MODIFIED LIGAND-GATED ION CHANNELS AND METHODS OF USE
20180009862 · 2018-01-11
Inventors
- Scott Sternson (Chevy Chase, MD, US)
- Peter Lee (Chevy Chase, MD, US)
- Christopher Magnus (Chevy Chase, MD, US)
Cpc classification
C07K14/70571
CHEMISTRY; METALLURGY
A61P21/00
HUMAN NECESSITIES
C07K14/4713
CHEMISTRY; METALLURGY
C07K14/705
CHEMISTRY; METALLURGY
C12N15/00
CHEMISTRY; METALLURGY
International classification
C07K14/705
CHEMISTRY; METALLURGY
C12N15/00
CHEMISTRY; METALLURGY
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.
Claims
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 substitution at residue 175; and a glycine receptor ion pore domain; wherein an exogenous ligand selectively binds to and activates the chimeric LGIC, wherein the exogenous ligand is varenicline or tropisetron; and wherein the chimeric LGIC has reduced binding with endogenous acetylcholine.
32. The homomeric chimeric LGIC of claim 31, wherein the amino acid substitution at residue 175 is a G175K substitution.
33. The homomeric chimeric LGIC of claim 32, wherein each α7-nAChR LBD further comprises a Y115F amino acid substitution.
34. The homomeric chimeric LGIC of claim 32, wherein each α7-nAChR LBD further comprises a Q79G amino acid substitution and a Y115F amino acid substitution.
35. The homomeric chimeric LGIC of claim 32, wherein each α7-nAChR LBD further comprises a W77F amino acid substitution and a Q79G amino acid substitution.
Description
DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0035] This document provides modified LGICs and methods of using them. For example, this document provides modified LGICs including at least one modified LGIC subunit having a LBD and an IPD, and having at least one modified amino acid (e.g., an amino acid substitution). In some cases, a modified LGIC can be a chimeric LGIC. For example, a chimeric LGIC can include a LBD from a first LGIC and an IPD from a second LGIC. In some cases, the modified amino acid can confer pharmacological selectivity to the modified LGIC. For example, the modified amino acid can confer the modified LGIC with selective binding of an exogenous LGIC ligand. For example, the modified amino acid can confer the modified LGIC with reduced (minimized or eliminated) binding of an unmodified LGIC subunit (an LGIC subunit lacking the modification and/or an endogenous LGIC subunit). For example, the modified amino acid can confer the modified LGIC with reduced (minimized or eliminated) binding of an endogenous LGIC ligand.
[0036] Modified LGICs provided herein can be used, for example, in methods for treating channelopathies (e.g., a neural channelopathy or a muscle channelopathy). For example, a modified LGIC, and an exogenous LGIC ligand that can bind to and activate the modified LGIC, can be used to treat a mammal having a channelopathy. In some cases, a modified LGIC and an exogenous LGIC ligand can be used to modulate (e.g., activate or inhibit) ion transport across the membrane of a cell of a mammal. In some cases, a modified LGIC and an exogenous LGIC ligand can be used to modulate (e.g., increase or decrease) the excitability of a cell in a mammal.
Modified LGICs
[0037] As used herein a “modified” LGIC is an LGIC that includes at least one LGIC subunit. A modified LGIC subunit can include at least one modified amino acid (e.g., an amino acid substitution) in the LBD and/or at least one modified amino acid (e.g., an amino acid substitution) in the IPD. A modified LGIC subunit described herein can be a modification of an LGIC from any appropriate species (e.g., human, rat, mouse, dog, cat, horse, cow, goat, pig, or monkey). In some cases, a modified LGIC can include at least one chimeric LGIC subunit having a non-naturally occurring combination of a LBD from a first LGIC and an IPD from a second LGIC.
[0038] A modified LGIC can be a homomeric (e.g., having any number of the same modified LGIC subunits) or heteromeric (e.g., having at least one modified LGIC subunit and any number of different LGIC subunits). In some cases, a modified LGIC described herein can be a homomeric modified LGIC. A modified LGIC described herein can include any suitable number of modified LGIC subunits. In some cases, a modified LGIC can be a trimer, a tetramer, a pentamer, or a hexamer. For example, a modified LGIC described herein can be a pentamer.
[0039] A modified LGIC subunit described herein can be a modification of any appropriate LGIC. The LGIC can conduct anions, cations, or both through a cellular membrane in response to the binding of a ligand. For example, the LGIC can transport sodium (Na+), potassium (K+), calcium (Ca2+), and/or chloride (Cl−) ions through a cellular membrane in response to the binding of a ligand. Examples of LGICs include, without limitation, Cys-loop receptors (e.g., AChR such as a nAChR (e.g., a muscle-type nAChR or a neuronal-type nAChR), gamma-aminobutyric acid (GABA; such as GABA.sub.A and GABA.sub.A-ρ (also referred to as GABA.sub.C) receptors, GlyR, GluCl receptors, and 5HT3 receptors), ionotropic glutamate receptors (iGluR; such as AMPA receptors, kainate receptors, NMDA receptors, and delta receptors), ATP-gated channels (e.g., P2X), and phosphatidylinositol 4,5-bisphosphate (PIP2)-gated channels. In cases where a modified LGIC described herein is a chimeric LGIC, the chimeric LGIC can include a LBD selected from any appropriate LGIC and an IPD selected from any appropriate LGIC. In cases where a LGIC includes multiple different subunits (for example, a neuronal-type nAChR includes α4, β2, and α7 subunits), the LBD and/or IPD can be selected from any of the subunits. For example, a LBD from a nAChR can be a α7 LBD. A representative rat α7 nAChR amino acid sequence (including both a LBD and an IPD) is as follows.
TABLE-US-00001 SEQ ID NO: 12 MGGGRGGIWLALAAALLHVSLQGEFQRRLYKELVKNYNPLERPVANDSQP LTVYFSLSLLQIMDVDEKNQVLTTNIWLQMSWTDHYLQWNMSEYPGVKNV RFPDGQIWKPDILLYNSADERFDATFHTNVLVNASGHCQYLPPGIFKSSC YIDVRWFPFDVQQCKLKFGSWSYGGWSLDLQMQEADISSYIPNGEWDLMG IPGKRNEKFYECCKEPYPDVTYTVTMRRRTLYYGLNLLIPCVLISALALL VFLLPADSGEKISLGITVLLSLTVFMLLVAEIMPATSDSVPLIAQYFAST MIIVGLSVVVTVIVLRYHHHDPDGGKMPKWTRIILLNWCAWFLRMKRPGE DKVRPACQHKPRRCSLASVELSAGAGPPTSNGNLLYIGFRGLEGMHCAPT PDSGVVCGRLACSPTHDEHLMHGAHPSDGDPDLAKILEEVRYIANRNRCQ DESEVICSEWKFAACVVDPLCLMAFSVFTIICTIGILMSAPNFVEAVSKD FA
[0040] In some cases, a modified LGIC subunit described herein can include a LBD from a α7 nAChR. Examples of α7 nAChR LBDs include, without limitation, a human α7 nAChR LBD having the amino acid sequence set forth in SEQ ID NO:1, a human α7 nAChR LBD having the amino acid sequence set forth in SEQ ID NO:2, and a human α7 nAChR LBD having the amino acid sequence set forth in SEQ ID NO:11. In some cases, a α7 nAChR LBD can be a homolog, orthologue, or paralog of the human α7 nAChR LBD set forth in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:11. In some cases, a α7 nAChR LBD can be have at least 75 percent sequence identity (e.g., at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97% or at least 99% sequence identity) to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:11.
TABLE-US-00002 SEQ ID NO: 1 MRCSPGGVWLALAASLLHVSLQGEFQRKLYKELVKNYNPLERPVANDSQP LTVYFSLSLLQIMDVDEKNQVLTTNIWLQMSWTDHYLQWNVSEYPGVKTV RFPDGQIWKPDILLYNSADERFDATFHTNVLVNSSGHCQYLPPGIFKSSC YIDVRWFPFDVQHCKLKFGSWSYGGWSLDLQMQEADISGYIPNGEWDLVG IPGKRSERFYECCKEPYPDVTFTV SEQ ID NO: 2 MRCSPGGVWLALAASLLHVSLQGEFQRKLYKELVKNYNPLERPVANDSQP LTVYFSLSLLQIMDVDEKNQVLTTNIWLQMSWTDHYLQWNVSEYPGVKTV RFPDGQIWKPDILLYNSADERFDATFHTNVLVNSSGHCQYLPPGIFKSSC YIDVRWFPFDVQHCKLKFGSWSYGGWSLDLQMQEADISGYIPNGEWDLVG IPGKRSERFYECCKEPYPDVTFTVTMRRR SEQ ID NO: 11 MRCSPGGVWLALAASLLHVSLQGEFQRKLYKELVKNYNPLERPVANDSQP LTVYFSLSLLQIMDVDEKNQVLTTNIWLQMSWTDHYLQWNVSEYPGVKTV RFPDGQIWKPDILLYNSADERFDATFHTNVLVNSSGHCQYLPPGIFKSSC YIDVRWFPFDVQHCKLKFGSWSYGGWSLDLQMQEADISGYIPNGEWDLVG IPGKRSERFYECCKEPYPDVTFTVTMRRRTLYY
[0041] In some cases, a modified LGIC subunit described herein can include a IPD from a 5HT3 receptor. Examples of 5HT3 IPDs include, without limitation, a murine 5HT3 IPD having the amino acid sequence set forth in SEQ ID NO:3, and a human 5HT3 IPD having the amino acid sequence set forth in SEQ ID NO:4. In some cases, a 5HT3 IPD can be a homolog, orthologue, or paralog of a 5HT3 IPD set forth in SEQ ID NO:3 or SEQ ID NO:4. In some cases, a 5HT3 IPD can be have at least 75 percent sequence identity (e.g., at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97% or at least 99% sequence identity) to SEQ ID NO:3 of SEQ ID NO:4.
TABLE-US-00003 SEQ ID NO: 3 IIRRRPLFYAVSLLLPSIFLMVVDIVGFCLPPDSGERVSFKITLLLGYSV FLIIVSDTLPATIGTPLIGVYFVVCMALLVISLAETIFIVRLVHKQDLQR PVPDWLRHLVLDRIAWILCLGEQPMAHRPPATFQANKTDDCSGSDLLPAM GNHCSHVGGPQDLEKTPRGRGSPLPPPREASLAVRGLLQELSSIRHFLEK RDEMREVARDWLRVGYVLDRLLFRIYLLAVLAYSITLVTLWSIWHYS SEQ ID NO: 4 LFYVVSLLLPSIFLMVMDIVGFYLPPNSGERVSFKITLLLGYSVFLIIVS DTLPATAIGTPLIGVYFVVCMALLVISLAETIFIVRLVHKQDLQQPVPAW LRHLVLERIAWLLCLREQSTSQRPPATSQATKTDDCSAMGNHCSHMGGPQ DFEKSPRDRCSPPPPPREASLAVCGLLQELSSIRQFLEKRDEIREVARDW LRVGSVLDKLLFHIYLLAVLAYSITLVMLWSIWQYA
[0042] In some cases, a modified LGIC subunit described herein can include an IPD from a GlyR. Examples of GlyR IPDs include, without limitation, a murine GlyR IPD having the amino acid sequence set forth in SEQ ID NO:5. In some cases, a GlyR IPD can be a homolog, orthologue, or paralog of the human GlyR IPD set forth in SEQ ID NO:5. In some cases, a GlyR IPD can be have at least 75 percent sequence identity (e.g., at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97% or at least 99% sequence identity) to SEQ ID NO:5.
