Modified ligand-dependent ion channels and methods of use

Abstract

This invention provides materials and methods for regulating ligand-gated ion channel (LGIC) activity. [Solution] A modified LGIC is provided, comprising at least one LGIC subunit having a modified ligand-binding domain (LBD) and / or a modified ion-pore domain (IPD). Also provided are an exogenous LGIC ligand capable of binding to and activating the modified LGIC, as well as a method for regulating ion transport across the membrane of mammalian cells, a method for regulating cellular excitability in mammals, and a method for treating mammals with channel disorders.

Description

[Technical Field]

[0001] Cross-reference with related applications This application claims the interests of U.S. Patent Application No. 62 / 584,428, filed November 10, 2017, and U.S. Patent Application No. 62 / 729,716, filed September 11, 2018. The disclosures of these prior applications are deemed to be part of the disclosure of this application (and are incorporated by reference in the disclosure of this application).

[0002] 1. Technical field This specification relates to materials and methods for modulating ligand-gated ion channel (LGIC) activity. For example, this specification provides modified LGICs comprising at least one LGIC subunit having a modified ligand-binding domain (LBD) and / or a modified ion pore domain (IPD). Exogenous LGIC ligands that can bind to and activate modified LGICs are also provided. In certain cases, modified LGICs and exogenous ligands can be used to treat mammals having channel disorders (e.g., neural channel disorders or muscle channel disorders). In certain cases, modified LGICs and exogenous LGIC ligands can be used to modulate (e.g., activate or inhibit) ion transport across the membrane of mammalian cells. In certain cases, modified LGICs and exogenous LGIC ligands can be used to modulate (e.g., increase or decrease) cellular excitability in mammals. [Background technology]

[0003] 2. Background information Ion channels mediate ion flux within cells, which deeply influences their biological functions. A prominent example of this is in neurons, where ion channels control electrical signaling within and / or between neurons to affect physiology, sensation, behavior, mood, and cognition.

[0004] Different LGICs have distinct ligand-binding properties and specific ion-conducting properties (Hille 2001 Ion Channels of Excitable Membranes. pp. 814. Sunderland, MA: Sinauer Associates (Non-Patent Literature 1); Kandel et al 2000 Principles of Neural Science. USA: McGraw-Hill Co. 1414 pp (Non-Patent Literature 2)). For example, nicotinic acetylcholine receptors (nAChRs) bind to the endogenous ligand acetylcholine (ACh), which activates the conductance of the cation, typically depolarizing the cell and thereby increasing its excitability. In contrast, glycine receptors (GlyRs) bind to the endogenous ligand glycine, which activates the conductance of the chloride anion, typically decreasing the cell's excitability by hyperpolarization and / or electroshunting of cell membrane resistance. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Hille 2001 Ion Channels of Excitable Membranes. pp. 814. Sunderland, MA: Sinauer Associates. [Non-Patent Document 2] Kandel et al 2000 Principles of Neural Science. USA: McGraw-Hill Co. 1414 pp [Overview of the project]

[0006] overview The levels of endogenous LGIC ligands (e.g., agonists) such as ACh are not easily controlled. This specification provides materials and methods for modulating LGIC activity (e.g., increasing the sensitivity of LGIC to exogenous ligands and / or decreasing its sensitivity to endogenous ligands such as ACh). For example, this specification provides modified LGIC comprising at least one modified LGIC subunit having LBD and IPD and at least one modified amino acid (e.g., amino acid substitution). Exogenous LGIC ligands that can bind to and modulate (e.g., activate) modified LGIC are also provided. In certain cases, modified LGIC and exogenous ligands can be used to treat mammals having channel disorders (e.g., neural channel disorders or muscle channel disorders). In certain cases, modified LGIC and exogenous LGIC ligands can be used to modulate (e.g., activate or inhibit) ion transport across the membrane of mammalian cells. In certain cases, modified LGIC and exogenous LGIC ligands can be used to modulate (e.g., increase or decrease) cellular excitability in mammals.

[0007] The ability to control LGIC activity offers a unique and yet-to-be-realized opportunity for controlling intracellular ion transport. For example, a modified LGIC with increased sensitivity to one or more exogenous LGIC ligands can be used to provide temporal and spatial control of ion transport and / or cellular excitability based on the delivery of exogenous LGIC ligands. For example, a modified LGIC with reduced sensitivity to endogenous LGIC ligands prevents undesirable activation of the modified LGIC and allows for selective control of the modified LGIC by exogenous ligands. Furthermore, an exogenous LGIC ligand with enhanced potency against the modified LGIC improves the selectivity of targeting the modified LGIC to endogenous ion channels. Therefore, the modified LGIC and exogenous LGIC ligands provided herein are useful for achieving therapeutic effects while mitigating side effects from small molecules targeting unintended targets.

[0008] As described herein, one or more mutations in modified LGIC can enhance the potency against exogenous LGIC ligands. Mutations in the α7 LBD of α7-GlyR at residue L131 (e.g., substituting Leu with Gly or Ala) reduced ACh potency against α7-GlyR (-6.4x) while increasing potency against varenicline (16x) and tropisetron (3.6x). Mutations in the α7 LBD of α7-GlyR at residue G175 (e.g., G175K) or P216 (e.g., P216I) enhanced potency against ACh, nicotine, tropisetron, varenicline, and other quinuclidines and tropane agonists. Combining a mutation at residue G175K with a mutation that reduces the potency of the endogenous agonist ACh (e.g., Y115F) produced α7-GlyR Y115F G175K, which increased potency against tropisetron (5.5 times) and decreased potency from ACh (-8 times). Furthermore, combining mutations in the α7 LBD at residues 77 (e.g., Trp replaced with Phe or Tyr) and / or 79 (e.g., Gln replaced with Gly, Ala, or Ser) and / or 131 (e.g., Leu replaced with Gly or Ala) and / or 141 (e.g., Leu replaced with Phe or Pro) in these chimeric channels with potency-enhancing mutations at residue G175 (e.g., G175K) or P216 (e.g., P216I) increased potency against different ligands and / or decreased ACh potency. For example, chimeric α7-GlyR LGIC having α7 nAChR LBD (α7 LBD) with mutations at residue 79 (e.g., Gln replaced with Gly), residue 115 (e.g., Tyr replaced with Phe), and residue 175 (e.g., Gly replaced with Lys) exhibits more than 100-fold increased sensitivity to exogenous tropane LGIC ligand compound 723 (tropane) and decreased ACh sensitivity (-15-fold) compared to unmodified chimeric α7-GlyR LGIC.Furthermore, modified LGICs containing at least one chimeric LGIC subunit having a mutation at residue 79 (e.g., substituting Gln with Ala, Gly, or Ser) in the α7 nAChR LBD (α7 LBD) and a mutation at residue 298 (e.g., substituting Ala with Gly) in the GlyR IPD exhibited nearly 20-fold increased sensitivity to exogenous LGIC ligands such as quinuclidines or tropanes. Further mutations at residues 27 (e.g., substituting Arg with Asp) and 41 (e.g., substituting Glu with Arg) of the α7 LBD reduced the association of modified chimeric LGICs with unmodified ion channels. Further mutations in residues 115 (e.g., substituting Tyr with Phe), 139 (e.g., substituting Gln with Gly or Leu), 210 (e.g., substituting Tyr with Phe), 217 (e.g., substituting Tyr with Phe), and / or 219 (e.g., substituting Asp with Ala) of the α7 LBD reduced the sensitivity of the chimeric LGIC to the endogenous ligand ACh. These chimeric LGICs enable highly selective regulation of cellular function in mammalian cells while minimizing cross-reactivity with endogenous signaling pathways in mammals.

[0009] In general, one aspect of this specification features a modified LGIC having at least one modified LGIC subunit comprising an amino acid modified LBD and an IPD, wherein an exogenous LGIC ligand activates the modified LGIC. The modified LGIC may be a chimeric LGIC having an LBD from a first LGIC and an IPD from a second LGIC. The LBD may be an alpha-7 nicotinic acetylcholine receptor (α7-nAChR) LBD. The modified LGIC according to claim 3, wherein at least one modified amino acid in the α7-nAChR LBD comprises an amino acid substitution at an amino acid residue selected from the group consisting of residues 77, 79, 131, 139, 141, 175, and 216 of the α7-nAChR LBD. The amino acid substitution may be at residue 79 of the α7 LBD, and the amino acid substitution may be Q79A, Q79G, or Q79S. For example, the amino acid substitution at residue 79 of the α7 LBD may be Q79G. IPD can be a serotonin 3 receptor (5HT3) IPD, a glycine receptor (GlyR) IPD, a gamma-aminobutyric acid (GABA) receptor IPD, or an α7-nAChR IPD. An IPD can be a GlyR IPD, which may contain an amino acid substitution (e.g., A298G substitution) at residue 298 of the chimeric LGIC. An IPD can be a GABA IPD, which may contain an amino acid substitution (e.g., W298A substitution) at residue 298 of the modified LGIC. The modified LGIC may be a chimeric LGIC containing an α7 LBD with the Q79G amino acid substitution and a GlyR IPD with the A298G amino acid substitution. The exogenous LGIC ligand may be a synthetic exogenous LGIC ligand selected from the group consisting of quinuclidine, tropane, 9-azabicyclo[3.3.1]nonane, 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, and 1,4-diazabicyclo[3.2.2]nonane. If the synthetic exogenous LGIC ligand is tropane, then tropane may be tropisetron, pseudotropisetron, nortropisetron, compound 723, compound 725, compound 737, or compound 745.If the synthetic exogenous LGIC ligand is quinuclidine, the quinuclidine may be PNU-282987, PHA-543613, compound 0456, compound 0434, compound 0436, compound 0354, compound 0353, compound 0295, compound 0296, compound 0536, compound 0676, or compound 702. If the synthetic exogenous LGIC ligand is 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, the ligand may be compound 765 or compound 770. If the synthetic exogenous LGIC ligand is 1,4-diazabicyclo[3.2.2]nonane, the ligand may be compound 773 or compound 774. In certain cases, the LBD may be an α7 LBD, which may also contain at least one modified amino acid that confers selective binding to another α7 LBD having at least one modified amino acid, superior to binding to unmodified LGIC. The unmodified LGIC may be an endogenous LGIC (e.g., endogenous α7-nAChR). The at least one modified amino acid in the α7 LBD resulting in reduced binding to the unmodified LGIC may include amino acid substitutions at residue 27 (e.g., R27D substitution) and / or residue 41 (e.g., E41R substitution). In some cases, the IPD may be a 5HT3 IPD, which may contain at least one modified amino acid that confers increased ionic conductivity (conductance) to the modified LGIC. At least one modified amino acid in 5HT3 IPD that confers increased ionic conductivity to modified LGIC may include amino acid substitutions at amino acid residues 425 (e.g., R425Q substitution), 429 (e.g., R429D substitution), and / or 433 (e.g., R433A substitution).

[0010] In another embodiment, this specification features a modified LGIC having at least one modified LGIC subunit comprising an LBD and an IPD having at least one modified amino acid, wherein the at least one modified amino acid in the LBD reduces binding to the endogenous LGIC ligand. The modified LGIC may be a chimeric LGIC having an LBD from a first LGIC and an IPD from a second LGIC. The endogenous LGIC ligand may be ACh. The modified LGIC may have an EC50 greater than 20 μM relative to ACh. At least one modified amino acid may include an amino acid substitution at residues 115, 139, 210, 217, and / or 219. If at least one modified amino acid includes an amino acid substitution at residue 115, the amino acid substitution may be a Y115F substitution. If at least one modified amino acid includes an amino acid substitution at residue 139, the amino acid substitution may be a Q139G or Q139L substitution. If at least one modified amino acid includes an amino acid substitution at residue 210, the amino acid substitution may be a Y210F substitution. If at least one modified amino acid contains an amino acid substitution at residue 217, the amino acid substitution may be a Y217F substitution. If at least one modified amino acid contains an amino acid substitution at residue 219, the amino acid substitution may be a D219A substitution. In certain cases, a modified LGIC may contain a modified α7-nAChR LBD having an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution. A modified LGIC (for example, a modified LGIC containing a modified α7-nAChR LBD having an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution) may also contain an endoplasmic reticulum transport sequence. The endoplasmic reticulum transport sequence may contain the amino acid sequence: FCYENEV (Sequence ID: 16). For example, a modified LGIC containing a modified α7-nAChR LBD having the L131G amino acid substitution, the Q139L amino acid substitution, and the Y217F amino acid substitution, and also containing an endoplasmic reticulum transport sequence, may contain the amino acid sequence described in SEQ ID NO: 13. A modified LGIC (for example, a modified LGIC containing a modified α7-nAChR LBD having the L131G amino acid substitution, the Q139L amino acid substitution, and the Y217F amino acid substitution) may contain a signal sequence (for example, a CHRNB4 signal sequence).The CHRNB4 signal sequence is the amino acid sequence: This may include TIFF2026065133000002.tif4128. For example, a modified LGIC containing a modified α7-nAChR LBD having the L131G amino acid substitution, the Q139L amino acid substitution, and the Y217F amino acid substitution, and also containing a CHRNB4 signal sequence, may contain the amino acid sequence described in SEQ ID NO: 14. A modified LGIC (for example, a modified LGIC containing a modified α7-nAChR LBD having the L131G amino acid substitution, the Q139L amino acid substitution, and the Y217F amino acid substitution) may also contain a somatic cell targeting sequence (for example, a KCNB1 somatic cell targeting sequence). The KCNB1 somatic cell targeting sequence is an amino acid sequence: This may include TIFF2026065133000003.tif12158. For example, a modified LGIC containing a modified α7-nAChR LBD having an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution, and also containing a KCNB1 somatic cell targeting sequence, may contain the amino acid sequence described in SEQ ID NO: 15.

[0011] In another aspect, this specification features a ligand having increased potency for a modified ligand-gated ion channel (LGIC), wherein the ligand is of formula I: TIFF2026065133000004.tif32128 (wherein X1, X2, and X3 can independently be CH, CH2, O, NH, or NMe, each n can independently be 0 or 1, Y is O or S, A is an aromatic substituent, and R is H or pyridinyl methylene) It contains. The aromatic substituents may be 1H-indole, 4-(trifluoromethyl)benzene, 2,5-dimethoxybenzene, 4-chloroaniline, aniline, 5-(trifluoromethyl)pyridine-2-yl, 6-(trifluoromethyl)nicotinic, or 4-chlorobenzene.

[0012] In certain cases, the LGIC ligand can be a quinuclidine, as shown in formula II: TIFF2026065133000005.tif18128 (wherein X3 is O, NH, or CH2, Y is O or S, A is an aromatic substituent, and R is H or pyridinylmethylene) The structure may be as shown. The aromatic substituents may be 1H-indole, 4-(trifluoromethyl)benzene, 4-chlorobenzene, 2,5-dimethoxybenzene, 4-(trifluoromethyl)benzene, 4-chloroaniline, aniline, 5-(trifluoromethyl)pyridine-2-yl, 6-(trifluoromethyl)nicotine, 3-chloro-4-fluorobenzene, or 1H-indole. The quinuclidine may be PNU-282987, PHA-543613, compound 0456, compound 0434, compound 0436, compound 0354, compound 0353, compound 0295, compound 0296, compound 0536, compound 0676, or compound 702.

[0013] In certain cases, the LGIC ligand may be tropane, as shown in formula III: TIFF2026065133000006.tif29128 (wherein X2 is NH or NMe, X3 is O, NH, or CH2, Y is O or S, and A is an aromatic substituent) The structure may be as shown. The aromatic substituent may be 1H-indole, 7-methoxy-1H-indole, 7-methyl-1H-indole, 5-chloro-1H-indole, or 1H-indazole. The tropane may be tropisetron, pseudo-tropisetron, nortropisetron, compound 723, compound 725, compound 737, or compound 745.

[0014] In certain cases, the LGIC ligand can be 9-azabicyclo[3.3.1]nonane, and formula IV: TIFF2026065133000007.tif27128 (wherein X1 may be CH, X2 may be NH or NMe, X3 may be O, NH, or CH, Y may be O or S, and A may be an aromatic substituent) It may have the structure shown. The aromatic substituent may be 4-chlorobenzene. 9-azabicyclo[3.3.1]nonane may be compound 0536.

[0015] In another embodiment, this specification provides a ligand having increased potency against a modified LGIC, wherein the ligand may be 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, and formula V: The ligand is characterized by having a structure that may be shown in TIFF2026065133000008.tif14128 (wherein R1 may be H, phenyl, 2-toluyl, 3-pyridyl, 4-pyridyl, trifluoromethyl, methoxy, N,N-dimethylamino, N,N-diethylamino, imidazole, pyrrole, pyrazole, triazole, or isoxazole-3-amine, and R2 may be H, methyl, or phenyl). The 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine may be varenicline, compound 0765, compound 0770, compound 0780, compound 0782, compound 0785, compound 0788, compound 0782, compound 0789, compound 0791, compound 0793, compound 0794, compound 0795, compound 0798, compound 0799, compound 0800, compound 0801, compound 0802, compound 0803, compound 0804, compound 0805, compound 0807, compound 0808, compound 0812, compound 0813, compound 815, and compound 817.

