Extracellular small molecule activators of the atrophin channel for the treatment of disease

Abstract

This article describes a method for activating one or more paralogs of tartaric acid, the method comprising contacting the paralogs of tartaric acid with a composition comprising one or more paralogs of tartaric acid.

Description

[0001] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 693,686, filed September 11, 2024, and U.S. Provisional Patent Application No. 63 / 591,019, filed October 17, 2023; the entire contents of each of these applications are incorporated herein by reference.

[0002] All patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. The disclosures of these publications are incorporated herein by reference in their entirety to provide a more complete description of the state of the prior art known to those skilled in the art up to the date of the invention described herein.

[0003] Government support This invention was carried out with government support, based on patents GM149252 and GM127652 granted by the National Institutes of Health (NIH). The government holds certain rights to this invention. Background Technology

[0004] bestrophins are Ca 2+ The activating anion channel family consists of four members (Best1-Best4) found in mammals. They are widely distributed in various human organs, including the airways, colon, kidneys, pancreas, and central nervous system, but they are best known for their physiological roles in the eye. Specifically, Best1 is primarily expressed in the retinal pigment epithelium (RPE) and is genetically associated with a range of retinal degenerative disorders collectively known as bestrophinopathies. More than 350 different Best1 mutations have been identified that cause bestrophinopathies. Exemplary bestrophinopathies include Best vitrectomyelitis (BVMD), adult-onset bestrophinopathies (AVMD), autosomal recessive bestrophinopathies (ARB), autosomal dominant vitreoretinal choroidal disease (ADVIRC), and retinitis pigmentosa (RP). Furthermore, bestrophinopathies play a role in multiple physiological aspects. Patients are prone to progressive vision loss, which can eventually lead to blindness, and there is no cure. On the other hand, Best2 resides in the non-pigmented epithelium (NPE) that regulates intraocular pressure (IOP). Since both high and low IOP are harmful conditions, it is essential to maintain IOP appropriately at all times. Therefore, pigmentin is a potential drug target for various human diseases, especially eye diseases. Summary of the Invention

[0005] We identified γ-aminobutyric acid (GABA) as the activator of the interaction between Best1 and Best2. The structures of GABA-bound Best1 and Best2 were resolved by cryo-electron microscopy (cryoEM), and the conserved GABA binding site was located extracellularly within the channel. Functionally, we demonstrated that nanomolar levels of GABA are sufficient to activate Best1 and Best2. Then, through function-based screening, we identified three groups of small molecules as activators of the cantharidin channel, including: 1) GABA analogs such as isoguvacine, pregabalin, gabapentinenacarbil, acanolic acid, muscarinic acid, aminohexenoic acid, gabapentine, and aminooxyacetic acid; 2) benzoic acid derivatives such as 4-aminobenzoic acid (PABA) and its analogs 2-amino-5-methoxybenzoic acid, 3,4-diaminobenzenesulfonic acid, 3-amino-5-methoxybenzoic acid, 4-amino-2-fluorobenzoic acid, 4-hydroxybenzoic acid, 4-nitrobenzoic acid, 4-amino-2-(hydroxymethyl)benzoic acid, 4-isobutoxybenzoic acid, 4-(aminomethyl)benzoic acid, 2-amino-5-pyrimidinecarboxylic acid, and 2-(dimethylamino)-5-aminobenzoic acid. -Pyrimidine carboxylic acid, 4-[(2-hydroxyethyl)amino]benzoic acid, 4-[(2-amino-2-oxoethyl)amino]benzoic acid, 4-[(2-aminoethyl)amino]-3-nitrobenzoic acid, 3,4-diaminobenzoic acid, 4-amino-3-hydroxybenzoic acid, 4-amino-N-(phenylmethyl)benzamide, 4-amino-N-(2-amino-2-oxoethyl)benzamide, 4-amino-N-(2-hydroxyethyl)benzamide, 4-amino-N-(2-methylpropyl)benzamide, lisocaine, 4-amino-N-propylbenzamide, 4-amino-1-naphthylcarboxylic acid, 6-amino-4-quinolinecarboxylic acid, 8-amino-4-quinolinecarboxylic acid and 1H-indole-5-carboxylic acid; 3) pantothenic acid and 5-hydroxyindole-2-carboxylic acid, which are neither analogs of GABA nor PABA. We conducted further analysis on PABA, which exhibited potent activation of Best1 / Best2: structurally, the PABA-binding Best1 and Best2 structures were resolved, revealing the same binding sites as GABA; functionally, PABA treatment rescued the functional defects of Best1 mutations originating from patients with vitelloid macular degeneration and stimulated native Best1 and Best2-mediated currents in RPE and NPE cells, respectively. In summary, our results demonstrate the mechanisms and potential of small molecule activators (including FDA-approved GABA and PABA analogs for other indications) as clinically applicable medicines for vitelloidin-related diseases / conditions.