TABLE-US-00004 SEQ ID NO: 5 MGYYLIQMYIPSLLIVILSWISFWINMDAAPARVGLGITTVLTMTTQSSG SRASLPKVSYVKAIDIWMAVCLLFVFSALLEYAAVNFVSRQHKELLRFRR KRRHHKEDEAGEGRFNFSAYGMGPACLQAKDGISVKGANNSNTTNPPPAP SKSPEEMRKLFIQRAKKIDKISRIGFPMAFLIFNMFYWIIYKIVRREDVH NQ
[0043] In some cases, a modified LGIC subunit described herein can include an IPD from a GABA receptor (e.g., GABA.sub.A-ρ, also referred to as GABA.sub.C). Examples of GABA.sub.A-ρ IPDs include, without limitation, a human GABA.sub.A-ρ IPD having the amino acid sequence set forth in SEQ ID NO:9. In some cases, a GABA.sub.A-ρ IPD can be a homolog, orthologue, or paralog of the human GABA.sub.A-ρ IPD set forth in SEQ ID NO:9. In some cases, a GABA.sub.A-ρ IPD can be have at least 75 percent sequence identity (e.g., at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97% or at least 99% sequence identity) to SEQ ID NO:9.
TABLE-US-00005 SEQ ID NO: 9 LLQTYFPATLMVMLSWVSFWIDRRAVPARVPLGITTVLTMSTIITGVNAS MPRVSYIKAVDIYLWVSFVFVFLSVLEYAAVNYLTTVQERKEQKLREKLP CTSGLPPPRTAMLDGNYSDGEVNDLDNYMPENGEKPDRMMVQLTLASERS SPQRKSQRSSYVSMRIDTHAIDKYSRIIFPAAYILFNLIYWSIFS
[0044] In calculating percent sequence identity, two sequences are aligned and the number of identical matches of amino acid residues between the two sequences is determined. The number of identical matches is divided by the length of the aligned region (i.e., the number of aligned amino acid residues) and multiplied by 100 to arrive at a percent sequence identity value. It will be appreciated that the length of the aligned region can be a portion of one or both sequences up to the full-length size of the shortest sequence. It also will be appreciated that a single sequence can align with more than one other sequence and hence, can have different percent sequence identity values over each aligned region. The alignment of two or more sequences to determine percent sequence identity can be performed using the computer program ClustalW and default parameters, which calculates the best match between a query and one or more subject sequences, and aligns them so that identities, similarities and differences can be determined. See, e.g., Chenna et al., 2003, Nucleic Acids Res., 31(13):3497-500.
[0045] In cases where a modified LGIC subunit described herein is a chimeric LGIC subunit, the chimeric LGIC subunit can include a LBD and IPD from the same species or a LBD and IPD from different species. In some cases, a chimeric LGIC subunit can include a LBD from a human LGIC protein and an IPD from a human LGIC protein. For example, a chimeric LGIC subunit can include a human α7 LBD and a human GlyR IPD. In some cases, a chimeric LGIC subunit can include a LBD from a human LGIC protein and an IPD from a murine LGIC protein. For example, a chimeric LGIC subunit can include a human α7 LBD and a murine 5HT3 IPD.
[0046] In cases where a modified LGIC subunit described herein is a chimeric LGIC subunit, the chimeric LGIC subunit can include varied fusion points connecting the LBD and the IPD such that the number of amino acids in a LBD may vary when the LBD is fused with different IPDs to form a chimeric channel subunit. For example, the length of an α7 nAChR LBD used to form a chimeric LGIC subunit with a 5HTS IPD is different from the length of an α7 nAChR LBD used to form a chimeric LGIC subunit with a GlyR IPD (compare, for example,
[0047] A modified LGIC subunit described herein can include a LBD having at least one modified amino acid and/or an IPD having at least one modified amino acid. For example, a modified LGIC subunit described herein can include a α7 LBD having at least 75 percent sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12, and an amino acid substitution at amino acid residue 27, 41, 77, 79, 131, 139, 141, 175, 210, 216, 217, and/or 219. For example, a modified LGIC subunit described herein can include a GlyR IPD having at least 75 percent sequence identity to a sequence set forth in SEQ ID NO:5, and an amino acid substitution at amino acid residue 298 of an α7-GlyR chimeric receptor (e.g., SEQ ID NO:7). For example, a modified LGIC subunit described herein can include a GABA.sub.C IPD having at least 75 percent sequence identity to SEQ ID NO:9, and an amino acid substitution at amino acid residue 298 of an α7-GABAc chimeric receptor (e.g., SEQ ID NO:10). In some cases, a modified LGIC subunit described herein can include more than one (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more) amino acid modifications. The modification can be an amino acid substitution. In some cases, the modified amino acid can confer pharmacological selectivity to the modified LGIC. For example, the modified amino acid can confer the modified LGIC with selective binding of an exogenous LGIC ligand. For example, the modified amino acid can confer the modified LGIC with reduced (minimized or eliminated) binding of an unmodified LGIC subunit (an LGIC subunit lacking the modification and/or an endogenous LGIC subunit). For example, the modified amino acid can confer the modified LGIC with reduced (minimized or eliminated) binding of an endogenous LGIC ligand.
[0048] In some aspects, a modified LGIC subunit described herein can include at least one modified amino acid that confers the modified LGIC with selective binding (e.g., enhanced binding or increased potency) with an exogenous LGIC ligand. The binding with an exogenous LGIC ligand can be selective over the binding with an endogenous LGIC ligand. A modified LGIC subunit with selective binding with an exogenous LGIC ligand can include any appropriate LDB (e.g., a α7 LBD). In some aspects, the modified LGIC subunit can include a α7 LBD set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12, and the amino acid modification can be a substitution at amino acid residue 77, 79, 131 139, 141, 175, and/or 216. In some cases, the tryptophan at amino acid residue 77 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with a hydrophobic amino acid residue such as phenylalanine (e.g., W77F), tyrosine (e.g., W77Y), or methionine (e.g., W77M). For example, a modified LGIC subunit described herein can include a α7 LBD set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 and having a W77F substitution. In some cases, the glutamine at amino acid residue 79 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with an amino acid residue such as alanine (e.g., Q79A), glycine (e.g., Q79G), or serine (e.g., Q79S). For example, a modified LGIC subunit described herein can include a α7 LBD having a Q79G substitution. In some cases, the leucine at amino acid residue 131 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with an amino acid residue such as alanine (e.g., L131A), glycine (e.g., L131G), methionine (e.g., L131M), asparagine (e.g., L131N), glutamine (e.g., L131Q), valine (e.g., L131V), or phenylalanine (e.g., L131F). In some cases, the glycine at amino acid residue 175 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with an amino acid residue such as lysine (e.g., G175K), alanine (e.g., G175A), phenyalanine (e.g., G175F), histidine (e.g., G175H), methionine (e.g., G175m), arginine (e.g., G175R), serine (e.g., G175S), valine (e.g., G175V). In some cases, the proline at amino acid residue 216 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with an amino acid residue such as isoleucine (e.g., P216I). A modified LGIC subunit with selective binding with an exogenous LGIC ligand can include any appropriate IPD (e.g., a GlyR IPD or a GABA.sub.A-ρ IPD). In some aspects, the modified LGIC subunit can include a GlyR IPD set forth in SEQ ID NO:5, and the amino acid modification can be a substitution at amino acid residue 298 of an α7-GlyR chimeric receptor (e.g., SEQ ID NO:7). In some cases, the alanine at amino acid residue 298 of SEQ ID NO:7 can be substituted with an amino acid residue such as glycine (e.g., A298G). In some aspects, the modified LGIC subunit can include the a GABA.sub.A-ρ IPD set forth in SEQ ID NO:9, and the amino acid modification can be a substitution at amino acid residue 298 of an α7-GABA.sub.A-ρ chimeric receptor (e.g., SEQ ID NO:10). In some cases, the tryptophan at amino acid residue 298 of SEQ ID NO:10 can be substituted with an amino acid residue such as alanine (e.g., W298A).
[0049] In some cases, a modified LGIC subunit described herein can include more than one (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more) amino acid modifications. For example, a modified LGIC subunit described herein can have at least 75 percent sequence identity to SEQ ID NO:7 and can include a Q79G substitution and a A298G substitution. Additional examples of modifications that can confer the modified LGIC with selective binding of an exogenous LGIC ligand include modifications described elsewhere (see, e.g., U.S. Pat. No. 8,435,762).
[0050] A modified LGIC subunit that selectively binds (e.g., enhanced binding or increased potency) an exogenous LGIC ligand over an endogenous (e.g., a canonical) LGIC ligand can also be described as having enhanced potency for an exogenous ligand. In some cases, a modified LGIC subunit described herein that selectively binds an exogenous LGIC ligand can have at least 4 fold (e.g., at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, or at least 20 fold) enhanced potency for an exogenous ligand. In some cases, a modified LGIC subunit described herein that selectively binds an exogenous LGIC ligand can have about 4 fold to about 200 fold (e.g., about 4 fold to about 200 fold, about 5 fold to about 180 fold, about 6 fold to about 175 fold, about 7 fold to about 150 fold, about 8 fold to about 125 fold, about 9 fold to about 100 fold, about 10 fold to about 90 fold, about 11 fold to about 75 fold, about 12 fold to about 65 fold, about 13 fold to about 50 fold, about 14 fold to about 40 fold, or about 15 fold to about 30 fold) enhanced potency for an exogenous ligand. For example, a modified LGIC subunit described herein that selectively binds an exogenous LGIC ligand can have about 10 fold to about 100 fold enhanced potency for an exogenous ligand. For example, a modified LGIC subunit described herein that selectively binds an exogenous LGIC ligand can have about 10 fold to about 20 fold enhanced potency for an exogenous ligand.
[0051] In some aspects, a modified LGIC subunit described herein can include at least one modified amino acid that confers the modified LGIC with reduced (e.g., minimized or eliminated) binding with an unmodified LGIC subunit. The binding with a modified LGIC subunit having the same modification can be selective over the binding with an unmodified LGIC subunit. An unmodified LGIC subunit can be a LGIC subunit lacking the modification that confers the modified LGIC with reduced binding with an unmodified LGIC subunit or an unmodified LGIC can be an endogenous LGIC subunit. The modification that confers the modified LGIC with reduced binding with an unmodified LGIC subunit can be a charge reversal modification. A modified LGIC subunit with reduced binding with an unmodified LGIC subunit can include any appropriate LBD (e.g., a α7 LBD). In some aspects, the modified LGIC subunit can include a α7 LBD set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12, and the amino acid modification can be a substitution at amino acid residue 27 and/or 41. For example, the arginine at amino acid residue 27 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with an aspartic acid (e.g., R27D). For example, the glutamic acid at amino acid residue 41 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with an arginine (e.g., E41R). In some cases, a modified LGIC subunit described herein can include a α7 LBD having a R27D substitution and a E41R.