[0016] In another embodiment, this specification relates to a ligand having increased potency against a modified LGIC, wherein the ligand is 2-(pyridine-3-yl)-1,5,6,7,8,9-hexahydro-5,9-methanoimidazo[4',5':4,5]benzo[1,2-d]azepine (compound 0786): It features a ligand that may be TIFF2026065133000009.tif12128.

[0017] In another embodiment, this specification provides a ligand having increased potency against modified LGIC, wherein the ligand may be 7,8,9,10-tetrahydro-1H-6,10-methanoazepino[4,5-g]quinoxaline-2(6H)-one, and formula VI: The ligand is characterized by having a structure that may be shown in TIFF2026065133000010.tif16128 (wherein R1 may be H or CH3, R2 may be H, CH3, or an aromatic substituent, and R3 may be O or S). Its 7,8,9,10-tetrahydro-1H-6,10-methanoazepino[4,5-g]quinoxaline-2(6H)-one may be compound 0783, compound 0784, compound 0790, or compound 0792.

[0018] In another embodiment, this specification features a ligand having increased potency against modified LGIC, where the ligand may be 1,4-diazabicyclo[3.2.2]nonane, formula VII: TIFF2026065133000011.tif25128 (In the formula, R can be H, F, or NO2) The structure may be as shown. 1,4-Diazabicyclo[3.2.2]nonane may be 3-(1,4-Diazabicyclo[3.2.2]nonane-4-yl)dibenzo[b,d]thiophene 5,5-dioxide, compound 0773, or compound 0774.

[0019] In another embodiment, the Specified herein features a method for treating channel dysfunction in mammals. The method comprises, or essentially comprises, administering modified LGIC to cells in a mammal, where an exogenous LGIC ligand selectively binds to the modified LGIC. The modified LGIC has at least one modified LGIC subunit comprising an LBD and an IPD containing at least one modified amino acid. The exogenous ligand is then administered to the mammal. Channel dysfunction can manifest as Bartter syndrome, Brugada syndrome, catecholamine-mediated polymorphic ventricular tachycardia (CPVT), congenital hyperinsulinemia, 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 myasthenia gravis, long QT syndrome (e.g., Romano-Ward syndrome), short QT syndrome, malignant hyperthermia, mucolipidosis type IV, myasthenia gravis, congenital myotonia, neuromyolitis optica, neuromyotonia, asymptomatic hearing loss, congenital paramyotonia, retinitis pigmentosa, Timothy syndrome, tinnitus, seizures, trigeminal neuralgia, and multiple sclerosis.

[0020] In another embodiment, this specification features a method for modulating ion transport across mammalian cell membranes. This method comprises, or essentially consists of, administering modified LGIC to cells, where an exogenous LGIC ligand selectively binds to the modified LGIC. The modified LGIC has at least one modified LGIC subunit comprising an LBD and an IPD containing at least one modified amino acid. The exogenous ligand is then administered to a mammal. The modulation may comprise activating or inhibiting ion transport. The cells may be neurons, glial cells, muscle cells, stem cells, endocrine cells, or immune cells. Administration of modified LGIC to cells may be in vivo or ex vivo. Administration of modified LGIC to cells may comprise administration of nucleic acids encoding the modified LGIC.

[0021] In another embodiment, this specification features a method for modulating cellular excitability in mammals. This method comprises, or essentially consists of, administering modified LGIC to mammalian cells, where an exogenous LGIC ligand selectively binds to the modified LGIC. The modified LGIC has at least one modified LGIC subunit comprising an LBD and an IPD containing at least one modified amino acid. The exogenous ligand is then administered to a mammal. The modulation may include increasing or decreasing cellular excitability. The cells may be excitatory cells. The cells may be neurons, glial cells, muscle cells, stem cells, endocrine cells, or immune cells. The administration of modified LGIC to cells may be in vivo or ex vivo. The administration of modified LGIC to cells may include the administration of nucleic acids encoding the modified LGIC.

[0022] In another embodiment, this specification features a method for modulating cellular activity in mammals. This method comprises, or essentially consists of, administering modified LGIC to cells, where an exogenous LGIC ligand selectively binds to the modified LGIC. The modified LGIC has at least one modified LGIC subunit comprising an LBD and an IPD containing at least one modified amino acid. The exogenous ligand is then administered to a mammal. The modulation may involve increasing or decreasing cellular activity. The activity may be ion transport, passive transport, excitation, inhibition, or exocytosis. The cells may be neurons, glial cells, muscle cells, stem cells, endocrine cells, or immune cells. The administration of modified LGIC to cells may be in vivo or ex vivo. The administration of modified LGIC to cells may involve the administration of nucleic acids encoding the modified LGIC (e.g., via a viral vector such as an adeno-associated virus, herpes simplex virus, or lentivirus).

[0023] In other embodiments, this document features a method for identifying ligands that selectively bind to modified LGICs. This method comprises, or essentially comprises, providing one or more candidate ligands for the modified LGICs described herein, and detecting binding between the candidate ligands and the modified LGICs, thereby identifying ligands that selectively bind to the modified LGICs. The modified LGICs may be homomeric modified LGICs.

[0024] In other embodiments, this specification features a method for detecting modified LGIC. This method comprises, or essentially comprises, providing one or more modified LGIC subunits as described herein, providing an agent that selectively binds to the modified LGIC, and detecting the binding between the modified LGIC and the agent that selectively binds to the modified LGIC. The agent that selectively binds to the modified LGIC may be an antibody, a protein (e.g., bungarotoxin), or a small molecule (e.g., a positron emission tomography (PET) ligand). The agent that selectively binds to the modified LGIC may include a detectable label (e.g., a fluorescent label, a radioactive label, or a positron emission label).

[0025] In another embodiment, this specification features a synthetic nucleic acid construct comprising a nucleic acid sequence having at least 75% sequence identity to the sequence described in SEQ ID NO: 27. The nucleic acid sequence having at least 75% sequence identity to the sequence described in SEQ ID NO: 27 may include a nucleic acid sequence capable of encoding an amino acid modified LBD. The modified LBD may be an α7-nAChR LBD having amino acid substitution residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, and / or 219 of an α7-nAChR LBD. The nucleic acid sequence having at least 75% sequence identity to the sequence described in SEQ ID NO: 27 may include a nucleic acid sequence capable of encoding an IPD. The IPD may be a 5HT3 IPD, a GlyR IPD, a GABA receptor IPD, or an α7-nAChR IPD. The IPD may be a 5HT3 IPD.

[0026] In another embodiment, this specification features a synthetic nucleic acid construct comprising a nucleic acid sequence having at least 75% sequence identity with respect to the sequence described in Sequence ID No. 28.

[0027] Nucleic acid sequences having at least 75% sequence identity to the sequence described in Sequence ID No. 28 may include nucleic acid sequences capable of encoding an amino acid-modified LBD. The modified LBD may be an α7-nAChR LBD having amino acid substitution residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, and / or 219 of the α7-nAChR LBD. Nucleic acid sequences having at least 75% sequence identity to the sequence described in Sequence ID No. 28 may include nucleic acid sequences capable of encoding an IPD. The IPD may be a 5HT3 IPD, a GlyR IPD, a GABA receptor IPD, or an α7-nAChR IPD. The IPD may be a GlyR IPD with amino acid modification. The modified GlyR IPD may have an amino acid substitution at amino acid residue 298 of the GlyR IPD.

[0028] In another embodiment, this specification features a synthetic nucleic acid construct comprising a nucleic acid sequence having at least 75% sequence identity to the sequence described in SEQ ID NO: 29. The nucleic acid sequence having at least 75% sequence identity to the sequence described in SEQ ID NO: 29 may include a nucleic acid sequence capable of encoding an amino acid modified LBD. The modified LBD may be an α7-nAChR LBD having amino acid substitution residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, and / or 219 of an α7-nAChR LBD. The nucleic acid sequence having at least 75% sequence identity to the sequence described in SEQ ID NO: 29 may include a nucleic acid sequence capable of encoding an IPD. The IPD may be a 5HT3 IPD, a GlyR IPD, a GABA receptor IPD, or an α7-nAChR IPD. The IPD may be a GABA IPD with amino acid modification. The modified GABA IPD may have an amino acid substitution at amino acid residue 298 of a GABA IPD.

[0029] In another embodiment, this specification features a synthetic nucleic acid construct having the sequence described in Sequence ID No. 33.

[0030] In another embodiment, this specification features a synthetic nucleic acid construct having the sequence described in Sequence ID No. 34.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Methods and materials are described herein for use in this disclosure. Other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative and not intended to limit. All publications, patent applications, patents, sequences, database entries, and other references referenced herein are incorporated in their entirety by citation. In case of any conflict, including definitions, this specification shall prevail.

[0032] Details of one or more embodiments of the present invention are described in the accompanying drawings and the following description. Other features, purposes, and advantages of the present invention will become apparent from the description and drawings and from the claims. [Brief explanation of the drawing]

