Modified ligand-gated ion channels and methods of use

Patent metadata

Field	Value
Publication number	US-2018009862-A1
Application number	US-201715644295-A
Country	US
Kind code	A1
Filing date	Jul 7, 2017
Priority date	Jul 7, 2016
Publication date	Jan 11, 2018
Grant date	—

Abstract

This document relates to materials and methods for controlling ligand gated ion channel (LGIC) activity. For example, modified LGICs including at least one LGIC subunit having a modified ligand binding domain (LBD) and/or a modified ion pore domain (IPD) are provided. Also provided are exogenous LGIC ligands that can bind to and activate the modified LGIC, as well as methods of modulating ion transport across the membrane of a cell of a mammal, methods of modulating the excitability of a cell in a mammal, and methods of treating a mammal having a channelopathy.

First claim

What is claimed is:

1 . A modified ligand gated ion channel (LGIC) comprising at least one modified LGIC subunit, said modified LGIC subunit comprising:
an alpha7 nicotinic acetylcholine receptor (α7-nAChR) ligand binding domain (LBD) comprising an amino acid modification, and an ion pore domain (IPD).

2 . The modified LGIC of claim 1 , wherein the α7 nAChR LBD comprises a sequence having at least 75 percent sequence identity to a sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11.

3 . The modified LGIC of claim 2 , wherein the α7-nAChR LBD comprises an amino acid substitution at one or more of amino acid residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, and 219.

4 . The modified LGIC of claim 3 , wherein the amino acid substitution at residue 77 is W77F or W77Y; wherein the amino acid substitution at residue 79 is Q79A, Q79Q or Q79S; wherein the amino acid substitution at residue 115 is Y115F; wherein the amino acid substitution at residue 131 is L131A or L131G; wherein the amino acid substitution at residue 139 is Q139G or Q139L; wherein the amino acid substitution at residue 175 is G175K; wherein the amino acid substitution at residue 210 is Y210F; wherein the amino acid substitution at residue 216 is P216I; wherein the amino acid substitution at residue 217 is Y217F; and wherein the amino acid substitution at residue 219 is D219A.

5 . The modified LGIC of claim 4 , wherein the α7-nAChR LBD comprises a L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution.

6 . The modified LGIC of claim 4 , wherein the α7-nAChR LBD comprises a W77F amino acid substitution, a Q79G amino acid substitution, and a G175K amino acid substitution.

7 . The modified LGIC of claim 4 , wherein the α7-nAChR LBD comprises a Q79G amino acid substitution, a Y115F amino acid substitution, and a G175K amino acid substitution.

8 . The modified LGIC of claim 4 , wherein the α7-nAChR LBD comprises a Y115F amino acid substitution and a G175K amino acid substitution.

9 . The modified LGIC of claim 4 , wherein the α7-nAChR LBD comprises a Q79G amino acid substitution and a 216I amino acid substitution.

10 . The modified LGIC of claim 1 , wherein the α7-nAChR LBD further comprises a R27D amino acid substitution and/or a E41R amino acid substitution.

11 . The modified LGIC of claim 1 , wherein the α7-nAChR LBD has reduced binding with endogenous acetylcholine (ACh).

12 . The modified LGIC of claim 1 , wherein the IPD is an IPD from a receptor selected from the group consisting of a serotonin 3 receptor (5HT3) IPD, a glycine receptor (GlyR) IPD, a gamma-aminobutyric acid (GABA) receptor IPD, and an alpha7 nicotinic acetylcholine receptor (α7-nAChR) IPD.

13 . The modified LGIC of claim 12 , wherein the IPD comprises an amino acid substitution at residue 298.

14 . The modified LGIC of claim 13 , wherein the IPD is a GlyR IPD, and wherein the amino acid substitution is an A298G substitution.

15 . The modified LGIC of claim 13 , wherein the IPD is a GABA IPD, and wherein the amino acid substitution is a W298A substitution.

16 . The modified LGIC of claim 1 , wherein the IPD is a human 5HT3 IPD, and wherein the human 5HT3 IPD further comprises a R420Q amino acid substitution, a R424D amino acid substitution, and/or a R428A amino acid substitution.