[0052] In some aspects, a modified LGIC subunit described herein can include at least one modified amino acid that confers the modified LGIC with reduced (e.g., minimized or eliminated) binding of an endogenous LGIC ligand. The endogenous LGIC ligand can be ACh. A modified LGIC subunit with reduced binding of an endogenous LGIC ligand can include any appropriate IPD (e.g., a GlyR LBD). For example, the modified LGIC subunit can include a α7 LBD set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12, and the amino acid modification can be a substitution at amino acid residue 115, 131, 139, 210, 217 and/or 219. In some cases, the tyrosine at amino acid residue 115 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with a phenylalanine (e.g., Y115F). In some cases, the leucine at amino acid residue 131 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with an amino acid residue such as alanine (e.g., L131A), glycine (e.g., L131G), methionine (e.g., L131M), asparagine (e.g., L131N), glutamine (e.g., L131Q), valine (e.g., L131V), or phenylalanine (e.g., L131F). In some cases, the glutamine at amino acid residue 139 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with a glycine (e.g., Q139G) or a leucine (e.g., Q139L). In some cases, the tyrosine at amino acid residue 210 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with a phenylalanine (e.g., Y210F). In some cases, the tyrosine at amino acid residue 217 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with a phenylalanine (e.g., Y217F). In some cases, the aspartate at amino acid residue 219 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with an alanine (e.g., D219A).
[0053] In some aspects, a modified LGIC subunit described herein can include at least one modified amino acid that confers the modified LGIC with increased ion conductance. In some cases, the modified LGIC subunit can include a 5HT3 IPD set forth in SEQ ID NO:3, and the amino acid modification can be a substitution at amino acid residue 425, 429, and/or 433. For example, a modified LGIC subunit described herein can include a 5HT3 IPD having a R425Q substitution, a R429D substitution, and a R433A substitution. In some cases, the modified LGIC subunit can include a 5HT3 IPD set forth in SEQ ID NO:4, and the amino acid modification can be a substitution at amino acid residue 420, 424, and/or 428. For example, a modified LGIC subunit described herein can include a 5HT3 IPD having a R420Q substitution, a R424D substitution, and a R428A substitution.
[0054] In some cases, a modified LGIC described herein can include at least one chimeric α7-5HT3 LGIC subunit (SEQ ID NO:6) having a human α7 nAChR LBD (SEQ ID NO:1) with a Q79G amino acid substitution and a Y115F amino acid substitution, and a murine 5HT3 IPD (SEQ ID NO:3).
[0055] In some cases, a modified LGIC described herein can include at least one chimeric α7-5HT3 LGIC subunit (SEQ ID NO:6) having a human α7 nAChR LBD (SEQ ID NO:1) with a Q79G amino acid substitution and a Q139G amino acid substitution, and a murine 5HT3 IPD (SEQ ID NO:3).
[0056] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD (SEQ ID NO:2) with a Q79G amino acid substitution and a Y115F amino acid substitution, and a human GlyR IPD (SEQ ID NO:5) with a A298G amino acid substitution.
[0057] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD (SEQ ID NO:2) with a Q79G amino acid substitution and a Q139G amino acid substitution, and a human GlyR IPD (SEQ ID NO:5) with a A298G amino acid substitution.
[0058] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD (SEQ ID NO:2) with a R27D amino acid substitution, a E41R amino acid substitution, a Q79G amino acid substitution, and a Y115F amino acid substitution, and a human GlyR IPD (SEQ ID NO:5) with a A298G amino acid substitution.
[0059] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD (SEQ ID NO:2) with a substitution at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N), and a human GlyR IPD (SEQ ID NO:5).
[0060] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD (SEQ ID NO:2) with a substitution at amino acid residues 131 (e.g., L131G, L131A, L131M, or L131N) and Y115 (e.g., Y115F), and a human GlyR IPD (SEQ ID NO:5).
[0061] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD (SEQ ID NO:2) with a substitution at amino acid residues 131 (e.g., L131G, L131A, L131M, or L131N) and 139 (e.g., Q139L), and a human GlyR IPD (SEQ ID NO:5).
[0062] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD (SEQ ID NO:2) with a substitution at amino acid residues 131 (e.g., L131G, L131A, L131M, or L131N) and 217 (e.g., Y217F), and a human GlyR IPD (SEQ ID NO:5).
[0063] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD (SEQ ID NO:2) with a substitution at amino acid residues 131 (e.g., L131G, L131A, L131M, or L131N), 139 (e.g., Q139L), and 217 (e.g., Y217F), and a human GlyR IPD (SEQ ID NO:5).
[0064] In some cases, a modified LGIC described herein can include at least one chimeric α7-5HT3 LGIC subunit having a human α7 nAChR LBD (SEQ ID NO:2) with a substitution at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N), and a human 5HT3 IPD (SEQ ID NO:4).
[0065] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD (SEQ ID NO:2) with a substitution at amino acid residue 175 (e.g., G175K), and a human GlyR IPD (SEQ ID NO:5).
[0066] In some cases, a modified LGIC described herein can include at least one chimeric α7-5HT3 LGIC subunit having a human α7 nAChR LBD (SEQ ID NO:2) with a substitution at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) and 139 (e.g., Q139L), and a human 5HT3 IPD (SEQ ID NO:4) with a R420Q substitution, a R424D substitution, and a R428A substitution.
[0067] In some cases, a modified LGIC described herein can include at least one chimeric α7-5HT3 LGIC subunit having a human α7 nAChR LBD (SEQ ID NO:2) with a substitution at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) and 139 (e.g., Q139L) and 217 (e.g., Y217F), and a human 5HT3 IPD (SEQ ID NO:4) with a R420Q substitution, a R424D substitution, and a R428A substitution.
[0068] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD (SEQ ID NO:2) with a substitution at amino acid residues 175 (e.g., G175K) and 115 (e.g., Y115F), and a human GlyR IPD (SEQ ID NO:5).
[0069] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD (SEQ ID NO:2) with a substitution at amino acid residues 175 (e.g., G175K) and 115 (e.g., Y115F) and 79 (e.g., Q79G), and a human GlyR IPD (SEQ ID NO:5).
[0070] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD (SEQ ID NO:2) with a substitution at amino acid residues 175 (e.g., G175K) and 77 (e.g., W77F) and 79 (e.g., Q79G), and a human GlyR IPD (SEQ ID NO:5).
[0071] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD (SEQ ID NO:2) with a substitution at amino acid residue 216 (e.g., P216I), and a human GlyR IPD (SEQ ID NO:5).
[0072] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD (SEQ ID NO:2) with a substitution at amino acid residues 216 (e.g., P216I) and 79 (e.g., Q79G), and a human GlyR IPD (SEQ ID NO:5).
[0073] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO:10) having a human α7 nAChR LBD (SEQ ID NO:2) with a substitution at amino acid residue 131 (e.g., L131A, L131G, L131M, L131N, L131Q, L131V, or L131F), and a human GABA.sub.C IPD (SEQ ID NO:9).
[0074] In cases where a LBD and/or a IPD is a homolog, orthologue, or paralog of a sequence set forth herein (e.g., SEQ ID NOs:1-5 and/or 9), it is understood that reference to a particular modified amino acid residue can shift to the corresponding amino acid in the homolog, orthologue, or paralog. For example, residues 425, 429, and 433 in a murine 5HT3 IPD set forth in SEQ ID NO:3 correspond to residues 420, 424, and 428 in a human 5HT3 IPD set forth in SEQ ID NO:4, and the R425Q, R429D, and R433A substitutions in a murine 5HT3 IPD correspond to R420Q, R424D, and R428A substitutions in a human 5HT3 IPD.
[0075] Any method can be used to obtain a modified LGIC subunit described herein. In some cases, peptide synthesis methods can be used to make a modified LGIC subunit described herein. Examples of methods of peptide synthesis include, without limitation, liquid-phase peptide synthesis, and solid-phase peptide synthesis. In some cases, protein biosynthesis methods can be used to make a modified LGIC subunit described herein. Examples of methods of protein biosynthesis include, without limitation, transcription and/or translation of nucleic acids encoding a phosphorylation-mimicking peptide provided herein. Similar modified LGIC subunits (e.g., modified subunits having essentially the same modifications and/or having essentially the same amino acid sequence) will self-assemble through interactions between the LBDs to form a modified LGIC.
[0076] This document also provides nucleic acids encoding modified LGIC subunits described herein as well as constructs (e.g., plasmids, non-viral vectors, viral vectors (such as adeno-associated virus, a herpes simplex virus, or lentivirus vectors)) for expressing nucleic acids encoding modified LGIC subunits described herein. Nucleic acids encoding modified LGIC subunits described herein can be operably linked to any appropriate promoter. A promoter can be a native (i.e., minimal) promoter or a composite promoter. A promoter can be a ubiquitous (i.e., constitutive) promoter or a regulated promoter (e.g., inducible, tissue specific, cell-type specific (e.g., neuron specific, muscle specific, glial specific), and neural subtype-specific). Examples of promoters that can be used to drive expression of nucleic acids encoding modified LGIC subunits described herein include, without limitation, synapsin, CAMKII, CMV, CAG, enolase, TRPV1, POMC, NPY, AGRP, MCH, and Orexin promoters. In some cases, a nucleic acid encoding a modified LGIC subunit described herein can be operably linked to a neuron specific promoter.
[0077] This document also provides cells (e.g., mammalian cells) having a modified LGIC described herein. Mammalian cells having a modified LGIC described herein can be obtained by any appropriate method. In some cases, a pre-assembled modified LGIC can be provided to the cell. In some cases, a nucleic acid encoding a modified LGIC subunit described herein can be provided to the cell under conditions in which a modified LGIC subunit is translated and under conditions in which multiple (e.g., three, four, five, six, or more) modified LGIC subunits can assemble into a modified LGIC described herein.
