 4 and samples are transferred to ice.
• An aliquot is spotted onto Whatman P81 chromatography paper, which retains the phosphorylated protein.
• the chromatography paper is washed and counted for '-P in a liquid scintillation counter.
• the cell extract is incubated with gamma-32-ATP, an ATP regenerating system, and biotinylated myelin basic protein bound by streptavidin to a filter support.
• the myelin basic protein is a substrate for activated MAP kinase.
• the phosphorylation reaction is carried out for 10 min at 30°C.
• the extract can then be aspirated through the filter, which retains the phosphorylated myelin basic protein.
• the filter is washed and counted for 32 P by liquid scintillation counting.
• Cell proliferation assay Activation of a G protein coupled receptor may lead to a mitogenic or proliferative response which can be monitored via [ 3 H] -thymidine uptake.
• the thymidine translocates into the nuclei where it is phosphorylated to thymidine triphosphate .
• the nucleotide triphosphate is then incorporated into the cellular DNA at a rate that is proportional to the rate of cell growth.
• cells are grown in culture for 1-3 days. Cells are forced into quiescence by the removal of serum for 24 hrs . A mitogenic agent is then added to the media.
• the cells are incubated with [ J H] -thymidine at specific activities ranging from 1 to 10 ⁇ Ci/ml for 2-6 hrs.
• Harvesting procedures may involve trypsinization and trapping of cells by filtration over GF/C filters with or without a prior incubation in TCA to extract soluble thymidine.
• the filters are processed with scintillant and counted for 3 H by liquid scintillation counting.
• adherent cells are fixed in MeOH or TCA, washed in water, and solubilized in 0.05% deoxycholate/0.1 N NaOH.
• the soluble extract is transferred to scintillation vials and counted for 3 H by liquid scintillation counting.
• Xenopus laevis Female Xenopus laevis (Xenopus-1, Ann Arbor, MI) are anesthetized in 0.2% tricain (3-aminobenzoic acid ethyl ester, Sigma Chemical Corp.) and a portion of ovary is removed using aseptic technique (Quick and Lester, 1994) .
• Oocytes are defolliculated using 2 mg/ml collagenase (Worthington Biochemical Corp., Freehold, NJ) in a solution containing 87.5 mM NaCl, 2 mM KCl, 2 mM MgCl_ and 5 M HEPES, pH 7.5.
• Oocytes may be injected (Nanoject, Drummond Scientific, Broomall, PA) with mammalian mRNA.
• Other oocytes may be injected with a mixture of mammalian mRNA and mRNA encoding the genes for G-protein-activated inward rectifiers (GIRK1 and GIRK4, U.S. Patent Nos. 5,734,021 and 5,728,535).
• GIRK1 and GIRK4 G-protein-activated inward rectifiers
• the upstream primer contained a BamHI site and the downstream primer contained an EcoRI site to facilitate cloning of the PCR product into pcDNAl-Amp (Invitrogen) .
• the transcription template for the mammalian receptor may be similarly obtained.
• mRNAs are prepared from separate ' DNA plasmids containing the complete coding regions of the mammalian receptor, GIRKl, and GIRK4. Plasmids are linearized and transcribed using the T7 polymerase ("Message Machine", Ambion) . Alternatively, mRNA may be translated from a template generated by PCR, incorporating a T7 promoter and a poly A * tail.
• Each oocyte receives 2 ng each of GIRKl and GIRK4 mRNA in combination with 25 ng of mammalian receptor mRNA. After injection of mRNA, oocytes are incubated at 16° C on a rotating platform for 3-8 days. Dual electrode voltage clamp ("GeneClamp", Axon Instruments Inc., Foster City, CA) is performed using 3 M KCl-filled glass microelectrodes having resistances of 1-3 Mohms . Unless otherwise specified, oocytes are voltage clamped at a holding potential of -80 mV.
