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WO2012168474A1 - Nouveaux procédés - Google Patents

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Publication number
WO2012168474A1
WO2012168474A1 PCT/EP2012/061023 EP2012061023W WO2012168474A1 WO 2012168474 A1 WO2012168474 A1 WO 2012168474A1 EP 2012061023 W EP2012061023 W EP 2012061023W WO 2012168474 A1 WO2012168474 A1 WO 2012168474A1
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WIPO (PCT)
Prior art keywords
serca3
compound
glp
activity
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2012/061023
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English (en)
Inventor
Liquan Huang
Naoko Iguchi
Jiang Xu
Jay Patrick Slack
Ping Zhong
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Givaudan SA
Monell Chemical Senses Center
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Givaudan SA
Monell Chemical Senses Center
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Publication of WO2012168474A1 publication Critical patent/WO2012168474A1/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the field relates to methods of screening, methods of modulating taste, appetite, metabolism and digestion, and methods of treating metabolic diseases.
  • the field relates to methods of modulation of GLP- l activity by inhibiting SERCA3, thereby enhancing GLP-l release induced by a lipid, sweetener or spice, e.g., a sweetener or spice compound. Furthermore, the field relates to methods of screening designed to identify compounds that modulate SERCA3 activity or production.
  • Metabolic disturbances may also be associated with poor digestion and abnormal intestinal motility, with symptoms such as abdominal pain, vomiting, constipation, diarrhoea, and poor absorption of nutrients, as well as serious diseases such as upper gastrointestinal disorders, e.g., gastrointestinal reflux disorder and peptic ulcers; and disorders of the lower bowel such as functional bowel diseases, e.g., irritable bowel disease; colitis, e.g., ulcerative colitis; and diverticulitis or diverticuiosis.
  • SERCA is an acronym for Sarcoplasmic or Endoplasmic Reticulum Ca++-
  • SERCA enzymes Muscle endoplasmic reticulum is called sarcoplasmic reticulum.
  • the function of SERCA enzymes is to catalyze the hydrolysis of ATP coupled with the translocation of calcium, from the cytosol to the endoplasmic reticulum lumen.
  • SERCA enzymes are involved in calcium sequestration associated with muscular excitation and contraction or with secretion. Alternative splicing of the enzyme results in multiple
  • SERCA3 is a Ca 2+ transporting ATPase, which is encoded by ATP2a3.
  • RCA 3 is one of the three intracellular ER Ca 2+ pumps located in the endoplasmic reticula of muscle cells, The sequence divergences between SERCA3 and the other
  • SERCAs, SERCA 1 and SERCA2 are mainly located in the N- and C-terminal domains.
  • SERCA3 is generally non-muscle SERCA isoform and is found in a number of tissues, most abundantly in thymus, trachea, salivary gland, spleen, bone marrow, lymph node, peripheral leukocytes, pancreas and colon. It is found to some extent in cardiomyocytes, although SERCA3 lacks the putative interacting domain for phospholamban, a modulator that regulates the Ca 2+ pump in cardiac muscle cells, and as a consequence, is not modulated by the presence of phospholamban.
  • SERCA3 is selectively expressed in some endothelial and excretory cells, for example, pancreatic ⁇ cells, platelets, and mast cells, Ozog.A.. PouzefB., Bobe,R. and Lompre,A. . Characterization of the 3' end of the mouse SERCA 3 gene and tissue distribution of mR A spliced variants.
  • GLP-1 10005
  • GLP- l belongs to a larger hormone group that is referred to as the glucagon superfamily of peptide hormones.
  • the hormones of this superfamily are categorized in the same family due, in large part, to their close sequence homology with one another.
  • This superfamily includes the following peptides: GLP-1 ( 1 -37), (7-37) and -(7-36) amide, GIP, exendin-3 and -4, secretin, peptide histidine-methionine amide (PHM), GLP-2, helospectin- 1 and-2, hclodcrmin.
  • GLP- 1 functions to stimulate insulin releasing factor secretion whereas GLP-2 operates to regulate the distinct function of growth of intestinal epithelial cells, Id.
  • GLP- 1 is a naturally occurring 37 amino acid fragment of a polypeptide hormone proglucagon.
  • the full length GLP- 1 is thought to be biologically inactive.
  • the six N-terminal amino acids are cleaved to give the active forms of the peptide, GIT 1 - 1 (7- 37) and GLP-1 (7-36). This cleavage seems to be activated by eating, and the truncated peptide is active in stimulating post— prandial insulin secretion, suppressing glucose- dependent glucagons secretion, delaying gastric emptying, and stimulating beta-cell proliferation.
  • GLP- 1 Given its mechanism of action, GLP- 1 has not surprisingly received considerable attention as a potential panacea for Type II diabetes.
  • GLP-1 is not orally active and has an extremely short plasma half life (about 90 seconds), making it impractical for use as a pharmaceutical.
  • Eli Lilly's exenatide (Byetta®) is a 39 amino acid peptide having a sequence derived from a peptide in Gila monster saliva, having some sequence identity to GLP- I , which binds the GLP- 1 receptor. See, e.g., US 5,424,286.
  • GLP- 1 operates to regulate gastric motility. Secreted by L cells, in response to nutrients, GLP- 1 has already been shown in vivo to have dual roles in decreasing glucagon and stimulating insulin secretion.
  • GLP- 1 The ability to modulate GLP- 1 is potentially critical to human weight control as the slowing or inhibition of gastric emptying functions to prolong the periods where a given individual perceives satiation. Thus, when an individual feels satiated for longer periods this will decrease the perceived need to eat, which, in turn, will reduce an individual's overall intake of calories and promote weight loss or maintain healthy body weight.
  • GLP- l activity can be enhanced by inhibitors of dipeptidyl peptidase 4 (DPP-4), the enzyme that deactivates GLP- l in plasma.
  • DPP-4 inhibitors or glyptins are a class of oral hypoglycemics that block DPP-4. They include for example silagliptin and vi ldagliptin and are used to treat diabetes mellitus type 2. Their mechanism of action is thought to result from increased incretin levels (GLP- l and GIP), which inhibit glucagon release, which increases insulin secretion, decreases gastric emptying, and decreases blood glucose levels.
  • one aspect of the invention provides a method for preventing or reducing weight gain in mammals, e.g., humans, by administering an effective amount of a SERCA3 inhibitor, Moreover, insulin production is stimulated, which may be desirable in, e.g., people suffering from or at high risk of Type II diabetes.
  • one aspect of the invention also relates to a method of screening for potential SERCA3 inhibitors. It is still further contemplated that these same SERCA3 inhibitors are useful to increase GLP- 1 production.
  • the invention also contemplates a SERCA3 inhibitor that may modulate GLP- 1 levels in order to prevent excessive weight gain or to treat excessive weight gain or maintain healthy body weight.
  • the invention also contemplates a SERCA3 inhibitor that may modulate GLP-1 levels in order to promote normal intestinal motility and for treatment or prophylaxis of diseases and disorders mediated by abnormal intestinal motility, for example to slow down gut motility and thereby mitigate discomfort caused by abnormal gut tissue contraction; and a method of treatment or prophylaxis of di eases and disorders mediated by abnormal intestinal motility comprising administering an effective amount of a SER.CA3 inhibitor to a patient in need thereof.
  • the invention also contemplates a SERCA3 inhibitor that may modulate GLP- 1 levels in order to treat Type II diabetes, by enhancing insulin secretion mediated by GLP- 1 , either as sole agent or in combination with one or more anti-diabetic drugs.
  • a SERCA3 inhibitor that may modulate GLP- 1 levels in order to treat Type II diabetes, by enhancing insulin secretion mediated by GLP- 1 , either as sole agent or in combination with one or more anti-diabetic drugs.
  • the invention contemplates combining a SERCA3 inhibitor with another modulator of GLP- I production in order to increase overall production of GLP-1 .
  • a SERCA3 inhibitor be taken in conjuction with a GLP- 1 mimetic, such as exenatide, or in order increase the production and/or concentration of GLP-1 levels in a given individual.