LGIC Ligands
[0078] This document also provides LGIC ligands that can bind to and activate modified LGICs described herein. A LGIC ligand that can bind to and activate modified LGICs described herein can be exogenous or endogenous. A LGIC ligand that can bind to and activate modified LGICs described herein can be naturally occurring or synthetic. A LGIC ligand that can bind to and activate modified LGICs described herein can be canonical or non-canonical. A LGIC ligand that can bind to and activate modified LGICs described herein can be an agonist or an antagonist. In some cases, an LGIC ligand is an exogenous LGIC agonist. Examples of LGIC ligands include, without limitation, ACh, nicotine, epibatatine, cytisine, RS56812, tropisetron, nortropisetron, PNU-282987, PHA-543613, compound 0353, compound 0354, compound 0436, compound 0676, compound 702, compound 723, compound 725, granisetron, ivermectin, mequitazine, promazine, varenicline, compound 765, compound 770, 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-dioxide, compound 773, and compound 774 (see, e.g.,
[0079] A LGIC ligand that can bind to and activate modified LGICs described herein can have selective binding (e.g., enhanced binding or increased potency) for a modified LGIC described herein. In some cases, a LGIC ligand that can bind to and activate modified LGICs described herein does not bind to and activate endogenous receptors. A LGIC ligand that selectively binds to and activates a modified LGIC (e.g., a modified LGIC having at least one amino acid modification that confers pharmacological selectivity to the modified LGIC) described herein over an unmodified LGIC ligand can also be described as having enhanced potency for a modified LGIC. In some cases, a modified LGIC subunit described herein that selectively binds an exogenous LGIC ligand can have at least 5 fold (e.g., at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 95 fold, at least 100 fold, at least 125 fold, at least 150 fold, at least 200 fold, at least 250 fold, or at least 300 fold) enhanced potency for a modified LGIC. For example, a LGIC ligand that selectively binds to and activates a modified LGIC can have about 10 fold to about 300 fold (e.g., about 10 fold to about 250 fold, about 10 fold to about 200 fold, about 10 fold to about 150 fold, about 10 fold to about 100 fold, about 25 fold to about 300 fold, about 50 fold to about 300 fold, about 100 fold to about 300 fold, about 200 fold to about 300 fold, about 25 fold to about 250 fold, about 50 fold to about 200 fold, or about 100 fold to about 150 fold) enhanced potency for a modified LGIC. In some cases, a LGIC ligand that binds to and activates a modified LGIC described herein can have a ligand potency of less than 25 nM (e.g., less than 22 nM, less than 20 nM, less than 17 nM, less than 15 nM, less than 13 nM, less than 12 nM, less than 11 nM, less than 10 nM, less than 5 nM, less, than 2 nM, or less than 1 nM). For example, a LGIC ligand that binds to and activates a modified LGIC described herein can have a ligand potency of less than 15 nM. In some cases, a LGIC ligand can have an EC50 of less than 25 nM (e.g., less than 22 nM, less than 20 nM, less than 17 nM, less than 15 nM, less than 13 nM, less than 12 nM, less than 11 nM, or less than 10 nM) for a modified LGIC subunit described herein. For example, a LGIC ligand (e.g., tropisetron) can have an EC50 of about 11 nM for a modified LGIC subunit described herein (e.g., α7.sup.Q79G-GlyR.sup.A298G). For example, a LGIC ligand (e.g., nortropisetron) can have an EC50 of about 13 nM for a modified LGIC subunit described herein (e.g. α7.sup.Q79G,Y115F-GlyR.sup.A298G). In some cases, a LGIC ligand can have an EC50 of greater than 20 μM (e.g., greater than 22 μM, greater than 25 μM, greater than 35 μM, greater than 50, greater than 65 μM, greater than 80 μM, or greater than 100 μM) for a modified LGIC subunit described herein. For example, a LGIC ligand (e.g., ACh) can have an EC50 of greater than 100 μM for a modified LGIC subunit described herein (e.g. α7.sup.Q79G,Y115F-GlyR.sup.A298G).
[0080] In some aspects, a LGIC ligand can be a synthetic ligand that can bind to and activate modified LGICs described herein can be a quinuclidine, a tropane, a 9-azabicyclo[3.3.1]nonane, or a 2-phenyl-7,8,9,10-tetrahydro-6H-6,10-methanoazepino[4,5-g]quinoxaline.
[0081] A LGIC ligand that can be to and activate a modified LGIC described herein can have Formula I:
##STR00007##
where X1 and X2 can independently be CH, CH2, O, NH, or NMe; each n can independently be 0 or 1; Y can be O or S; A can be an aromatic substituent; and R can be H or pyridinylmethylene. Examples of aromatic substituents include, without limitation, 4-chloro-benzene, 1H-indole, 4-(trifluoromethyl) benzene, 4-chloro benzene, 2,5-dimethoxy benzene, 4-chloroaniline, aniline, 5-(trifluoromethyl) pyridin-2-yl, 6-(trifluoromethyl) nicotinic, and 4-chloro-benzene.
[0082] A LGIC ligand that can bind to and activate a modified LGIC described herein can be a quinuclidine. A quinuclidine can have the structure of Formula II:
##STR00008##
where X3 can be O, NH, or CH2; Y can be O or S; A can be an aromatic substituent; and R can be H or pyridinylmethylene. Examples of aromatic substituents include without limitation, 1H-indole, 4-(trifluoromethyl) benzene, 4-chloro benzene, 2,5-dimethoxy benzene, 4-(trifluoromethyl) benzene, 4-chloroaniline, aniline, 5-(trifluoromethyl) pyridin-2-yl, 6-(trifluoromethyl) nicotinic, 3-chloro-4-fluoro benzene, 4-chloro-benzene, and 1H-indole. Examples of quinuclidines include, without limitation, compounds PNU-282987, PHA-543613, 0456, 0434, 0436, 0354, 0353, 0295, 0296, and 0676 (see, e.g.,
[0083] A LGIC ligand that can bind to and activate a modified LGIC described herein can be a tropane. A tropane can have the structure of Formula III:
##STR00009##
where X2 can be NH or NMe; X3 can be O, NH, or CH2; Y can be O or S; and A can be an aromatic substituent. Example of aromatic substituents include, without limitation, 1H-indole, 7-methoxy-1H-indole, 7-methyl-1H-indole, 5-chloro-1H-indole, and 1H-indazole. Examples of tropanes include, without limitation, tropisetron, pseudo-tropisetron, nortropisetron, compound 737, and compound 745 (see, e.g.,
[0084] A LGIC ligand that can bind to and activate a modified LGIC described herein can be a 9-azabicyclo[3.3.1]nonane. A 9-azabicyclo[3.3.1]nonane can have the structure of Formula IV:
##STR00010##
where X1 can be CH, X2 can be NH or NMe, X3 can be O, NH, or CH; Y can be O or S; and A can be an aromatic substituent. An example of an aromatic substituent is, without limitation, 4-chloro-benzene. Examples of 9-azabicyclo[3.3.1]nonanes include, without limitation, compound 0536, compound 0749, compound 0751, compound 0760, and compound 0763 (see, e.g.,
[0085] In some cases, a LGIC ligand can be an a 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine and can have a structure shown in Formula V:
##STR00011##
where R=H or CH3; and where A=H or an aromatic substituent. Examples of 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepines include, without limitation, varenicline, compound 0765, and compound 0770 (see, e.g.,
[0086] In some cases, a LGIC ligand can be a 1,4-diazabicyclo[3.2.2]nonane and can have a structure shown in Formula VI:
##STR00012##
where R=H, F, NO.sub.2. Examples of 1,4-diazabicyclo[3.2.2]nonanes include, without limitation, 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-dioxide, compound 0773, and compound 0774 (see, e.g.,
Methods of Using
[0087] This document also provides methods of using a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein. A LGIC ligand that can bind to and activate the modified LGIC can be used to activate a modified LGIC with temporal and/or spatial control based on delivery of the ligand.
[0088] In some aspects, a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein can be used to identify a ligand that selectively binds to a modified LGIC described herein. For example, such screening methods can include providing one or more candidate ligands to a modified LGIC described herein, and detecting binding between the candidate ligand and the modified LGIC.
[0089] Any appropriate method can be used to detect binding between a candidate ligand and the modified LGIC and any appropriate method can be used to detect activity of a modified LGIC. For example, the ability of a ligand to bind to and activate a modified LGIC can be measured by assays including, but not limited to, membrane potential (MP) assay (e.g., a fluorescence MP assay), radioactive binding assays, and/or voltage clamp measurement of peak currents and sustained currents.
[0090] In some aspects, a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein can be used to treat a mammal having a channelopathy (e.g., a neural channelopathy or a muscle channelopathy). For example, a mammal having a channelopathy can be treated by administering a modified LGIC described herein, and then administering a LGIC ligand that can bind to and activate the modified LGIC. For example, a mammal having a channelopathy can be treated by administering a modified LGIC described herein (e.g., including at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO:6) having a human α7 nAChR LBD (SEQ ID NO:2) with a R27D amino acid substitution, a E41R amino acid substitution, a Q79G amino acid substitution, and a Y115F amino acid substitution, and a human GlyR IPD (SEQ ID NO:5) with a A298G amino acid substitution), and then administering tropisetron. For example, a mammal having a channelopathy can be treated by administering a modified LGIC described herein including a modified human α7 nAChR LBD (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12) with an L131 amino acid substitution (e.g., L131G, L131A, L131M, or L131N) and, optionally, a Q79S amino acid substitution, a Q139L amino acid substitution, and/or a Y217F amino acid substitution, and then administering varenicline, tropisetron, and/or compound 765.
[0091] Any type of mammal can be treated using a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein. For example, humans and other primates such as monkeys can be treated using a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein. In some cases, dogs, cats, horses, cows, pigs, sheep, rabbits, mice, and rats can be treated using a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein.
[0092] Any appropriate method can be used to identify a mammal having a channelopathy and/or a mammal at risk of developing a channelopathy. For example, genetic testing can be used to identify a mammal having a channelopathy and/or a mammal at risk of developing a channelopathy.
[0093] Once identified as having a channelopathy and/or a mammal at risk of developing a channelopathy, the mammal can be administered or instructed to self-administer a modified LGIC described herein, and then administered or instructed to self-administer a LGIC ligand that can bind to and activate the modified LGIC as described herein. A modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein can be administered together or can be administered separately.
[0094] When treating a mammal having a channelopathy and/or a mammal at risk of developing a channelopathy using the materials and methods described herein, the channelopathy can be any channelopathy. As used herein, a channelopathy can be any disease or disorder caused by aberrant ion channel function and/or aberrant ligand function, or which could be alleviated by modulated ion channel function and/or altered cellular ion flux (e.g., calcium ion flux). A channelopathy can be congenital or acquired. Examples of channelopathies include, without limitation, 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. Alternatively, or in addition, the materials and methods described herein can be used in other applications including, without limitation, pain treatment, cancer cell therapies, appetite control, spasticity treatment, muscle dystonia treatment, tremor treatment, and movement disorder treatment.
[0095] In some cases, a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein can be used to modulate the activity of a cell. The activity of the cell that is modulated using a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein can be any cellular activity. Examples of cellular activities include, without limitation, active transport (e.g., ion transport), passive transport, excitation, inhibition, ion flux (e.g., calcium ion flux), and exocytosis. The cellular activity can be increased or decreased. For example, a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein can be used to modulate (e.g., increase) ion transport across the membrane of a cell. For example, a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein can be used to modulate (e.g., increase) the excitability of a cell.
[0096] A modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein can be used to modulate the activity of any type of cell in a mammal. The cell can be a neuron, a glial cell, a myocyte, an immune cell (e.g., neutrophils, eosinophils, basophils, lymphocytes, and monocytes), an endocrine cell, or a stem cell (e.g., an embryonic stem cell). In some cases, the cell can be an excitable cell. The cell can be in vivo or ex vivo.
[0097] A modified LGIC described herein can be administered by any appropriate method. A modified LGIC can be administered as modified LGIC subunits or as pre-assembled modified LGICs. A modified LGIC can be administered as a nucleic acid encoding a modified LGIC. A modified LGIC can be administered as a nucleic acid encoding a modified LGIC subunit described herein. For example, a nucleic acid can be delivered as naked nucleic acid or using any appropriate vector (e.g., a recombinant vector). Vectors can be a DNA based vector, an RNA based, or combination thereof. Vectors can express a nucleic acid in dividing cells or non-dividing cells. Examples of recombinant vectors include, without limitation, plasmids, viral vectors (e.g., retroviral vectors, adenoviral vectors, adeno-associated viral vectors, and herpes simplex vectors), cosmids, and artificial chromosomes (e.g., yeast artificial chromosomes or bacterial artificial chromosomes). In some cases, a nucleic acid encoding a modified LGIC subunit described herein can be expressed by an adeno-associated viral vector.