• oocytes are bathed in continuously flowing (2-5 ml/min) medium containing 96 mM NaCl, 2 mM KCl, 2 mM CaCl 2 , 2 mM MgCl_., and 5 mM HEPES, pH 7.5 (“ND96”) , or, in the case of oocytes expressing GIRKl and GIRK4, elevated K + containing 96 mM KCl, 2 mM NaCl, 2 mM CaCl 2 , 2 mM MgCl 2 , and 5 mM HEPES, pH 7.5 ("hK”) . Drugs are applied by switching from a series of gravity fed perfusion lines.
• Heterologous expression of GPCRs in Xenopus oocytes has been widely used to determine the identity of signaling pathways activated by agonist stimulation (Gundersen et al., 1983; Takahashi et al . , 1987).
• Activation of the phospholipase C (PLC) pathway is assayed by applying test compound in ND96 solution to oocytes previously injected with mRNA for the mammalian receptor and observing inward currents at a holding potential of -80 mV.
• GIRK inwardly rectifying K + (potassium) channel
• oocytes that have been co- injected with mRNAs encoding the mammalian receptor, GIRKl, and GIRK4.
• the two GIRK gene products co-assemble to form a G-protein activated potassium channel known to be activated (i.e., stimulated) by a number of GPCRs that couple to Gi or G 0 (Kubo et al . , 1993; Dascal et al . , 1993) .
• Oocytes expressing the mammalian receptor plus the two GIRK subunits are tested for test compound responsivity by measuring K" currents in elevated K" solution (hK) .
• Activation of inwardly rectifying currents that are sensitive to 300 ⁇ M Ba 'r+ signifies the mammalian receptor coupling to a G ⁇ .or G r pathway in the oocytes.
• a strategy for determining whether MCHl can couple preferentially to selected G proteins involves co- transfection of MCHl receptor cDNA into a host cell together with the cDNA for a G protein alpha sub-unit.
• G alpha sub-units include members of the G ⁇ i/G ⁇ o- class (including G ⁇ t2 and G ⁇ z) , the G ⁇ q class, the G s class, and the G ⁇ l2/13 class.
• a typical procedure involves transient transfection into a host cell such as COS-7. Other host cells may be used.
• a key consideration is whether the cell has a downstream effector (a particular adenylate cyclase, phospholipase C, or channel isoform, for example) to.
• G protein beta gamma sub-units native to the cell are presumed to complete the G protein heterotrimer; otherwise specific beta and gamma sub-units may be co-transfected as well. Additionally, any individual or combination of alpha, beta, or gamma subunits may be co-transfected to optimize the functional signal mediated by the receptor.
• the receptor/G alpha co-transfected cells are evaluated in a binding assay, in which case the radioligand binding may be enhanced by the presence of the optimal G protein coupling or in a functional assay designed to test the receptor/G protein hypothesis.
• the MCHl receptor may be hypothesized to inhibit cAMP accumulation through coupling with G alpha sub-units of the G ⁇ i/G ⁇ o class.
• Host cells co-transfected with the MCHl receptor and appropriate G alpha sub-unit cDNA are stimulated with forskolin +/- MCHl agonist, as described above in cAMP methods. Intracellular cAMP is extracted for analysis by radioimmunoassay .
• GTP ⁇ 35 S binding assays examples include GTP ⁇ 35 S binding assays and inositol phosphate hydrolysis assays.
• Host cells transfected with MCHl minus G alpha or with G alpha minus MCHl would be tested simultaneously as negative controls.
• MCHl receptor expressio in transfected cells may be confirmed in radioligand binding studies using membranes from transfected cells.
• G alpha expression in transfected cells may be confirmed by Western blot analysis of membranes from transfected cells, using antibodies specific for the G protein of interest.
• the efficiency of the transient transfection procedure is a critical factor for signal to noise in an inhibitory assay, much more so than in a stimulatory assay. If a positive signal present in all cells (such as forskolin- stimulated cAMP accumulation) is inhibited only in the fraction of cells successfully transfected with receptor and G alpha, the signal to noise ratio will be poor.
• One method for improving the signal to noise ratio is to create a stably transfected cell line in which 100% of the cells express both the receptor and the G alpha subunit.