  • the invention provides a mixture in either food or beverage forms or in capsule, powder, liquid or pill forms that contain at least one
  • SERCA3 inhibitor and at least one lipid, sweetener and/or spice as well as the use of such mixture to regulate gut motility through GLP-I regulation.
  • the invention provides for administration of a
  • SERCA-3 inhibitor in conjunction with a DPP-4 blocker and optionally a lipid, sweeter or a spice compound, as a single composition or simultaneously or sequentially.
  • X may consist exclusively of X or may include something additional e.g. X + Y.
  • Figure I The expression of Serca3 in mouse enteroendocrine cells.
  • Mouse small intestine sections are stained with anti-Serca3 antibody. A subset of endocrine cells is immunoreactive to the antibody (green, top and bottom rows). The same sections are also stained with DAP1 to visualize nuclei (middle row). Overlapping of the top and middle images shows that most of the SERCA3 staining is concentrated in the cytosol,
  • Figure 2 lmmunostaining of a human duodenal section with anti-
  • SERCA3 antibody A subset of human enteroendocrine cells expressed SERCA3 (green). The magnified inset shows that most of the staining are concentrated in the bottom half of the cells.
  • FIG. 3 Co-expression of SERCA3 with TRPM5 in mouse small intestine. Expression of SERCA3 in mouse small intestinal is visualized using anti-
  • TRPM5 is a taste signalling protein that has been found to be co-expressed in taste receptors and other taste signalling components such as a-gusducin, Gb3, Gg l 3 and PLC b2(Jang et al, PNAS, 104: 105069-74, 2007).
  • TrpM5 and SERCA3 are Co-expressed in human duodenal enteroendocrine cells. Double immunostaining of a human duodenal section with anti- ⁇ 5 (green) and anti-SERCA.3 (red) antibodies indicates that these two proteins are co-expressed in the same set of chemosensory cells in human gut, DAP! was used to slain nuclei (blue),
  • FIG. 6 GLP- 1 fold change in response to l QmM saccharin.
  • GLP-1 secretion from mouse small intestine, in response to 10 mM Saccharin shows an increase in SERCA3 knock-out mice.
  • Secretion of GLP-1 from wild-type and SERCA3-knockout gut tissue was increased in response to 10 mM Saccharin. The increase was about 3-4 fold in the wild-type tissue whereas 9-1 0 fold increase was observed in the KO tissue. The increased GLP- 1 levels are maintained for over 20 min.
  • FIG. 7 SERCA3 gene knockout further increased the GLP- 1 release from mouse intestinal tissue. Thymol and isoeugenol increased the GLP-1 release from the wild-type tissue by 2-3 fold. The increase was 6 fold in the SERCA3-knockout tissue,
  • Figure 8 GLP- 1 response of gul villi and crypts to lipid administration in SBRCA3 wild type and knockout mice.
  • the Serca-3 inhibitor should be administered with lipid, sweetener or spice, e.g., a sweetener or spice, molecule at about the same time,
  • Figure 10 The combination of BHQ and Saccharin maximized the GLP- 1 release from the gut tissue.
  • the mixture of 0.05% DMSO and 5 mM Saccharin increases the GLP- 1 level (over 2-fold).
  • the mixture of 5 mM Saccharin, 30 mM BHQ and 0.05% DMSO induced the maximum GLP-1 level (3.5 to 4.5 fold increase).
  • the invention provides for Method 1, wherein Method
  • I is a method of identifying a compound that modulates SERCA3 activity, comprising;
  • steps i and ii are conducted simultaneously or sequentially, e.g.
  • Method 1 .3. The method of Method 1. 1 .1 , 1.2, wherein said cell is prokaryotic.
  • 1 .5. The method of 1 .4 wherein the eukaryotic cell is a yeast, insect, amphibian or mammalian cell.
  • 1 .6 The method of 1 .4 wherein the eukaryotic cell is a CHO cell, HEK-293 r xenopus oocyte.
  • Method 1.9 wherein the label is a ehemi luminescent label.
  • Method 1.9 wherein the label is a fluorescent label.
  • Method 1 . 14 wherein the determination of GLP-1 levels is done by measurement of said measurable gene product.
  • GLP- 1 levels is done by measurement of Gl.P- 1 mRNA.
  • GLP-1 levels is done by measurement of GLP-1 protein.
  • GLP- I levels is done by measurement of the mRNA of a second gene product.
  • GLP-l levels is done by measurement of insulin.
  • test compound binds to a gene or gene product: wherein the binding of said test compound to said gene or gene product modulates SERCA3.
  • Method 1 .39. The method of Method 1 or any of the foregoing methods, wherein the steps of Method 1 are carried out in vivo.
  • the invention provides for Method II, wherein
  • Method II is a method of treatment, mitigation or prophylaxis of an abnormal condition mediated by GLP-1 activity, comprising administering an effective amount of a
  • Method 11 wherein the condition mediated by GLP- 1 activity is Type I I diabetes.
  • Method I I wherein the condition mediated by GLP-1 is abnormal intestinal motility.
  • SERCA3 inhibits the activity of SERCA3 is administered in conjunction with known treatments for diseases or disorders caused or characterized by abnormal intestinal motility.
  • drugs selected from the group consisting of bisguanides, e.g., metformin, a sulfonylurea, thiazolidinediones such as pioglitazone or rosiglitazone, DPP-4 inhibitors, e.g., sitagliptin (Januvia), megtitinides, e.g. repaglinide (Prandin) or nateglinide (Starlix), GLP-1 mimetics, e.g., cxenatide. and/or insulin or an insulin analog, for example a long acting basal insulin analogue, for example glargine (l.antus).
  • bisguanides e.g., metformin, a sulfonylurea, thiazolidinediones such as pioglitazone or rosiglitazone
  • DPP-4 inhibitors e.g., sitagliptin (Januvia), megtitinides,
  • SERCA3 2,5-di-(tert-butyI)- f ,4-hydroquinone.
  • SERCA3 inhibits the activity of SERCA3 is a cyclosporin, e.g., cyclosporinc A.
  • Method II or any of the foregoing methods wherein the compound that inhibits the activity of SERCA3 is an antibody to SERCA3, e.g., a monoclonal human, humanized or chimeric antibody to S RCA3.
  • Method II or any of the foregoing methods wherein the compound that inhibits the activity of SERCA3 is selected from antisense oligonucleotides, triple helix DNA. RNA aptamers, ribozymes and double stranded RNA directed to a nucleic acid sequence of SERCA3.
  • Method II or any of the foregoing methods, wherein the mammal to be treated is a human.
  • the invention provides an effective amount of a compound that inhibits the activity of SERCA3 for use as a medicament.
  • the invention provides a further medical use I (FMU I) wherein FMU I is an effective amount of a compound that inhibits the activity of SERCA3 for use in the treatment or prophylaxis of an abnormal condition mediated by GLP-1 activity in a mammal in need thereof
  • the FM.LJ I wherein the condition mediated by GLIM activity is a pre- diabetic condition.
  • the FMU 1 wherein the condition mediated by GLP- 1 activity is a
  • the FMU 1 wherein the condition mediated by GLP-1 is obesity.
  • FMU I comprises co-administration (concurrently or
  • drugs selected from the group consisting of bisguanides, e.g., metformin, a sulfonylurea, thiazolidinediones such as pioglitazone or rosiglitazone, DPP-4 inhibitors, e.g., sitagliptin (Januvia), meg litin ides, e.g. repaglinide (Prandin) or nateglinide (Starlix), GLP- 1 mimetics, e.g., exenatide, and/or insulin or an insulin analog, for example a long acting basal insulin analogue, for example glargine (Lantiis).
  • bisguanides e.g., metformin, a sulfonylurea, thiazolidinediones such as pioglitazone or rosiglitazone
  • DPP-4 inhibitors e.g., sitagliptin (Januvia), meg litin ides,
  • the FMU I or any one of the previous FMU I (3.1 - 3.1.0), wherein the compound that inhibits the activity of SERCA3 is administered in conjunction with a sweetener.
  • antihistamine is known to enhance SERCA3 expression; antihistamine is believed to inhibit histamine-mediated SERCA3 expression].
  • SERCA3 is a cyclosporin, e.g.,
  • SERCA3 is an antibody to SERCA3, e.g., a monoclonal human, humanized or chimeric antibody to SERCA3.