[0006] In some aspects, this document describes a small molecule activator capable of binding to one or more paralogs of cantharidin, wherein the one or more paralogs of cantharidin include Best1, wherein the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best1 and the main-chain nitrogen atoms of V275 and F276 of Best1; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best1. In some embodiments, Best1 is human Best1.

[0007] In some aspects, this document describes a small molecule activator capable of binding to one or more paralogs of cantharidin, wherein the one or more paralogs of cantharidin include Best2, wherein the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best2 and the main-chain nitrogen atoms of I275 and F276 of Best2; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best2. In some embodiments, Best2 is human Best2.

[0008] In some respects, this document describes a small molecule activator capable of binding to one or more paralogs of cantharidin, wherein the one or more paralogs include Best3, wherein the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best3 and the main-chain nitrogen atoms of I275 and F276 of Best3; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best3. In some embodiments, Best3 is human Best3.

[0009] In some respects, this document describes a small molecule activator capable of binding to one or more paralogs of cantharidin, wherein the one or more paralogs of cantharidin include Best4, wherein the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best4 and the main-chain nitrogen atoms of L290 and T291 of Best4; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of S72 of Best4. In some embodiments, Best4 is human Best4.

[0010] In some aspects, this document describes a small molecule activator capable of binding to one or more paralogs of cantharidin, wherein the small molecule activator activates the one or more paralogs of cantharidin by contacting a pocket formed at the N-terminal helical dipole of the helix S4a of the one or more paralogs of cantharidin. In some embodiments, the one or more paralogs of cantharidin include Best1, Best2, Best3, or Best4. In some embodiments, Best1, Best2, Best3, or Best4 is human Best1, human Best2, human Best3, or human Best4.

[0011] In some embodiments, the small molecule activator is an amino acid. In some embodiments, the amino acid is a non-proteinogenic amino acid. In some embodiments, the small molecule activator is GABA or a GABA analog. In some embodiments, the small molecule activator is GABA. In some embodiments, the small molecule activator is a GABA analog. In some embodiments, the GABA analog is desmethylarecanthine, pregabalin, gabapentin, enalacarbinol, acampalic acid, muscarinic acid, vigabatrin, gabapentin, or aminooxyacetic acid. In some embodiments, the small molecule activator is PABA or a PABA analog. In some embodiments, the small molecule activator is PABA. In some embodiments, the small molecule activator is a PABA analog. In some embodiments, the PABA analogues are 2-amino-5-methoxybenzoic acid, 3,4-diaminobenzenesulfonic acid, 3-amino-5-methoxybenzoic acid, 4-amino-2-fluorobenzoic acid, 4-hydroxybenzoic acid, 4-nitrobenzoic acid, 4-amino-2-(hydroxymethyl)benzoic acid, 4-isobutoxybenzoic acid, 4-(aminomethyl)benzoic acid, 2-amino-5-pyrimidinecarboxylic acid, 2-(dimethylamino)-5-pyrimidinecarboxylic acid, 4-[(2-hydroxyethyl)amino]benzoic acid, 4-[(2-amino-2-oxoethyl)amino]benzoic acid, 4 -[(2-aminoethyl)amino]-3-nitrobenzoic acid, 3,4-diaminobenzoic acid, 4-amino-3-hydroxybenzoic acid, 4-amino-N-(phenylmethyl)benzamide, 4-amino-N-(2-amino-2-oxoethyl)benzamide, 4-amino-N-(2-hydroxyethyl)benzamide, 4-amino-N-(2-methylpropyl)benzamide, lisocaine, 4-amino-N-propylbenzamide, 4-amino-1-naphthylcarboxylic acid, 6-amino-4-quinolinecarboxylic acid, 8-amino-4-quinolinecarboxylic acid, or 1H-indole-5-carboxylic acid. In some embodiments, the small molecule activator is not GABA, a GABA analog, PABA, or a PABA analog. In some embodiments, the small molecule activator is pantothenic acid or 5-hydroxyindole-2-carboxylic acid.