• Another method involves transient co-transfection with a third cDNA for a G protein-coupled receptor which positively regulates the signal which is to be inhibited.
• a positive signal may be elevated selectively in transfected cells using a receptor-specific agonist.
• An example involves co-transfection of COS-7 cells with 5-HT4 receptor, MCHl receptor, and a G alpha sub-unit. Transfected cells are stimulated with a 5-HT4 agonist +/- MCHl agonist. Cyclic AMP is expected to be elevated only in the cells also expressing MCHl and the G alpha subunit of interest, and a MCHl-dependent inhibition may be measured with an improved signal to noise ratio.
• cell lines described herein are merely illustrative of the methods used to evaluate the binding and function of the mammalian receptors of the present invention, and that other suitable cells may be used in the assays described herein.
• a GPCR which normally might prefer to couple through a specific signaling pathway (e.g. G s , Gi, G q , G 0 , etc.), can be made to couple through the pathway defined by the promiscuous G a subunit and upon agonist activation produce the second messenger associated with that subunit' s pathway.
• G l6 and/or G aqz this would involve activation of the G q pathway and production of the second messenger inositol phosphate.
• it is possible to bias receptor signaling through pathways producing other second messengers such as Ca + ⁇ , cAMP, K + currents, etc.
• microphysiometric measurements of cell metabolism can in principle provide a generic assay of cellular activity arising from the activation of any receptor regardless of the specifics of the receptor' s proximal signaling pathway.
• a typical protocol employing transiently transfected CHO cells is as follows; 24 hours prior to recording, transfected cells are harvested and counted. 3 x 10 5 cells are seeded into cell culture capsules (Costar) , and allowed to attach to the capsule membrane. 10 hours later (14 hours prior to recording) the cell media is switched to serum free F-12 complete to minimize ill-defined metabolic stimulation caused by assorted serum factors.
• the cell capsules are transferred to the microphysiometer (Cytosensor, Molecular Devices Corporation, Sunnyvale, CA) and allowed to equilibrate in recording media (low buffered RPMI 1640, no bicarbonate, no serum) with 0.1% BSA (essentially fatty acid free) , during which a baseline measurement of basal metabolic activity is established.
• the recording paradigm consists of a 100 ⁇ l/min flow rate, with a 2 min pump cycle which includes a 30 sec flow interruption during which the rate measurement is taken. Challenges involve a 1 min 20 sec exposure to a drug just prior to the first post challenge rate measurement being taken, followed by two additional pump cycles for a total of 5 min 20 sec drug exposure. Drug is then washed out and rates allowed to return to basal. Reported extracellular acidification rates are expressed as a percentage increase of the peak response over the baseline rate observed just prior to challenge .
• GPCR ligand library Functional assays of new receptors such as MCHl may include a preliminary test of a small library of compounds containing representative agonists for all known GPCRs as well as other compounds which may be agonists for prospective GPCRs or which may be effectors for targets peripherally involved with GPCRs.
• the collection used in this study comprises approximately 180 compounds (including small molecules, hormones, preprohormones, peptides, etc.) for more than 45 described classes of GPCRs (serotonin, dopamine, noradrenaline, opioids, etc.) and additionally includes ligands for known or suspected but not necessarily pharmacological characterized or cloned GPCR families (such as MCH) .
• the diversity of the library can be expanded to include agonist and antagonist compounds specific for GPCR subtypes, combinatorial peptide and/or small molecule libraries, natural product collections, and the like.
• the substances are distributed as either separate or pooled compound concentrates in 96 well plates and stored frozen as ready to use reagent plates.
• RNA polymerase promoter sites The full length cDNA encoding the rat MCHl will be digested with Pst 1, (nucleotides 905-1194) and this 289 nucleotide fragment will be cloned into the Pst I site of pGEM 3z, containing both sp6 and T7 RNA polymerase promoter sites .
• the construct will be sequenced to confirm sequence identity and orientation.
• this construct will be linearized with Hind III or Eco Rl (depending on orientation) and T7 or sp6 RNA polymerase will be used to incorporate radiolabeled nucleotide as described below.