  • compound that inhibits the activity of SERCA3 is a monoclonal antibody binding to the same epitope as monoclonal antibody PL/IM4302 (See, Chandraseicera, CP., el aL Jour. Bio. Chem. (2003) 278 ( 14): 12482-12488, incorporated herein by reference), e.g., wherein the antibody has the complementarity determining regions (CDRs) of monoclonal antibody PL/1IVI43Q2, for example a chimeric or humanized form of monoclonal antibody PL/IM4302.
  • CDRs complementarity determining regions
  • compound that inhibits the activity of SERCA3 is selected from antisense oligonucleotides, triple helix DNA, RNA aptamers, ribo/ymes and double stranded RNA directed to a nucleic acid sequence of SERCA3.
  • modulation of the mammal's metabolism occurs by inhibition of gastric motility or slowing inhibition of gastric emptying.
  • SERCA3 is locally active in the gastrointestinal tract, such that systemic activity is reduced or eliminated, e.g., substantially free of activity other than on GLP- 1. .
  • the invention provides for the use of an effective amount of a compound that inhibits the activity of SERCA in the manufacture of a medicament for the treatment or prophylaxis of an abnormal condition mediated by GLP-l activity in a mammal, in need thereof.
  • GLP-1 activity is Type 11 diabetes.
  • GLP-1 activity is a pre-diabetic condition.
  • GLP-1 activity is a metabolic disease or disorder.
  • GLP- 1 is obesity.
  • SERCA3 is used in conjunction with known treatments for diseases or disorders caused or characterized by abnormal intestinal motility.
  • medicament is co-administered (concurrently or sequentially) with an effective amount of one or more drags selected from the group consisting of bisguanides, e.g., metformin, a sulfonylurea, thia olidinediones such as pioglitazone or rosigl itazone, DPP-4 inhibitors, e.g., sitagl iptin (Januvia), megl itinides. e.g.
  • repaglinide Prandin
  • nateglinide Starlix
  • GLP-1 mimetics e.g., exenatide
  • insulin or an insulin analog for example a long acting basal insulin analogue, for example glargine (l .antus).
  • antihistamine is known to enhance SERCA3 expression; antihistamine is believed to inhibit histamine-mediateil SERCA3 expression].
  • a compound that inhibits the activity of SERCA3 is a cyclosporin, e.g., cyclosporine A.
  • SERCA3 is an antibody to SERCA3, e.g., a monoclonal human, humanized or chimeric antibody to SERCA3.
  • compound that inhibits the activity of SERCA3 is a monoclonal antibody binding to the same epitope as monoclonal antibody PL/IM4302 (See, Chandrasekera, CP., et al.. Jour. Bio. Chem. (2003) 278 (14): 12482- 12488, incorporated herein by reference), e.g., wherein the antibody has the complementarity determining regions (CDRs) of monoclonal antibody PL/IM4302, for example a chimeric or humanized form of monoclonal antibody PE/1M4302.
  • CDRs complementarity determining regions
  • any one of 4.0— 4.22 wherein the compound that inhibits the activity of SERCA3 is selected from antisense oligonucleotides, triple helix DNA, RNA aptamers, ribozymes and double stranded R ' NA directed to a nucleic acid sequence of SERCA3.
  • medicament is administered within 1-2 hours of a meal.
  • the invention further provides a compound that inhibits the activity of
  • the invention further provides the use of a compound that inhibits the activity of SERCA3 in the manufacture of a formulation for use in any of Method II or 2.01 -2.28.
  • the invention further provides a composition for oral administration, e.g, a food, beverage, capsule, pill, powder, food additive, or beverage additive which contains at least one SERCA3 inhibitor and at least one lipid, sweetener or spice, e.g., a sweetener or spice ingredient, e.g., a non-sugar sweetener selected from stevia,
  • a composition for oral administration e.g, a food, beverage, capsule, pill, powder, food additive, or beverage additive which contains at least one SERCA3 inhibitor and at least one lipid, sweetener or spice, e.g., a sweetener or spice ingredient, e.g., a non-sugar sweetener selected from stevia,
  • a SERCA3 modulator is carried out by treating the animals with regular food plus, for example, a Serca3 inhibitor and then checking the plasma insulin level or glucose level to assess the effective of the potential inhibitor on plasma insulin or glucose levels.
  • the invention provides for administration of a
  • SERCA-3 inhibitor in conjunction with a DPP-4 blocker and optionally a sweeter or a spice compound, as a single composition or simultaneously or sequentially,
  • Lipids for use in the methods and compositions described herein include palatable triglycerides and fatty acids, e.g., fish oil or vegetable oils.
  • Sweeteners include non-sugar sweeteners selected from stevia, aspartame, sucralose, neotame, acesulfame potassium, saccharin, sugar alcohols, and combinations thereof.
  • Spices include edible compounds having a spicy flavor as used to season foods, beverages and compositions for oral administration, e.g., essential oils from plants, as well as dried seed, fruit, root, bark, or vegetative materials, for example thymol and/or eugenol, as well as pepper, cinnamon, and so forth.
  • SERCA3 refers to gene products of naturally occurring variants a-f of the SERCA3 gene or additional functional variants that are expressed in cells in vertebrate species, for example, without limitation, SERCA3 isoforms from mouse, rat, rabbit, ape, monkey, pig, dog, cat, cattle, and frog. These include in particular, without limitation, animals commonly used in laboratories such as Mus museulus, Rattus norvegicus, and Xenopus lacvis.
  • rat SERCA3a (SEQ ID: I , SEQ ID: 2), mouse SERCA3a (SEQ ID: 3, SEQ ID: 4), mouse SERCA3b (SEQ ID: 5, SEQ ID: 6), and human SERCA3c (SEQ ID:7 and SEQ ID:8), Human SERCA3a (SEQ ID: 9, SEQ ID: 10), Human SERCA3b (SEQ ID: 1 1 , SEQ ID: 12), Human SERCA3d (SEQ ID: 13, SEQ ID: 14), Human SERCA3e (SEQ ID: 1 5, SEQ ID: 16), Human SERCA3f (SEQ I D: 1 7. SEQ ID: 18), and their SERCA3 gene products.
  • SERCA3 nucleic acids or polypeptides may be synthesized chemically as is well known in the art based on known sequences. Alternatively they may be amplified using clones comprising one or more SERCA3 sequences. For transfection and expression, a cDNA clone may be used. Clones that are known to comprise Serca3 may be selected and obtained from commercial sources, or a cDNA library may be screened using all or part of the known Serca3 sequence. Serca3 may be amplified using clones and/or polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • SERCA3 may then be introduced into one of many well known expression systems to produce the SERCA3 gene product/protein.
  • SERCA3 may be heterologous! ⁇ expressed in various cells including, without limitation, vertebrate cells or mammalian cells, as is well known in the art.
  • Vertebrates comprise amphibians, reptiles, birds, and mammals (including humans).
  • SERCA homologue genes are known in various vertebrates, including various vertebrates commonly employed in the lab, for example, without limitation, Xenopus laevis (X. laevis), Mus musculm (M muscul s), Ratt s mrwegicus (R. norvegicus), and other rhodents.
  • Invertebrates having one or more SERCA homologue may also be used, for example Caenorhabditis elegam (C. elegam ⁇ , and the fruit fly Drosophila
  • the SERCA3 expressing cells may be used, or alternatively, an isolated SERCA3 protein may be used.
  • Isolated SERCA3 protein may, for example, be integrated into an artificial lipid bilayer as is well known in the art and the resulting SERCA3 lipid bilayer can be used for further methods as is well known in the art.
  • Effects on SERCA3 may be direct or indirect, for example, through an effect on SERCA3 regulation and/or expression.
  • the effects on GEP-I may be direct or indirect.
  • the ability of a substance to '"modulate" SERCA3 refers to, but is not limited to, the ability of a substance to inhibit the biological activity of SERCA3, or modulate the subcellular localization of the protein, or alter the stability of the protein (e.g., by post- translational modification such as phosphorylation, glycosylation, etc..) and/or inhibit SERCA3 gene expression. Such modulation could also involve affecting the ability of other proteins to interact with SERCA3,
  • a SERCA3 inhibitor may bind to and/or block SERCA3, or may inhibit expression of SERCA.3.