[0033] [Figure 1A]Figure 1 shows an exemplary amino acid sequence of a chimeric LGIC. Mutations at amino acid residue 77 (e.g., W77F or W77Y) resulted in sensitivity to granisetron and tropisetron. Mutations at amino acid residue 79 (e.g., Q79G) were most effective against several agonists. Mutations at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. Combining LBD mutations with mutations at amino acid residue 298 of GlyR or GABAC IPD significantly enhanced potency. Combining α7 nAChR LBD mutations with mutations at amino acid residues G175 and P216 also enhanced potency. A) Amino acid sequence of the α7-5HT3 chimeric receptor (SEQ ID NO: 6) containing human α7 nAChR LBD (SEQ ID NO: 1) and mouse 5HT3 IPD (SEQ ID NO: 3) components. [Figure 1B] Figure 1 shows an exemplary amino acid sequence of a chimeric LGIC. Mutations at amino acid residue 77 (e.g., W77F or W77Y) resulted in sensitivity to granisetron and tropisetron. Mutations at amino acid residue 79 (e.g., Q79G) were most effective against several agonists. Mutations at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. Combining LBD mutations with mutations at amino acid residue 298 of GlyR or GABAC IPD significantly enhanced potency. Combining α7 nAChR LBD mutations with mutations at amino acid residues G175 and P216 also enhanced potency. B) Amino acid sequence of the α7-GlyR chimeric receptor (SEQ ID NO: 7), which includes components of human α7nAChR LBD (SEQ ID NO: 2) and human GlyR IPD (SEQ ID NO: 5). [Figure 1C]Figure 1 shows an exemplary amino acid sequence of a chimeric LGIC. Mutations at amino acid residue 77 (e.g., W77F or W77Y) resulted in sensitivity to granisetron and tropisetron. Mutations at amino acid residue 79 (e.g., Q79G) were most effective against several agonists. Mutations at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. Combining LBD mutations with mutations at amino acid residue 298 of GlyR or GABAC IPD significantly enhanced potency. Combining α7 nAChR LBD mutations with mutations at amino acid residues G175 and P216 also enhanced potency. C) Amino acid sequence of the α7-5HT3 chimeric receptor (SEQ ID NO: 8) containing human α7 nAChR LBD (SEQ ID NO: 1) and human 5HT3 IPD (SEQ ID NO: 4) components. [Figure 1D] Figure 1 shows an exemplary amino acid sequence of a chimeric LGIC. Mutations at amino acid residue 77 (e.g., W77F or W77Y) resulted in sensitivity to granisetron and tropisetron. Mutations at amino acid residue 79 (e.g., Q79G) were most effective against several agonists. Mutations at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. Combining LBD mutations with mutations at amino acid residue 298 of GlyR or GABAC IPD significantly enhanced potency. Combining α7 nAChR LBD mutations with mutations at amino acid residues G175 and P216 also enhanced potency. D) Amino acid sequence of the α7-GABAc chimeric receptor (SEQ ID NO: 10) containing components of human α7 nAChR LBD (SEQ ID NO: 11) and human GABAC IPD (SEQ ID NO: 9). [Figure 1E]Figure 1 shows an exemplary amino acid sequence of a chimeric LGIC. Mutations at amino acid residue 77 (e.g., W77F or W77Y) resulted in sensitivity to granisetron and tropisetron. Mutations at amino acid residue 79 (e.g., Q79G) were most effective against several agonists. Mutations at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. Combining the LBD mutation with a mutation at amino acid residue 298 of GlyR or GABAC IPD significantly enhanced potency. Combining the α7 nAChR LBD mutation with mutations at amino acid residues G175 and P216 also enhanced potency. E) Amino acid sequence of the rat nAChR sequence (SEQ ID NO: 12). [Figure 2] Figure 2 shows the EC50 of tropisetron against α7-5HT3 chimeric LGICs and variants of chimeric LGICs with LBD mutations at the positions shown in Figure 1. Multiple mutations at Gln79 showed similar or improved potency compared to the unmodified α7-5HT3 channel (arrows). [Figure 3A] Figure 3 shows the relative potency of known nAChR agonists against α7-5HT3 chimeric LGIC. A) Graph of EC50 normalized for unmodified α7-5HT3 chimeric channels (logarithmic scale). *P<0.05, statistically significant change in potency is observed (ANOVA and subsequent Dunn test). [Figure 3B] Figure 3 shows the relative potency of known nAChR agonists against α7-5HT3 chimeric LGIC. B) Chemical structures of known nAChR agonists. [Figure 4]Figure 4 shows the relative potency of known nAChR agonists against α7-GlyR chimeric LGIC. A) Graph of EC50 for the Q79 LBD mutant normalized to the unmodified α7-GlyR chimeric channel (logarithmic scale). B) Graph of EC50 for the A298G IPD mutant normalized to the unmodified α7-GlyR chimeric channel (logarithmic scale). C) Graph of EC50 for α7-GlyRA298G, normalized to the unmodified α7-GlyR chimeric channel and compared to the dual mutant channel α7Q79G-GlyRA298G (logarithmic scale). *P<0.05, statistically significant change in potency observed (ANOVA and subsequent Dunn test). [Figure 5A] Figure 5 shows schematic structures of LGIC agonists with substitution patterns best suited to enhancing potency for α7Q79G-5HT3 and α7Q79G-GlyRA298G. A) Generalized structure showing properties related to enhanced potency. [Figure 5B] Figure 5 shows schematic structures of LGIC agonists that have the substitution patterns best suited to enhancing the potency of α7Q79G-5HT3 and α7Q79G-GlyRA298G. The specific pharmacophores represented in B)(A) are quinuclidine, tropane, and 9-azabicyclo[3.3.1]nonane core structures. [Figure 5C] Figure 5 shows the schematic structure of an LGIC agonist with the substitution pattern best suited to enhancing the potency of α7Q79G-5HT3 and α7Q79G-GlyRA298G. C) Exemplary synthetic molecules showing high potency against α7Q79G-GlyRA298G, α7Q79G, Y115F, G175K-GlyR, α7W77F, Q79G, and G175K-GlyR. [Figure 6A]Figure 6 shows a mutation that reduces the association between the chimeric LCIG α7nAChR LBD and the unmodified LBD. A) Schematic potential configuration of charge reversal in transfection of two epitope-tagged (HA and V5) constructs encoding α7-5HT3 (upper side), or two constructs encoding α7-5HT3-HA and α7R21D,E41R-5HT3-V5. Here, association between two different epitope-tagged subunits would be undesirable due to charge reversal mutations at the subunit interface. [Figure 6B] Figure 6 shows a mutation that reduces the association between the chimeric LCIG α7nAChR LBD and the unmodified LBD. B) Whole-cell recordings in HEK cells expressing α7R21D,E41R-5HT3 with the V5 epitope tag show a strong response to PNU-282987. [Figure 6C] Figure 6 shows mutations that reduce the association between chimeric LCIG α7nAChR LBD and unmodified LBD. C) The association of α7-5HT3 LGIC with HA and V5 epitope tags in HEK cells was examined by HA immunoprecipitation (left) or total lysate isolation, followed by Western blotting with either anti-HA (top) or anti-V5 antibody (bottom). In cells co-expressing HA and V5 epitopes and channels, anti-HA IP and subsequent anti-V5 immunoblotting showed co-immunoprecipitation of each type of unmodified channel, but charge reversal mutations in LBDα7R21D and E41R-5HT3-V5 did not immunoprecipitate. The MW of α7-5HT3 is approximately 48 kD (arrow). [Figure 7] Figure 7 shows that chimeric LGICs can be controlled using exogenous ligands. Cortical neurons from mouse brains transduced with the α7Q79G-GlyRA298G chimeric LGIC via an adeno-associated virus (AAV) vector fire action potentials in response to a 40 pA current injection (PRE) which is strongly suppressed by 30 nM tropisetron. After tropisetron wash, neuronal firing is restored. [Figure 8A]Figure 8 shows the agonist activity against the chimeric LGIC with the G175K mutation. A) Graph (logarithmic scale) of EC50 for the Q79G G175K LBD mutant against known agonists, normalized to the unmodified α7-GlyR chimeric channel. [Figure 8B] Figure 8 shows the agonist activity against chimeric LGICs with the G175K mutation. B) Graphs of EC50 for ACh and tropisetron for channels with mutations in α7-GlyR chimeric LGICs. Mutations resulting in channels with high potency for tropisetron and low potency for the endogenous ligand acetylcholine (ACh) are optimal (gray shading). Unmod: Unmodified α7-GlyR chimeric LGIC. [Figure 8C] Figure 8 shows the agonist activity against chimeric LGIC with the G175K mutation. C) Action potentials of cortical neurons from mouse brains transduced with α7Q79G, Y115F, G175K-GlyR chimeric LGIC. Neurons fire in response to current injection (PRE) and are strongly inhibited by 100 nM tropisetron. After tropisetron wash, neuronal firing is restored. [Figure 9A] Figure 9 shows the activity of agonists against chimeric LGICs with the L131G mutation. A) Graph (logarithmic scale) of EC50 for L131 LBD mutants against known agonists normalized to unmodified α7-GlyR chimeric channels. [Figure 9B] Figure 9 shows the agonist activity against chimeric LGICs with the L131G mutation. B) Graphs of ACh and tropisetron EC50 for channels with mutations in α7L131G-GlyR chimeric LGICs. [Figure 9C] Figure 9 shows the agonist activity against chimeric LGICs with the L131G mutation. C) Graph (gray shading) showing that the optimal mutation is one that results in a channel with high potency for varenicline and low potency for the endogenous ligand acetylcholine (ACh). Unmod: Unmodified α7-GlyR chimeric LGIC. [Figure 9D] Figure 9 shows the agonist activity against chimeric LGICs with the L131G mutation. D) Graph showing activation of channels with the α7L131G-GlyR mutation by Ach and varenicline after short-term channel antagonism with picrotoxin (PTX). The solid line represents the duration of molecular administration. [Figure 9E] Figure 9 shows the agonist activity against chimeric LGICs with the L131G mutation. E) Action potentials of cortical neurons derived from mouse brains transduced with α7L131G, Q139L, Y217F-GlyR chimeric LGIC. Neurons fire in response to current injection (PRE) and are strongly suppressed by 10 nM varenicline even at injection currents more than 6 times stronger. After varenicline washing (WASH), neuronal firing is restored. [Figure 10] Figure 10 includes a graph showing that varenicline can activate chimeric receptors in mice to induce behavioral effects. In this case, the expression of α7L131G, Q139L, and Y217F-GlyR in unilateral VGAT-expressing neurons in the substantia nigra pars reticularis, and subsequent varenicline administration, induces contralateral rotation, which is consistent with varenicline-induced silencing of α7L131G, Q139L, and Y217F-GlyR-expressing neurons. [Figure 11A] Figure 11 shows the chemical structures of exemplary LGIC agonists. A) Chemical structure of an LGIC agonist having the substitution pattern best suited to enhancing the potency of α7Q79G, Y115F, G175K-GlyR. [Figure 11B] Figure 11 shows the chemical structures of exemplary LGIC agonists. B) Chemical structures of LGIC agonists having the substitution pattern best suited for potency enhancement of α7L131G, Q139L, Y217F-GlyR, α7L131G, Q139L, Y217F-5HT3, or α7L131G, Q139L, Y217F-5HT3 HC. [Figure 11C]Figure 11 shows the chemical structures of exemplary LGIC agonists. C) Chemical structures of LGIC agonists having the substitution pattern best suited for potency enhancement of α7L131G, Q139L, Y217F-GlyR, or α7L131G, Q139L, Y217F-5HT3 HC. [Figure 12] Figure 12 shows chemogenetic perturbations of cortical neuron activity. A-B) Varenicline strongly suppressed action potential firing in neurons expressing PSAM4-GlyR by decreasing input resistance (A) and increasing baseline current (B). C) Cortical latitude 2 / 3 neuronal membrane properties were similar in PSAM4-GlyR-expressing neurons and mixed untransfected control neurons. D-E) Varenicline depolarized (D) and induced firing (E) in neurons expressing PSAM4-5HT3 HC. F) Cortical latitude 2 / 3 neuronal membrane properties were similar in PSAM4-5HT3 HC-expressing neurons and mixed untransfected control neurons. Data are mean ± SEM. Mann-Whitney U test, nsP > 0.05, ***P < 0.001. [Figure 13A] Figure 13 shows silencing of PSAM4-GlyR neurons in mice. A) PSAM4-GlyR-IRES-EGFP unilaterally targeted SNr. Inset: Schematic diagram of unilateral SNr transmission shows the result of counter-rotational silencing. Asterisk: Non-specific immunofluorescence. [Figure 13B] Figure 13 shows silencing of PSAM4-GlyR neurons in mice. B) Low doses of intraperitoneal varenicline induce contraversive rotation in mice that express PSAM4-GlyR but have undergone sham surgery or mice that express only EGFP. [Figure 13C] Figure 13 shows silencing of PSAM4-GlyR neurons in mice. C) Two doses of varenicline separated by 5 hours yielded a similar rate of total rotation, indicating the absence of tachyphylaxis in the chemogenetic response. [Figure 13D]Figure 13 shows silencing of PSAM4-GlyR neurons in mice. D) Duration of chemogenetic silencing monitored by the time course of rotational response normalized to maximal rotation for each mouse. Pink (thin) arrows: amphetamine injection, cyan (thick) arrows: varenicline injection. Mann-Whitney U test, nsP>0.05, **P<0.01. [Figure 14-1] Figure 14 shows ultrapotent chemogenetic agonists (uPSEM). A) Comparison of uPSEM agonist EC50 with IC50 of α4β2 nAChR at the PSAM4 channel and endogenous varenicline target, and IC50 with 1 μM ACh. LED: Minimum effective dose in mice in the SNr rotation assay. Unit: nM; Parentheses: SEM. Selectivity for PSAM4-GlyR in bold. [Figure 14-2] Figure 14 shows ultrapotent chemogenetic agonists (uPSEMs). B,C) Dose-response curves for PSAM4-GlyR, α4β2 nAChR, and 5HT3-R. uPSEM792(B) is a 10% partial agonist of α4β2 nAChR, and uPSEM817(C) inhibits α4β2 nAChR. [Figure 14-3] Figure 14 shows an ultrapotent chemogenetic agonist (uPSEM). D) Current response to uPSEM (2 μM) and Ach (10 μM) in HEK cells expressing α7 nAChR. uPSEM does not activate α7 nAChR, but Ach does. (E) Response amplitude normalized to ACh. [Figure 14-4] Figure 14 shows ultrapotent chemogenetic agonists (uPSEMs). F~J) uPSEM792, uPSEM793, uPSEM815, and uPSEM817 strongly suppress the firing of cortical neurons expressing PSAM4-GlyR by reducing the current required to fire an action potential (base current) (G~J). [Figure 14-5] Figure 14 shows ultrapotent chemogenetic agonists (uPSEMs). F~J) uPSEM792, uPSEM793, uPSEM815, and uPSEM817 strongly suppress the firing of cortical neurons expressing PSAM4-GlyR by reducing the current required to fire an action potential (base current) (G~J). [Figure 15A] Figure 15 shows the in vivo uPSEM dose-response in mice unilaterally expressing PSAM4-GlyR in SNr. Behavioral responses and time courses for uPSEM792 (A,B), uPSEM793 (C,D), uPSEM815 (E,F), and uPSEM817 (G,H). Time courses of rotational responses normalized to the maximum rotation of each mouse. Pink (thin) arrows: amphetamine injection, blue (thick) arrows: uPSEM injection. [Figure 15B] See the explanation in Figure 15A. [Figure 15C] See the explanation in Figure 15A. [Figure 15D] See the explanation in Figure 15A. [Figure 15E] See the explanation in Figure 15A. [Figure 15F] See the explanation in Figure 15A. [Figure 15G] See the explanation in Figure 15A. [Figure 15H] See the explanation in Figure 15A. [Figure 16] Figure 16 shows an exemplary amino acid sequence (SEQ ID NO: 13) of PSAM4-GlyR-KirM3M4. The PSAM4 mutation is highlighted. The Kir2.1(KCNJ2) endoplasmic reticulum (ER) transport sequence is underlined and shown in blue. [Figure 17] Figure 17 shows an exemplary amino acid sequence (SEQ ID NO: 14) of PSAM4-GlyR-B4sig. The PSAM4 mutation is highlighted. The β4 nAChR subunit (CHRNB4) signal sequence is underlined and shown in blue. [Figure 18]Figure 18 shows an exemplary amino acid sequence (SEQ ID NO: 15) of PSAM4-GlyR-Kv2M3M4-soma. The PSAM4 mutation is highlighted. The somatic cell targeting sequence of Kv2.1(KCNB1) is underlined and shown in blue. [Figure 19A] Figure 19 shows an exemplary construct containing a nucleic acid sequence encoding a modified LGIC subunit. (A) Plasmid map of the AAV-Syn :: PSAM4-GlyR-IRES-EGFP-WPRE construct containing a nucleic acid sequence encoding a modified LGIC subunit (SEQ ID NO: 33). [Figure 19B-1] Figure 19 shows an exemplary construct containing a nucleic acid sequence encoding a modified LGIC subunit. (B) Nucleic acid sequence of the AAV-Syn :: PSAM4-GlyR-IRES-EGFP-WPRE construct containing a nucleic acid sequence encoding a modified LGIC subunit. [Figure 19B-2] This is a continuation of Figure 19B-1. [Figure 19B-3] This is a continuation of Figure 19B-2. [Figure 20A] Figure 20 shows an exemplary construct containing a nucleic acid sequence encoding a modified LGIC subunit. (A) Plasmid map of the AAV-CamkII :: PSAM4-GlyR-IRES-EGFP-WPRE construct containing a nucleic acid sequence encoding a modified LGIC subunit (SEQ ID NO: 34). [Figure 20B-1] Figure 20 shows an exemplary construct containing nucleic acid sequences encoding the modified LGIC subunit. (B) Nucleic acid sequences of the AAV-CamkII :: PSAM4-GlyR-IRES-EGFP-WPRE construct containing nucleic acid sequences encoding the modified LGIC subunit. [Figure 20B-2] This is a continuation of Figure 20B-1. [Figure 20B-3] This is a continuation of Figure 20B-2. [Modes for carrying out the invention]

[0034] Detailed explanation This specification provides modified LGICs and methods for using them. For example, this specification provides a modified LGIC comprising at least one modified LGIC subunit having an LBD and an IPD and having at least one modified amino acid (e.g., an amino acid substitution). In certain cases, the modified LGIC may be a chimeric LGIC. For example, a chimeric LGIC may comprise an LBD from a first LGIC and an IPD from a second LGIC. In certain cases, the modified amino acid can confer pharmacological selectivity to the modified LGIC. For example, the modified amino acid can confer selective binding of exogenous LGIC ligands to the modified LGIC. For example, the modified amino acid can confer a reduction (e.g., minimization or exclusion) of binding of unmodified LGIC subunits (e.g., unmodified LGIC subunits and / or endogenous LGIC subunits) to the modified LGIC. For example, the modified amino acid can confer a reduction (e.g., minimization or exclusion) of binding of endogenous LGIC ligands to the modified LGIC.

[0035] Modified LGICs provided herein may be used, for example, in methods for treating channel disorders (e.g., neural channel disorders or muscle channel disorders). For example, modified LGICs and exogenous LGIC ligands that can bind to and activate modified LGICs may be used to treat mammals having channel disorders. In certain cases, modified LGICs and exogenous LGIC ligands may be used to modulate (e.g., activate or inhibit) ion transport across the membrane of mammalian cells. In certain cases, modified LGICs and exogenous LGIC ligands may be used to modulate (e.g., increase or decrease) cellular excitability in mammals.

[0036] Qualified LGIC As used herein, “modified” LGIC is an LGIC comprising at least one LGIC subunit. Modified LGIC may also refer to a pharmacologically selective actuator module (PSAM). A modified LGIC subunit may contain at least one modified amino acid (e.g., amino acid substitution) in the LBD and / or at least one modified amino acid (e.g., amino acid substitution) in the IPD. Modified LGIC subunits as described herein may be modifications of LGICs derived from any suitable species (e.g., human, rat, mouse, dog, cat, horse, cattle, goat, pig, or monkey). In particular, a modified LGIC may contain at least one chimeric LGIC subunit having a non-spontaneous combination of an LBD derived from a first LGIC and an IPD derived from a second LGIC.

[0037] A modified LGIC (e.g., an LGIC containing one or more modified LGIC subunits) may be a homomer (e.g., having any number of the same modified LGIC subunits) or a heteromer (e.g., having at least one modified LGIC subunit and any number of different LGIC subunits). In certain cases, the modified LGIC described herein may be a homomeric modified LGIC. The modified LGIC described herein may contain any appropriate number of modified LGIC subunits. In certain cases, the modified LGIC may be a trimer, tetramer, pentamer, or hexamer. For example, the modified LGIC described herein may be a pentamer.

[0038] The modified LGIC subunits described herein may be any suitable modification of LGIC. LGIC can conduct anions, cations, or both across the cell membrane in response to ligand binding. For example, LGIC can conduct sodium (Na) across the cell membrane in response to ligand binding. + ), potassium (K + ), calcium (Ca 2+ ), and / or chloride (Cl -) can transport ions. Examples of LGICs include Cys-loop receptors (e.g., AChRs such as nAChRs (e.g., muscular nAChRs or neural nAChRs)), gamma-aminobutyric acid (GABA; e.g., GABA) A and GABA A Examples of modified LGICs include, but are not limited to, ρ (also known as GABAC) receptors, GlyR, GluCl receptors, and 5HT3 receptors), ion channel-type glutamate receptors (iGluR, e.g., AMPA receptor, kainate receptor, NMDA receptor, and delta receptor), ATP-dependent channels (e.g., P2X), and phosphatidylinositol 4,5-bisphosphate (PIP2)-dependent channels. If the modified LGIC described herein is a chimeric LGIC, the chimeric LGIC may include a LBD selected from any suitable LGIC and an IPD selected from any suitable LGIC. If the LGIC contains multiple different subunits (e.g., a neuronal nAChR contains α4, β2, and α7 subunits), the LBD and / or IPD can be selected from any subunit. For example, the LBD from an nAChR may be an α7 LBD. A representative rat α7 nAChR amino acid sequence (containing both LBD and IPD) is as follows: TIFF2026065133000012.tif68158

[0039] In certain cases, the modified LGIC subunits described herein may include LBDs derived from α7 nAChR. Examples of α7 nAChR LBDs include, but are not limited to, human α7 nAChR LBDs having the amino acid sequence described in SEQ ID NO: 1, human α7 nAChR LBDs having the amino acid sequence described in SEQ ID NO: 2, and human α7 nAChR LBDs having the amino acid sequence described in SEQ ID NO: 11. In certain cases, the α7 nAChR LBD may be a homolog, orthologue, or paralog of the human α7 nAChR LBD described in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 11. In certain cases, the α7 nAChR LBD may have at least 75% sequence identity with respect to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 11 (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). It may have. TIFF2026065133000013.tif118158

[0040] In certain cases, the modified LGIC subunits described herein may include IPDs derived from the 5HT3 receptor. Examples of 5HT3 IPDs include, but are not limited to, mouse 5HT3 IPDs having the amino acid sequence described in SEQ ID NO: 3, and human 5HT3 IPDs having the amino acid sequence described in SEQ ID NO: 4. In certain cases, a 5HT3 IPD may be a homolog, ortholog, or paralog of a 5HT3 IPD described in SEQ ID NO: 3 or SEQ ID NO: 4. In certain cases, a 5HT3 IPD may have at least 75% sequence identity with respect to SEQ ID NO: 3 or SEQ ID NO: 4 (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). TIFF2026065133000014.tif76158

[0041] In certain cases, the modified LGIC subunit described herein may include an IPD derived from a GlyR. Examples of GlyR IPDs include, but are not limited to, a mouse GlyR IPD having the amino acid sequence set forth in SEQ ID NO: 5. In certain cases, the GlyR IPD may be a homolog, ortholog, or paralog of the human GlyR IPD set forth in SEQ ID NO: 5. In certain cases, the GlyR IPD may have at least 75% 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. TIFF2026065133000015.tif33158

[0042] In certain cases, the modified LGIC subunit described herein may include an IPD derived from a GABA receptor (e.g., GABA A -ρ, also referred to as GABAc). GABA A -ρ IPD examples include, but are not limited to, a human GABA A -ρ IPD having the amino acid sequence set forth in SEQ ID NO: 9. In certain cases, the GABA A -ρ IPD may be a homolog, ortholog, or paralog of the human GABA A -ρ IPD set forth in SEQ ID NO: 9. In certain cases, the GABA A -ρ IPD may have at least 75% 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. TIFF2026065133000016.tif32158

[0043] To calculate the percentage of sequence identity, two sequences are aligned, and the number of identical 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 the value of the percentage of sequence identity is obtained by multiplying by 100. It will be understood that the length of the aligned region can be a portion of one or both sequences up to the full length of the shortest sequence. It will also be understood that a single sequence can be aligned with two or more other sequences, and therefore can have different percentage of sequence identity values ​​across each aligned region. Alignment of two or more sequences to determine the percentage of sequence identity can be performed using the computer program ClustalW and its default parameters, which calculates the best match between a query and one or more target sequences, and aligns them so that identity, similarity, and difference can be determined. See, for example, Chenna et al., 2003, Nucleic Acids Res., 31(13):3497-500.