• GAPDH rat glyceraldehyde 3-phosphate dehydrogenase
• MCHl and GAPDH cDNA sequences preceded by phage polymerase promoter sequences will be used to synthesize radiolabeled riboprobes.
• Conditions for the synthesis of riboprobes will be: 0.25-1.0 ⁇ g linearized DNA plasmid template, 1.5 ⁇ l of ATP, GTP, UTP (10 mM each), 3 ⁇ l dithiothreitol (0.1 M) , 30 units RNAsin RNAse inhibitor, 0.5-1.0 ⁇ l (15-20 units/ ⁇ l) RNA polymerase, 7.0 ⁇ l transcription buffer (Promega Corp.), and 12.5 ⁇ l ⁇ 32 P-CTP (specific activity 3, OOOCi/mmol) .
• 0.1 mM CTP (0.02-1.0 ⁇ l) will be added to the reactions, and the volume will be adjusted to 35 ⁇ l with DEPC-treated water. Labeling reactions will be incubated at 37°C for 60 min, after which 3 units of RQ1 RNAse-free DNAse (Promega Corp.) will be added to digest the template. Riboprobes will be separated from unincorporated nucleotides using Microspin S-300 columns (Pharmacia Biotech) . TCA precipitation and liquid scintillation spectrometry will be used to measure the amount of label incorporated into the probe.
• a fraction of all riboprobes synthesized will be size-fractionated on 0.25 mm thick 7M urea, 4.5% acrylamide sequencing gels. These gels will be apposed to storage phosphor screens and the resulting autoradiograph scanned using a phoshorimager (Molecular Dynamics, Sunnyvale, CA) to confirm that the probes synthesized were full-length and not degraded.
• Solution hybridization/ribonuclease protection assay For solution hybridization 2.0 ⁇ g of mRNA isolated from tissues will be used. Negative controls consisted of 30 ⁇ g transfer RNA (tRNA) or no tissue blanks. All mRNA samples will be placed in 1.5-ml microfuge tubes and vacuum dried. Hybridization buffer (40 ⁇ l of 400 mM NaCl, 20 mM Tris, pH 6.4, 2 mM EDTA, in 80% formamide) containing 0.25-2.0 E 6 counts of each probe will be added to each tube. Samples will be heated at 95°C for 15 min, after which the temperature will be lowered to 55°C for hybridization .
• Hybridization buffer 40 ⁇ l of 400 mM NaCl, 20 mM Tris, pH 6.4, 2 mM EDTA, in 80% formamide
• RNA/probe mixtures will be digested with RNAse A (Sigma) and RNAse Tl (Life Technologies) .
• a mixture of 2.0 ⁇ g RNAse A and 1000 units of RNAse Tl in a buffer containing 330 mM NaCl, 10 mM Tris (pH 8.0) and 5 mM EDTA (400 ⁇ l) will be added to each sample and incubated for 90 min at room temperature.
• 20 ⁇ l of 10% SDS and 50 ⁇ g proteinase K will be added to each tube and incubated at 37°C for 15 min. Samples will be extracted with phenol/chloroform: isoamyl .
• RT-PCR For the detection of RNA encoding human MCHl, RT-PCR was carried out on mRNA extracted from human tissue. Reverse transcription and PCR reactions were carried out in 50 ml volumes using EZrTth DNA polymerase (Perkin Elmer) . Primers with the following sequences were used:
• Reverse primer (RA/ SLCla MCH B) ; CTT GGA CTT CTT CAC GAC (SEQ ID NO: 15)
• primers will amplify a 248 base pair fragment from nucleotide 169 to 417.
• Each reaction contained 0.1 ⁇ g mRNA and 0.3 ⁇ M of each primer. Concentrations of reagents in each reaction were:
• Positive controls for PCR reactions consisted of amplification of the target sequence from a plasmid construct, as well as reverse transcribing and amplifying a known sequence.
• Negative controls consisted of mRNA blanks, as well as primer and mRNA blanks. To confirm that the mRNA was not contaminated with genomic DNA, samples were digested with RNAses before reverse transcription. Integrity of RNA was assessed by amplification of mRNA coding for GAPDH.