  • the assays described herein may be used to screen libraries for agents that modulate Serca3 activity.
  • the assays may be designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to the assays, which are typically run in parallel (for example in microliter formats on microtiter plates in robotic assays).
  • Assays may be run in high throughput screening methods (whereby it is possible to screen up to several thousand potential modulators in a single day) that involve providing a combinatorial chemical or peptide library containing a large number of potential enhancers.
  • Such libraries are then screened in one or more assays described herein-above to identify those library agents (particular chemical species or subclasses) that display the activity described herein-above.
  • the enhancers thus identified can be directly used or may serve as leads to identify further modulators by making and testing derivatives.
  • Synthetic compound libraries are commercially available from a number of companies including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.I 1.). and Microsource (New Mil ford. Conn.).
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • a rare-chemical library is available from Aldrich (Milwaukee, Wis.). Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are commercially available for example from Pan Laboratories (Bothell. Wash.) or ycoSearch (NC), or are readily producible by methods well known in the art.
  • libraries include protein/expression libraries, cDNA libraries from natural sources, including, for example, foods, plants, animals, bacteria, libraries expressing randomly or systematically mutated variants of one or more polypeptides, and genomic libraries in viral vectors that are used to express the mRNA content of one cell or tissue.
  • the invention provides a compound that modulates Serca3 activity.
  • Won limiting examples of modulators of Serca 3 activity include a protein, a peptide, a small molecule, an antihistamine; a cyclosporinc; an antibody to Serca3; an ant ⁇ sense oligonucleotides, triple helix DNA, RNA aptamers, ribozynies or double stranded RNA directed to a nucleic acid sequence of SERCA3, taste receptor ligands such as a bitter receptor ligand, a sweet receptor ligand, an umami receptor ligand, a fat receptor liga d, a sour receptor ligands,.
  • amami receptor ligands as used herein also includes compounds that enhance umami taste.
  • umami receptor ligands include, but are not limited to: 2-(3- phenylpropy pyridine, L-GIu (glutamic acid, glutamate, for example in the form of its salts such as monosodium glutamate, monopotassium glutamate, monoammonium glutamate, calcium diglutamate, magnesium diglutamate), L-Asp (L-asparagine, ), 5 * - ribonucleotides or their salts including, without limitation, calcium S'-ribonucleotides. di sod in m 5'-ribonucleotides, and dipotassium 5 ' -ribonucleotides (e.g.
  • inosinic acid guanylic acid, adenosinic acid, inosinates, guanylates. and adenylates, including guanosine 5 '-monophosphate, inosine 5 '-monophosphate, and 5'-adenylate and their salts such as disodium guanylate, disodium inosinate, disodium adenylate; dipotassium guanylate, dipotassium inosinate, dipotassium adenylate, calcium guanylate, calcium inosinate, calcium adenylate), maltol, ethyl maltol, glycine, L-!eucine, autolyzed or hydroly/ed proteins (e.g. autolyzed yeast, hydrolyzed yeast, hydrolyzcd vegetable proteins)
  • Many more urn ami receptor ligands other than those listed herein are known to those of skill in the art and still more can be identified using methods
  • Non-limiting examples of fat receptor ligands include linoleie acids, oleic acids,
  • Non-limiting examples of sour receptor ligands include citric acid and hydroxycitric acid. Many more sour receptor ligands other than those listed herein are known to those of skill in the art, and still more can be identified using methods known in the art and described herein.
  • Non-limiting bitter receptor l igands include flavanones, flavones, fiavonols, flavans, phenolic flavonoids, isoflavones, limonoid aglycones, glucosinolates or hydrolysis product thereof, caffeine, quinine, metformin, metformin hydrochloride, extracts of
  • Non-limiting sweet receptor ligands include nutritive and non nutritive sweeteners e.g. sucrose, fructose, glucose, high fructose corn syrup, com syrup, xylose, arabinosc, rhamnose, erythritol, xylitoi, mannitol, sorbitol, inositol, saccharine, swingle extract, rubus extract, rebaudiosides e.g. Rebaudioside A, Rcbaudioside B, and Rebaudioside C, methyl chavicol.
  • nutritive and non nutritive sweeteners e.g. sucrose, fructose, glucose, high fructose corn syrup, com syrup, xylose, arabinosc, rhamnose, erythritol, xylitoi, mannitol, sorbitol, inositol, saccharine, swingle extract, rubus extract, rebaudiosides e
  • Theasaponin El Acesulfame K, Alitame, Aspartame, CM 401 , Dulcin, Neotame, sodium Cyclamate, Sucralose.
  • Carnosifiosi.de V Carnosifioside VI, D. cumminsii, Cyctocarioside A, Cyclocarioside 1, Dulcoside A, Glycyrrhizic Acid, Hern and uicin, I lcrnandulcin, 4 be! a-h yd roxy-I lesperilin- 7-G!ucoside Dihydrochalcone, Huangqioside E, Huangqiosidc E, 3-Hydroxyphloridzin, 2,3-Dihydro-6-Methoxy 3-0-Acet:ate 3 Mabinlin Maltosyl-Alpha-(l ,6)-Neohesperidin
  • Neomogroside Osladin
  • Periandrin 1 Periandrin II
  • Periandrtn III Periandrin IV
  • Periandrin V Phlomisoside I
  • Phlorizin Phlorizin
  • Phyllodulein Polypodoside A
  • Potassium magnesium calcium glycyrrhizin Pterocaryosides A, Pterocaryosides B, Rubusoside, Scandenoside 6, Siamenoside I, Sodium glycyrrhizinatc.
  • Steviolbioside Stevioside, alpha-Glycosyl Suavioside A, Suavioside B, Suavioside G, Suaviosidc H, Suavioside I, Suavioside J, Thaumatin, Triammonium Glycyrrhizinate (TAG), Trilobatin Curculin, Strogin I , Strogin 2, Strogin 4, Miraculin, Hoduleirt, Jujubasaponin II, Jujubasaponin ill, Abrusoside E, Periandrin ic acd I, monoglucuronide, Periandrinic acid II,
  • Non limiting examples of particular modulators of Serca 3 include 1 ,4-Dihydroxy-2,5-di- tert-butylbenzene (BHQ); 2.5-d i -(tert-buty 1 )- 1 ,4-hydroquinone; 2,5-di(t-butyI)- 1 ,4- benzohydroq uinone: thapsigargin and structurally similar compounds to any of the foregoing.
  • Non limiting examples of structurally similar compounds to the foregoing include:
  • Rj to R are independently selected from the group consisting of H, and COR5 and, wherein Rs is a hydrocarbon residue having 1 to 7 carbon atoms.
  • Rj is straight or branched C1 -C7 alky I, C 2 -C 7 alkenyl or C 2 -C 7 alkynyl.
  • Rj is COC4H7
  • R 3 ⁇ 4 is COCH3
  • R and R 4 are independently selected from the group consisting of straight or branched C1-C7 alkyl, C 2 -C7 alkcnyl.
  • Non limiting examples of compounds of formula (I) are Thapsigargicine and
  • the invention provides a cell line which overexpresses
  • SERCA3 in order to detect a compounds ability to affect GLP- l production.
  • SERCA3 may be overexpressed by transfcction of plasmid D A that contains a SERCA3 coding sequence, a eukaryotic promoter and a polyadenylation site sequence into a mammalian cell line. Any suitable cells that allow SERCA3 expression and a chosen detection method to measure binding to or interaction with SERCA3 can be used.
  • COS cells derived from kidney cells of the African green monkey are useful mammalian cells.
  • Various eukaryotic promoters may be used, a useful promoter is the C V
  • the SERCA3-transfected ce ls can be used for detection of GLP- l levels therein.
  • transfcction could be performed using any suitable cells or cell lines, for example, any mammalian cells (all mammalians have the three SERCA genes including SERCA3), or any vertebrate cells (which have SERCA3 homologous genes). Invertebrate cells may be suitable as well
  • GLP- 1 refers to gene products of natural ly occurring variants GLP- l (1 -37), GLP-l (7-37) and -(7-36) amide and additional variants that are expressed in gut cells in vertebrate species. These include in particular, without limitation, animals commonly used in laboratories such as Mus mmc hts, Rattus norvegicus, and Xenopus laevis.