[0012] In some embodiments, the small molecule activator activates one or more paracellular homologs of cantharidin by binding to their extracellular space. In some embodiments, the small molecule activator promotes neck opening of one or more paracellular homologs of cantharidin. In some embodiments, neck opening results in the side chains of gate-forming residues being displaced from the channel pores of one or more paracellular homologs of cantharidin. In some embodiments, the gate-forming residues are I76, F80, and F84 of one or more paracellular homologs of cantharidin.

[0013] In some respects, this document describes a method for activating one or more paralogs of tartaric acid, the method comprising contacting the paralogs of tartaric acid with a composition comprising a small molecule activator of one or more paralogs of tartaric acid.

[0014] In some embodiments, the small molecule activator is an amino acid. In some embodiments, the amino acid is a non-proteinogenic amino acid. In some embodiments, the small molecule activator is GABA or a GABA analog. In some embodiments, the small molecule activator is GABA. In some embodiments, the small molecule activator is a GABA analog. In some embodiments, the GABA analog is desmethylarecanthine, pregabalin, gabapentin, enalacarbinol, acampalic acid, muscarinic acid, vigabatrin, gabapentin, or aminooxyacetic acid. In some embodiments, the small molecule activator is PABA or a PABA analog. In some embodiments, the small molecule activator is PABA. In some embodiments, the small molecule activator is a PABA analog. In some embodiments, the PABA analogues are 2-amino-5-methoxybenzoic acid, 3,4-diaminobenzenesulfonic acid, 3-amino-5-methoxybenzoic acid, 4-amino-2-fluorobenzoic acid, 4-hydroxybenzoic acid, 4-nitrobenzoic acid, 4-amino-2-(hydroxymethyl)benzoic acid, 4-isobutoxybenzoic acid, 4-(aminomethyl)benzoic acid, 2-amino-5-pyrimidinecarboxylic acid, 2-(dimethylamino)-5-pyrimidinecarboxylic acid, 4-[(2-hydroxyethyl)amino]benzoic acid, 4-[(2-amino-2-oxoethyl)amino]benzoic acid, 4 -[(2-aminoethyl)amino]-3-nitrobenzoic acid, 3,4-diaminobenzoic acid, 4-amino-3-hydroxybenzoic acid, 4-amino-N-(phenylmethyl)benzamide, 4-amino-N-(2-amino-2-oxoethyl)benzamide, 4-amino-N-(2-hydroxyethyl)benzamide, 4-amino-N-(2-methylpropyl)benzamide, lisocaine, 4-amino-N-propylbenzamide, 4-amino-1-naphthylcarboxylic acid, 6-amino-4-quinolinecarboxylic acid, 8-amino-4-quinolinecarboxylic acid, or 1H-indole-5-carboxylic acid. In some embodiments, the small molecule activator is not GABA, a GABA analog, PABA, or a PABA analog. In some embodiments, the small molecule activator is pantothenic acid or 5-hydroxyindole-2-carboxylic acid.

[0015] In some embodiments, the small molecule activator activates one or more paracellular homologs of cantharidin by binding to the extracellular side of the paracellular surface of the cantharidin. In some embodiments, the small molecule activator activates one or more paracellular homologs of cantharidin by contacting a pocket formed at the N-terminal helical dipole of the helix S4a of the paracellular homolog. In some embodiments, the small molecule activator promotes neck opening of one or more paracellular homologs of cantharidin. In some embodiments, neck opening results in the side chains of the gate-forming residues being displaced from the channel pores of one or more paracellular homologs of cantharidin. In some embodiments, the gate-forming residues are I76, F80, and F84 of one or more paracellular homologs of cantharidin.