• the second major product to elute was (-) -5- (benzyloxycarbonyl) -4-ethyl-l, 6-dihydro-l- ⁇ N- [2-phenyl) ethyl] ⁇ carboxamido-2-methoxy-6- ( 3, 4-difluoroph enyl) pyr-imidine .
• 5-METHYLBENZFUROXAN 4-Methyl-2-nitroaniline (100 g, 0.650 mol) was suspended in saturated methanolic sodium hydroxide solution (1.50 L) . This suspension was cooled (5 C) and aqueous sodium hypochlorite until the red color disappeared. The resulting fluffy yellow precipitate was filtered, washed with cold water and recrystallized from ethanol, giving 5-methylbenzfuroxan (88.2 g, 89 % yield) as a pale yellow solid: X H NMR d 2.39 (s, 3 H) , 6.90-7.40 (br m. 3 H) .
• 5-METHYLBENZOFURAZAN To 5-Methylbenzfuroxan (88.2 g, 0.590 mol) in refluxing EtOH (75 mL) was added dropwise P(OEt) 3 (150 mL) . Heating was continued at reflux temperature for 1 h. The solvent was removed in vacuo and the residue was shaken with water (200 mL) and allowed to stand overnight at (0-5 °C) . The resulting brown solid was filtered, washed with water.
• 5-DIBROMOMETHYLBENZOFURAZAN An anhydrous solution of 5-methylbenzofurazan (70.0 g, 0.520 mol), N-bromosuccinamide (325 g) , and benzoyl peroxide (0.50 g) in carbon tetrachloride (1.5 L) was heated at reflux temperature with stirring for 30 h. The reaction mixture was washed with water (2 X 500 mL) , dried (NaS0 4 ) , and the solvent was removed in vacuo .
• METHYL ESTER A solution of 10% aqueous HCl (5 mL) was added to a stirring solution of
• the reaction mixture was concentrated in vacuo, re-dissolved in hexanes :EtOAc (9:1), passed through a plug of alumina and the alumina plug was washed with hexanes : EtOAc .(9:1).
• the combined extracts were concentrated to yield 16.5 g of the desired product that was contaminated with some starting Tf 2 NPh.
• TERT-BUTYI 4- [3- (AMINO) PHENYL] -1-PIPERIDINECARBOXYLATE
• a mixture of 3.10 g of tert-butyl 4- (3-aminophenyl) - 1, 2, 3, 6-tetrahydropyridine-l-carboxylate (11.3 mmol) ' and 1.0 g of 10% Pd/C in 200 mL of ethanol was hydrogenated at room temperature using the balloon method for 2 days. The reaction mixture was filtered and washed with ⁇ ethanol.
• 1,2,3, 6-TETRAHYDRO-4- (3-NITROPHENYL) PYRIDINE Into a stirred solution of 5.00 g (16.0 mmol) of tert-butyl 1,2,3, 6-tetrahydro-4- (3-nitrophenyl) pyridine-1- carboxylate in 100 ml of 1,4-dioxane at 0°C was bubbled HCl gas for 10 minutes. The reaction mixture was allowed to warm to room temperature and the bubbling of the HCl gas was continued for an additional 1 hour. The solvent was removed in vacuo, the residue was dissolved in 50 mL of water and was neutralized by the addition of KOH pellets.
• TERT-BUTYL 3- (4- (3-NITROPHENYL) -3 , 6-DIHYDRO-l (2H) - PYRIDINYL) ROPYLCARBAMATE : A mixture of 2.80 g (14.0 mmol) of 1, 2, 3, 6-tetrahydro-4- (3-nitrophenyl) yridine, 3.60 g (15.0 mmol) of tert-butyl N-(3- bromopropyl) carbamate, 11.6 g (84.0 mmol) of K 2 C0 3 , 14.6 mL (84.0 mmol) of diisopropylethylamine and 0.78 g (2.00 mmol) of tetrabutylammonium iodide in 250 mL of 1,4- dioxane was heated at reflux temperature for 14 hours .