  • GLP-1 nucleic acids or polypeptides may be synthesized chemically as is well known in the art based on known sequences. Alternatively they may be amplified using clones comprising one or more GLP-1 sequences. For transfcction and expression, a cD A clone may be used. Clones that are known to comprise GLP-1 may be selected and obtained from commercial sources, or a cDNA library may be screened using all or part of the known GLP-I sequence. GLP- 1 may be amplified using clones and/or polymerase chain reaction (PGR),
  • PGR polymerase chain reaction
  • GLP-1 may be heterologous ly expressed in various cells including, without limitation, vertebrate ceils or mammalian cells, as is well known in the art.
  • the GLP- 1 expressing cells may be used.
  • the cells may express GLP-1 endogenous! ⁇ ' or may express GLP- 1 after being subject to recombinant techniques used to introduce a nucleic acid encoding for GLP- 1 into the cell.
  • the term "test compound” as used herein may be used singly or as a mixture of multiple test compounds. Multiple test compounds can be used to test more efficiently and to detect synergistic effects of the test compounds on SERCA3 activity.
  • Test compounds may include any compound including, without limitation, bioaetive peptides, polypeptides, antibodies.
  • the test compounds can be naturally occurring or synthetically produced, the latter are available as synthetic combinatorial libraries.
  • GLP- 1 is well conserved among mammalian species.
  • invertebrate cells may be suitable as well.
  • GLP- 1 If measurement of GLP- 1 is used to detect test compound binding to or interaction with SERCA3, the cells chosen for transfection need to be capable of GLP- 1 production in response to an exogenous stimulus. For example, without limitation, it is well known in the field that certain enterocndocrine cells are able to produce GLP-1 .
  • nucleic acid sequence encoding GLP-1 may be recombinant! ⁇ ' introduced into COS and HEK-293 cells, for example, and many other suitable cells thai are well known in the art. GLP-1 measurement may then be conducted by any method known in the art or disclosed herein.
  • the compound that inhibits SERCA3 activity is an antibody.
  • Methods utilized to generate antibodies include hybridoma technology, ribosome display, bacterial and yeast display, and others known in the art.
  • the vast majority of monoclonal antibodies (ni Abs) are of rodent origin. When such antibodies are administered in a different species, patients can mount their own antibody response to such xenogenic antibodies, which may result in the eventual neutralization and elimination of the antibody.
  • This problem for humans is minimized by using antibodies which have fully human sequences, for example made by phage display, or by engineering antibodies so thai only the critical binding residues are of rodent origin, while the constant domain and optionally the framework regions of the variable domain are predominantly of human origin.
  • Chimeric antibodies are generally antibodies wherein the constant region is of human origin and the variable region is predominantly murine.
  • Humanized antibodies are human antibodies in which residues from the complementarity determining regions (CDR) are replaced by residues from a CDR of a non-human species such as mouse, having the desired properties such as specificity, affinity, and potency.
  • CDR complementarity determining regions
  • FR framework region residues of the human immunoglobulin are replaced by corresponding non- human residues.
  • This humanizalion strategy is generally referred to as "CDR grafting", see, e.g.. Winter, U.S. Pat. No. 5,225,539. Back mutation of selected target framework residues to the corresponding donor residues might be required lo restore and / or improved affinity.
  • Structure-based methods may also be employed for humanization and affinity maturation, for example as described for humanization in U.S. Patent Appl'n Ser. No. 10/ 153, 159.
  • Comparison of the essential framework residues required in humanizalion of several antibodies, as well as computer modeling based on antibody crystal structures revealed a set of framework residues termed as "Vernier zone residues" (Foote, J Mol
  • humanized antibodies may contain the CDRs from a non-human sequence grafted into pools (e.g. libraries) of individual human framework regions. This newly engineered antibody is able to bind to the same antigen as the original antibody.
  • the antibody constant region is derived from a human antibody.
  • the methodology for performing this aspect is generally described as framework shuffling (Dall'Acqua, Methods, 36:43-60 (2005)).
  • the humanized antibody may contain sequences from two or more framework regions derived from at least two human antibody germline sequences with high homology to the donor species.
  • Antibodies designed using this method are described as hybrid antibodies (Rother el aL U.S. Pat, No. 7,393,648). Any techniques wherein the affinity and specificity is adequately retained are encompassed herein.
  • Nucleic acid sequence refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin that may be single or double stranded, and represent the sense or antisense strand.
  • Polynucleotide refers to DNA or RNA.
  • double stranded RNA as used herein is meant to encompass any and all conventional techniques for RNAi-mediated gene silencing. Such techniques are familiar to one of skill in the art and may include, but. are not limited to, use of dsRNA, si RNA and shRNA to knockdown gene expression.
  • antisense refers to nucleotide sequences that are complementary to a specific DNA or RNA sequence.
  • antisense strand is used in reference to a nucleic acid strand that is complementary to the "sense' strand.
  • Antisense molecules may be produced by any method, including synthesis by ligating the gene(s) of interest in a reverse orientation to a viral promoter that permits the synthesis of a complementary strand. Once introduced into a cell, this transcribed strand combines natural sequences produced by the cell to form duplexes. These duplexes then block either the further transcription or translation.
  • the designation "negative " is sometimes used in reference to the antisense strand, and "positive” is sometimes used in reference to the sense strand.
  • antisense oligonucleotides, triple helix DNA, RNA aptamers, ribozymes and double stranded RNA are "directed to a nucleic acid sequence of SERCA3" such that the nucleotide sequence of SERCA3 chosen will produce gene- specific inhibition of SERCA3 gene expression.
  • SERCA3 nucleic acid sequence of SERCA3
  • knowledge of the SER.CA3 nucleotide sequence may be used to design an antisense molecule which gives strongest hybridization to the mRNA.
  • ribozymes can be synthesized to recognize specific nucleotide sequences of SERCA3 and cleave it (Cech, J. Amer. Med Assn.
  • compounds useful in the present invention are locally active in the gastroinlentinal tract and have minimal systemic activity. Such compounds may be administered orally or rectal ly. In other embodiments, the compounds may administered by any suitable means, depending on the particular properties of the compound, e.g., orally, parenteraily (including intravenous, intramuscular, intraperitoneal, intraosseus, or subcutaneous injection), transdermal ly, by inhalation,, rcctally, vaginally or topically (including buccal and sublingual administration).
  • the compounds useful in the invention may generally be provided in the form of tablets or capsules, as a powder or granules, or as an aqueous solution or suspension.
  • Tablets for oral use may include the active ingredients mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives.
  • suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents.
  • Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc, If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
  • Dosages of the compounds of the invention will vary depending upon the condition to be treated or prevented and on the identity of the inhibitor being used.
  • Estimates of effective dosages and in vivo half-lives for the individual compounds encompassed by the invention can. be made on the basis of in vivo testing using an animal model, such as the mouse model described herein or an adaptation of such method to larger mammals.
  • Appropriate dosage may range from, e.g., 0.0 !mg to 5000 mg, and is suitably adjusted to achieve a target blood level roughly corresponding to the effective level observed in vitro.
  • the compounds are administered within one to two hours before a meal.
  • Compounds useful according to the invention to inhibit SE CA3 can be administered in combination or in conjunction with other known therapeutic agents useful for treating Type II diabetes, obesity, pre-diabetic conditions and other metabolic diseases and disorders.
  • the administering physician can adjust the amount and timing of drug administration on the basis of results observed.
  • the following invention will now be further described only by way of example. The following examples are presented only to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
  • Example 1 GLP-1 Release in Wild-Type and SERCA3 Knock-out Mice and in human small intestine tissue
  • Tissue preparation C57BL/6J mice are sacrificed, and small intestine excised. The tissue is then rinsed in I PBS, and fixed in 4% paraformaldehyde in PBS for one hour on ice, and cryoprotected in 20% sucrose in PBS overnight. Frozen tissues are further sliced into 12- micron thick sections. Duodenum tissue sections of human small intestine are purchased from IMGE EX (San Diego, CA; Catalogue number IMH- 1028).