[0016] In some embodiments, one or more paralogs of cantharidin include Best1, wherein the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best1 and the main-chain nitrogen atoms of V275 and F276 of Best1; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best1. In some embodiments, Best1 is human Best1. In some embodiments, one or more paralogs of cantharidin include Best2, wherein the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best2 and the main-chain nitrogen atoms of I275 and F276 of Best2; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best2. In some embodiments, Best2 is human Best2.

[0017] In some embodiments, one or more paralogs of cantharidin include Best3, wherein the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best3 and the main-chain nitrogen atoms of I275 and F276 of Best3; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best3. In some embodiments, Best3 is human Best3. In some embodiments, one or more paralogs of cantharidin include Best4, wherein the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best4 and the main-chain nitrogen atoms of L290 and T291 of Best4; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of S72 of Best4. In some embodiments, Best4 is human Best4.

[0018] In some respects, this document describes a method for treating or preventing a disease or condition in a subject of need, the method comprising administering a pharmaceutical composition to the subject, wherein the composition comprises a small molecule activator of one or more paralogs of cantharidin. In some embodiments, the subject is a human being.

[0019] In some embodiments, the disease or condition is caused by an imbalance of one or more phytotoxic proteins. In some embodiments, the disease or condition is a phytotoxic protein (Best)-related disease. In some embodiments, phytotoxic protein-related diseases are caused by a selection from... BEST1 , BEST2 , BEST3 and BEST4 It is caused by one or more mutations in the gene. In some embodiments, the macula-associated disease is vitrectomyeloid macular degeneration. In some embodiments, the disease or condition is Best vitrectomyeloid macular dystrophy (BVMD), adult-onset vitrectomyeloid macular dystrophy (AVMD), autosomal recessive vitrectomyeloid macular degeneration (ARB), autosomal dominant vitreoretinopathy and choroidal disease (ADVIRC), autosomal dominant microkeratosis, cone-rod dystrophy, early-onset cataract, posterior staphyloma syndrome (MRCS syndrome), age-related macular degeneration (AMD), retinitis pigmentosa (RP), or a combination thereof.

[0020] In some implementations, plaquefoil-related disease is associated with an increase or decrease in the subject's intraocular pressure (IOP). In some implementations, the subject's IOP is higher than 21 mmHg. In some implementations, plaquefoil-related disease is glaucoma, myopia, dispersive pigment syndrome, pseudoexfoliation syndrome, age-related macular degeneration, or a combination thereof.

[0021] In some implementations, the disease or symptom is Alzheimer's disease.

[0022] In some embodiments, the small molecule activator is an amino acid. In some embodiments, the amino acid is a non-proteinogenic amino acid. In some embodiments, the small molecule activator is GABA or a GABA analog. In some embodiments, the small molecule activator is GABA. In some embodiments, the small molecule activator is a GABA analog. In some embodiments, the GABA analog is desmethylarecanthine, pregabalin, gabapentin, enalacarbinol, acampalic acid, muscarinic acid, vigabatrin, gabapentin, or aminooxyacetic acid. In some embodiments, the small molecule activator is PABA or a PABA analog. In some embodiments, the small molecule activator is PABA. In some embodiments, the small molecule activator is a PABA analog. In some embodiments, the PABA analogues are 2-amino-5-methoxybenzoic acid, 3,4-diaminobenzenesulfonic acid, 3-amino-5-methoxybenzoic acid, 4-amino-2-fluorobenzoic acid, 4-hydroxybenzoic acid, 4-nitrobenzoic acid, 4-amino-2-(hydroxymethyl)benzoic acid, 4-isobutoxybenzoic acid, 4-(aminomethyl)benzoic acid, 2-amino-5-pyrimidinecarboxylic acid, 2-(dimethylamino)-5-pyrimidinecarboxylic acid, 4-[(2-hydroxyethyl)amino]benzoic acid, 4-[(2-amino-2-oxoethyl)amino]benzoic acid, 4 -[(2-aminoethyl)amino]-3-nitrobenzoic acid, 3,4-diaminobenzoic acid, 4-amino-3-hydroxybenzoic acid, 4-amino-N-(phenylmethyl)benzamide, 4-amino-N-(2-amino-2-oxoethyl)benzamide, 4-amino-N-(2-hydroxyethyl)benzamide, 4-amino-N-(2-methylpropyl)benzamide, lisocaine, 4-amino-N-propylbenzamide, 4-amino-1-naphthylcarboxylic acid, 6-amino-4-quinolinecarboxylic acid, 8-amino-4-quinolinecarboxylic acid, or 1H-indole-5-carboxylic acid. In some embodiments, the small molecule activator is not GABA, a GABA analog, PABA, or a PABA analog. In some embodiments, the small molecule activator is pantothenic acid or 5-hydroxyindole-2-carboxylic acid.