[0044] If the modified LGIC subunit described herein is a chimeric LGIC subunit, the chimeric LGIC subunit may contain LBD and IPD from the same species, or LBD and IPD from different species. In certain cases, the chimeric LGIC subunit may contain LBD derived from human LGIC protein and IPD derived from human LGIC protein. For example, the chimeric LGIC subunit may contain human α7 LBD and human GlyR IPD. In certain cases, the chimeric LGIC subunit may contain LBD derived from human LGIC protein and IPD derived from mouse LGIC protein. For example, the chimeric LGIC subunit may contain human α7 LBD and mouse 5HT3 IPD.

[0045] If the modified LGIC subunit described herein is a chimeric LGIC subunit, the chimeric LGIC subunit may include various fusion sites connecting the LBD and the IPD such that the number of amino acids in the LBD may differ when the LBD fuses with different IPDs to form a chimeric channel subunit. For example, the length of the α7 nAChR LBD used to form a chimeric LGIC subunit with a 5HTS IPD is different from the length of the α7 nAChR LBD used to form a chimeric LGIC subunit with a GlyR IPD (see, for example, Figure 1A and Figure 1C with Figure 1B).

[0046] The modified LGIC subunits described herein may include LBDs having at least one modified amino acid and / or IPDs having at least one modified amino acid. For example, the modified LGIC subunits described herein may include an α7 LBD having at least 75% 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 residues 27, 41, 77, 79, 131, 139, 141, 175, 210, 216, 217, and / or 219. For example, the modified LGIC subunits described herein may include a GlyR IPD having at least 75% sequence identity to the sequence described in SEQ ID NO: 5, and an amino acid substitution at amino acid residue 298 of the α7-GlyR chimeric receptor (e.g., SEQ ID NO: 7). For example, the modified LGIC subunit described herein may include a GABAc IPD having at least 75% sequence identity with SEQ ID NO: 9 and having an amino acid substitution at amino acid residue 298 of the α7-GABAc chimeric receptor (e.g., SEQ ID NO: 10). In certain cases, the modified LGIC subunit described herein may include more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more) amino acid modifications. The modifications may be amino acid substitutions. In certain cases, the modified amino acid can confer pharmacological selectivity to the modified LGIC. For example, the modified amino acid can confer selective binding of exogenous LGIC ligands to the modified LGIC. For example, the modified amino acid can result in a reduction (minimization or exclusion) of binding of unmodified LGIC subunits (LGIC subunits lacking modification and / or endogenous LGIC subunits) to the modified LGIC. For example, the modified amino acid can confer a reduction (minimization or exclusion) of binding of endogenous LGIC ligands to the modified LGIC.

[0047] In certain embodiments, the modified LGIC subunit described herein may comprise at least one modified amino acid that confers selective binding (e.g., enhanced binding or enhanced potency) to the modified LGIC with an exogenous LGIC ligand. Binding to an exogenous LGIC ligand may be more selective than binding to an endogenous LGIC ligand. Modified LGIC subunits having selective binding to an exogenous LGIC ligand may comprise any suitable LDB (e.g., α7 LBD). In certain embodiments, the modified LGIC subunit may comprise the α7 LBD described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12, where the amino acid modification may be a substitution at residues 77, 79, 131, 139, 141, 175, and / or 216. In certain 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 may 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, the modified LGIC subunit described herein may include an α7 LBD shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12 and having the W77F substitution. In certain 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 may be substituted with an amino acid residue such as alanine (e.g., Q79A), glycine (e.g., Q79G), or serine (e.g., Q79S). For example, the modified LGIC subunit described herein may include an α7 LBD having the Q79G substitution. In certain cases, the leucine at amino acid residue 131 in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12 may 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 certain cases, the glycine at amino acid residue position 175 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12 can be substituted with amino acid residues such as lysine (e.g., G175K), alanine (e.g., G175A), phenylalanine (e.g., G175F), histidine (e.g., G175H), methionine (e.g., G175M), arginine (e.g., G175R), serine (e.g., G175S), or valine (e.g., G175V). In certain 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 amino acid residues such as isoleucine (e.g., P216I). Modified LGIC subunits having selective binding to exogenous LGIC ligands can be substituted with any suitable IPD (e.g., GlyR IPD or GABA). A -ρIPD) may be included. In certain embodiments, the modified LGIC subunit may include GlyR IPD as described in SEQ ID NO: 5, and its amino acid modification may be a substitution at amino acid residue 298 of the α7-GlyR chimeric receptor (e.g., SEQ ID NO: 7). In certain cases, the alanine at amino acid residue 298 of SEQ ID NO: 7 may be substituted with an amino acid residue such as glycine (e.g., A298G). In certain embodiments, the modified LGIC subunit may include GABA as described in SEQ ID NO: 9. A - May contain ρIPD, and its amino acid modification is α7-GABA A -ρ chimeric receptors (e.g., SEQ ID NO: 10) may have a substitution at amino acid residue 298. In certain 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).

[0048] In certain cases, the modified LGIC subunits described herein may comprise two or more amino acid modifications (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more). For example, the modified LGIC subunits described herein may have at least 75 percent sequence identity with SEQ ID NO: 7 and may comprise Q79G and A298G substitutions. Further examples of modifications that can confer selective binding of exogenous LGIC ligands to modified LGIC include those described elsewhere (see, for example, U.S. Patent No. 8,435,762).

[0049] A modified LGIC subunit that selectively binds to an exogenous LGIC ligand (e.g., enhances binding or increases potency) more than an endogenous (e.g., canonical) LGIC ligand may also be described as having enhanced potency of the exogenous ligand. In particular cases, a modified LGIC subunit described herein that selectively binds to an exogenous LGIC ligand may have at least four times (e.g., at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least eleven times, at least twelve times, at least thirteen times, at least fourteen times, at least fifteen times, at least sixteen times, at least seventeen times, at least eighteen times, at least nineteen times, or at least twenty times) enhanced potency with respect to the exogenous ligand. In certain cases, the modified LGIC subunits described herein that selectively bind to exogenous LGIC ligands may have an enhanced potency of about 4 to 200 times (e.g., about 4 to 200 times, about 5 to 180 times, about 6 to 175 times, about 7 to 150 times, about 8 to 125 times, about 9 to 100 times, about 10 to 90 times, about 11 to 75 times, about 12 to 65 times, about 13 to 50 times, about 14 to 40 times, or about 15 to 30 times) compared to the exogenous ligand. For example, the modified LGIC subunits described herein that selectively bind to exogenous LGIC ligands may have an enhanced potency of about 10 to 100 times compared to the exogenous ligand. For example, the modified LGIC subunit described herein, which selectively binds to an exogenous LGIC ligand, may have approximately 10 to 20 times enhanced potency against the exogenous ligand.

[0050] In certain embodiments, the modified LGIC subunit described herein may include at least one modified amino acid that confers a reduced (e.g., minimized or eliminated) bond to the modified LGIC with an unmodified LGIC subunit. Bonding with a modified LGIC subunit having the same modification may be more selective than bonding with an unmodified LGIC subunit. An unmodified LGIC subunit may be an LGIC subunit lacking a modification that confers a reduced bond to the unmodified LGIC subunit, or the unmodified LGIC may be an endogenous LGIC subunit. The modification that confers a reduced bond to the unmodified LGIC subunit may be a charge inversion modification. A modified LGIC subunit with reduced bond to an unmodified LGIC subunit may include any suitable LBD (e.g., α7 LBD). In certain embodiments, the modified LGIC subunit may include the α7 LBD described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12, and the amino acid modification may be a substitution at amino acid residues 27 and / or 41. For example, the arginine at amino acid residue 27 in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12 may be substituted with aspartic acid (e.g., R27D). For example, the glutamic acid at amino acid residue 41 in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12 may be substituted with arginine (e.g., E41R). In certain cases, the modified LGIC subunit described herein may include an α7 LBD having the R27D substitution and E41R.

[0051] In certain embodiments, the modified LGIC subunits described herein may comprise at least one modified amino acid that confers a reduction (e.g., minimization or elimination) of the binding of the endogenous LGIC ligand to the modified LGIC. The endogenous LGIC ligand may be ACh. The modified LGIC subunit with reduced binding of the endogenous LGIC ligand may comprise any suitable IPD (e.g., GlyR LBD). For example, the modified LGIC subunit may comprise the α7 LBD shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12, and the amino acid modification may be a substitution at amino acid residues 115, 131, 139, 210, 217, and / or 219. In certain cases, the tyrosine at amino acid residue 115 in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12 may be substituted with phenylalanine (e.g., Y115F). In certain cases, the leucine at amino acid residue 131 in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12 can be substituted with amino acid residues 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 certain cases, the glutamine at amino acid residue 139 in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12 can be substituted with glycine (e.g., Q139G) or leucine (e.g., Q139L). In certain cases, the tyrosine at amino acid residue 210 in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12 can be substituted with phenylalanine (e.g., Y210F). In certain cases, the tyrosine at amino acid residue 217 in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12 may be substituted with phenylalanine (e.g., Y217F). In certain cases, the aspartic acid at amino acid residue 219 in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12 may be substituted with alanine (e.g., D219A).

[0052] In certain embodiments, the modified LGIC subunits described herein may include at least one modified amino acid that confers an increase in ion conductance to the modified LGIC. In certain cases, the modified LGIC subunit may include the 5HT3 IPD shown in SEQ ID NO: 3, where the amino acid modification may be a substitution at amino acid residues 425, 429, and / or 433. The modified LGIC subunits described herein may include the 5HT3 IPD having R425Q substitution, R429D substitution, and R433A substitution. In certain cases, the modified LGIC subunit may include the 5HT3 IPD shown in SEQ ID NO: 4, where the amino acid modification may be a substitution at amino acid residues 420, 424, and / or 428. The modified LGIC subunits described herein may include the 5HT3 IPD having R420Q substitution, R424D substitution, and R428A substitution.

[0053] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-5HT3 LGIC subunit (SEQ ID NO: 6) having a human α7 nAChR LBD (SEQ ID NO: 1) with Q79G and Y115F amino acid substitutions, and a mouse 5HT3 IPD (SEQ ID NO: 3).

[0054] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-5HT3 LGIC subunit (SEQ ID NO: 6) having a human α7 nAChR LBD (SEQ ID NO: 1) with Q79G amino acid substitutions and Q139G amino acid substitutions, and a mouse 5HT3 IPD (SEQ ID NO: 3).

[0055] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) having a Q79G amino acid substitution and a Y115F amino acid substitution, and a human GlyR IPD (SEQ ID NO: 5) having an A298G amino acid substitution.

[0056] In certain cases, the modified LGIC described herein may include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) having a Q79G amino acid substitution and a Q139G amino acid substitution, and a human GlyR IPD (SEQ ID NO: 5) having an A298G amino acid substitution.

[0057] In certain cases, the modified LGIC described herein may include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) having an R27D amino acid substitution, an E41R amino acid substitution, a Q79G amino acid substitution, and a Y115F amino acid substitution, as well as a human GlyR IPD (SEQ ID NO: 5) having an A298G amino acid substitution.

[0058] In certain cases, the modified LGIC described herein may comprise 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).

[0059] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions 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).

[0060] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions 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).

[0061] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions 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).

[0062] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions 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).

[0063] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-5HT3 LGIC subunit having a human α7 nAChR LBD (SEQ ID NO: 2) having a substitution at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) and a human 5HT3 IPD (SEQ ID NO: 4).

[0064] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) having a substitution at amino acid residue 175 (e.g., G175K) and a human GlyR IPD (SEQ ID NO: 5).

[0065] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-5HT3 LGIC subunit having human α7 nAChR LBD (SEQ ID NO: 2) having substitutions at amino acid residues 131 (e.g., L131G, L131A, L131M, or L131N) and 139 (e.g., Q139L), and human 5HT3 IPD (SEQ ID NO: 4) having R420Q substitution, R424D substitution, and R428A substitution.

[0066] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-5HT3 LGIC subunit having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions at amino acid residues 131 (e.g., L131G, L131A, L131M, or L131N), 139 (e.g., Q139L), and 217 (e.g., Y217F), as well as a human 5HT3 IPD (SEQ ID NO: 4) with R420Q substitution, R424D substitution, and R428A substitution.

[0067] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions at amino acid residues 175 (e.g., G175K) and 115 (e.g., Y115F), and a human GlyR IPD (SEQ ID NO: 5).

[0068] In certain cases, the modified LGIC described herein may include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions at amino acid residues 175 (e.g., G175K), 115 (e.g., Y115F), and 79 (e.g., Q79G), as well as a human GlyR IPD (SEQ ID NO: 5).

[0069] In certain cases, the modified LGIC described herein may include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions at amino acid residues 175 (e.g., G175K), 77 (e.g., W77F), and 79 (e.g., Q79G), as well as a human GlyR IPD (SEQ ID NO: 5).

[0070] In certain cases, the modified LGIC described herein may include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) having a substitution at amino acid residue 216 (e.g., P216I) and a human GlyR IPD (SEQ ID NO: 5).

[0071] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions at amino acid residues 216 (e.g., P216I) and 79 (e.g., Q79G) and a human GlyR IPD (SEQ ID NO: 5).

[0072] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 10) having a human α7 nAChR LBD (SEQ ID NO: 2) having a substitution at amino acid residue 131 (e.g., L131A, L131G, L131M, L131N, L131Q, L131V, or L131F) and a human GABAc IPD (SEQ ID NO: 9).

[0073] In certain cases, the modified LGIC described herein may comprise one or more additional polypeptide sequences. These polypeptide sequences may be transport sequences (e.g., transport (export) sequences and / or signal sequences). Examples of transport (export) sequences include, but are not limited to, ER transport sequences (e.g., FCYENEV (SEQ ID NO: 16)). Examples of signal sequences include, but are not limited to, CHRNB4 signal sequences (e.g., This includes TIFF2026065133000017.tif4128. Polypeptide sequences can be targeting sequences. Examples of targeting sequences, but not limited to, include KCNB1 somatic cell targeting sequences (e.g., Includes TIFF2026065133000018.tif12158. One or more additional polypeptide sequences may be included in the modified LGIC at any appropriate position. In certain cases, the additional polypeptide sequence may be a terminal (e.g., C-terminal or N-terminal) polypeptide sequence. In certain cases, the additional polypeptide sequence may be an insertion. In certain cases, the additional polypeptide sequence may be a substitution.

[0074] In certain cases, the modified LGICs described herein may include transport sequences. For example, a modified LGIC may include an ER transport sequence (e.g., FCYENEV (SEQ ID NO: 16)). An exemplary modified LGIC including an α7 nAChR LBD having an L131G substitution, a Q139L substitution, and a Y217F (e.g., each of these corresponding to the residue numbers shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12), and a GlyR IPD having an ER transport sequence inserted between residues 142 and 143 (e.g., corresponding to the residue numbers shown in SEQ ID NO: 5), is shown in Figure 16.

[0075] In certain cases, the modified LGIC described herein may include a signal sequence. For example, the modified LGIC may include a CHRNB4 signal sequence (e.g., Exemplary modified LGICs, including TIFF2026065133000019.tif4128), which may include a CHRNB4 signal sequence substituting residues 1-22, an L131G substitution, a Q139L substitution, and a Y217F (for example, each of these being for residue numbers shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12), as well as a GlyR IPD, are shown in Figure 17.