  • Tissue sections are blocked first and incubated with rabbit polyclonal anti-TrpM5 antibody, followed by Alexa488-conjugated goat-anti- rabbit antibody containing DAP1 to stain nuclei.
  • the rabbit primary antibody against SER.CA3 is first labeled with Alexa.647 using Zenon Tricolor Rabbit IgG labeling kit (Invitrogen), and the labeled primary antibody is used to incubate the tissue sections for 2 ho rs at room temperature, followed by post- fixation in 4% paraformaldehyde/PBS for 10 minutes. I mages are taken using a con local microscope.
  • SERCA3 proteins Most of the proteins concentrate in the cytosol or in the bottom half of the human cells. Since these cells are known to contain peptide hormone-filled granules, the subcellular localisation of Serca3 suggests that this protein may be directly involved in the regulation of the hormone secretion by controlling the uptake of the free calcium into the endoplasmic reticulum and thus the intracellular concentrations of free calcium ions that triggers the secretion.
  • Enteroendocrine cells- containing villi and crypts are collected from duodenal segment and cultured in 3-ml low- glucose DM EM medium. After cells are settled in the medium for 1 5 min, 0.2 ml supernatant is taken immediately prior to the application of stimuli, and this sample is referred to as the "time 0 " sample. The same amount of supernatant is collected at specified time points subsequent to the stimulus application. The quantity of GEP- 1 in these samples is determined using an ELISA kit. The data are normalized by dividing GLP- 1 amount at later time points by that at time 0, and expressed as RE I J (relative fold unit) (Figure 6).
  • Enzyme-linked immunoabsorbent assays ELISA
  • RlA radioimmunoassays
  • Tissue is then cultured in low-glucose DM EM with antibiotics and 5%COi at 37 °C.
  • the cultured tissue is challenged with stimulus including saccharin, thymol and isoeugenol, linolenic acid, and the culture supernatant is collected at different post-stimulation time points.
  • Dipeptidyl peptidase I V inhibitor is added to the supernatants, and GLP- 1 levels in these samples is then determined using an ELISA (Millipore).
  • BHQ ( l,4-Dihydroxy-2,5-di-tert-butylbenzene), is tested on the appropriate gut samples (Figure 9).
  • the GLP- 1 level was stable for the duration of the test, for example 20 minutes, during which period the GLP- 1 in the BHQ-free control started to decline.
  • Saccharin (5 mM) in combination with 0.05% DMSO' causes over a 2-fold increase.
  • the mixture of Saccharin and BHQ increase the GLP- 1 level by 3-4,5 fold; this effect lasls for at least 20 minutes,
  • TRP 5-expressing enteroendoctrine cells also express Serca3, As in taste bud cells, TRPM5 is also involved in tastant -elicited cellular responses in the gastrointestinal tract, regulating the release of gut hormones such as glucagon-like peptide 1 (GLP-1 ).
  • GLP-1 assays showed that knocking out Serca3 boosted the amount of GLP- 1 released from, the endoctritie cells stimulated by such tastants as saccharin, which is apparently due to the impairment of the intracellular clearance in these cells, thus prolonging the duration of GLP-1 release.
  • knocking out Scrca3 also significantly augmented the spicy compound ⁇ thymol and tsoeugenol) but not the fatty acid (linolenic acid) -induced GLP- 1 release from mouse small intestine,
  • Example 5 Characterisation, of the effect of Serca3 inhibition by BHQ on the calcium response of human duodena! cells upon stimulation by tastants and spices.
  • Biopsies of human duodenal tissue are briefly treated with calcium/magnesium- free medium and dissociated into individual cells which are then cultured in complete DMEM medium (Dulbeeco's Modified Eagle medium.) containing 10% fetal bovine serum. Once attached to the covers! ip these cells are loaded with a calcium sensitive dye such as Fura- 2 AM, and stimulated with tastants and spices; saccharin, thymol and isoeugenol. The calcium responses of these cells to these stimuli are recorded in the presence and absence of the SERCA3 inhibitor BHQ. The effect of BHQ on the calcium response amplitude and duration is determined.
  • DMEM medium Dulbeeco's Modified Eagle medium.
  • the cells are fixed and immunohistochernistry is carried out to determine the expression of SBRCA3, taste receptors and signalling molecules such as TRPM5 in the responsive cells.
  • Example 6 Characterisation of the effect of Serca3 inhibition by BHQ on the GLP- 1 release from human duodenal cells upon stimulation by tastants and spices.
  • HBSS Hormon's balanced salt solution
  • Saccharin, thymol and isoeugenol in the absence or presence of the SERCA3 inhibitor BHQ is added to the medium.
  • the GLIM level in the medium before and after addition of these compounds is quantitatively determined using HI .
  • Example 7 Characterisation of the effect of Serca3 modulators on the calcium response of the GLP-1 release from human duodenal cells upon stimulation by tastants and spices.
  • DMEM DMEM
  • Artificial sweeteners such as saccharin that are pre-dissolved in DMEM are applied to the tissue culture. An aliquot of the medium is taken at time 0, followed by additional aliquots taken every 5 minutes. The concentration of GLP- 1 in each aliquot is determined using an ELISA assays. The effect of the artificial sweetener on GLP-1 release is determined by fold change; that is, the GLP-1 concentration in the later aliquot is divided by that in the aliquot at time 0.
  • Negative control experiments are also performed in the same manner as described above, except that the modulator alone, or neither the modulator nor the saccharin, are applied.
  • the GLP- 1 fold changes from these four experiments are compared to determine the effect of the potential modulator.
  • Example 8 Characterisation of Serea3's role in gut chemosensory cells' response to sweet compounds
  • !GF-I insulin like growth factor 2
  • GF-II insulin like growth factor 2
  • Serotonin cholecystokinin
  • CCK cholecystokinin
  • fat receptors are also expressed in the gut chemosensory cells. Dietary fat or metabolites produced by gut microbiota can regulate gut hormone release.
  • SercaB knockout affects dietary fat- mediated gut hormone release
  • WT and O mice are fasted and then fed with a mixture of short-, medium- and long chain fatty acids (capric, lauric, stearic, and linolenic acids), and plasma concentrations of the hormones GLP-I, 1GF-I, IGF-ll, Serotonin, and CCK are determined using ELISA or RIA assays. The data is comparatively analysed between the WT and KO samples.
  • Spice is an important flavour component.
  • Spicy compounds such as thymol, eugenof abd isoeugenol are known to stimulate enterocyles, triggering the release of gut hormones such as serotonin.
  • Gut hormones such as serotonin.
  • WT and KO mice are led with a mixture of thymol, eugenol and isoeugenol, and plasma concentrations of the hormones GLP-1, 1GF-I, IGF-ll, Serotonin, and CCK are determined using ELISA or RIA assays. The data is comparatively analysed between the WT and KO samples.
  • Example 11 Administration of a Serca3 modulator in Diabetic mammals
  • diabetic rat models Numerous established and accepted diabetic rat models exist for the assessment of therapies for the treatment of diabetes.
  • a suitable diabetic animal model is a hypoinsulinemic animal model. This model is useful because it provides for a low insulin level in the blood and so it facilitates the assessment of any therapies on the release of insulin, and in particular, the effect of any therapies on an increase in the release of insulin in the blood stream.
  • Diabetic rat models are commercially available from the The Jackson Laboratory, More information can be found on the Jackson Laboratory website www.jax.org/jaxmice
  • a single Serca3 modulator e.g. BHQ
  • BHQ Serca3 modulator
  • Diabetic rats and Wistar rats are selected for administration of the Serca3 modulator (e.g. BHQ) for the treatment of diabetes.
  • Animals are grouped according to dosage, and increasing dosages (range of 0.01 - l OOmg/kg) are utilised.
  • the Serca.3 modulator is instilled into the animal via silastic tubing inserted into the duodenum through the mouths of the lightly anesthetised animals.