[0098] A modified LGIC described herein can be detected (e.g., to confirm its presence in a cell) by any appropriate method. In some cases, an agent that selectively binds a modified LGIC can be used to detect the modified LGIC. Examples of agents that can be used to bind to a modified LGIC described herein include, without limitation, antibodies, proteins (e.g., bungarotoxin), and small molecule ligands (e.g., PET ligands). An agent that selectively binds a modified LGIC can include a detectable label (e.g., fluorescent labels, radioactive labels, positron emitting labels, and enzymatic labels). Methods to detect LGIC expression in a cell can include fluorescence imaging, autoradiography, functional MRI, PET, and SPECT.
[0099] A modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein can be administered to a mammal having a channelopathy and/or at risk of developing a channelopathy as a combination therapy with one or more additional agents/therapies used to treat a channelopathy. For example, a combination therapy used to treat a mammal having a channelopathy as described herein can include administering a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein and treating with acetazolaminde, dichlorphenamide, mexilitine, glucose, calcium gluconate, L-DOPA, muscle stimulation, spinal stimulation, brain stimulation, and/or nerve stimulation.
[0100] In embodiments where a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein are used in combination with additional agents/therapies used to treat a channelopathy, the one or more additional agents can be administered at the same time or independently. For example, a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein first, and the one or more additional agents administered second, or vice versa. In embodiments where a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein are used in combination with one or more additional therapies used to treat a channelopathy, the one or more additional therapies can be performed at the same time or independently of the administration of a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein. For example, a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein can be administered before, during, or after the one or more additional therapies are performed.
[0101] In some cases, a modified LGIC described herein and/or a LGIC ligand that can bind to and activate the modified LGIC as described herein can be formulated into a pharmaceutically acceptable composition for administration to a mammal having a channelopathy or at risk of developing a channelopathy. For example, a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and/or a LGIC ligand that can bind to and activate the modified LGIC as described herein can be formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. A pharmaceutical composition can be formulated for administration in solid or liquid form including, without limitation, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules.
[0102] Pharmaceutically acceptable carriers, fillers, and vehicles that may be used in a pharmaceutical composition described herein include, without limitation, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
[0103] A pharmaceutical composition containing a modified LGIC described herein and/or a LGIC ligand that can bind to and activate the modified LGIC as described herein can be designed for oral, parenteral (including subcutaneous, intracranial, intraarterial, intramuscular, intravenous, intracoronary, intradermal, or topical), or inhaled administration. When being administered orally, a pharmaceutical composition containing a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and/or a LGIC ligand that can bind to and activate the modified LGIC as described herein can be in the form of a pill, tablet, or capsule. Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Compositions for inhalation can be delivered using, for example, an inhaler, a nebulizer, and/or a dry powder inhaler. The formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
[0104] A pharmaceutically acceptable composition including a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and/or a LGIC ligand that can bind to and activate the modified LGIC as described herein can be administered locally or systemically. In some cases, a composition containing a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and/or a LGIC ligand that can bind to and activate the modified LGIC as described herein can be administered systemically by venous or oral administration to, or inhalation by a mammal (e.g., a human). In some cases, a composition containing a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and/or a LGIC ligand that can bind to and activate the modified LGIC as described herein can be administered locally by percutaneous, subcutaneous, intramuscular, intracranial, or open surgical administration (e.g., injection) to a target tissue of a mammal (e.g., a human).
[0105] Effective doses can vary depending on the severity of the channelopathy, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents, and the judgment of the treating physician.
[0106] The frequency of administration can be any frequency that improves symptoms of a channelopathy without producing significant toxicity to the mammal. For example, the frequency of administration can be from about once a week to about three times a day, from about twice a month to about six times a day, or from about twice a week to about once a day. The frequency of administration can remain constant or can be variable during the duration of treatment. A course of treatment with a composition containing a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and/or a LGIC ligand that can bind to and activate the modified LGIC as described herein can include rest periods. For example, a composition containing a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and/or a LGIC ligand that can bind to and activate the modified LGIC as described herein can be administered daily over a two week period followed by a two week rest period, and such a regimen can be repeated multiple times. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the channelopathy may require an increase or decrease in administration frequency.
[0107] An effective duration for administering a composition containing a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and/or a LGIC ligand that can bind to and activate the modified LGIC as described herein can be any duration that improves symptoms of a channelopathy without producing significant toxicity to the mammal. For example, the effective duration can vary from several days to several weeks, months, or years. In some cases, the effective duration for the treatment of a channelopathy can range in duration from about one month to about 10 years. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the channelopathy being treated.
[0108] In certain instances, a course of treatment and the symptoms of the mammal being treated for a channelopathy can be monitored. Any appropriate method can be used to monitor the symptoms of a channelopathy.
[0109] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1: Potency-Enhancing Ligand Binding Domain Mutations
[0110] A screen was performed with a panel of 41 α7-5HT3 chimeric channels having mutant LBDs against a panel of 51 clinically used drugs with chemical similarity to nicotinic receptor agonists. Mutations were at residues highlighted in
TABLE-US-00006 TABLE 1 Potency of nAChR agonists against chimeric cation channels mutated at Gln79 in HEK cells. Mean EC50, SEM in parentheses (μM). Agonist α7-5HT3 α7.sup.Q79A-5HT3 α7.sup.Q79G-5HT3 α7.sup.Q79S-5HT3 Acetylcholine 7.0 (0.8) 9.2 (1.8) 6.7 (0.6) 6.2 (1.4) Nicotine 3.9 (0.4) 4.1 (1.3) 3.1 (0.5) 2.1 (0.4) Epibatidine 0.053 (0.006) 0.067 (0.022) 0.050 (0.008) 0.044 (0.006) Varenicline 0.92 (0.16) 0.76 (0.21) 0.91 (0.12) 0.47 (0.07) Cytisine 8.2 (0.3) 4.0 (0.9) 1.7 (0.2) 4.4 (1.0) RS56812 10 (1.8) 6.8 (1.9) 1.4 (0.2) 5.7 (0.8) Tropisetron 0.24 (0.03) 0.08 (0.02) 0.035 (0.002) 0.11 (0.02) Nortropisetron 0.061 (0.021) 0.010 (0.002) 0.006 (0.001) 0.019 (0.007) PNU-282987 0.22 (0.03) 0.037 (0.009) 0.018 (0.003) 0.023 (0.004)
[0111] These mutated LBDs were used to generate α7-GlyR chimeric channels having enhanced potency for most of these ligands up to 6-fold (
[0112] Another relationship that was observed in the small molecule screen was that mutations at Trp77 conferred agonist activity for the drug granisetron at the α7.sup.W77F-5HT3 (EC50: 1.2 μM), α7.sup.W77Y-5HT3 (EC50: 1.1 μM), and α7.sup.W77F-GlyR (EC50: 0.66 μM) receptors. Granisetron is a 5HT3 receptor antagonist granisetron, which does not activate α7-5HT3 or α7-GlyR.
[0113] These results show that mutation of Q79 (to A, G, or S) in the α7 nAChR LBD enhanced binding of known LGIC ligands to modified LGICs.
Example 2: Potency Enhancing Ion Pore Domain Mutations
[0114] α7-GlyR channels having IPD mutations previously established in full length glycine receptor channels (T258S and A288G, GlyR numbering; equivalent to T268S and A298G for α7-GlyR numbering) were examined for enhanced potency for the allosteric agonist ivermectin. Channels having α7-GlyR.sup.T268S were found to have substantial ligand-free open probability, which rendered them unsuitable for ligand-controlled manipulations of cells. Mutations at α7-GlyR.sup.A298G, which were effective for enhancing ivermectin potency at the full length glycine receptor, led to modest change in open probability in the absence of the ligand; thus this channel was examined for activity against a panel of known agonists. For canonical agonists ACh, nicotine, and epibatidine, as well as for varenicline and tropisetron, the agonist potency was not significantly enhanced in α7-GlyR.sup.A298G. A subset of α7 nAChR agonists did show up to a modest 4-fold increase in potency: RS56812, cytisine, PNU-282987, and nortropisetron were significantly more potent. Therefore, the effect of the IPD A298G mutation improved ligand potency, but depended on ligand structure and was not as effective as mutations in the LBD.
[0115] The Q79G mutation in the LBD and the A298G IPD mutation for α7-GlyR was examined (Table 2). The double mutant chimeric channel, α7.sup.Q79G-GlyR.sup.A298G, led to synergistic enhancement of potency showing up to 18-fold enhancement of potency relative to α7-GlyR to α7 nAChR agonists. The enhancement from this double mutant channel was greater than that from the individual mutations for agonists RS56812, tropisetron, nortropisetron, and PNU-282987. Further underscoring the unexpected structural sensitivity of this combination of mutations, multiple agonists, such as ACh, nicotine, epibatidine, varenicline, and cytisine were not significantly changed between α7-GlyR and α7.sup.Q79G-GlyR.sup.A298G. Therefore, combination of the LBD mutation Q79G with the IPD mutation A298G led to a synergistic effect where potency for some but not all nicotinic agonists was greatly increased by ˜10-20-fold.
TABLE-US-00007 TABLE 2 Potency of nAChR agonists against mutated chimeric chloride channels. Mean EC50 and SEM in parentheses (μM) for agonist activity in HEK cells expressing chimeric channels. Agonist α7 GlyR α7.sup.Q79A-GlyR α7.sup.Q79G-GlyR α7.sup.Q79S-GlyR α7-GlyR.sup.A298G α7.sup.Q79G-GlyR.sup.A298G Acetylcholine 6.4 (1.2) 7.6 (1.7) 7.1 (1.2) 4.5 (1.2) 6.4 (1.8) 4.8 (0.5) Nicotine 5.0 (1.8) 2.6 (0.7) 4.1 (0.3) 1.4 (0.4) 3.1 (1.8) 2.2 (0.6) Epibatidine 0.062 (0.021) 0.038 (0.005) 0.069 (0.011) 0.024 (0.003) 0.018 (0.001) 0.032 (0.007) Varenicline 0.62 (0.2) 0.48 (0.08) 1.1 (0.25) 0.28 (0.06) 0.25 (0.04) 0.33 (0.08) Cytisine 6.4 (2.0) 4.5 (0.6) 5.6 (2.1) 2.5 (0.7) 2.1 (0.28) 2.8 (1.0) RS56812 6.5 (1.8) 3.5 (0.5) 2.0 (0.15) 2.8 (0.5) 2.3 (0.1) 0.61 (0.14) Tropisetron 0.15 (0.045) 0.044 (0.008) 0.038 (0.003) 0.040 (0.009) 0.065 (0.026) 0.011 (0.002) Nortropisetron 0.022 (0.007) 0.004 (0.001) 0.008 (0.003) 0.005 (0.001) 0.005 (0.001) 0.002 (0.001) PNU-282987 0.13 (0.038) 0.022 (0.004) 0.026 (0.005) 0.014 (0.002) 0.035 (0.005) 0.007 (0.001)
[0116] These results show that mutation of Q79 (to A, G, or S) in the α7 nAChR LBD and/or mutation of A298 (to G) in the GlyR IPD further enhanced selective binding of known LGIC ligands to modified LGICs.