[0023] In some embodiments, the small molecule activator activates one or more paracellular homologs of cantharidin by binding to the extracellular side of the paracellular side of the cantharidin. In some embodiments, the small molecule activator activates one or more paracellular homologs of cantharidin by contacting a pocket formed at the N-terminal helical dipole of the helix S4a of the paracellular side of the cantharidin. In some embodiments, the small molecule activator binds to the extracellular side of the cantharidin. In some embodiments, the small molecule activator promotes neck opening of the cantharidin. In some embodiments, neck opening results in the side chains of the gate-forming residues being displaced from the channel pores of the cantharidin. In some embodiments, the gate-forming residues are I76, F80, and F84 of the cantharidin.

[0024] In some embodiments, one or more paralogs of cantharidin include Best1, wherein the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best1 and the main-chain nitrogen atoms of V275 and F276 of Best1; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best1. In some embodiments, Best1 is human Best1. In some embodiments, one or more paralogs of cantharidin include Best2, wherein the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best2 and the main-chain nitrogen atoms of I275 and F276 of Best2; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best2. In some embodiments, Best2 is human Best2.

[0025] In some embodiments, one or more paralogs of cantharidin include Best3, wherein the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best3 and the main-chain nitrogen atoms of I275 and F276 of Best3; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best3. In some embodiments, Best3 is human Best3. In some embodiments, one or more paralogs of cantharidin include Best4, wherein the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best4 and the main-chain nitrogen atoms of L290 and T291 of Best4; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of S72 of Best4. In some embodiments, Best4 is human Best4.

[0026] In some embodiments, the subject has a Best1 mutation. In some embodiments, the Best1 mutation is hereditary. In some embodiments, the Best1 mutation is a loss-of-function (LOF) mutation. In some embodiments, the composition is administered to salvage Best1 function.

[0027] In some embodiments, the pharmaceutical composition is administered via ophthalmic delivery, oral delivery, or parenteral delivery. In some embodiments, the pharmaceutical composition is administered via ophthalmic delivery, wherein the pharmaceutical composition is applied topically. In some embodiments, the pharmaceutical composition is administered as eye drops.

[0028] In some respects, this article describes a method for maintaining intraocular pressure (IOP) in subjects in need, comprising administering a pharmaceutical composition wherein the composition increases Best2-mediated Ca2+ in non-pigmented epithelial (NPE) cells. 2+ Dependence Cl - Electric current. In some implementations, the subject is a human being.

[0029] In some embodiments, the subject suffers from a disease caused by an imbalance of one or more macular degeneration proteins. In some embodiments, the subject suffers from a macular degeneration protein-related disease. In some embodiments, the macular degeneration protein-related disease is associated with an increase or decrease in the subject's intraocular pressure (IOP). In some embodiments, the subject suffers from either high or low intraocular pressure. In some embodiments, the subject's IOP is greater than 21 mmHg. In some embodiments, the macular degeneration protein-related disease is glaucoma, myopia, dispersive pigment syndrome, pseudoexfoliation syndrome, age-related macular degeneration, or a combination thereof.