• TETRAHYDRO-5-PYRIMIDINECARBOXYLATE A mixture of 3.02 g (6.33 mmol) 5-methyl 1- (4-nitrophenyl) (6S) -6- (3, 4- difluorophenyl) -4- (methoxymethyl) -2-oxo-3, 6-dihydro-
• the residue was dissolved in 100 mL of anhydrous ethanol containing 0.50 g 10% Pd/C and the reaction mixture was stirred under a hydrogen balloon for 24 hours.
• the reaction mixture was passed through a column of Celite 545 filtering agent, washed with ethanol, the filtrate was dried (MgS0 4 ) and concentrated in vacuo .
• the residue was purified by column chromatography (silica, 9.5 : 0.5 , dichloromethane : methanol + 1% isopropyl amine) to afford 1.65 g (52.0% yield) of the desired product.
• BENZYL 6- (3, 4-DIFLUOROPHENYL) -4-ETHYL-2-METHOXY-1 , 6- DIHYDRO-5-PYRIMIDINECARBOXYLATE.
• (+) -BENZYL 6- (3 , 4-DIFLUOROPHENYL) -4-ETHYL-2-METHOXY-1 , 6- DIHYDRO-5-PYRIMIDINECARBOXYLATE.
• (+) -benzyl 6- (3, 4-difluorophenyl) -4-ethyl-2-methoxy- 1- ( ⁇ [ (IR) -1-phenylethyl] amino ⁇ carbonyl) -1, 6-dihydro-5- pyrimidinecarboxylate (17.1 mmol, 9.35 g) in CH 2 C1 2 was added 1, 8-diazabicyclo [5, 4 , 0] -undec-7-ene (17.1 mmol, 2.56 mL) and stirring was continued for 16 h at room temperature.
• (+) -benzyl 6- (3, 4- difluorophenyl) -4-ethyl-2-methoxy-l, 6-dihydro-5- pyrimidinecarboxylate (6.4 g, 16.0 mmol) and pyridine (1.5 mL) in CH 2 C1 2 (150 mL) was added 4-nitrophenyl chloroformate (3.41 g, 19.2 mmol) at room temperature.
• PYRIMIDINEDICARBOXYLATE Into a well-stirred solution of 6- (3, 4-Difluorophenyl) -1, 6-dihydro-2-methoxy-5- methoxycarbonyl-4-methyl-l- [ (4- nitrophenyloxy) carbonyl] pyrimidine (1.5 mmol, 0.66 g) in 5 mL of chloroform was added a solution of bromine (1.5 mmol, 0.09 mL) in 3 mL of chloroform at 0 °C and the solution was allowed to attain room temperature over 1.5 h. The solvent was removed in vacuo and the residue was again dissolved in CHC1 3 (20 L) and washed with brine.
• reaction mixture was cooled to -78 °C and tert- butyl 4-oxo-l-piperidinecarboxylate (40.0 mmol) in THF (40 mL) was added dropwise to the reaction mixture and stirred for 30 minutes.
• Tf 2 NPh (15.0 g, 42.0 mmol) in THF (40 mL) was added dropwise to the reaction mixture and the mixture was stirred at 0 °C overnight.
• the reaction mixture was concentrated in vacuo, re-dissolved in hexanes/EtOAc (9/1), passed through a plug of alumina and washed with hexanes/EtOAc (9/1) .
• Nl- [3- (4-PIPERIDYL) PHENYL] ACETAMIDE A solution of HCl in dioxane (4N, 5 mL) was added to tert-butyl 4- [3- (acetylamino) -phenyl] -1-piperidinecarboxylate ( 660 mg) in dry dichloromethane (15 L) . The reaction mixture was stirred at room temperature overnight and concentrated in vacuo, giving the desired product (550 mg) : mp 102-104
• 1-BENZYL-4-METHYL-4-PHENYLPIPERIDINE l-Benzyl-4-methyl- piperidin-4-ol (4.81 g, 23.4 mmol) was added to a suspension of A1C1 3 (15.62 g, 117 mmol) in benzene (100 mL) at room temperature under argon. The mixture was stirred at reflux for 24 hours, then cooled and poured cautiously into ice water (100 g of ice, 50 L of water) . The aqueous phase was adjusted to pH 11-12 by addition of 6 N aqueous NaOH at 0 °C, and extracted with EtOAc (3 x 100 mL) .