[0076] In certain cases, the modified LGIC described herein may include a targeting sequence. For example, the modified LGIC may include a KCNB1 somatic cell targeting sequence (e.g., This may include TIFF2026065133000020.tif11158. An exemplary modified LGIC, including an α7 nAChR LBD having an L131G substitution, a Q139L substitution, and Y217F (for example, each of these being for the residue number shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12), and a GlyR IPD having a KCNB1 somatic cell-targeting sequence inserted between residues 142 and 143 (for example, for the residue number shown in SEQ ID NO: 5), is shown in Figure 18. If the LBD and / or IPD are homologs, orthologues, or paralogs of the sequences described herein (e.g., SEQ ID NOs: 1-5 and / or 9), it is understood that references to specific modified amino acid residues may shift to the corresponding amino acids in the homolog, orthologue, or paralog. For example, residues 425, 429, and 433 in mouse 5HT3 IPD described in SEQ ID NO: 3 correspond to residues 420, 424, and 428 in human 5HT3 IPD described in SEQ ID NO: 4, and substitutions of R425Q, R429D, and R433A in mouse 5HT3 IPD correspond to substitutions of R420Q, R424D, and R428A in human 5HT3 IPD.

[0077] Any method can be used to obtain the modified LGIC subunits described herein. In certain cases, peptide synthesis methods can be used to produce the modified LGIC subunits described herein. Examples of peptide synthesis methods include, but are not limited to, liquid-phase peptide synthesis and solid-phase peptide synthesis. In certain cases, protein biosynthesis methods can be used to produce the modified LGIC subunits described herein. Examples of protein biosynthesis methods include, but are not limited to, the transcription and / or translation of nucleic acids encoding phosphorylation-mimicking peptides provided herein. Similar modified LGIC subunits (e.g., modified subunits having essentially the same modifications and / or essentially the same amino acid sequence) will self-assemble through LBD-to-LBD interactions to form modified LGICs.

[0078] This specification also provides nucleic acids encoding the modified LGIC subunits described herein, as well as constructs for expressing the nucleic acids encoding the modified LGIC subunits described herein (e.g., plasmids, nonviral vectors, synthetic constructs such as viral vectors (e.g., adeno-associated virus, herpes simplex virus, or lentiviral vectors)).

[0079] Nucleic acid sequences encoding modified LGIC subunits described herein may encode any LGIC described herein. In certain cases, nucleic acid sequences provided herein may encode LBDs from any LGIC described herein. In certain cases, nucleic acid sequences provided herein may encode IPDs from any LGIC described herein. If a nucleic acid sequence provided herein encodes a chimeric LGIC, the chimeric LGIC may include an LBD selected from any suitable LGIC and an IPD selected from any suitable LGIC.

[0080] In certain cases, nucleic acid sequences may encode LGICs as described herein. For example, nucleic acid sequences may encode nAChRs (e.g., α7 nAChRs). Representative nucleic acid sequences encoding rat α7 nAChR amino acid sequences (including both LBD and IPD) are as follows:

[0081] TIFF2026065133000021.tif183158

[0082] In certain cases, nucleic acid sequences encoding modified LGIC subunits described herein may encode LBDs from α7 nAChR. Examples of nucleic acid sequences encoding α7 nAChR LBDs include, but are not limited to, the nucleic acid sequence shown in SEQ ID NO: 20, the nucleic acid sequence shown in SEQ ID NO: 21, and the nucleic acid sequence shown in SEQ ID NO: 22. In certain cases, nucleic acid sequences encoding α7 nAChR LBDs may have at least 75% sequence identity to SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22 (for example, 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).

[0083] TIFF2026065133000022.tif126158TIFF2026065133000023.tif154158

[0084] In certain cases, nucleic acid sequences encoding modified LGIC subunits as described herein may encode IPD from the 5HT3 receptor. Examples of nucleic acid sequences encoding 5HT3 IPD include, but are not limited to, the nucleic acid sequence shown in SEQ ID NO: 23 and the nucleic acid sequence shown in SEQ ID NO: 24. In certain cases, nucleic acid sequences encoding 5HT3 IPD may have at least 75% sequence identity to SEQ ID NO: 23 or SEQ ID NO: 24 (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).

[0085] TIFF2026065133000024.tif191158

[0086] In certain cases, nucleic acid sequences encoding modified LGIC subunits described herein may encode IPD from GlyR. Examples of nucleic acid sequences encoding GlyR IPD include, but are not limited to, the nucleic acid sequence shown in SEQ ID NO: 5. In certain cases, GlyR IPD may be a homolog, ortholog, or paralog of the human GlyR IPD shown in SEQ ID NO: 5. In certain cases, GlyR IPD may have at least 75% sequence identity to SEQ ID NO: 25 (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).

[0087] TIFF2026065133000025.tif75158

[0088] In certain cases, the nucleic acid sequences encoding the modified LGIC subunits described herein are GABA receptors (e.g., GABA C Also known as GABA A-ρ ,) can code IPD. GABA A-ρExamples of nucleic acid sequences encoding IPD include, but are not limited to, the nucleic acid sequence shown in SEQ ID NO: 26. In specific cases, GABA A-ρ IPD may have at least 75% sequence identity with respect to sequence number 26 (for example, 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).

[0089] TIFF2026065133000026.tif75158

[0090] In calculating the percentage of sequence identity (%), two sequences are aligned, and the number of identical 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 nucleic acid residues) and multiplied by 100 to obtain the percentage of sequence identity (%) value. It will be understood that the length of the aligned region may be part of one or both sequences up to the full length of the shortest sequence. It will also be understood that a single sequence can be aligned with multiple other sequences, and therefore may have different percentage of sequence identity (%) values ​​across each aligned region. Alignment of two or more sequences to determine the percentage of sequence identity (%) can be performed using the computer program ClustalW with default parameters, which aligns the query and one or more target sequences so that the best match between them can be calculated and identity, similarity, and difference can be determined. See, for example, Chenna et al., 2003, Nucleic Acids Res., 31(13):3497-500.

[0091] Nucleic acid sequences encoding modified LGICs as described herein may comprise at least one modified nucleic acid such that the nucleic acid sequence can encode an LBD having at least one modified amino acid and / or an IPD having at least one modified amino acid. In particular cases, nucleic acid sequences encoding modified LGICs as described herein may comprise more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more) modified nucleic acids. For example, a nucleic acid sequence may encode a modified LGIC subunit comprising an α7 LBD having at least 75% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12, and amino acid substitutions at amino acid residues 27, 41, 77, 79, 131, 139, 141, 175, 210, 216, 217, and / or 219. Examples of nucleic acid codons at codon numbers 27, 41, 77, 79, 131, 139, 141, 175, 210, 216, 217, and / or 219 that may cause amino acid substitutions at amino acid residues 27, 41, 77, 79, 131, 139, 141, 175, 210, 216, 217, and / or 219 are shown below.

[0092] (Table 12) Codons that produce LBD amino acid substitutions TIFF2026065133000027.tif80163

[0093] For example, the nucleic acid sequence may encode a GlyR IPD having at least 75% sequence identity with the sequence shown in SEQ ID NO: 5, and a modified LGIC subunit including an amino acid substitution at amino acid residue 298 of the α7-GlyR IPD. Examples of nucleic acid codons at codon number 298 that may result in an amino acid substitution at amino acid residue 298 are shown below.

[0094] (Table 13) Codons used that cause GlyR IPD amino acid substitutions TIFF2026065133000028.tif11163* The numbering corresponds to the residue number listed in Sequence ID: 7.

[0095] For example, a nucleic acid sequence may encode a GABAC IPD with at least 75% sequence identity to SEQ ID NO: 9, and a modified LGIC subunit including an amino acid substitution at amino acid residue 298 of the α7 GABAC chimeric receptor. Examples of nucleic acid codons at codon number 298 that may result in an amino acid substitution at amino acid residue 298 are shown below.

[0096] (Table 14) GABAC IPD codons that cause amino acid substitution TIFF2026065133000029.tif11163* The numbering corresponds to the residue number listed in Sequence ID No. 10.

[0097] In certain cases, the nucleic acid sequences encoding the modified LGIC described herein may encode the α7-5HT3 chimeric receptor shown in SEQ ID NO: 6 (for example, including the human α7 nAChR LBD (SEQ ID NO: 1) and mouse 5HT3 IPD (SEQ ID NO: 3) components). Examples of nucleic acid sequences encoding the α7-5HT3 chimeric receptor, including but not limited to the human α7 nAChR LBD and mouse 5HT3 IPD, include the nucleic acid sequence shown in SEQ ID NO: 27.

[0098] TIFF2026065133000030.tif169158

[0099] In certain cases, the nucleic acid sequences encoding the modified LGIC described herein may encode the α7-GlyR chimeric receptor shown in SEQ ID NO: 7 (e.g., human α7 nAChR LBD (SEQ ID NO: 2) and human GlyR IPD (SEQ ID NO: 5)). Examples of nucleic acid sequences encoding the α7-GlyR chimeric receptor, including but not limited to human α7 nAChR LBD and human GlyR IPD, include the nucleic acid sequence shown in SEQ ID NO: 28.

[0100] TIFF2026065133000031.tif154158

[0101] In certain cases, the nucleic acid sequences encoding the modified LGIC described herein may encode the α7-GABAC chimeric receptor shown in SEQ ID NO: 10 (e.g., human α7 nAChR LBD (SEQ ID NO: 2) and human GABAC IPD (SEQ ID NO: 9)). Examples of nucleic acid sequences encoding the chimeric receptor including human α7 nAChR LBD (SEQ ID NO: 2) and human GABAC IPD (SEQ ID NO: 9) include, but are not limited to, the nucleic acid sequence shown in SEQ ID NO: 29.

[0102] TIFF2026065133000032.tif154158

[0103] In certain cases, the nucleic acid sequences encoding the modified LGIC described herein may include nucleic acid sequences encoding one or more additional polypeptide sequences (e.g., transport sequences, e.g., transport (export) sequences and / or signal sequences, or targeting sequences). Examples of nucleic acid sequences encoding transport (export) sequences include, but are not limited to, nucleic acid sequences encoding ER transport sequences (e.g., This includes TIFF2026065133000033.tif4128. Examples of nucleic acid sequences encoding signal sequences include, but are not limited to, nucleic acid sequences encoding CHRNB4 signal sequences (for example, This includes TIFF2026065133000034.tif12159. Examples of nucleic acid sequences encoding targeting sequences include, but are not limited to, nucleic acid sequences encoding KCNB1 somatic cell targeting sequences (e.g., This includes TIFF2026065133000035.tif26159.

[0104] In certain cases, nucleic acids encoding the modified LGIC subunits described herein can be ligated (e.g., actionably ligated) to one or more regulatory elements. For example, nucleic acids encoding the modified LGIC subunits described herein can be actionably ligated to any suitable promoter. The promoter may be a native (i.e., minimal) promoter or a compound promoter. The promoter may 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 neuronal subtype-specific). Examples of promoters that can be used to drive the expression of nucleic acids encoding the modified LGIC subunits described herein include, but are not limited to, synapsin (SYN), CAMKII, CMV, CAG, enolase, TRPV1, POMC, NPY, AGRP, MCH, and orexin promoters. In certain cases, nucleic acids encoding the modified LGIC subunits described herein can be actionably ligated to a neuron-specific promoter.

[0105] Where a nucleic acid encoding a modified LGIC subunit described herein is present in the construct, the construct may be any suitable construct. The construct may be a nucleic acid (e.g., DNA, RNA, or a combination thereof) construct. Examples of constructs include, but are not limited to, plasmids, nonviral vectors, and viral vectors (e.g., adeno-associated virus vectors, herpes simplex virus vectors, or lentiviral vectors). In certain cases, a construct containing a nucleic acid encoding a modified LGIC subunit described herein may express the modified LGIC subunit. In certain cases, the construct may include an internal ribosome entry site (IRES, e.g., a bicistronic IRES). In certain cases, the construct may include a nucleic acid sequence encoding a detectable marker (e.g., a fluorescent polypeptide, e.g., green fluorescent polypeptide (GFP; e.g., high-sensitivity GFP (EGFP))). In certain cases, the construct may include a nucleic acid sequence providing a selectable marker (e.g., an antibiotic resistance marker, e.g., ampicillin resistance) to the construct. Figure 19 shows an exemplary plasmid map and nucleic acid sequence of a construct containing nucleic acid sequences encoding the modified LGIC subunit described herein. Figure 20 shows another exemplary plasmid map and nucleic acid sequence of a construct containing nucleic acid sequences encoding the modified LGIC subunit described herein.

[0106] This specification also provides cells (e.g., mammalian cells) having the modified LGIC described herein. Mammalian cells having the modified LGIC described herein can be obtained by any suitable method. In certain cases, pre-assembled modified LGIC can be provided to cells. In certain cases, nucleic acids encoding the modified LGIC subunit described herein can be provided to cells under conditions in which the modified LGIC subunit is translated and under conditions in which multiple (e.g., 3, 4, 5, 6, or more) modified LGIC subunits can be assembled into the modified LGIC described herein.

[0107] LGIC ligand This specification also provides LGIC ligands that can bind to and activate the modified LGIC described herein. The LGIC ligand may also refer to a pharmacologically selective effector module (PSEM). The LGIC ligands described herein that can bind to and activate the modified LGIC may be exogenous or endogenous. The LGIC ligands described herein that can bind to and activate the modified LGIC may be spontaneous or synthetic. The LGIC ligands described herein that can bind to and activate the modified LGIC may be canonical or non-canonical. The LGIC ligands described herein that can bind to and activate the modified LGIC may be agonists or antagonists. In certain cases, the LGIC ligand is an exogenous LGIC agonist. Examples of LGIC ligands include, but are not limited to, ACh, nicotine, epivatatin, cyticine, RS56812, tropisetron, nortopisetron, 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, for example, Figures 3B, 5C, 11A, 11B, and 11C).

[0108] LGIC ligands that can bind to and activate modified LGICs as described herein may have selective binding (e.g., enhanced binding or increased potency) to the modified LGICs described herein (e.g., compared to unmodified LGICs). In certain cases, LGIC ligands that can bind to and activate modified LGICs as described herein may not bind to or activate endogenous receptors (e.g., endogenous LGICs). LGIC ligands that selectively bind to and activate modified LGICs as described herein rather than to unmodified LGIC ligands (e.g., modified LGICs having at least one amino acid modification that confers pharmacological selectivity to the modified LGIC) may be described as having enhanced potency for the modified LGIC. In certain cases, the modified LGIC subunit described herein, which selectively binds to an exogenous LGIC ligand, may have at least 5 times (e.g., at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 65 times, at least 70 times, at least 75 times, at least 80 times, at least 85 times, at least 95 times, at least 100 times, at least 125 times, at least 150 times, at least 200 times, at least 250 times, or at least 300 times) enhanced potency compared to the modified LGIC. For example, an LGIC ligand that selectively binds to and activates modified LGIC may have an enhanced potency of approximately 10 to 300 times (e.g., approximately 10 to 250 times, approximately 10 to 200 times, approximately 10 to 150 times, approximately 10 to 100 times, approximately 25 to 300 times, approximately 50 to 300 times, approximately 100 to 300 times, approximately 200 to 300 times, approximately 25 to 250 times, approximately 50 to 200 times, or approximately 100 to 150 times) compared to modified LGIC. In certain cases, the LGIC ligands described herein that bind to and activate the modified LGIC may have 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, an LGIC ligand that binds to and activates a modified LGIC as described herein may have a ligand potency of less than 15 nM. In certain cases, an LGIC ligand may 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) relative to a modified LGIC subunit as described herein. For example, an LGIC ligand (e.g., tropisetron) binds to a modified LGIC subunit (e.g., α7) as described herein. Q79G -GlyR A298G ) may have an EC50 of approximately 11 nM. For example, an LGIC ligand (e.g., nortropisetron) may have a modified LGIC subunit (e.g., α7) as described herein. Q79G、Y115F -GlyR A298G ) may have an EC50 of approximately 13 nM. In certain cases, an LGIC ligand may have an EC50 of more than 20 μM (e.g., more than 22 μM, more than 25 μM, more than 35 μM, more than 50, more than 65 μM, more than 80 μM, or more than 100 μM) with respect to the modified LGIC subunits described herein. For example, an LGIC ligand (e.g., ACh) may have an EC50 of more than 20 μM (e.g., more than 22 μM, more than 25 μM, more than 35 μM, more than 50, more than 65 μM, more than 80 μM, or more than 100 μM) with respect to the modified LGIC subunits described herein. Q79G、Y115F -GlyR A298G ) may have an EC50 exceeding 100 μM.