[0030] In some embodiments, the composition comprises a small molecule activator of Best2. In some embodiments, the small molecule activator is an amino acid. In some embodiments, the amino acid is a non-proteinogenic amino acid. In some embodiments, the small molecule activator is GABA or a GABA analog. In some embodiments, the small molecule activator is GABA. In some embodiments, the small molecule activator is a GABA analog. In some embodiments, the GABA analog is desmethylarecanthine, pregabalin, gabapentin, enalacarbinol, acampalic acid, muscarinic acid, vigabatrin, gabapentin, or aminooxyacetic acid. In some embodiments, the small molecule activator is PABA or a PABA analog. In some embodiments, the small molecule activator is PABA. In some embodiments, the small molecule activator is a PABA analog. In some embodiments, the PABA analogues are 2-amino-5-methoxybenzoic acid, 3,4-diaminobenzenesulfonic acid, 3-amino-5-methoxybenzoic acid, 4-amino-2-fluorobenzoic acid, 4-hydroxybenzoic acid, 4-nitrobenzoic acid, 4-amino-2-(hydroxymethyl)benzoic acid, 4-isobutoxybenzoic acid, 4-(aminomethyl)benzoic acid, 2-amino-5-pyrimidinecarboxylic acid, 2-(dimethylamino)-5-pyrimidinecarboxylic acid, 4-[(2-hydroxyethyl)amino]benzoic acid, 4-[(2-amino-2-oxoethyl)amino]benzoic acid, 4 -[(2-aminoethyl)amino]-3-nitrobenzoic acid, 3,4-diaminobenzoic acid, 4-amino-3-hydroxybenzoic acid, 4-amino-N-(phenylmethyl)benzamide, 4-amino-N-(2-amino-2-oxoethyl)benzamide, 4-amino-N-(2-hydroxyethyl)benzamide, 4-amino-N-(2-methylpropyl)benzamide, lisocaine, 4-amino-N-propylbenzamide, 4-amino-1-naphthylcarboxylic acid, 6-amino-4-quinolinecarboxylic acid, 8-amino-4-quinolinecarboxylic acid, or 1H-indole-5-carboxylic acid. In some embodiments, the small molecule activator is not GABA, a GABA analog, PABA, or a PABA analog. In some embodiments, the small molecule activator is pantothenic acid or 5-hydroxyindole-2-carboxylic acid.

[0031] In some embodiments, the small molecule activator activates Best2 by binding to the extracellular side of Best2. In some embodiments, the small molecule activator activates Best2 by contacting a pocket formed at the N-terminal helical dipole of helix S4a of Best2. In some embodiments, the small molecule activator promotes neck opening of Best2. In some embodiments, neck opening results in the side chains of the gate-forming residues moving away from the channel pores of Best2. In some embodiments, the gate-forming residues are I76, F80, and F84 of Best2. In some embodiments, the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best2 and the main-chain nitrogen atoms of I275 and F276 of Best2; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best2. In some embodiments, the Best1 mutation is A243T, A10T, R218H, L234P, Q293K, or D302A. In some embodiments, the composition is applied to rescue Best1 function.

[0032] In some embodiments, the pharmaceutical composition is administered via ophthalmic delivery, oral delivery, or parenteral delivery. In some embodiments, the pharmaceutical composition is administered via ophthalmic delivery, wherein the pharmaceutical composition is applied topically. In some embodiments, the pharmaceutical composition is administered as eye drops.

[0033] In some embodiments, the small molecule activator comprises a 5-membered ring or a 6-membered ring. In some embodiments, the small molecule activator comprises a 5-membered ring. In some embodiments, the small molecule activator comprises a 6-membered ring.

[0034] In some embodiments, one or more paralogs of taraxacin include Best1, and the small molecule activator is GABA or a GABA analogue, wherein: the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best1 and the main-chain nitrogen atoms of V275 and F276 of Best1; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best1; the α carbon (C2) of the small molecule activator forms a van der Waals contact with the β carbon of P274 of Best1; the β carbon of the small molecule activator forms a van der Waals contact with the C3 carbon of the ring of the side chain of Y72 of Best1; and the nitrogen atom of the small molecule activator is adjacent to the hydroxyl group of the side chain of Y72 of Best1.

