Identification of TRPML3 (MCOLN3) as a salty taste receptor and use in assays for identifying taste (salty) modulators and/or therapeutics that modulate sodium transport, absorption or excretion and/or aldosterone and/or vasopressin production or release

A taste receptor, salty taste technology, applied in the identification of TRPML3 (MCOLN3) as a salty taste receptor and in the identification of the regulation of sodium transport, absorption or excretion and/or production of aldosterone and/or vasopressin or Field of use in the determination of released taste (salt) modulators and/or therapeutic agents

Inactive Publication Date: 2011-03-30
SENOMYX INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0043] However, to the best of the inventors' knowledge, no one has previously shown that TRPML3 or the related genes TRPML1 or TRPML2 are involved in salty taste or encode taste receptor polypeptides that sense and respond to salty stimuli in different mammals or other vertebrates.

Method used

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  • Identification of TRPML3 (MCOLN3) as a salty taste receptor and use in assays for identifying taste (salty) modulators and/or therapeutics that modulate sodium transport, absorption or excretion and/or aldosterone and/or vasopressin production or release
  • Identification of TRPML3 (MCOLN3) as a salty taste receptor and use in assays for identifying taste (salty) modulators and/or therapeutics that modulate sodium transport, absorption or excretion and/or aldosterone and/or vasopressin production or release
  • Identification of TRPML3 (MCOLN3) as a salty taste receptor and use in assays for identifying taste (salty) modulators and/or therapeutics that modulate sodium transport, absorption or excretion and/or aldosterone and/or vasopressin production or release

Examples

Experimental program
Comparison scheme
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preparation example Construction

[0421] The preparation and screening of combinatorial chemical libraries are well known to those skilled in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, for example, U.S. Patent No. 5,010,175, Furka, Int J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature 354:84-88 ( 1991)). Other chemicals that produce chemical diversity libraries can also be used. Such chemical substances include, but are not limited to: peptoids (for example, PCT Publication No. WO 91 / 19735), coded peptides (for example, PCT Publication No. WO 93 / 20242), random biological oligomers (for example, PCT Publication No. WO 92 / 00091), benzodiazepines (for example, U.S. Patent No. 5,288,514), such as hydantoin, benzodiazepines and dipeptide diversomer (Hobbs et al., Proc. Nat. Acad. Sci. USA90: 6909-6913 (1993)), ethylene polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114: 6568 (1992)), non-peptide mimetic peptides with glucose scaffolds...

Embodiment 1

[0521] This example relates to the experimental and molecular biology data contained in Figure 1, which shows that TRPML3 is a taste-specific gene. The human (left) and monkey (right) taste buds (taste) and tongue epithelial cells (tongue) collected by laser capture microdissection were subjected to RT-PCR. Figure 1 shows that TRPML3 is only expressed in taste cells, similar to the known taste-specific genes T1R2 and TRPM5. The figure also shows that the housekeeping gene β-actin is expressed in taste and tongue cells, indicating that the RNA from both samples is of high quality. ‘+’ indicates that reverse transcription was performed, and ‘-’ indicates that reverse transcription was not performed (negative control). Only bands with reverse transcription were observed. All bands were cloned and sequenced to confirm the identity of the gene.

Embodiment 2

[0523] This example contains the electrophysiological assay included in Figure 2, which reveals that TRPML 3 forms sodium channels. Whole cell patch clamp electrophysiology of cells expressing human TRPML3 was performed as described therein. It can be seen that TRPML3 produces a sodium leakage current that is suppressed when sodium is removed and replaced with a large impermeable cation NMDG. The upper trace in the same figure shows the current at a holding potential of -60mV. The middle trace of Figure 2 shows the current-voltage traces from -100 mV to +60 mV in the presence (NaCl) and absence (NMDG-Cl) of sodium. The bottom graph in the figure shows the current-voltage curve in the presence (dark blue line; diamond) and absence (red-purple line; square) of sodium. It can be seen that TRPML3 exhibits inward rectification (stronger current under negative voltage compared to positive voltage).

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Abstract

The present invention relates to high-throughput mammalian and medium-throughput oocyte-based electrophysiological assays for identifying human TRPML3 modulators, preferably TRPML3 enhancers. Compounds that modulate TRPML3 function in the assay are expected to affect salty taste in humans. The inventive electrophysiological assays, such as the two-electrode voltage-clamp technique, facilitate the identification of compounds which specifically modulate human TRPML3. The assays of the invention provide a robust screen useful to detect compounds that facilitate (enhance) or inhibit TRPML3 function. Compounds that enhance or block TRPML3 channel activity should thereby modulate salty taste. In addition, these compounds may be used to regulate sodium excretion, urinary output and other biological functions relating to sodium levels. This invention relates to the elucidation that TRPML3 is involved in salty taste perception in primates including humans and likely other mammals (given the significance of sodium and other ions to physiological functions and conditions this phenotype is likely strongly conserved in different animals). The TRPML3 gene also modulates one or more of sodium metabolism, sodium excretion, blood pressure, fluid retention, cardiac function and urinary functions such as urine production and excretion. The inventors have identified TRPML3 as encoding a salty taste receptor in primates and humans (and likely other mammals) based on gene expression assays which have determined that TRPML3 is expressed specifically in taste bud cells and not in lingual epithelial cells, similar assays that have determined that TRPML3 is specifically expressed or enriched in the top half of taste bud cells in a subset of taste cells which do not express TRPM5 or PKD2L1, prior reports that document the expression of TRPML3 in other sensory organs such as the ear (therefore further substantiating the role of TRPML3 as a 'professional' sensory gene).

Description

[0001] Related temporary and non-temporary applications [0002] This application relates to a provisional application previously filed by the assignee Senomyx Inc concerning new principles for identifying primate taste-specific genes and particularly for identifying primate salty taste receptor genes. These provisional applications: the U.S. application serial number 60 / 929,017 filed on June 8, 2007; the U.S. application serial number 60 / 929,007 filed on June 8, 2007; the U.S. application serial number filed on June 29, 2007 No. 60 / 947,052; U.S. Application Serial No. 60 / 935,297 filed on August 3, 2007; U.S. Application Serial No. 60 / 987,611 filed on November 13, 2007; U.S. Application Serial Filed on November 19, 2007 No. 60 / 988,938; US application serial No. 60 / 991,274 filed on November 30, 2007; US application serial No. 60 / 991,289 filed on November 30, 2007; US application filed on December 5, 2007 Serial No. 60 / 992,502; US Application Serial No. 60 / 992,517 filed on December...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07C07K14/705A01K67/027C12Q1/68C12Q1/02A61K38/17A61P1/16A61P9/12A61P7/00A61P3/06A61P5/00A61P3/04A61P9/10A61P5/50A61P27/02A61P15/08A61P5/20A61P5/22A61P5/18A61P17/00G01N33/68A23L1/227A23L27/21
Inventor B·莫耶尔A·兹洛托尼克P·赫维兹H·索托鲁敏高娜G·瑟文特P·布鲁斯特M·威廉姆斯D·卡拉贝特E·C·怀特D·达罕B·莱塔M·萨加尼奇
Owner SENOMYX INC
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