[0109] In certain embodiments, the LGIC ligand may be a synthetic ligand capable of binding to and activating the modified LGIC described herein, and may be quinuclidine, tropane, 9-azabicyclo[3.3.1]nonane, or 2-phenyl-7,8,9,10-tetrahydro-6H-6,10-methanoazepino[4,5-g]quinoxaline.

[0110] The LGIC ligands that can bind to and activate the modified LGIC described herein are of formula I: TIFF2026065133000036.tif32128 (wherein 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; R can be H or pyridinylmethylene) It may have the following: Examples of aromatic substituents include, but are not limited to, 4-chlorobenzene, 1H-indole, 4-(trifluoromethyl)benzene, 4-chlorobenzene, 2,5-dimethoxybenzene, 4-chloroaniline, aniline, 5-(trifluoromethyl)pyridine-2-yl, 6-(trifluoromethyl)nicotine, and 4-chlorobenzene.

[0111] The LGIC ligands that can bind to and activate the modified LGIC described herein may be quinuclidines. Quinuclidines are given by formula II: TIFF2026065133000037.tif18128 (wherein X3 can be O, NH, or CH2; Y can be O or S; A can be an aromatic substituent; R can be H or pyridinylmethylene) The structure may have the following characteristics. Examples of aromatic substituents include, but are not limited to, 1H-indole, 4-(trifluoromethyl)benzene, 4-chlorobenzene, 2,5-dimethoxybenzene, 4-(trifluoromethyl)benzene, 4-chloroaniline, aniline, 5-(trifluoromethyl)pyridine-2-yl, 6-(trifluoromethyl)nicotine, 3-chloro-4-fluorobenzene, 4-chlorobenzene, and 1H-indole. Examples of quinuclidines include, but are not limited to, compounds PNU-282987, PHA-543613, 0456, 0434, 0436, 0354, 0353, 0295, 0296, and 0676 (see, for example, Figure 5C, Table 3, and Table 6).

[0112] An LGIC ligand capable of binding to and activating the modified LGIC described herein may be tropane. Tropane is given by formula III: TIFF2026065133000038.tif29128 (wherein X2 can be NH or NMe; X3 can be O, NH, or CH2; Y can be O or S; A can be an aromatic substituent) The structure may be as follows. Examples of aromatic substituents include, but are not limited to, 1H-indole, 7-methoxy-1H-indole, 7-methyl-1H-indole, 5-chloro-1H-indole, and 1H-indazole. Examples of tropanes include, but are not limited to, tropisetron, pseudo-tropisetron, nortropisetron, compound 737, and compound 745 (see, for example, Figure 5C, Table 3, and Table 6).

[0113] An LGIC ligand capable of binding to and activating the modified LGIC described herein may be 9-azabicyclo[3.3.1]nonane. 9-azabicyclo[3.3.1]nonane is given by formula IV: TIFF2026065133000039.tif27128 (wherein X1 may be CH, X2 may be NH or NMe, X3 may be O, NH, or CH; Y may be O or S; A may be an aromatic substituent) It may have the following structure. Examples of aromatic substituents are, but are not limited to, 4-chlorobenzene. Examples of 9-azabicyclo[3.3.1]nonanes include, but are not limited to, compounds 0536, 0749, 0751, 0760, and 0763 (see, for example, Figure 5C, Table 3, and Table 6).

[0114] In certain cases, the LGIC ligand may be 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, formula V: The structure may be as shown in TIFF2026065133000040.tif14128 (wherein R1 is H, an aromatic substituent, a methyl-containing group, an ethyl-containing group, or another aliphatic group, an alkoxy-containing group (e.g., a methoxy-containing group, an ethoxy-containing group, a propoxy-containing group, and an isopropoxy-containing group); or an amino-containing group; R2 is H, an aliphatic substituent (e.g., methyl), or an aromatic substituent (e.g., phenyl)). Examples of the R1 group, but not limited to these, include phenyl, 2-toluyl, 3-pyridyl, 4-pyridyl, imidazole, pyrrole, pyrazole, triazole, isoxazole-3-amine, trifluoromethyl, methoxy, N,N-dimethylamino, and N,N-diethylamino. In certain cases, R1 and R2 can be joined to form a ring. Examples of 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepines, but not limited to these, include varenicline, compound 0765, compound 0770, compound 0780, compound 0782, compound 0785, compound 0788, compound 0782, compound 0789, compound 0791, compound 0793, compound 0794, compound 0795, compound 0798, compound 0799, compound 0800, compound 0801, compound 0802, compound 0803, compound 0804, compound 0805, compound 0807, compound 0808, compound 0812, compound 0813, compound 815, compound 816, and compound 817 (see, for example, Figures 11A, 14A, Tables 3, 9, 10, and 11).

[0115] In certain cases, the LGIC ligand is 2-(pyridine-3-yl)-1,5,6,7,8,9-hexahydro-5,9-methanoimidazo[4',5':4,5]benzo[1,2-d]azepine: This could be TIFF2026065133000041.tif12128. An example of 2-(pyridine-3-yl)-1,5,6,7,8,9-hexahydro-5,9-methanoimidazo[4',5':4,5]benzo[1,2-d]azepines is compound 0786 (see, for example, Tables 10 and 11).

[0116] In certain cases, the LGIC ligand may be 7,8,9,10-tetrahydro-1H-6,10-methanoazepino[4,5-g]quinoxaline-2(6H)-one, formula VI: It may have the structure shown in TIFF2026065133000042.tif14128 (wherein R1 is H or an aliphatic substituent (e.g., methyl), R2 is H, an aliphatic substituent (e.g., methyl), or an aromatic substituent, and R3 is O or S). Examples of 7,8,9,10-tetrahydro-1H-6,10-methanoazepino[4,5-g]quinoxaline-2(6H)-one include, but are not limited to, compounds 0783, 0784, 0790, or 0792 (see, for example, Figure 11B, Table 10, and Table 11). For example, LGIC is, It may be 7,8,9,10-tetrahydro-1H-6,10-methanoazepino[4,5-g]quinoxaline-2(6H)-one having the structure of TIFF2026065133000043.tif17128.

[0117] In certain cases, the LGIC ligand can be 1,4-diazabicyclo[3.2.2]nonane, formula VII: TIFF2026065133000044.tif25128 (in the formula, R=H, F, NO2) It may have the structure shown. Examples of 1,4-diazabicyclo[3.2.2]nonanes include, but are not limited to, 3-(1,4-diazabicyclo[3.2.2]nonane-4-yl)dibenzo[b,d]thiophene 5,5-dioxide, compound 0773, and compound 0774 (see, for example, Figure 11C, Table 6, and Table 9).

[0118] How to use This specification also provides methods using the modified LGIC described herein and the LGIC ligands that can bind to and activate the modified LGIC described herein. The LGIC ligands that can bind to and activate the modified LGIC can be used to activate the modified LGIC, along with temporal and / or spatial control based on ligand delivery.

[0119] In certain embodiments, the modified LGIC described herein and the LGIC ligands that can bind to and activate the modified LGIC described herein can be used to identify ligands that selectively bind to the modified LGIC described herein. For example, such a screening method may include providing one or more candidate ligands to the modified LGIC described herein and detecting binding between the candidate ligands and the modified LGIC.

[0120] Any suitable method can be used to detect binding between the candidate ligand and the modified LGIC, and any suitable method can be used to detect the activity of the modified LGIC. For example, the ability of a ligand to bind to and activate the modified LGIC can be measured by assays including, but are not limited to, membrane potential (MP) assays (e.g., fluorescence MP assays), radiobinding assays, and / or voltage clamp measurements of peak and sustained currents.

[0121] In certain embodiments, the modified LGICs described herein and the LGIC ligands that can bind to and activate the modified LGICs described herein can be used to treat mammals having channel disorders (e.g., neural channel disorders or muscle channel disorders). For example, a mammal having a channel disorder can be treated by administering the modified LGICs described herein, and then by administering the LGIC ligands that can bind to and activate the modified LGICs. For example, a mammal having a channel disorder can be treated by administering the modified LGICs described herein (including, for example, human α7 nAChR LBD (SEQ ID NO: 2) with R27D amino acid substitutions, E41R amino acid substitutions, Q79G amino acid substitutions, and Y115F amino acid substitutions, and human GlyR IPD (SEQ ID NO: 5) with an A298G amino acid substitution) and then by administering tropisetron. For example, mammals with channel disorders can be treated by administering a modified LGIC as described herein, comprising 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, followed by administration of varenicline, tropisetron, and / or compound 765.

[0122] Any type of mammal can be treated with the modified LGIC described herein and the LGIC ligands that can bind to and activate the modified LGIC described herein. For example, humans and other primates such as monkeys can be treated with the modified LGIC described herein and the LGIC ligands that can bind to and activate the modified LGIC described herein. In specific cases, dogs, cats, horses, cattle, pigs, sheep, rabbits, mice, and rats can be treated with the modified LGIC described herein and the LGIC ligands that can bind to and activate the modified LGIC described herein.

[0123] Any suitable method can be used to identify mammals with channel disorders and / or mammals at risk of developing channel disorders. For example, genetic testing can be used to identify mammals with channel disorders and / or mammals at risk of developing channel disorders.

[0124] If a mammal is identified as having channel dysfunction and / or being at risk of developing channel dysfunction, that mammal may be administered or instructed to self-administer the modified LGIC described herein, and then an LGIC ligand capable of binding to and activating the modified LGIC described herein, or instructed to self-administer it. The modified LGIC described herein and the LGIC ligand capable of binding to and activating the modified LGIC described herein may be administered together or separately.

[0125] When using the materials and methods described herein to treat mammals with channel dysfunction and / or mammals at risk of developing channel dysfunction, the channel dysfunction may be any channel dysfunction. As used herein, the channel dysfunction may be any disease or disorder caused by abnormal ion channel function and / or abnormal ligand function, or mitigated by regulated ion channel function and / or modified cellular ion flux (e.g., calcium ion flux). The channel dysfunction may be congenital or acquired. Examples of channel disorders include, but are not limited to, Bartter syndrome, Brugada syndrome, catecholamine-mediated polymorphic ventricular tachycardia (CPVT), congenital hyperinsulinemia, cystic fibrosis, Dravet syndrome, transient ataxia, acroerythromelalgia, 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, congenital myotonia, neuromyotonia, non-symptomatic hearing loss, congenital paramyotonia, retinitis pigmentosa, Timothy syndrome, tinnitus, seizures, trigeminal neuralgia, and multiple sclerosis. Alternatively, or furthermore, the materials and methods described herein may be used for other purposes, including but not limited to, pain treatment, cancer cell therapy, appetite control, spasticity treatment, muscular dystonia treatment, tremor treatment, and movement disorder treatment.

[0126] In certain cases, the modified LGIC described herein and the LGIC ligands that can bind to and activate the modified LGIC described herein can be used to modulate cellular activity. The cellular activity modulated using the modified LGIC described herein and the LGIC ligands that can bind to and activate the modified LGIC described herein may be any cellular activity. Examples of cellular activities, but not limited to, include active transport (e.g., ion transport), passive transport, excitation, inhibition, ion flux (e.g., calcium ion flux), and exocytosis. Cellular activity may be increased or decreased. For example, the modified LGIC described herein and the LGIC ligands that can bind to and activate the modified LGIC described herein can be used to modulate (e.g., increase) ion transport across the cell membrane. For example, the modified LGIC described herein and the LGIC ligands that can bind to and activate the modified LGIC described herein can be used to modulate (e.g., increase) cellular excitability.

[0127] The modified LGIC described herein, and the LGIC ligands that can bind to and activate the modified LGIC described herein, can be used to modulate the activity of any type of cell in mammals. The cells may be neurons, glial cells, muscle cells, immune cells (e.g., neutrophils, eosinophils, basophils, lymphocytes, and monocytes), endocrine cells, or stem cells (e.g., embryonic stem cells). In certain cases, the cells may be excitatory cells. The cells may be in vivo or ex vivo.

[0128] The modified LGIC described herein can be administered by any suitable method. The modified LGIC can be administered as a modified LGIC subunit or as a pre-assembled modified LGIC. The modified LGIC can be administered as a nucleic acid encoding the modified LGIC subunit described herein. For example, the nucleic acid can be delivered as a naked nucleic acid or using any suitable vector (e.g., a recombinant vector). The vector may be a DNA-based vector, an RNA-based vector, or a combination thereof. The vector can express the nucleic acid in dividing or non-dividing cells. Examples of recombinant vectors, but not limited to, include plasmids, viral vectors (e.g., retroviral vectors, adenovirus vectors, adeno-associated virus vectors, and herpes simplex vectors), cosmids, and artificial chromosomes (e.g., yeast artificial chromosomes or bacterial artificial chromosomes). In certain cases, the nucleic acid encoding the modified LGIC subunit described herein can be expressed by an adeno-associated virus vector.

[0129] The modified LGICs described herein can be detected by any suitable method (for example, to confirm their presence in cells). In certain cases, agents that selectively bind to modified LGICs can be used to detect them. Examples of agents that can be used to bind to the modified LGICs described herein, but are not limited to, antibodies, proteins (e.g., bungarotoxin), and small molecule ligands (e.g., PET ligands). Active agents that selectively bind to modified LGICs may include detectable labels (e.g., fluorescent labels, radioactive labels, positron emission labels, and enzymatic labels). Methods for detecting LGIC expression in cells may include fluorescence imaging, autoradiography, functional MRI, PET, and SPECT.

[0130] The modified LGICs described herein and the LGIC ligands that can bind to and activate the modified LGICs described herein may be administered to mammals having and / or at risk of developing channel disorders as combination therapies with one or more additional agents / therapies used to treat channel disorders. For example, combination therapies used to treat mammals having channel disorders as described herein may include administering the modified LGICs described herein and the LGIC ligands that can bind to and activate the modified LGICs described herein, and then being treated with acetazoleamide, dichlorophenamide, mexilitzin, glucose, calcium gluconate, L-DOPA, muscle stimulation, spinal cord stimulation, brain stimulation, and / or nerve stimulation.

[0131] In embodiments in which the modified LGIC described herein and the LGIC ligands that can bind to and activate the modified LGIC described herein are used in combination with additional agents / therapies used to treat channel dysfunction, one or more additional agents may be administered at the same time or independently. For example, the modified LGIC described herein and the LGIC ligands that can bind to and activate the modified LGIC described herein may be administered first, followed by one or more additional agents, or vice versa. In embodiments in which the modified LGIC described herein and the LGIC ligands that can bind to and activate the modified LGIC described herein are used in combination with one or more additional treatments used to treat channel dysfunction, one or more additional treatments may be administered simultaneously with or independently of the administration of the modified LGIC described herein and the LGIC ligands that can bind to and activate the modified LGIC described herein. For example, the modified LGIC described herein and the LGIC ligands that can bind to and activate the modified LGIC described herein may be administered before, during, or after one or more additional treatments.

[0132] In certain cases, the modified LGICs and / or LGIC ligands described herein that can bind to and activate the modified LGICs described herein may be formulated in pharmaceutically acceptable compositions for administration to mammals having or at risk of developing channel dysfunction. For example, a therapeutically effective amount of the modified LGICs described herein (e.g., nucleic acids encoding the modified LGICs described herein) and / or LGIC ligands that can bind to and activate the modified LGICs described herein may be formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. Pharmaceutical compositions may be formulated for administration in solid or liquid forms, including but not limited to sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules.

[0133] Pharmaceutically acceptable carriers, fillers, and vehicles that can be used in the pharmaceutical compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphate, 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, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0134] Pharmaceutical compositions comprising the modified LGIC described herein and / or LGIC ligands capable of binding to and activating the modified LGIC described herein may be designed for oral, parenteral (including subcutaneous, intracranial, intra-arterial, intramuscular, intravenous, intra-coronary, intradermal, or topical) or inhalation administration. For oral administration, a therapeutically effective amount of the pharmaceutical composition comprising the modified LGIC described herein (e.g., nucleic acids encoding the modified LGIC described herein) and / or LGIC ligands capable of binding to and activating the modified LGIC described herein may be in the form of pills, tablets, or capsules. Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that are isotonic with the blood of the recipient to whom the formulation is intended, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners. Compositions for inhalation may be delivered, for example, using inhalers, nebulizers, and / or dry powder inhalers. The formulations can be supplied in unit or multi-dose containers, such as sealed ampoules and vials, and can be stored in a lyophilized state requiring only the addition of a sterile liquid carrier, such as water for injection, immediately before use. Immediate injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0135] A pharmaceutically acceptable composition comprising a therapeutically effective amount of the modified LGIC described herein (e.g., nucleic acid encoding the modified LGIC described herein) and / or an LGIC ligand capable of binding to and activating the modified LGIC described herein may be administered topically or systemically. In certain cases, a composition comprising a therapeutically effective amount of the modified LGIC described herein (e.g., nucleic acid encoding the modified LGIC described herein) and / or an LGIC ligand capable of binding to and activating the modified LGIC described herein may be administered systemically to mammals (e.g., humans) by intravenous or oral administration or by inhalation thereof. In certain cases, a composition comprising a therapeutically effective amount of the modified LGIC described herein (e.g., nucleic acid encoding the modified LGIC described herein) and / or an LGIC ligand capable of binding to and activating the modified LGIC described herein may be administered topically to target tissues of mammals (e.g., humans) by transdermal, subcutaneous, intramuscular, intracranial, or open surgical administration (e.g., injection).