  • Blood samples are collected via cannulation of the tail vein, and samples are withdrawn at baseline, 15, 30, 60 and 120 minutes post-instillation. Blood samples are collected in collection tubes containing standard cocktails of peptidase inhibitors and preservatives, and samples are stored at -25.degree. C. until assayed. Blood samples are assayed for the presence of the hormone GLP-1 . Assays for the hormone are performed using standard ELISA methodologies. Results are analyzed for efficacy of SercaS modulators administration for the treatment of diabetic rats.
  • Metabolites and other analyte concentrations including glucose, free fatty acids, triglycerides, calcium, potassium, sodium, magnesium, phosphate, are also assessed. Circulating concentrations of GLP-1 are expected to increase.
  • Example 12 Administration, of a Serea3 modulator in Obese mammals
  • Obesity rat models are commercially available from the The Jackson Laboratory. re information can be found on the Jackson Laboratory website www.jax.org/jaxmice
  • a single Serca3 modulator (e.g. BHQ) can be assayed in the obesity rat model detailed herein below.
  • industry standard diet induced obesity rats and applicable controls (healthy rats) are administered with a Serca3 modutator (e.g. BHQ) for the treatment of obesity following the procedure described above in example 1 1 .
  • a Serca3 modutator e.g. BHQ
  • animals are grouped according to dosage, and increasing dosages (range of 0.01 - 1 OOmg/kg) are utilised.
  • Serca3 protein is set forth as SEQ ID NO: 2 (GenBank Accession number: NMJ) 12914.1 ).
  • Preferred Serca3 proteins for use with the invention comprise an araino acid sequence: (a) having 50% or more identity (e, g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99. 5% or more) to SEQ ID NO: 2; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 2, wherein n is 7 or more (e. g.
  • SERCA3 proteins include variants (e. g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 2,
  • Fragments may lack one or more amino acids (e. g. 1 ,2, 3, 4,5, 6,7, 8,9, 1 0, 15, 20,25 or more) from the C -terminus and/or one or more amino acids (c. g. 1 ,2, 3,4, 5,6, 7, 8, 9,10, 15,20, 25 or more) from the N-terminus of SEQ ID NO: 2.
  • Other fragments may omit one or more domains of the protein (eg. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • Serca3 nucleotide sequence encoding a Serca3 protein is set forth as SEQ ID NO: 1 (GenBank Accession number: NM_012914.1 ).
  • Preferred Serca3 nucleotide sequences for use with the invention comprise a nucleotide sequence: (a) having 50% or more identity (e. g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99. 5% or more) to SEQ ID NO: 1 ; and/or (b) which is a fragment of at least n consecutive nucleotides of SEQ ID NO: 1 , wherein n is 7 or more (e.
  • Serca3 nucleotide sequenes include variants (e. g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 1 .
  • Serca3 protein is set forth as SEQ ID NO: 4 (GenBank Accession number: NM_001 163336.1 ).
  • Preferred Serca3 proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e, g. 60%, 65%, 70%, 75%, 80%. 85%, 90%. 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. 99. 5% or more) to SEQ ID NO: 4; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ 11) NO: 4, wherein n is 7 or more (e.g.
  • Serca3 proteins include variants (e» g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 4. Fragments may lack one or more amino acids (e. g. 1 ,2, 3, 4,5, 6,7, 8,9, 10, 15, 20,25 or more) from the C -terminus and/or one or more amino acids (e. g. 1 ,2, 3,4, 5,6, 7, 8, 9, 1 , 15,20, 25 or more) from the N-lerminus of SEQ ID NO: 4. Other fragments may omit one or more domains of the protein (eg. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • Serca3 nucleotide sequences for use with the invention comprise a nucleotide sequence: (a) having 50% or more identity (e. g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%. 94%, 95%, 96%, 97%, 98%, 99%, 99. 5% or more) to SEQ ID NO: 3; and/or (b) which is a fragment of at least n consecutive nucleotides of SEQ ID NO: 3, wherein n is 7 or more (e.
  • Serca3 nucleotide sequences include variants (e. g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 3.
  • Serca3 protein is set forth as SEQ ID " NO: 6 (GenBank Accession number: NM_ 016745.3).
  • Preferred Serca.3 proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e. g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99. 5% or more) to SEQ ID NO: 6; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 6, wherein n is 7 or more (e. g.
  • Serca3 proteins include variants ⁇ e. g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO; 6.
  • Fragments may lack one or more amino acids (e. g. 1,2, 3, 4,5, 6,7, 8,9, 10, 15, 20,25 or more) from the C-terminus and/or one or more amino acids (e. g. 1 ,2, 3,4, 5,6, 7, 8, 9, 10, 15,20, 25 or more) from the N-terminus of SEQ ID NO: 6, Other fragments may omit one or more domains of the protein (eg. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • SEQ ID NO: 5 GenBank Accession number: NM_016745.3
  • Preferred Serca3 nucleotide sequences for use with the invention comprise a nucleotide sequence: (a) having 50% or more identity (e. g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99. 5% or more) to SEQ ID NO: 5; and/or (b) which is a fragment of at least n consecutive nucleotides of SEQ ID NO: 5, wherein n is 7 or more (e. g, 8, 10, 12, 14, 16, 1 8, 20,25, 30,35, 40,50, 60,70, 80,90, 1 00, 150, 200,250 or more).
  • These Serca3 nucleotide sequences include variants ⁇ e.g. allelic variants, homo logs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 5.
  • Serea3 proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%», 90%», 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99, 5% or more) to SEQ I D NO: 8; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 8, wherein n is 7 or more (e.g.
  • Serca3 proteins include variants (e. g, allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 8.
  • Fragments may lack one or more amino acids (e.g. 1 ,2, 3, 4,5, 6,7, 8,9, 10, 15, 20,25 or more) from the C -terminus and/or one or more amino acids (e.g. 1 ,2, 3,4, 5.6, 7, 8, 9,10, 15,20, 25 or more) from the -terminus of SEQ ID NO; 8.
  • Other fragments may omit one or more domains of the protein (eg. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • Serca3 nucleotide sequence encoding a Serca3 protein is set forth as SEQ ID NO: 7 (GenBank Accession number: NM 1 74958.2).
  • Preferred Serca3 nucleotide sequences for use with the invention comprise a nucleotide sequence: (a) having 50% or more identity (e. g. 60%, 65%, 70%, 75%, 80%, 85%,. 90%, 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99. 5% or more) to SEQ ID NO: 7; and/or (b) which is a fragment of at least n consecutive nucleotides of SEQ ID NO: 7, wherein n is 7 or more (e. g.
  • Serca3 nucleotide sequences include variants (e. g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 7.
  • Serca3 protein is set forth as SEQ I D NO: 10 (GenBank Accession number: N _0051 73).
  • Preferred Serca3 proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (c. g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 1 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99. 5% or more) to SEQ ID NO: 10; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 10, wherein n is 7 or more (e.
  • Serca3 proteins include variants (e. g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 10. Fragments may lack one or more amino acids (e. g. 1 ,2, 3, 4,5, 6,7, 8,9, 10, 15, 20,25 or more) from the C-terminus and/or one or more amino acids (e. g. 1 ,2, 3,4, 5,6, 7, 8, 9, 10, 15,20, 25 or more) from the N-lerminus of SEQ ID NO: 10. Other fragments may omit one or more domains of the protein (eg. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • Serca3 nucleotide sequence encoding a Serca3 protein is set forth as SEQ ID NO: 9 (GenBank Accession number: NM_005 I 73).
  • Preferred Serca3 nucleotide sequences for use with the invention comprise a nucleotide sequence: (a) having 50% or more identity (e. g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99%, 99. 5% or more) to SEQ I D NO: 9; and/or (b) which is a fragment of at least n consecutive nucleotides of SEQ ID NO: 9, wherein n is 7 or more (e. g.
  • Scrca3 nucleotide sequences include variants (e. g. allelic variants, homologs. orthologs, paralogs, mutants, etc.) of SEQ I D NO: 9.
  • Serca3 protein is set forth as SEQ ID NO: 12 (GenBank Accession number: NM 174955), Preferred Serca3 proteins for use with the invention comprise an amino add sequence: (a) having 50% or more identity (e. g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 1 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99. 5% or more) to SEQ ID NO: 12; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 12, wherein n is 7 or more (e. g.