• Oxalyl chloride (1.1 equivalents) was added dropwise to a mixture of 4- [[ (tert-butoxycarbonyl) -amino] methyl] cyclohexanecarboxylic acid (1 equivalent, Maybridge) in toluene .
• the reaction mixture was stirred at room temperature for 2-6 h.
• the solvent was removed in vacuo, the residue was dissolved in acetone and the resulting mixture was added dropwise to an aqueous solution of sodium azide (1.2 equivalents) at a rate such as to maintain a temperature of 10-15 °C .
• the reaction mixture was extracted with ethyl acetate, the combined extracts were dried and concentrated in vacuo.
• TERT-BUTYL N-(4-[ ( 1-NAPHTHYLCARBONYL) AMINO] CYCLOHEXYLMETHYL) -CARBAMATE A mixture of 1-naphthoic acid (1.00 mmol, 0.172 g) , DMAP (2.00 mmol, 0.250 g) and ECD (0.383 g, 2.00 mmol) in dry dichloromethane (20 mL) was stirred at room temperature for 0.5 h followed by the addition of tert-butyl (4-amino) cyclohexyl )methyl-carbamate amine (1.09 mmol, 0.250 g) .
• the reaction mixture was stirred at room temperature for 12 h.
• the reaction mixture was quenched with aqueous 6 N HCl .
• the reaction mixture was concentrated to a small volume, partitioned between dichloromethane and water (100 mL each) , the mixture was adjusted to pH 8 by addition of NaC0 3 , the layers were separated, and the aqueous layer was extracted with dichloromethane (3 x 30 mL) .
• the combined organic extracts were dried (Na 2 S0 4 ) and the product was chromatographed, giving the desired product.
• the HCl salt was prepared by the addition of 1 N HCl in ether to a solution of the product in CH 2 C1 2 .
• the precipitated salt was filtered, washed with ether and d r i e d i n ' v a c u o , g i v i n g
• Example 5 l,2,3,4-TETRAHYDRO-l-OXO-2-NAPHTHACETIC ACID METHYL ESTER: Under argon, ⁇ -tetralone (5.00 g, 34.2 mmol) in dry THF (300 mL) was treated with LDA in THF (2 M, 18.8 mL) at -78 °C. The solution was stirred at -78 °C for 1 h. Methyl bromoacetate (15.7 g, 0.103 mole) was then added to the solution, the mixture was stirred overnight and allowed to warm to room temperature..
• the compound of Example 10 may also be prepared via hydrogenation of the compoun of example 2 (H 2 balloon method, methanol, Pd/C, overnight) .
• a synthetic path analogous to the latter route (Scheme 11) was used in the preparation of the tritiated analog, which in turn, was used as a radioligand in the MCH pharmacological assays.
• Example 12 3- ⁇ (3, 4, 5-TRIFLUOROPHENYL) METHYLENE ⁇ -2, 4-PENTANEDIONE: A stirring mixture of 3, 4 , 5-trifluorobenzaldehyde (4.2 g, 26.2 mmol), 2, 4-pentanedione (2.62 • -g, 26.2 mmol), piperidine (0.430 g, 5 mmol) in benzene (150 mL) was heated at reflux temperature (equipped with a Dean-Stark trap) for 8 h. The benzene was evaporated, the yellow oily residue, 2- ⁇ (3,4,5-trifluorophenyl)- methylene ⁇ -2, 4-pentanedione, was used in the next step without further purification.
• 3-AMINOPROPYL-4- (2-PYRIDYL) PIPERIDINE A suspension of 3- (3 ' , 6 ' -dihydro-2 ' -H- [2,4' ] bipyridinyl-1 ' -yl) -propylamine (3.48 g crude, 15.9 mmol) and Pearlman's catalyst (1.0 g) in MeOH (40 mL) was hydrogenated under 120 psi for 10 h, after which the reaction mixture was filtered through a pad of Celite and the solvent was removed.