Example 3: Molecules Exhibiting Enhanced Potency
[0117] Based on the structure activity relationship of known agonists that showed enhanced potency with α7.sup.Q79G-GlyR.sup.A298G, a variety of synthetic molecules comprised of either quinuclidine, tropane, or 9-azabicyclo[3.3.1]nonane pharmacophores with one or more aromatic side chain substituents were tested. In addition, the known α7 nAChR agonist PHA-543613 (Walker et al 2006, Wishka et al 2006) was also tested and showed exceptional potency for α7.sup.Q79G-GlyR.sup.A298G. These molecules generally showed enhanced potency 10-fold to 100-fold (Table 3), indicating that, for these pharmacophores, a range of structural features were compatible with improved potency for α7.sup.Q79G-GlyR.sup.A298G.
[0118] These results show that modified LGICs can be activated by synthetic quinuclidine-containing and tropane-containing LGIC ligands.
TABLE-US-00008 TABLE 3 Potency of compounds against chimeric channels. Mean EC50 and SEM in parentheses (μM) for agonist activity in HEK cells expressing chimeric channels. Partial refers to partial agonist activity. C—X Compound X.sub.1 X.sub.2 X.sub.3 Y C.sub.1 n C.sub.2 n C.sub.3 n config R PNU-282987 N CH.sub.2 NH O 0 1 0 R H Tropisetron C NMe O O 1 0 0 Endo H Pseudo- C NMe O O 1 0 0 Exo H tropisetron Nortropisetron C NH O O 1 0 0 Endo H PHA-543613 N CH.sub.2 NH O 0 1 0 R H 0542 C NMe NH S 1 0 0 Endo H 0026 N CH.sub.2 O O 0 1 0 S H 0456 N CH.sub.2 CH.sub.2—NH S 0 1 0 mix H 0434 N CH.sub.2 NH O 0 1 0 mix pyridin-3- ylmethyl 0436 N CH.sub.2 NH O 0 1 0 mix pyridin-3- ylmethyl 0354 N CH.sub.2 NH S 0 1 0 R H 0353 N CH.sub.2 NH O 0 1 0 S H 0295 N CH.sub.2 NH O 0 1 0 S H 0296 N CH.sub.2 NH O 0 1 0 S H 0536 C NMe NH S 1 0 1 Endo H 0676 N CH.sub.2 NH O 0 1 0 S H α7-5HT3 α7-GlyR α7.sup.Q79G-GlyR.sup.A298G Compound A EC.sub.50 (μM) EC.sub.50 (μM) EC.sub.50 (μM) PNU-282987 4-chloro-benzene 0.22 0.13 0.007 Tropisetron 1H-indole 0.24 0.15 0.011 Pseudo- 1H-indole 2 0.7 <0.2 tropisetron Nortropisetron 1H-indole 0.061 0.022 0.002 PHA-543613 furo[2,3]pyridine 0.046 0.039 0.004 0542 1H-indole 3.8 0.58 0.072 0026 4-(trifluoromethyl) — 13.7 1.43 benzene 0456 4-chloro benzene — 2.8 0.47 0434 2,5-dimethoxy >10 >10 0.19 benzene 0436 4-(trifluoromethyl) 0.84 0.31 0.006 benzene 0354 4-chloroaniline 1.4 partial 1.0 0.03 0353 aniline 0.65 0.27 0.01 0295 5-(trifluoromethyl) >100 >100 4.6 pyridin-2-yl) 0296 6-(trifluoromethyl) >100 — 0.45 nicotinic 0536 4-chloro-benzene >33 >100 9.1 0676 1H-indole 0.03 0.018 0.002
Example 4: Mutations that Reduce Acetylcholine Responsiveness
[0119] The α7 nAChR has relatively low sensitivity to ACh compared to other nAChR isoforms, and potency enhancing mutations for tropane and quinuclidine ligands did not substantially alter the potency of acetylcholine at these channels. Thus, the chimeric channels were further modified to reduce acetylcholine responsiveness of these channels. Acetylcholine responsiveness was considerably reduced to more than 100 μM in some cases with additional LBD mutations Y115F and Q139G that that only modestly reduced the potency of some agonists for α7.sup.Q79G,Y115F-5HT3, α7.sup.Q79G,Q139G-5HT3, α7.sup.Q79G,Q139G-GlyR.sup.A298, α7.sup.Q79G,Y115F-GlyR.sup.A298G. For example, α7.sup.Q79G,Y115F-GlyR.sup.A298G has an EC50 of 13 nM for nortropisetron and >100 μM for ACh (Table 4).
TABLE-US-00009 TABLE 4 Potency of nAChR agonists against mutated chimeric chloride channels with low acetylcholine responsiveness. Mean EC50 and SEM in parentheses (μM) for activity in HEK cells expressing chimeric channels. α7.sup.Q79G, Y115F- α7.sup.Q79G, Q139G- α7.sup.Q79G, Y115F- α7.sup.Q79G, Q139G- α7.sup.R27D, E41R, Q79G, Y115F- 5HT3 5HT3 GlyR.sup.A298G GlyR.sup.A298G GlyR.sup.A298G Acetylcholine >100 36 (2) >100 73 (27) >100 Nicotine 34 (4) 24 (4) 22 (3) 30 (8) 7.5 (1.3) Tropisetron 0.10 (0.12) 0.31 (0.06) 0.086 (0.043) 0.26 (0.04) 0.035 (0.021) Nortropisetron 0.028 (0.005) 0.047 (0.013) 0.013 (0.001) 0.031 (0.006) 0.003 (0.001) PNU-282987 0.35 (0.07) 0.16 (0.04) 0.22 (0.04) 0.18 (0.04) 0.066 (0.010)
[0120] These results show that Y115F and/or Q139G mutations in the α7 nAChR LBD reduced binding of the endogenous LGIC ligand Ach to the modified LGIC.
Example 5: Mutations that Reduce Associations with Endogenous Receptor Subunits
[0121] Assembly of α7 nAChRs is based on associations of five homomeric subunits through interactions between the LBDs (Celie et al 2004 Neuron 41: 907-14). To minimize undesired associations with endogenous α7 nAChR subunits and/or unwanted associations of chimeric channels, potential inter-subunit salt bridges were identified by examining the crystal structure of the acetylcholine binding protein and identifying nearby inter-subunit residues with opposite charge that also have homologous ionic amino acids in the α7 nAChR receptor LBD. Charge reversal mutations (switching the acidic member of a potential salt bridge to a basic residue and its basic partner to an acidic residue) were designed to disrupt inter-subunit interactions with unmodified subunits but preserve interactions between the subunits with charge reversal mutations (
[0122] These results show that R27D and E41R mutations in α7 nAChR LBD reduced association of the modified LGIC subunits with other modified and/or endogenous LGIC subunits.
Example 6: LBD Mutations that Increase Ligand Potency
[0123] Mutations in Gly.sup.175 and Pro.sup.216 of the α7 nAChR LBD in α7-GlyR chimeric channels were tested. Mutation of Gly.sup.175 to Lys (α7.sup.G175K-GlyR) showed increased potency for ACh (5-fold) (Table 5). For α7.sup.G175K-GlyR, it was also found that nicotine potency was enhanced 10-fold relative to the unmodified α7-GlyR chimeric channel (Table 5). Mutation of Pro.sup.216 to Ile (α7.sup.P216I-GlyR) did not substantially alter ACh potency (Table 5). However, α7.sup.P216I-GlyR showed increased nicotine potency by >4-fold relative to unmodified α7-GlyR (Table 5). These potency enhancing mutations in α7.sup.G175K-GlyR and α7.sup.P216I-GlyR also affected potency of several other α7-GlyR agonists up to 30-fold (Table 5). For α7.sup.G175K-GlyR, greater than 10-fold potency enhancement over α7-GlyR was seen for the clinically used drugs tropisetron, varenicline, cytisine, granisetron, and epibatidine. For α7.sup.P216I-GlyR, potency enhancement was approximately 3-fold (Table 5).
TABLE-US-00010 TABLE 5 Agonist potency enhancement by G175K and P216I mutations at a7GlyR chimeric channels. α7GlyR α7GlyR α7GlyR α7GlyR α7GlyR α7GlyR Y115F G175K W77F Q79G Compound a7GlyR G175K P216I G175K Y210F G175K G175K Acetylcholine 6.4 (1.2) 1.2 (0.41) 4.0 (0.5) 52 (6.6) 93 (1.3) 6.8 (1.6) 4.5 (1.3) Nicotine 5.0 (1.8) 0.5 (0.25) 1.4 (0.1) 4.1 (1.4) .sup. 6 (0.5) 1.3 (0.4) 1.1 (0.1) Epibatidine 0.062 (0.021) 0.005 (0.001) 0.03 (0.01) 0.036 (0.006) 0.65 (0.11) 0.04 (0) 0.037 (0.013) Varenicline 0.62 (0.2) 0.056 (0.014) 0.18 (0.06) 5.0 (1.7) 4.3 (0.6) 0.57 (0.18) 0.42 (0.1) Cytisine 6.4 (2.0) 0.4 (0.05) 1.9 (0.2) 7.1 (1.2) >10 1.5 (0.6) 2.5 (1.1) PNU-282987 0.13 (0.038) 0.005 (0.001) 0.04 (0.004) 0.1 (0.01) 0.7 (0.3) 0.67 (0.35) 0.06 (0.05) Tropisetron 0.15 (0.045) 0.011 (0.002) 0.05 (0.003) 0.027 (0.004) 1.1 (0.2) 0.04 (0.01) 0.01 (0.001) Nortropisetron 0.022 (0.007) 0.003 (0.002) 0.006 (0.0004) 0.007 (0.001) 0.28 (0.09) 0.004 (0.001) 0.0008 (0.0001) PHA-543613 0.03 (0.01) 0.001 (0.0001) 0.009 (0.001) 0.02 (0.007) 0.26 (0.08) 0.041 (0.016) 0.003 (0.0004) Granisetron >100 3.3 (0.1) 6.1 (0.9) 1.6 (0.6) 1.4 (0.1) 0.18 (0.02) >100 Ivermectin nd nd nd nd nd nd α7GlyR α7GlyR α7GlyR α7GlyR W77F α7GlyR α7GlyR Q79G Q79G α7GlyR α7GlyR W77F Q79G W77F Q79G Y115F Y115F Y115F Q79G Q79G Y115F G175K G175K G175K G175K G175K Q139L Compound G175K G175K Y210F Y115F Y210F K322L L141F G175K Acetylcholine 41 (3.1) 143 (13) 80 (31) 98 (10) >1000 >200 58 53 Nicotine 2.6 (0.7) 6.1 (2.0) 4.2 13 (0.2) >100 14.5 3 5.8 Epibatidine 2.6 (2.3) 0.33 0.38 0.22 (0.015) >10 0.27 0.144 0.144 Varenicline 3.3 (1.0) >10 >9 >10 >30 >30 >8.1 0.96 Cytisine 6.9 (1.2) 4.02 5.1 >10 >30 >30 4.74 3.24 PNU-282987 0.5 (0.2) >1 >40 0.08 (0.01) >1 0.018 0.51 0.05 Tropisetron 0.024 (0.004) 0.1 (0.04) >1 0.027 (0.002) 0.717 0.066 0.117 0.105 Nortropisetron 0.0026 (0.0004) 0.014 >12 0.012 (0.001) >0.3 0.069 0.075 0.001 PHA-543613 0.12 (0.04) >0.3 >3 0.036 (0.006) >1 0.111 0.057 0.024 Granisetron 1.6 (0.4) 0.2 0.06 (0.01) 6.8 (1.7) 4.8 >30 0.84 >30 Ivermectin nd nd nd 0.21 nd nd nd nd nd = not determined
[0124] For use in organisms that produce ACh, it is important to reduce the endogenous ACh potency at these channels comprised of the α7 nAChR LBD. Mutation G175K could be further combined with other mutations that reduced sensitivity to ACh, such as Y115F and Y210F. For α7.sup.Y115F,G175K-GlyR, high potency for agonists based on tropane or quinuclidine core structures were found for tropisetron, granisetron, nortropisetron, PNU-282987, and PHA-543613, and greatly reduced potency for varenicline and cytisine (Table 5). For α7.sup.G175K,Y210F-GlyR, potency for most agonists was considerably reduced, however potency enhancement for granisetron was observed (Table 5).