[0136] The effective dose may vary depending on the severity of the channel dysfunction, the route of administration, the patient's age and general health status, the use of excipients, the possibility of concomitant use of other therapeutic measures such as other medications, and the judgment of the treating physician.

[0137] The frequency of administration may be any frequency that improves the symptoms of channel dysfunction without causing significant toxicity to mammals. For example, the frequency of administration may be about once a week to about three times a day, about twice a month to about six times a day, or about twice a week to about once a day. The frequency of administration may remain constant or may be variable during the course of treatment. The course of treatment with a composition comprising a therapeutically effective amount of the modified LGIC described herein (e.g., nucleic acid encoding the modified LGIC described herein) and / or an LGIC ligand that can bind to and activate the modified LGIC described herein may include rest periods. For example, a composition comprising a therapeutically effective amount of the modified LGIC described herein (e.g., nucleic acid encoding the modified LGIC described herein) and / or an LGIC ligand that can bind to and activate the modified LGIC described herein may be administered daily for two weeks, followed by a two-week rest period, and such regimens may be repeated multiple times. As with the effective dose, various factors may influence the actual frequency of administration used for a particular application. For example, the effective dose, duration of treatment, use of multiple therapeutic agents, route of administration, and severity of channel dysfunction may necessitate an increase or decrease in the frequency of administration.

[0138] The effective period for administering a therapeutically effective amount of a composition comprising a modified LGIC (e.g., a nucleic acid encoding a modified LGIC as described herein) and / or an LGIC ligand capable of binding to and activating a modified LGIC as described herein is any period of time during which the symptoms of the channel disorder are improved without causing significant toxicity to the mammal. For example, the effective period can vary from a few days to several weeks, months, or even years. In particular cases, the effective period for treating a channel disorder may range from about one month to about ten years. Several factors influence the actual effective period used for a particular treatment. For example, the effective period may vary depending on the frequency of administration, the effective dose, the use of multiple therapeutic agents, the route of administration, and the severity of the channel disorder being treated.

[0139] In certain cases, the course of treatment and the symptoms of the treated mammal can be monitored for channel dysfunction. Any appropriate method can be used to monitor the symptoms of channel dysfunction.

[0140] The present invention will be further illustrated in the following examples, but these examples are not intended to limit the scope of the invention as described in the claims. [Examples]

[0141] Example 1: Efficacy-enhancing ligand-binding domain mutation A panel of 51 clinically used drugs chemically similar to nicotinic receptor agonists was screened using a panel of 41 α7-5HT3 chimeric channels with mutant LBDs. The mutations are located at the residues highlighted in Figure 1. The screen revealed that Gln in α7 nAChR LBDs enhances the efficacy of the known nAChR agonist tropisetron. 79 Mutations in α7 nAChR were identified (Figure 2). These mutations (Q79A, Q79G, Q79S) reduce the size of the amino acid side chain. Several mutant ion channel-ligand combinations gave up to a 12-fold improvement in potency (Table 1, Figure 3). Standard α7 nAChR agonists, ACh, nicotine, epivatidine, and the smoking cessation drug varenicline were not significantly affected by the Q79A, Q79G, or Q79S mutations. However, a subset of α7 nAChR agonists showed enhanced potency with several mutations. Cyticine, RS56812, tropisetron, nortropisetron, and PNU-282987 were found to be α7 nAChR agonists. Q79G They showed significantly improved efficacy for -5HT3. Furthermore, nortropisetron and PNU-282987 showed significantly improved efficacy for α7 Q79A -5HT3 and α7 Q79S-5HT3 showed significantly enhanced potency. Generally, agonists based on quinuclidines or tropane pharmacophores having a bound aromatic structure that interacts with the complementary binding surface of the ligand-binding domain showed improved potency with Gln79 substitution with smaller amino acid residues Ala, Gly, or Ser. For most agonists, α7 Q79G -5HT3 was the most preferred mutant chimera channel.

[0142] (Table 1) Efficacy of nAChR agonists against Gln79-mutated chimeric cation channels in HEK cells. Mean EC50, SEM (μM) in parentheses. TIFF2026065133000045.tif54157

[0143] These mutant LBDs were used to generate α7-GlyR chimeric channels with up to 6-fold enhanced potency for most of these ligands (Figure 4A). Similar to the α7-5HT3 mutations, these mutations in Gln79 did not significantly affect the potency of ACh, nicotine, epivathidine, varenicline, or cyticine. However, tropisetron, nortropisetron, and RS56812 showed enhanced potency for α7 Q79G It showed significantly enhanced efficacy against -GlyR. Similar to LBD mutations against α7-5HT3, nortropisetron showed α7 Q79A -It has significantly enhanced efficacy against GlyR, and PNU-282987 is α7 Q79S -It showed significantly enhanced efficacy against GlyR. For most agonists, α7 Q79G -GlyR was the most preferred mutant chimera channel.

[0144] Another relationship observed in small molecule screening was that mutations in Trp77 were associated with α7 W77F -5HT3 (EC50: 1.2μM), α7 W77Y -5HT3 (EC50: 1.1μM), and α7 W77FThe study showed that the GlyR (EC50: 0.66 μM) receptor induced agonist activity against the drug granisetron. Granisetron is a 5HT3 receptor antagonist and does not activate α7-5HT3 or α7-GlyR.

[0145] These results indicate that mutations in Q79 (to A, G, or S) in the α7 nAChR LBD enhanced the binding of known LGIC ligands to modified LGIC.

[0146] Example 2: Potency-enhancing ion pore domain mutation α7-GlyR channels with previously established IPD mutations in full-length glycine receptor channels (T258S and A288G, GlyR numbering; equivalent to T268S and A298G for α7-GlyR numbering) were examined for enhanced efficacy against the allosteric agonist ivermectin. T268S Channels possessing this characteristic have been found to have virtually ligand-free open probability, which makes them unsuitable for manipulating cellular ligand regulation. α7-GlyR was effective in enhancing ivermectin potency at full-length glycine receptors. A298G Mutations in this channel resulted in a moderate change in the open probability in the absence of the ligand. Therefore, this channel was examined for activity against a panel of known agonists: standard agonists ACh, nicotine, and epivathidine, as well as varenicline and tropisetron, and α7-GlyR A298G In this case, agonist potency was not significantly enhanced. A subset of α7 nAChR agonists showed a moderate increase in potency up to 4-fold: RS56812, cyticine, PNU-282987, and nortropisetron were significantly more potent. Therefore, the effect of the IPD A298G mutation improved ligand potency, but was not as effective as the mutation in LBD, depending on the ligand structure.

[0147] We examined the Q79G mutation and A298G IPD mutation in the LBD of α7-GlyR (Table 2). Double mutant chimeric channel, α7 Q79G -GlyR A298G This resulted in a synergistic potency enhancement, showing up to an 18-fold potency increase compared to α7-GlyR with α7 nAChR agonists. The enhancement from this dual mutant channel was greater than the enhancement from individual mutations for the agonists RS56812, tropisetron, nortropisetron, and PNU-282987. Further highlighting the unexpected structural sensitivity of this mutation combination, multiple agonists, including ACh, nicotine, epivathidine, varenicline, and cyticine, exhibited synergistic potency enhancements against α7-GlyR and α7 Q79G -GlyR A298G There was no significant change between the two. Therefore, the combination of the LBD mutation Q79G and the IPD mutation A298G resulted in a synergistic effect that significantly increased the potency of some, though not all, nicotinic agonists by approximately 10 to 20 times.

[0148] (Table 2) Efficacy of nAChR agonists against mutant chimeric chloride channels. Mean EC50 and SEM (μM) in parentheses for agonist activity in HEK cells expressing chimeric channels. TIFF2026065133000046.tif59168

[0149] These results indicate that mutations at Q79 (to A, G, or S) in α7 nAChR LBD and / or mutations at A298 (to G) in GlyR IPD further enhanced the selective binding of known LGIC ligands to modified LGIC.

[0150] Example 3: Molecules exhibiting enhanced potency α7 Q79G -GlyR A298GBased on the structure-activity relationships of known agonists that showed enhanced efficacy, various synthetic molecules consisting of quinuclidines, tropanes, or 9-azabicyclo[3.3.1]nonanepharmacophores having one or more aromatic side-chain substituents were tested. Furthermore, the known α7 nAChR agonist PHA-543613 (Walker et al 2006, Wishka et al 2006) was also tested, and α7 Q79G -GlyR A298G These molecules showed exceptional efficacy against α7. These molecules generally exhibited 10 to 100 times enhanced efficacy (Table 3), suggesting that for these pharmacophores, a specific range of structural characteristics is present in α7. Q79G -GlyR A298G This demonstrates compatibility with improved efficacy.

[0151] These results indicate that modified LGIC can be activated by synthetic quinuclidine-containing and tropane-containing LGIC ligands.

[0152] (Table 3) Efficacy of compounds against chimeric channels. Mean EC50 and SEM (μM) in parentheses for agonist activity in HEK cells expressing chimeric channels. "Partial" refers to partial agonist activity. TIFF2026065133000047.tif230125

[0153] Example 4: Mutation that reduces acetylcholine responsiveness The α7 nAChR is relatively less sensitive to ACh compared to other nAChR isoforms, and potency-enhancing mutations for tropane and quinuclidine ligands did not substantially alter the potency of acetylcholine in these channels. Therefore, the chimeric channels were further modified to reduce the acetylcholine responsiveness of these channels. Additional LBD mutations Y115F and Q139G significantly reduced acetylcholine responsiveness above 100 μM in certain cases, and α7 Q79G、Y115F -5HT3, α7 Q79G、Q139G -5HT3, α7 Q79G、Q139G -GlyR A298, α7 Q79G、Y115F -GlyR A298G Regarding this, it only moderately reduced the effectiveness of certain agonists. For example, α7 Q79G、Y115F -GlyR A298G It has an EC50 of 13 nM for nortropisetron and over 100 μM for ACh (Table 4).

[0154] (Table 4) Efficacy of nAChR agonists against mutant chimeric chloride channels with low acetylcholine responsiveness. Mean EC50 and SEM (μM) in parentheses for activity in HEK cells expressing the chimeric channel. TIFF2026065133000048.tif48161

[0155] These results indicate that Y115F and / or Q139G mutations in α7 nAChR LBD reduced the binding of endogenous LGIC ligand Ach to modified LGIC.

[0156] Example 5: Mutation that reduces association with endogenous receptor subunits The assembly of α7 nAChR is based on the association of five homomeric subunits via LBD-to-LBD interactions (Celie et al 2004 Neuron 41: 907-14). To minimize undesirable association with endogenous α7 nAChR subunits and / or undesirable association of chimeric channels, potential intersubunit crosslinks were identified by examining the crystal structure of the acetylcholine-binding protein and identifying nearby intersubunit residues with opposite charges that also have homologous ionic amino acids in the α7 nAChR receptor LBD. Charge inversion 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 interfere with intersubunit interactions with unmodified subunits but preserve interactions between subunits with charge inversion mutations (Figure 6A). Chimeric LGIC subunits with charge inversion mutations were able to selectively associate with each other without interacting with unmodified channels, e.g., endogenous α7 nAChR. Double mutations in R27D and E41R in α7 nAChR LBD resulted in functional channels (Figure 6B). Co-expression of these charge-reversal channels with α7-5HT3 channels containing unmodified sequences showed that the charge-reversal subunits did not co-immunoprecipitate with the unmodified channels (Figure 6C). Chimeric channel α7 R27D、E41R、Q79G、Y115F -GlyR A298G Combinations of potency-enhancing mutations and acetylcholine-blocking mutations to obtain these mutations revealed that some agonists retain high potency against their congener agonists (Table 4, right column).

[0157] These results indicate that R27D and E41R mutations in α7 nAChR LBD reduced the association of modified LGIC subunits with other modifications and / or endogenous LGIC subunits.

[0158] Example 6: LBD mutation that increases ligand potency Gly of α7 nAChR LBD in α7-GlyR chimeric channel 175 and Pro 216We tested mutations in Gly. 175 Mutation of Lys (α7 G175K -GlyR) showed increased efficacy against ACh (5 times) (Table 5). α7 G175K Regarding -GlyR, it was also found that its nicotine potency was enhanced 10-fold compared to the unmodified α7-GlyR chimeric channel (Table 5). 216 Mutation to Ile (α7 P216I -GlyR) did not substantially alter ACh potency (Table 5). However, α7 P216I -GlyR showed nicotine potency that was more than four times greater than that of unmodified α7-GlyR (Table 5). G175K -GlyR and α7 P216I These potency-enhancing mutations in α7-GlyR also affected the potency of several other α7-GlyR agonists up to 30-fold (Table 5). G175K -GlyR showed more than 10 times greater potency enhancement than α7-GlyR for clinically used drugs such as tropisetron, varenicline, cyticine, granisetron, and epivatidine. P216I -For GlyR, the potency enhancement was approximately threefold (Table 5).

[0159] (Table 5) Enhancement of agonist efficacy in the a7GlyR chimeric channel by G175K and P216I mutations. Unit: μM. Brackets: SEM. TIFF2026065133000049.tif232113nd=Not decided

[0160] For use in ACh-producing organisms, it is important to reduce endogenous ACh potency in these channels composed of α7 nAChR LBDs. The G175K mutation could be further combined with other mutations that reduce sensitivity to ACh, such as Y115F and Y210F. Y115F、G175K-For GlyR, tropisetron, granisetron, nortropisetron, PNU-282987, and PHA-543613 showed high efficacy against agonists based on tropane or quinuclidine core structures, while their efficacy against varenicline and cyticine was significantly reduced (Table 5). α7 G175K、Y210F Regarding GlyR, the efficacy against most agonists was significantly reduced, but an enhancement in efficacy against granisetron was observed (Table 5).

[0161] To develop a channel with reduced ACh responsiveness but high efficacy against other agonists, α7 G175K -GlyR was combined with further mutations that increased the potency of specific agonists. The combination with W77F reduced ACh potency and α7 W77F、G175K -GlyR showed increased potency compared to α7-GlyR for granisetron, nortopisetron, and tropisetron, but not for PNU282-987, varenicline, cytidine, or PHA-543613 (Table 5). The combination of G175K with Q79G reduced ACh potency and α7 Q79G、G175K -GlyR showed increased potency with nortropisetron, PHA-543613, and tropisetron (Table 5). However, this potency enhancement was not observed with other agonists such as PNU282-987 or varenicline. α7 G175K、Q139L -GlyR reduced the potency of ACh and increased the potency of nortropisetron and tropisetron (Table 5).

[0162] By incorporating various combinations of mutations in W77F, Q79G, L141F, Y115F, G175K, and Y210F, further reduction of ACh potency was achieved while maintaining high potency for synthetic agonists, including those based on tropane and quinuclidine core structures. α7 Q79G、Y115F、G175K -GlyR reduced ACh responsiveness while maintaining a strong response to tropisetron (Table 5). These mutations also affected α7 Y115F、G175KCompared to -GlyR, and also compared to α7-5HT3 (representing endogenous α7 nAChR activity), responsiveness to other tropane and quinuclidine core structures, particularly quinuclidine thiourea 702 and 703, as well as tropane esters 723, 725, 726, 736, 737, 738, and 745, was also enhanced (Table 6). Q79G、Y115F、G175K -GlyR also showed high sensitivity to ivermectin (Table 5). α7 W77F、Q79G、G175K -GlyR reduced ACh responsiveness while maintaining a high potency response to tropisetron and nortropisetron (Table 5). α7 W77F、Q79G、G175K -GlyR also showed enhanced potency against compounds 723 and 725, as well as further tropane-based core structures such as the clinically used drugs mequitazine and promazine (Table 6). α7 W77F、G175K、Y210F -GlyR reduced ACh responsiveness but significantly improved efficacy against granisetron (Table 5). α7 L141F、Y115F、G175K -GlyR reduced ACh responsiveness while conferring sensitivity to granisetron (Table 5). α7 Q79G、Q139L、G175K -GlyR reduced ACh responsiveness but showed a strong response to nortropisetron (Table 5).