  • Serca.3 proteins include variants (e. g, allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 12, Fragments may lack one or more amino acids (e. g. 1 ,2, 3, 4,5, 6,7, 8,9, 10, 1 , 20,25 or more) from the C -terminus and/or one or more amino acids (e, g.
  • fragments may omit one or more domains of the protein (eg, omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • Serca3 nucleotide sequence encoding a Serca3 protein is set forth as SEQ ID NO: 1 1 (GenBank Accession number: NM_174955).
  • Preferred Serca3 nucleotide sequences for use with the invention comprise a nucleotide sequence: (a) having 50% or more identity (e. g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.
  • SEQ ID NO: 1 1 5% or more to SEQ ID NO: 1 1 ; and/or (b) which is a fragment of at least n consecutive nucleotides of SEQ ID NO: 1 1 , wherein n is 7 or more (e. g. 8, 10, 12, 14, 16, 18, 20,25, 30,35, 40,50, 60,70, 80,90, 100, 1 50, 200,250 or more).
  • Serca3 nucleotide sequences include variants (e. g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 1 1 ..
  • Serca3 protein is set forth as SEQ ID NO: 14 (GenBank Accession number: NM_174954)
  • Preferred Serca3 proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e. g. 60%, 65%, 70%, 75%, 80%. 85%, 90%, 9 1 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99. 5% or more) lo SEQ ID NO: 14; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO; 14, wherein, n is 7 or more (e. g. 8, 10, 12, 14, 16,18, 20,25, 30,35, 40,50.
  • Serca3 proteins include variants (e. g. allelic variants, homologs, orthologs,. paralogs, mutants, etc.) of SEQ ID NO: 14. Fragments may lack one or more amino acids (e. g. 1 ,2, 3, 4,5, 6,7, 8,9, 10, 15, 20,25 or more) from the C-term in s and/or one or more amino acids (e. g. 1 ,2, 3,4. 5,6, 7, 8, 9, 1 0, 15,20, 25 or more) from the N-terminus of SEQ ID NO: 14. Other fragments may omit one or more domains of the protein (eg, omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain),
  • Serca3 nucleotide sequence encoding a Serca3 protein is set forth as SEQ ID NO: 13 (GenBank Accession number: NM 1 74954 ).
  • Preferred Serca3 nucleotide sequences for use with the invention comprise a nucleotide sequence: (a) having 50% or more identity (e. g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99. 5% or more) to SEQ ID NO: 13; and/or (b) which is a fragment of at least n consecutive nucleotides of SEQ ID NO: 1 3, wherein n is 7 or more (e. g.
  • SE CA3 nucleotide sequences include variants (e. g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 13.
  • Serca3 protein is set forth as SEQ ID NO: 16 (GenBank Accession number; NM 1 74953 ).
  • Preferred Serca3 proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e. g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99. 5% or more) to SEQ ID NO: 16; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 16, wherein n is 7 or more (e. g.
  • Serca3 proteins include variants (e. g. allelic variants, homologs, ortho!ogs, paralogs, mutants, etc.) of SEQ ID NO: 16. Fragments may lack one or more amino acids (e. g, 1 ,2, 3, 4,5, 6,7, 8,9, 10, 15, 20,25 or more) from the C-terminus and/or one or more amino acids (e, g. 1 ,2, 3,4, 5,6, 7, 8, 9, 10, 15,20, 25 or more) from the N-terminus of SEQ ID NO: 16. Other fragments may omit one or more domains of the protein ⁇ eg. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • Serca3 nucleotide sequence encoding a Serca3 protein is set forth as SEQ ID NO: 15 (GenBank Accession number: NM_J 74953).
  • Preferred Serca3 nucleotide sequences for use with the invention comprise a nucleotide sequence: (a) having 50% or more identity (e. g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99. 5% or more) to SEQ ID NO: 15; and/or (b) which is a fragment of at least n consecutive nucleotides of SEQ ID NO: 1 , wherein n is 7 or more (e.
  • Serca3 nucleotide sequences include variants (e. g. allelic variants, homologs, orthotogs, paralogs, mutants, etc.) of SEQ ID NO: 15.).
  • Serca3 protein is set forth as SEQ I D NO: 18 (GenBank Accession number: N _174957).
  • Preferred Serca3 proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e. g, 60%,, 65%, 70%, 75%. 80%, 85%, 90%, i %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99. 5% or more) to SI Q ID NO: 18; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 1 8, wherein n is 7 or more (e, g.
  • Serca3 proteins include variants (e. g. al lelic variants, homologs, orthologs, para logs, mutants, etc.) of SEQ ID NO: 1 8
  • Fragments may lack one or more amino acids (c. g. 1 ,2, 3, 4,5, 6,7, 8,9, 10, 15, 20,25 or more) from the C-terminus and/or one or more amino acids (e. g. 1 ,2, 3,4, 5,6, 7, 8, 9, 10, 15,20, 25 or more) from the N-terminus of SEQ ID NO: 18.
  • Other fragments may omit one or more domains of the protein (eg. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • Serca3 nucleotide sequence encoding a Serca3 protein is set forth as SEQ ID NO: 17 (GenBank Accession number: MM_174957).
  • Preferred Serca3 nucleotide sequences for use with the invention comprise a nucleotide sequence: (a) having 50% or more identity (e. g, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99, 5% or more) to SEQ ID NO; 17; and/or (b) which is a fragment of at least n consecutive nucleotides of SEQ ID NO: 17, wherein n is 7 or more (e. g.
  • Serca3 nucleotide sequences include variants (c. g. allelic variants, homo logs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 17..
  • SEQ ID NO: 1 GenBank Accession number: NMJ ) 1 2 14.1
  • SEQ ID NO: 3 GenBank Accession number: NM_001 163336.1
  • SEQ ID NO: 5 GenBank Accession number:
  • nucleotide sequences for use with the invention comprise a nucleotide sequence that is substantially homologous to SEQ ID NO: I , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 1 7.
  • a "substantially homologous" nucleotide sequence of the present invention has 50% or more identity (e. g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99. 5% or more) to SEQ ID NO: I , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17.
  • identity refers to an exact nucleotide-to-nuclcotide or amino acid-to- amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity”. The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and
  • a preferred method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F, Collins and Shane S.Sturrok, and distributed by InteliiGenetics, Inc. (Mountain View, CA). From this suite of packages the Smith- Walcrman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the"Match "value refleets"scquence identity.
  • BLAST Altschul et al.
  • nucleotide sequence is considered “substantially homologous" nucleotide if it is capable of selectively hybridizing to the nucleotide sequences disclosed herein under, for example, stringent conditions, as defined for that particular system.
  • stringent hybridization conditions can include 50% formamide, 5x Denhardt's Solution, 5 SSC, 0. 1 % SDS and 100 pg/ml denatured salmon sperm D A and the washing conditions can include 2x SSC, 0. 1% SDS at 37 C followed by Ix. SSC, 0. 1% SDS at 68 C.
  • SEQ ID O: 12, SEQ ID NO: 14, SEQ ID NO: 16 and, SEQ ID NO: 1 8 is 90% or more (e. g. 1 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more).

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Abstract

L'invention concerne des procédés de criblage de composés qui modulent l'activité de SERCA3, et régulent ainsi la production de GLP-1, ainsi que des méthodes de traitement ou de prévention des maladies métaboliques, de l'obésité, des maladies et des troubles impliquant une motilité intestinale anormale et d'autres maladies et troubles induits par GLP-1, et des compositions comprenant des inhibiteurs de SERCA3. L'invention concerne également un modulateur de Serca3 utilisé comme médicament, un modulateur de Serca3 utilisé pour traiter les états pathologiques précités et l'utilisation d'un modulateur de Serca3 dans la préparation d'un médicament pour traiter les états pathologiques précités.
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EP4309515A4 (fr) * 2021-04-07 2025-07-09 Nagase Viita Co Ltd Composition favorisant la sécrétion de glp-1

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EP4309515A4 (fr) * 2021-04-07 2025-07-09 Nagase Viita Co Ltd Composition favorisant la sécrétion de glp-1

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