17 . The modified LGIC of claim 1 , wherein an exogenous LGIC ligand activates the modified LGIC, and wherein the exogenous LGIC ligand is selected from the group consisting of a synthetic exogenous LGIC ligand selected from the group consisting of a quinuclidine, a tropane, a 9-azabicyclo[3.3.1]nonane, a 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, and a 1,4-diazabicyclo[3.2.2]nonane.

18 . A method of treating a channelopathy in a mammal, the method comprising:
administering to a cell in the mammal the modified LGIC of claim 1 , wherein an exogenous LGIC ligand selectively binds to and activates the modified LGIC; and administering the exogenous ligand to the mammal.

19 . The method of claim 18 , wherein the channelopathy is selected from the group consisting of Bartter syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT), congenital hyperinsulinism, cystic fibrosis, Dravet syndrome, episodic ataxia, erythromelalgia, generalized epilepsy (e.g., with febrile seizures), familial hemiplegic migraine, fibromyalgia, hyperkalemic periodic paralysis, hypokalemic periodic paralysis, Lambert-Eaton myasthenic syndrome, long QT syndrome (e.g., Romano-Ward syndrome), short QT syndrome, malignant hyperthermia, mucolipidosis type IV, myasthenia gravis, myotonia congenital, neuromyelitis optica, neuromyotonia, nonsyndromic deafness, paramyotonia congenital, retinitis pigmentosa, timothy syndrome, tinnitus, seizure, trigeminal neuralgia, and multiple sclerosis.

20 . The method of claim 18 , wherein the administering the modified LGIC comprises administering a nucleic acid encoding the modified LGIC.

21 . A method of modulating the activity of a cell in a mammal, said method comprising:
administering to the cell the modified LGIC of claim 1 , wherein an exogenous LGIC ligand selectively binds to and activates the modified LGIC; and administering the exogenous ligand to the mammal.

22 . The method of claim 21 , wherein the modulating comprises increasing the activity of the cell.

23 . The method of claim 21 , wherein the modulating comprises decreasing the activity of the cell.

24 . The method of claim 21 , wherein the activity is selected from the group consisting of ion transport, passive transport, excitation, inhibition, and exocytosis.

25 . The method of claim 21 , wherein the cell is selected from the group consisting of a neuron, a glial cell, a myocyte, a stem cell, an endocrine cell, and an immune cell.

26 . The method of claim 21 , wherein the administering the modified LGIC comprises administering a nucleic acid encoding the modified LGIC.

27 . A homomeric chimeric ligand gated ion channel (LGIC) comprising chimeric LGIC subunits, each chimeric LGIC subunit comprising:
a alpha7 nicotinic acetylcholine receptor (α7-nAChR) ligand binding domain (LBD) having an amino acid substitution at residue 131; and an ion pore domain selected from the group consisting of a serotonin 3 receptor (5HT3) ion pore domain and a glycine receptor (GlyR) ion pore domain; wherein an exogenous ligand selectively binds to and activates the chimeric LGIC, wherein the exogenous ligand is tropisetron or granisetron; and wherein the chimeric LGIC has reduced binding with endogenous acetylcholine.

28 . The homomeric chimeric LGIC of claim 27 , wherein the amino acid substitution at residue 131 is a L131A substitution or a L131G substitution.

29 . The homomeric chimeric LGIC of claim 28 , wherein the amino acid substitution at residue 131 is a L131G substitution.

30 . The homomeric chimeric LGIC of claim 29 , wherein each α7-nAChR LBD further comprises a Q139L amino acid substitution and a Y217F amino acid substitution.

31 . A homomeric chimeric ligand gated ion channel (LGIC) comprising chimeric LGIC subunits, each chimeric LGIC subunit comprising:
an alpha7 nicotinic acetylcholine receptor (α7-nAChR) ligand binding domain (LBD) having an amino acid substi

Assignees

• Hughes Howard Med Inst

Inventors

• Sternson Scott
• Lee Peter
• Magnus Christopher

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• C07K14/70571 Primary

  for neuromediators, e.g. serotonin receptor, dopamine receptor · CPC title

• C07K2319/03

  containing a transmembrane segment · CPC title

• A61P21/00

  Drugs for disorders of the muscular or neuromuscular system · CPC title

• A61P25/00

  Drugs for disorders of the nervous system · CPC title