[0163] (Table 6) Enhancement of potency of tropane, quinuclidine agonists, 9-azabicyclo[3.3.1]nonane agonists, diazabicyclo[3.2.2]nonane agonists, and promazine by the G175K and P216I α7GlyR chimeric channels. Indole and indazole aromatic (A) substituents attached at the 3-position. Unit: μM. TIFF2026065133000050.tif235139TIFF2026065133000051.tif235148nd=Not determined; Parentheses:SEM

[0164] α7 G175K -GlyR and α7 P216I-GlyR was also compatible with non-associative mutations R27D, E41R, along with mutations at Q79G, Y115F, and G175K, and the GlyR IPD mutation A298G, which further enhanced ligand efficacy against granisetron, epibatidine, varenicline, cytosine, PNU-282987, tropisetron, nortropisetron, and PHA-543613 (Table 7). α7 R27D、E41R、Q79G、Y115F、G175K Combinations with non-associative mutations to form α7 had low ACh responsiveness and further improved efficacy for 702, 723, 725, and 726 (Table 6).

[0165] (Table 7) Agonist efficacy enhancement by G175K and A298G mutations in α7GlyR chimeric channels and by W298A in α7GABAc (also called GABA A -ρ) channels. Unit: μM. TIFF2026065133000052.tif79157nd = not determined; parentheses: SEM

[0166] α7 Y115F Further amino acid substitutions at Gly of the α7 nAChR LBD in the α7-GlyR chimeric channel also enhanced agonist efficacy. α7 175 The efficacy of tropisetron in the α7-GlyR chimeric channel was enhanced by additional mutations including 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). Y115F (Table 8) Agonist efficacy enhancement by G175 mutations in the α7GlyR Y115F chimeric channel. Unit: μM.

[0167] (表8)α7GlyR Y115FキメラチャネルにおけるG175突然変異によるアゴニスト効力増強。単位:μM。 TIFF2026065133000053.tif49148nd = not determined; parentheses: SEM

[0168] Leu to smaller amino acids 131The mutation was found to decrease the potency of the canonical agonists Ach and nicotine, while significantly increasing the potency of varenicline, tropisetron and several other agonists. α7 L131A -GlyR and α7 L131G -GlyR decreased ACh responsiveness (6-fold) and enhanced potency against varenicline (8-fold and 17-fold, respectively) and tropisetron (2.5-fold and 3.6-fold, respectively) (Table 9). α7 L131G -5HT3 HC decreased ACh responsiveness (5-fold) and enhanced potency against varenicline (16-fold) and tropisetron (2.3-fold) (Figure 9A and Table 9). α7 L131G、Q139L -GlyR and α7 L131G、Y217F -GlyR showed a similar enhancement of potency against varenicline as α7-GlyR (21-fold), but also decreased ACh sensitivity (-11-fold and -13-fold, respectively). α7 Q79S、L131G -GlyR further improved potency over α7-GlyR against varenicline (89-fold) and tropisetron (15-fold). α7 L131G、Q139L、Y217F -GlyR showed the greatest improvement in potency over α7-GlyR against varenicline (387-fold) and also showed a decrease in ACh potency (13-fold) (Figure 9B and Table 9). α7 L131G、Q139L、Y217F -GlyR also showed very high potency against compound 770 (0.001 μM), compound 773 (0.00034 μM), and compound 774 (0.00013 μM) (Figure 11). α7 Q79S、L131G、Q139L -GlyR also improved potency over α7-GlyR against varenicline (31-fold) and tropisetron (3-fold), but ACh potency (9-fold) was decreased (Figure 9B and Table 9). α7 L131M -GlyR, α7 L131Q -GlyR, and α7 L131V -GlyR decreased the ACh titer but enhanced potency against tropisetron, nor-tropisetron, PHA-543613, and granisetron (Table 9). α7 L131F-GlyR was found to substantially reduce ACh potency but did not improve the potency of other agonists (Table 8). α7 L131G -GABAc substantially reduced the potency of ACh but did not improve the potency of other agonists (Table 9). α7 L131G、Q139L、Y217F -5HT3 HC (Table 9) improved the efficacy of varenicline by 131 times compared to α7-5HT3 (Table 1). L131G、Q139L、Y217F -5HT3 HC also showed high potency for compound 770 (0.007 μM), compound 773 (0.002 μM), and compound 774 (0.004 μM) (Table 8).

[0169] (Table 9) Enhancement of agonist efficacy by chimeric channels with L131 mutations. Unit: μM. TIFF2026065133000054.tif235122nd=Not determined; Parentheses:SEM

[0170] Example 7: Chimeric LGIC in neurons α7 Q79G -GlyR A298G Or α7 Q79G、Y115F、G175K AAV or DNA plasmids containing nucleic acids encoding the -GlyR chimeric LGIC were transduced into mouse cortical neurons. Low concentrations of tropisetron (30 nM or 100 nM) were administered to the mouse cortical neurons. Neuronal activity was suppressed by the application of low concentrations of the agonist (Figures 7 and 8C).

[0171] α7 L131G、Q139L、Y217F A DNA plasmid containing nucleic acid encoding the -GlyR chimeric LGIC was transfected into mouse cortical neurons. Low concentrations of varenicline (10 nM) were administered to the mouse cortical neurons. Neuronal activity was suppressed by the application of low concentrations of the agonist (Figure 9C).

[0172] These results demonstrate that modified LGIC activity can be regulated in neurons using low concentrations of LGIC ligands, tropisetron and varenicline.

[0173] Example 8: Varenicline and varenicline derivatives and chimeric LGIC in therapeutic use The smoking cessation drug varenicline is a potent partial α4b2 agonist. Varenicline is also a moderate α7 nAChR agonist and a 5HT3 agonist. It has excellent brain permeability.

[0174] Chimera channel α7 L131G, Q139L, Y217F -GlyR and α7 L131G, Q139L, Y217F -5HT3 HC exhibits enhanced binding ability to the ligand varenicline compared to the unmodified chimeric channels, α7-GlyR and α7-5HT3. However, since varenicline activates endogenous ion channels, such as α7 nAChR, α4β2 nAChR, and serotonin receptor 3 (5HT3-R), it is desirable to obtain varenicline derivatives that exhibit high potency on the modified channels but reduced potency on these endogenous targets.

[0175] Using the crystal structure of varenicline bound to acetylcholine-binding protein, it was found that the molecular contact is located at V106, which is homologous to L131 in the α7 nAChR sequence. Mutation to L131G improved varenicline efficacy by 20 times and reduced Ach efficacy by 5 times (Figure 9A).

[0176] Additional mutations to L131G were made to reduce ACh efficacy and improve varenicline efficacy. In most cases, the change in efficacy was associated with both molecules, but a subset of mutations selectively enhanced varenicline efficacy 360-fold and reduced ACh efficacy 20-fold (Figures 9B and 9C). The response to 10 nM varenicline compared to 300 μM ACh can be seen on the right (Figure 9D). These chimeric channels exhibit characteristic slow activation that functions as a low-pass filter for transient fluctuations in ACh. It should also be noted that despite the high efficacy of the channels, they have no ligand-independent activity, as shown by the short application of the channel antagonist picrotoxin before the addition of these ligands. Ligand-independent activity is recorded as an outward current in this trace. When the chimeric channels were expressed in cortical neurons, 10 nM varenicline induced ultra-potent neuronal silencing (Figure 9E).

[0177] In vivo activity was also extremely potent in the unilateral substantia nigra (SNr) silencing experiment. Rotation on the contralateral side of the silenced SNr was seen at doses exceeding 0.1 milligram / kg (mpk). A greater than 10-fold improvement in varenicline efficacy over anti-nicotine activity was seen (Figure 10).

[0178] Varenicline derivatives were designed to reduce or eliminate endogenous varenicline activity. The efficacy of varenicline and each varenicline derivative was determined for various chimeric channels (Table 10).

[0179] (Table 10) Efficacy (EC50, μM) of varenicline and varenicline derivatives in chimeric channels and comparison with agonist efficacy at 5HT3-R and α4β2 nAChR. TIFF2026065133000055.tif109163TIFF2026065133000056.tif225164nd = not determined; parentheses: SEM

[0180] Table 10 shows the α7 L131G、Q139L、Y217F -GlyR or α7 L131G、Q139L、Y217F -5HT3 HC with specific chemical structures of LGIC agonists having substitution patterns compatible with high potency.

[0181] All molecules in Table 10 show a decrease in sensitivity for α7-GlyR and α7-5HT3 (functioning as a surrogate for α7 nAChR potency). Compounds 780, Compound 783, Compound 789, Compound 790, Compound 791, Compound 792, Compound 793, Compound 795, Compound 798, Compound 802, Compound 803, Compound 804, Compound 805, Compound 807, Compound 808, Compound 812, and Compound 813 show potency less than 30 nM for either α7 L131G、Q139L、Y217F -GlyR or α7 L131G、Q139L、Y217F -5HT3 HC. Compounds 780, Compound 783, Compound 791, Compound 792, Compound 793, Compound 798, Compound 802, Compound 803, Compound 807, or Compound 808 show potency less than 10 nM for either α7 L131G、Q139L、Y217F -GlyR or α7 L131G、Q139L、Y217F -5HT3 HC. Compounds 792, Compound 795, Compound 802, Compound 808 show potency less than 10 nM for either α7 L131G、Q139L、Y217F -GlyR or α7 L131G、Q139L、Y217F -5HT3 HC, as well as potency greater than 1 μM for 5HT3-R and greater than 10 μM for α4β2 nAChR.

[0182] Chemogenetic perturbation of cortical neuron activity was evaluated. PSAM 4 -GlyR-expressing neurons showed strong suppression of action potential firing by nicotine. Cortical layer 2 / 3 neuron membrane properties were similar in PSAM 4 -GlyR-expressing neurons and mixed untransfected control neurons. Nicotine depolarizes and induces firing in PSAM 4 -5HT3 HC-expressing neurons. Cortical layer 2 / 3 neuron membrane properties were 4Similarities are observed in -5HT3 HC-expressing neurons and mixed in with untransfected control neurons. See, for example, Figure 12.

[0183] PSAM 4 -GlyR neuron silencing was evaluated in mice. PSAM 4 -GlyR-IRES-EGFP targeted SNr unilaterally. Low-dose intraperitoneal varenicline was used in PSAM. 4 -In mice expressing GlyR, it induced a contraversive rotation, but not in sham-surgery mice or mice expressing only EGFP. Two doses of varenicline separated by 5 hours showed similar rates of total rotation, indicating the absence of tachyphylaxis of the chemogenetic response. The duration of chemogenetic silencing was monitored by the time course of the rotational response normalized to maximal rotation for each mouse. See, for example, Figure 13.

[0184] We evaluated a highly potent chemogenetic agonist. The uPSEM agonist EC50 was used in PSAM 4 The IC50 of channels and endogenous varenicline targets, as well as 1 μM ACh and α4β2 nAChR, were compared using uPSEM. 792 α4β2 nAChR and uPSEM 817 It was a 10% partial agonist and inhibited α4β2 nAChR. uPSEM 792 uPSEM 793 uPSEM 815 , and uPSEM 817 PSAM reduces the current required to ignite an action potential (rheobase). 4 -GlyR strongly suppressed firing in cortical neurons expressing GlyR. See, for example, Figure 14.

[0185] PSAM in SNr 4 -In vivo uPSEM dose-response was evaluated in mice unilaterally expressing GlyR. See, for example, Figure 15.

[0186] Varenicline derivatives were designed to reduce or eliminate endogenous varenicline activity. The potency of varenicline and each varenicline derivative was determined for various chimeric channels (Table 11).

[0187] (Table 11) EC50 of varenicline and derivatives in chimeric channels, with comparison of agonist efficacy in 5HT3-R and α4β2 nAChR MP (μM). In vivo potency determined in the SNr silencing rotation assay in mice. nd: Undetermined. nr: No response. Values ​​are mean ± SEM. TIFF2026065133000057.tif45161

[0188] Ethoxy and propoxy compounds (uPSEM) 815 and 817 are PSAM 4 - Sub-nanomole efficacy was achieved in GlyR. uPSEM 817 The selectivity of the agonist is excellent, PSAM 4 -GlyR exhibits 5,000 to 10,000 times greater selectivity than α7-GlyR, α7-5HT3, and 5HT3-R. uPSEM 815 and uPSEM 817 It did not show a clear α4β2 nAChR agonism up to 30 μM, and uPSEM 815 PSAM is better than 5HT3-R. 4 - It exhibits selectivity of over 2000 times compared to GlyR.

[0189] Example 9: Chimeric LGIC in treatment Chemogenetic tools offer an intriguing strategy for combining drug therapy and gene therapy. This is because the use of exogenously delivered ion channels selectively involved in drug administration allows for consistent modulation of cellular function across different cell types in various indications using the same ion channels and ligands. The identification of ion channels that are highly tolerant and opened / closed by clinically used drugs is particularly appealing for potentially extending chemogenetics to human therapeutic applications.

[0190] Regarding the drug tropisetron, we found that its EC50 is 11 nM, similar to the IC50 of 10 nM tropisetron reported for its therapeutic target, the 5HT3 receptor, and α7 Q79G -GlyR A298G It was found to activate (Combrink et al. 2009 Pharmacological reports: PR 61: 785-97).

[0191] Other Embodiments While this disclosure has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative and does not limit the scope of this disclosure as defined by the attached claims. Other aspects, advantages, and modifications are within the scope of the claims.

Claims

1. A nucleic acid encoding a modified ligand-gated ion channel (LGIC) comprising at least one modified LGIC subunit, wherein the modified LGIC subunit is (i) A modified alpha-7 nicotinic acetylcholine receptor (α7-nAChR) ligand-binding domain (LBD) having an amino acid substitution in one or more amino acid residues selected from the group consisting of residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, and 219, as indicated by the numbering in Sequence ID No. 2, (ii) Serotonin 3 receptor (5HT3) ion pore domain (IPD), Includes, An exogenous LGIC ligand selected from the group consisting of the above selectively binds to the modified LGIC subunit. Nucleic acid.

2. A synthetic nucleic acid construct encoding a modified ligand-gated ion channel (LGIC) subunit, comprising a modified alpha-7 nicotinic acetylcholine receptor (α7-nAChR) ligand-binding domain (LBD) including amino acid modifications and a serotonin 3 receptor (5HT3) ion pore domain (IPD), wherein the synthetic nucleic acid construct comprises the nucleic acid sequence described in Sequence ID No. 27, and the modified α7-nAChR LBD comprises, in the numbering in Sequence ID No. 2, (a) L131G amino acid substitution, (b) L131G amino acid substitution and Q139L amino acid substitution, (c) L131G amino acid substitution and Y217F amino acid substitution, or (d) L131G amino acid substitution, Q139L amino acid substitution and Y217F amino acid substitution.

3. The nucleic acid according to claim 1, wherein the 5HT3 IPD comprises an R420Q amino acid substitution at residue 420 in the numbering in SEQ ID NO: 8, an R424D amino acid substitution at residue 424 in the numbering in SEQ ID NO: 8, and / or an R428A amino acid substitution at residue 428 in the numbering in SEQ ID NO:

8.

4. The nucleic acid according to claim 1, wherein the 5HT3 IPD comprises R425Q substitution, R429D substitution, and / or R433A substitution in the numbering in Sequence ID No.

6.

5. The nucleic acid according to claim 1, wherein the modified α7-nAChR LBD comprises an amino acid substitution at amino acid residue 131, an amino acid substitution at amino acid residue 139, and / or an amino acid substitution at amino acid residue 217.

6. The nucleic acid according to claim 5, wherein the modified α7-nAChR LBD comprises an L131G amino acid substitution, a Q139L amino acid substitution, and / or a Y217F amino acid substitution.

7. The synthetic nucleic acid construct according to claim 2, or the nucleic acid according to claim 6, wherein the modified α7-nAChR LBD comprises an L131G amino acid substitution.

8. The synthetic nucleic acid construct according to claim 2, or the nucleic acid according to claim 6, wherein the modified α7-nAChR LBD comprises an L131G amino acid substitution and a Q139L amino acid substitution.

9. The synthetic nucleic acid construct according to claim 2, or the nucleic acid according to claim 6, wherein the modified α7-nAChR LBD comprises an L131G amino acid substitution and a Y217F amino acid substitution.

10. The synthetic nucleic acid construct according to claim 2, or the nucleic acid according to claim 6, wherein the modified α7-nAChR LBD comprises an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution.

11. A viral vector comprising the nucleic acid according to any one of claims 1, 3, 4, 5, and 6, or the synthetic nucleic acid construct according to claim 2.

12. The viral vector according to claim 11, wherein the viral vector is an adeno-associated virus vector.

13. The viral vector according to claim 12, wherein the synthetic nucleic acid construct further comprises a synapsin promoter.

Images