[0125] To develop channels with reduced ACh responsiveness but high potency for other agonists, α7.sup.G175K-GlyR was combined with additional mutations that increase the potency of specific agonists. Combination with W77F reduced ACh potency, and α7.sup.W77F,G175K-GlyR showed increased potency over α7-GlyR for granisetron, nortropisetron, and tropisetron but not for PNU282-987, varenicline, cytisine, or PHA-543613 (Table 5). Combination of G175K with Q79G reduced ACh potency, and α7.sup.Q79G,G175K-GlyR showed increased potency for nortropisetron, PHA-543613, and tropisetron (Table 5). However, this potency enhancement was not observed for other agonists, such as PNU282-987, or varenicline. α7.sup.G175K,Q139L-GlyR reduced ACh potency and increased potency for nortropisetron and tropisetron (Table 5).
[0126] Further reductions in ACh potency were achieved while maintaining high potency for with synthetic agonists, including those based on tropane and quinuclidine core structures, by incorporating mutations at W77F, Q79G, L141F, Y115F, G175K, and Y210F in various combinations. α7.sup.Q79G,Y115F,G175K-GlyR reduced ACh responsiveness while maintaining potent responses to tropisetron (Table 5). These mutations also enhanced responsiveness to other tropane and quinuclidine core structures relative to α7.sup.Y115F,G175K-GlyR as well as relative to α7-5HT3 (representative of endogenous α7 nAChR activity), especially quinuclidine thioureas 702 and 703 as well as tropane ester 723, 725, 726, 736, 737, 738, and 745 (Table 6). α7.sup.Q79G,Y115F,G175K-GlyR also showed high sensitivity to ivermectin (Table 5). α7.sup.W77F,Q79G,G175K-GlyR reduced ACh responsiveness while maintaining high potency responses to tropisetron, and nortropisetron (Table 5). α7.sup.W77F,Q79G,G175K-GlyR also showed enhanced potency for additional tropane-based core structures, such as compounds 723 and 725, as well as the clinically used drugs mequitazine and promazine (Table 6). α7.sup.W77F,G175K,Y210F-GlyR reduced ACh responsiveness but markedly improved potency to granisetron (Table 5). α7.sup.L141F,Y115F,G175K-GlyR reduced ACh responsiveness while conferring sensitivity to granisetron (Table 5). α7.sup.Q79G,Q139L,G175K-GlyR reduced ACh responsiveness but showed potent responses to nortropisetron (Table 5).
TABLE-US-00011 TABLE 6 Potency enhancement of tropane, quniuclidine agonists, 9-azabicyclo[3.3.1]nonane agonists, diazabicyclo[3.2.2]nonane agonists, and promazine by G175K and P216I α7GlyR chimeric channels. Indole and indazole aromatic (A) substituents attached at 3-position. C—X Aromatic substitution α7- Agonist class X.sub.1 X.sub.2 X.sub.3 Y C.sub.1 n C.sub.2 n C.sub.3 n Configuration R (A) Compound 5HT3 Quinuclidine N CH.sub.2 NH S 0 1 0 R H 3,5-dichloro-aniline 677 10.6 Quinuclidine N CH.sub.2 NH S 0 1 0 R H 3,4-dichloro-aniline 682 >100 Quinuclidine N CH.sub.2 NH S 0 1 0 R H 4- 684 >100 (trifluoromethoxy)aniline Quinuclidine N CH.sub.2 NH S 0 1 0 R H 4-fluoroaniline 699 2.8 Quinuclidine N CH.sub.2 NH S 0 1 0 R H 3-chloro-aniline 700 1.8 Quinuclidine N CH.sub.2 NH S 0 1 0 R H 3-chloro-2-fluoroaniline 701 >100 Quinuclidine N CH.sub.2 NH S 0 1 0 R H 3-chloro-4-fluoroaniline 702 >100 Quinuclidine N CH.sub.2 NH S 0 1 0 R H 5-chloro-2-fluoroaniline 703 >100 Quinuclidine N CH.sub.2 NH S 0 1 0 R H 3-chloro-4-methylaniline 704 0.7 Quinuclidine N CH.sub.2 NH S 0 1 0 R H 5-chloro-2-methylaniline 705 >100 Quinuclidine N CH.sub.2 NH S 0 1 0 S H 4- 713 >100 (trifluoromethoxy)aniline Tropane C NMe NH S 1 0 0 Endo H 1-methyl-1H-indole 622 >100 Tropane C NMe O O 1 0 0 Endo H 4-methoxy-1H-indole 721 0.5 Tropane C NMe O O 1 0 0 Endo H 6-methoxy-1H-indole 722 0.5 Tropane C NMe O O 1 0 0 Endo H 7-methoxy-1H-indole 723 12.8 Tropane C NMe O O 1 0 0 Endo H 4-methyl-1H-indole 724 1.2 Tropane C NMe O O 1 0 0 Endo H 7-methyl-1H-indole 725 12.2 Tropane C NMe O O 1 0 0 Endo H 4-chloro-1H-indole 726 4.2 Tropane C NMe O O 1 0 0 Endo H 5-methoxy-1H-indole 736 0.83 Tropane C NMe O O 1 0 0 Endo H 5-chloro-1H-indole 737 1 Tropane C NMe O O 1 0 0 Endo H 6-chloro-1H-indole 738 0.4 Tropane C NMe O O 1 0 0 Endo H 1H-indazole 745 1.2 9-azabi- CH NMe NH O 1 0 1 Endo H 1H-indole 749 6.6 cyclo[3.3.1]no- nane 9-azabi- CH NMe NH O 1 0 1 Endo H 1H-indazole 751 1.8 cyclo[3.3.1]no- nane 9-azabi- CH NMe NH O 1 0 1 Endo H 7-methoxy-1H-indazole 760 >100 cyclo[3.3.1]no- nane 9-azabi- CH NH O O 1 0 1 Endo H 1H-indole 763 1.9 cyclo[3.3.1]no- nane 1,4- F dibenzo[b, d]thiophene 773 0.135 diazabicyclo 5,5-dioxide [3.2.2]nonane 1,4- NO.sub.2 dibenzo[b, d]thiophene 774 0.03 diazabicyclo 5,5-dioxide [3.2.2]nonane Quinuclidine N CH.sub.2 CH.sub.2 0 1 0 R H 10H-phenothiazine Mequitazine >30 N,N- 10H-phenothiazine Promazine >100 dimethylpropyl amine α7GlyR Q79G α7Gly G175K α7GlyR α7GlyR α7GlyR Q79G Y115F W77F α7GlyR Q79G Y115F G175K R27D Q79G Agonist class α7-GlyR G175K G175K G175K Y115F E41R G175K Quinuclidine 4.4 0.66 (0.06) 0.86 (0.004) 3.7 (0.7) 0.98 (0.09) 0.58 (0.14) nd Quinuclidine 0.2 0.12 (0.1) 0.013 (0.001) 0.40 (0.01) 0.13 (0.01) 0.06 (0.012) nd Quinuclidine 1.6 0.23 (0.02) 0.078 (0.022) 3.0 (0.3) 0.79 (0.04) 0.4 (0.03) nd Quinuclidine 3.6 0.26 (0.11) 0.039 (0.009) 2.9 0.52 (0.09) 0.33 (0.1) nd Quinuclidine 1.9 0.081 (0.009) 0.012 (0.0002) 1.5 0.21 (0.04) 0.11 (0.02) nd Quinuclidine nd 0.47 (0.17) 0.086 (0.014) 5.46 1.0 (0.2) 0.58 (0.03) nd Quinuclidine 0.9 0.12 (0.004) 0.018 (0.003) 1.6 0.17 (0.03) 0.12 (0.02) nd Quinuclidine nd 0.52 (0.08) 0.03 (0.01) 12.7 1.2 (0.06) 1.1 (0.5) nd Quinuclidine nd 0.062 (0.008) 0.018 (0.002) 0.76 (0.01) 0.24 (0.02) 0.18 (0.06) nd Quinuclidine nd 9.6 0.67 (0.14) >10 4.8 (1.4) 4.5 (2.7) nd Quinuclidine nd 2.1 (0.2) 0.54 (0.06) >10 23.9 >10 nd Tropane nd 0.87 1.3 (0.2) 2.5 (0.4) 0.93 (0.02) 1.0 (0.2) 1.7 Tropane nd 0.027 (0) 0.015 (0.003) 0.080 (0.002) 0.020 (0.001) 0.016 (0.001) 0.04 Tropane nd 0.02 (0.001) 0.015 (0) 0.052 (0.008) 0.028 (0.008) 0.016 (0.001) 0.03 Tropane 4 0.31 (0.02) 0.02 (0) 0.71 (0.46) 0.07 (0.01) 0.024 (0.003) 0.02 Tropane nd 0.036 (0.003) 0.012 (0.002) 0.091 (0.013) 0.02 (0.006) 0.012 (0.002) 0.06 Tropane 8.1 0.022 (0.02) 0.069 (0.33) 0.042 (0.005) 0.022 (0.0001) 0.024 Tropane nd 0.58 (0.24) 0.016 (0.001) 0.51 (0.37) 0.044 (0.006) 0.018 (0) 0.03 Tropane nd 0.2 (0.01) 0.044 (0.002) 0.57 (0.21) 0.078 (0.018) 0.078 (0.024) 0.06 Tropane 0.9 0.082 (0.004) 0.013 (0.001) 0.16 (0.03) 0.033 (0.004) 0.016 (0.001) 0.101 Tropane nd 0.015 (0) 0.016 (0.002) 0.04 (0.014) 0.025 (0.002) 0.012 (0.001) 0.033 Tropane 1.3 0.069 0.026 (0.002) 0.26 (0.03) 0.089 (0.024) 0.043 (0.014) 0.05 9-azabi- nd nd nd nd 1.3 nd 1.9 cyclo[3.3.1]no- nane 9-azabi- 3.4 nd nd nd 3.2 nd 0.7 cyclo[3.3.1]no- nane 9-azabi- 9.8 nd nd nd 3 nd 1.3 cyclo[3.3.1]no- nane 9-azabi- 0.17 nd nd nd 0.3 nd 0.2 cyclo[3.3.1]no- nane 1,4- 0.001 nd nd 0.0003 0.00042 nd 0.0014 diazabicyclo [3.2.2]nonane 1,4- 0.006 nd nd 0.00078 0.03 nd 0.03 diazabicyclo [3.2.2]nonane Quinuclidine nd nd nd nd >10 nd 0.15 N,N- nd nd nd nd >100 nd 1.6 dimethylpropyl amine nd = not determined; parentheses: SEM
[0127] α7.sup.G175K-GlyR and α7.sup.P216I-GlyR along with mutations at Q79G, Y115F, and G175K were also compatible with non-association mutations R27D,E41R as well as the GlyR IPD mutation A298G, which further enhanced ligand potency for granisetron, epibatidine, varenicline, cytisine, PNU-282987, tropisetron, nortropisetron, and PHA-543613 (Table 7). Combination with non-association mutations to form α7.sup.R27D,E41R,Q79G,Y115F,G175K further improved the potency for 702, 723, 725, and 726, with low ACh responsiveness (Table 6).