• XO-6- (3, 4-DIFLUOROPHENYL) -PYRIMIDINE Copper (I) oxide (5.06 g, 0.035 mole) and acetic acid (2.05 mL) were added sequentially to a stirring solution of methyl 4-methoxyacetoacetate (50.0 g, 0.351 mol), 3, 4-difluorobenzaldehyde (51.4 g, 0.351 mmol), and urea (31.6 g, 0.527 mole) in THF (300 mL) at room temperature, followed by dropwise addition of boron trifluoride diethyl etherate (56.0 mL, 0.456 mole) .
• PROPYL] JCARBOXAMIDO-5-METHOXYCARBONYL-4-METHOXYMETHYL- 2-OXO- 1,2,3, 6-TETRAHYDROPYRIMIDINE DIHYDROCHLORIDE A solution of ( + ) -5-methoxycarbonyl-4-methoxymethyl-l, 2, 3, 6- tetrahydro-2-oxo-6- (3, 4-dif luorophenyl ) -1- [ (4-nitropheny loxy) carbonyl] pyrimidine (2.38 g, 5 mmol) ,
• the HCl salt was prepared by treatment of a solution of the free base in ether with 1 N HCl in ether.
• the white powder was dried under reduced pressure: 1 H NMR ⁇ 2.05-2.20 (m, 4H) , 2.77-2.88 (m, 2H) , 3.00-3.20 (m, 4H) , 3.35-3.47 (m, 2H) , 3.47 (s, 3H) , 3.64-3.70 (m, 2H) , 3.71 (s, 3H) , 4.05 (br t, IH) , 4.67 (s, 2H) , 6.59 (s, IH) , 7.05-7.20 (m, 3H) , 7.79 (t, IH) , 8.00 (d, IH) , 8.43 (dt, IH) , 8.96 (br t, IH, NH) , 12.4 (br s, IH) .
• Example 19 2- (3, 4-DIFLUOROPHENYL) 4 , 5-DIHYDROIMIDAZOLE-l-CARBOXYLIC A C I D ⁇ 3-[4-PHENYL-4- ( 4-BROMO-5-METHYLTHIOPNEN-2-YL) ] -PROPYL ⁇ - AMIDE: Anal. Calcd. for C 30 H 3n N 4 O 5 ClF 3 + HCl + 1.5 H ⁇ : C, 55.26; H, 6.03; N, 8.59. Found: C, 55.29; H, 5.95; N,
• Trif luoroacetic acid (1 ml) was added to l- tert-butoxycarbonyl-3- ( 4-spiro [isobenzo- furan-1 (3H) , 4 ' -piperidine] ) propylamine (0.180 g, 0.52 mmol) in dichloromethane (5 ml) ' and the resulting solution was stirred at room temperature for 1 hour. The solution was concentrated, neutralized with 10% KOH solution and extracted into dichloromethane (25 ml) . The organic layer was dried over sodium sulfate, filtered and concentrated, giving propylamine (0.156 g, 100%) which was used in the subsequent step without further purification.
• reaction mixture was stirred for another 1 h after addition of 2 mL of 6N HCl.
• the combined organic extracts were dried over sodium sulfate, filtered and concentrated.
• Step B 1- (3-AMINOPROPYL) SPIRO [ISOCHROMAN-3, 4 'PIPERIDIN] - 1-ONE: To 1- (3-tert-Butoxycarbonylaminopropyl) - spiro [isochroman-3, 4 ' -piperidin] -1-one (0.144 g, 0.375 mmol) in 5 mL of dichloromethane, 1 mL of trifluoroacetic acid was added and the solution stirred at room temperature for 1 h. The solution was concentrated, neutralized with 10 % KOH solution and extracted into 25 mL of dichloromethane. The organic layer was dried over sodium sulfate, filtered and concentrated, giving 0.110 g (100%) of the product which was used as such for the subsequent step.

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