TABLE-US-00012 TABLE 7 Agonist potency enhancement by G175K and A298G mutations at α7GlyR chimeric channels as well as W298A at α7GABAc (also referred to as GABA.sub.A-ρ) channels. α7GlyR α7GlyR α7GlyR Q79G Q79G α7GlyR α7GlyR α7GlyR Q79G α7GlyR A298G α7GABAc G175K R27D Q79G Q79G A298G Q79G Y115F Q79G Y115F E41R W77F G175K G175K A298G K395 L141F R27D, Q79G Compound A298G A298G Y115F P216I K396A W298A E41R Y115F Acetylcholine 45 0.66 31 5 90 52 52 (7.7) >500 Nicotine 3.8 0.11 3.3 1.6 16.5 16.2 4.8 (0.4) >39.8 Epibatidine 0.37 0.0023 0.011 0.05 0.15 0.42 0.059 (0.03) 0.267 Varenicline 3.66 0.022 2.37 0.18 >30 6.27 4.9 (0.3) >30 Cytisine 14.1 0.134 4.6 5.5 >30 13.3 4.8 (0.4) >30 PNU-282987 1.63 0.00036 0.009 0.25 0.11 0.12 0.05 (0.03) 0.34 Tropisetron 0.018 0.0006 0.0028 0.009 0.021 0.111 0.013 (0.005) >0.096 Nortropisetron 0.0024 0.00013 0.0084 0.0012 0.0063 0.009 0.003 (0.001) 0.102 PHA-543613 0.0066 0.00018 0.0039 0.003 0.0408 0.039 0.0054 0.156 Granisetron 1.2 nd nd nd >30 >100 2.4 (0.3) >30 nd = not determined; parentheses: SEM
Additional amino acid substitutions at Gly.sup.175 of the α7 nAChR LBD in α7.sup.Y115F-GlyR chimeric channels are also enhanced agonist potency. Potency for tropisetron at α7.sup.Y115F-GlyR chimeric channels was enhanced with additional mutations, which include G175A (7.1-fold), G175F (2-fold), G175H (2.3-fold), G175K (5.6-fold), G175M (2.6-fold), G175R (5.8-fold), G175S (9.3-fold), G175V (16.7-fold).
TABLE-US-00013 TABLE 8 Agonist potency enhancement by G175 mutations at α7GlyR Y115F chimeric channels. α7GlyR α7GlyR α7GlyR α7GlyR α7GlyR α7GlyR α7GlyR α7GlyR Y115F Y115F Y115F Y115F Y115F Y115F Y115F Y115F Compound a7GlyR G175K G175A G175F G175H G175M G175R G175S G175V Acetylcholine 6.4 (1.2) 52 (6.6) 24 67 79 71 29.5 31.5 15 Varenicline 0.62 (0.2) 5.0 (1.7) 5.9 13.6 12.7 14.1 7.6 9.7 4.6 Tropisetron 0.15 (0.045) 0.027 (0.004) 0.021 0.074 0.064 0.057 0.024 0.016 0.009 PHA-543613 0.03 (0.01) 0.02 (0.007) 0.027 0.173 0.12 0.25 0.11 0.12 0.037 nd = not determined; parentheses: SEM
[0128] Mutations for Leu.sup.131 to smaller amino acids were found to reduce the potency of canonical agonists ACh and nicotine, while markedly increasing potency of varenicline, tropisetron and several other agonists. α7.sup.L131A-GlyR and α7.sup.L131G-GlyR had reduced ACh responsiveness (6-fold) and enhanced potency for varenicline (8-fold and 17-fold, respectively) and tropisetron (2.5-fold and 3.6-fold, respectively) (Table 9). α7.sup.L131G-5HT3 HC had reduced ACh responsiveness (5-fold) and enhanced potency for varenicline (16-fold) and tropisetron (2.3-fold) (
TABLE-US-00014 TABLE 9 Agonist potency enhancement by chimeric channels with L131 mutations. α7GlyR α7GlyR α7GlyR L131G α7GlyR α7GlyR α7GlyR L131G L131G Q139L Q79G Compound a7GlyR L131A L131G Q139L Y217F Y217F L131G Acetylcholine 6.4 (1.2) 42 (21) 41 (11) 68 85 83 (20) >500 Nicotine 5.0 (1.8) 8.0 (3.2) 15 (3.5) 26 28 55 (18) >100 Epibatidine 0.062 (0.021) 0.027 0.009 (0.004) 0.012 0.015 0.021 (0.002) nd Varenicline 0.62 (0.2) 0.082 (0.068) 0.037 (0.026) 0.03 0.03 0.0016 (0.001) >10 Cytisine 6.4 (2.0) 20.6 (9.4) 13.1 (0.66) 12 30 nd >30 PNU-282987 0.13 (0.038) 0.055 (0.025) 0.034 (0.008) 0.063 0.054 0.16 (0.03) 0.096 Tropisetron 0.15 (0.045) 0.06 (0.021) 0.042 (0.01) 0.13 0.087 0.31 (0.05) 0.09 Nortropisetron 0.022 (0.007) 0.006 (0.003) 0.004 (0.001) 0.024 0.018 0.047 (0.006) 0.012 PHA-543613 0.03 (0.01) 0.012 (0.006) 0.008 (0.002) 0.021 0.016 0.045 (0.008) 0.066 Granisetron >100 17.2 (12.8) 6.7 (1.6) 4 4 nd nd 765 >100 nd nd nd nd 0.031 (0.02) 0.027 770 nd nd nd nd nd 0.001 (0.0003) nd 773 0.001 nd 0.00013 0.00004 nd 0.00034 0.00004 774 0.006 nd 0.00004 0.00004 nd 0.00018 0.00004 α7GlyR α7GlyR Q79S α7GlyR Q79S α7GlyR L131G α7GlyR Q79S L131G L131G α7GlyR Q139L α7GlyR Y115F Compound L131G Q139L D219A L131F Y217F L131M L131M Acetylcholine 21 (3.5) 58 210 92 (32) 67 (3) 29 >500 Nicotine 8.2 (0.8) 25 36 20 (6.3) 41 (8) 15 nd Epibatidine 0.007 (0.001) 0.012 0.16 0.24 (0.05) 0.022 (0.004) 0.042 nd Varenicline 0.007 (0.001) 0.02 0.78 2.6 (1.1) 0.003 (0.001) 0.53 >100 Cytisine 8.1 (0.3) 10 >30 10.5 (1.8) nd 7 nd PNU-282987 0.006 (0.002) 0.018 0.41 0.20 (0.04) 0.05 (0.01) 0.021 nd Tropisetron 0.01 (0.003) 0.045 0.36 0.39 (0.2) 0.084 (0.009) 0.024 0.035 Nortropisetron 0.004 (0.002) 0.006 0.07 0.027 (0.008) 0.014 (0.002) 0.006 nd PHA-543613 0.002 (0.0005) 0.009 0.038 0.04 (0.007) 0.015 (0.001) 0.009 0.028 Granisetron 4.2 (0.8) nd >30 >100 nd 4 nd 765 0.024 nd nd nd 0.034 (0.013) nd nd 770 nd nd nd 0.034 0.001 (0.0001) 0.03 nd 773 nd nd nd 0.0005 nd 0.00005 nd 774 nd nd nd 0.0013 nd 0.001 nd α75HT3 L131G α75HT3 Q139L α7- α7GlyR α7GlyR α7GlyR L131G Y217F GABA.sub.C Compound L131N L131Q L131V HC HC L131G Acetylcholine 5 (0.5) 58 16 (5) 35 39 >500 Nicotine nd 13 3.9 (0.7) 15 20 >500 Epibatidine nd 0.027 0.21 (0.04) 0.009 nd Varenicline 0.069 (0.027) 0.72 0.33 (0.21) 0.04 0.007 0.3 Cytisine nd >30 4.3 (0.7) 11 nd >500 PNU-282987 nd 0.048 0.064 (0.018) 0.033 0.015 0.12 Tropisetron 0.025 (0.005) 0.048 0.062 (0.013) 0.066 0.04 0.18 Nortropisetron nd 0.009 0.003 (0.001) 0.009 nd 0.021 PHA-543613 0.02 0.015 0.011 (0.002) 0.012 0.009 0.027 Granisetron nd 4 5.4 (1.3) 4 nd >500 765 >10 nd nd nd 0.11 nd 770 >10 >0.3 nd nd 0.007 nd 773 0.0004 0.006 nd nd 0.002 nd 774 0.0006 0.002 nd nd 0.004 nd nd = not determined; parentheses: SEM
Example 7: Chimeric LGICs in Neurons
[0129] AAVs or DNA plasmids containing nucleic acids encoding a α7.sup.Q79G-GlyR.sup.A298G, or α7Q79G,Y115F,G175K-GlyR chimeric LGICs were transduced into mouse cortical neurons. A low concentration of tropisetron (30 nM or 100 nM) was administered to mouse cortical neurons. Neuron activity was silenced by application of low concentration of agonist (
[0130] DNA plasmids containing nucleic acids encoding a α7L131G,Q139L,Y217F-GlyR chimeric LGICs were transfected into mouse cortical neurons. Low concentration of varenicline (10 nM) was administered to mouse cortical neurons. Neuron activity was silenced by application of low concentration of agonist (
[0131] These results show that modified LGIC activity can be controlled in neurons using low concentration of the LGIC ligands tropisetron and varenicline.
Example 8: Chimeric LGICs in Therapy
[0132] Chemogenetic tools offer an attractive strategy for combined drug and gene therapy. This is because cellular function can be modulated in a consistent manner across different cell types in various indications using the same ion channels and ligands by use of an exogenously delivered ion channel that is selectively engaged by administration of a drug. Identification of ion channels that are gated by well tolerated, clinically used drugs are especially attractive for potentially extending chemogenetics to human therapeutic use.
[0133] For the drug tropisetron, we have found that it activates α7.sup.Q79G-GlyR.sup.A298G with an EC50 of 11 nM, which is similar to the reported IC50 of 10 nM tropisetron for its therapeutic target, the 5HT3 receptor (Combrink et al 2009 Pharmacological reports: PR 61: 785-97).
Other Embodiments
[0134] It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.