[0035] In some embodiments, one or more paralogs of taraxacin include Best1, and the small molecule activator is PABA or a PABA analogue, wherein: the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best1 and the main-chain nitrogen atoms of V275 and F276 of Best1; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 of Best1 and the side-chain hydroxyl group of T277 of Best1; the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best1; the amino group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of H267 of Best1; the aromatic ring of the small molecule activator forms a π-stacking interaction with the side chain of F257 of Best1; and the aromatic ring of the small molecule activator forms a side-to-side interaction with the side chain of Y72 of Best1.

[0036] In some embodiments, one or more paralogs of taraxacin include Best2, and the small molecule activator is PABA or a PABA analogue, wherein: the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best2 and the main-chain nitrogen atoms of I275 and F276 of Best2; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 of Best2 and the side-chain hydroxyl group of T277; the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best2; the amino group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of H267 of Best2; the aromatic ring of the small molecule activator forms a π-stacking interaction with the side chain of F257 of Best2; and the aromatic ring of the small molecule activator forms a side-to-side interaction with the side chain of Y72 of Best2. Attached Figure Description

[0037] This patent or application document contains at least one drawing that was originally in color.

[0038] Figures 1a-f The structure of GABA-bound Best1 and the effect of GABA on Best1 function are shown. (a) Side view of Best1 in its fully open GABA-bound state, where the protein is depicted as a clear band and the GABA molecule as a blue space-filled sphere. The inset shows the location of the magnified region in b. (b) Left side A close-up of the GABA binding site, where hydrogen bonds are depicted as dashed lines and interacting residue side chains are marked. Middle and rightModel and map of the GABA binding site on Best1 in two views (45-degree rotation) (shown at sigma 5), ​​with local residues marked. (c) For the fully open state (FFFFF, Left Intermediate state 1 (PCCCC, Second from the left Intermediate state 2 (PCPCC, From left to right Three ) and fully closed state (CCCCC, right (d, e) Top views of Best1 in different conformations (from the cell's outer membrane), where GABA molecules are shown as blue spheres on the periphery, I76 as brown spheres, and F80 / F84 as pink spheres on the inner periphery (exemplary labeling provided for each case). In the fully closed state, I76 is positioned above F80 / 84 in the top view. 2+ ] i In standard external (d) or internal (e) solutions, 20 mM GABA replaces 20 mM Cl. - (f) The relationship between the normalized steady-state current density (Y-axis) at +100 mV and different concentrations of external GABA (X-axis), and fitted to the Hill equation; n=5-6. All error bars are represented as mean + / - SEM.

[0039] Figures 2a-1 illustrate the structure of GABA-binding Best2 and the effect of GABA on Best2 function. (a) Side view of GABA-binding Best2, where the GABA molecule is shown as a blue rod. PM, plasma membrane. The black box indicates the focal region in Figure b. (b) Close-up of the GABA binding site, where hydrogen bonds are depicted as dashed lines and the interacting residue side chains are labeled. (c, d) Top views of Best2 (from the extracellular side of the membrane) in fully open (FFFFF, c) and closed (CCCCC, d) states, where the GABA molecule is shown as a blue sphere on the periphery, I76 as a brown sphere, and F80 / F84 as a pink sphere on the inner periphery (exemplary labeling provided for each case). In the fully closed state, from the top view, I76 is positioned above F80 / F84. (e) When Cl - When it is the dominant anion in both internal and external solutions, at 1 μM [Ca 2+ ] i Below, the population steady-state current density-voltage (IV) relationship of Best2 in transiently transfected HEK293 cells, with or without 100 μM external GABA added (lighter red line) or not added (darker line); for each point, n=6–9; *Using two-tailed unpaired studentst The test, compared to no GABA, p <0.05. (f) Normalized steady-state current density (Y-axis) at +100 mV versus external GABA concentrations (X-axis), fitted to the Hill equation; for each point, n=6-9. (g, h) at 0 (hollow box), 1, and 10 μM [Ca 2+ ] i Below, the IV relationship of Best2 in transiently transfected HEK293 cells (the 10 μM line is higher than the 1 μM line), where Cl - It is the main anion in both the internal and external solutions, and the external solution contains 100 μM GABA (g), or glutamate and Cl-. - These are the major anions (h) in the internal and external solutions, respectively; for each data point, n = 5–7, *using two-tailed unpaired students. t Test, with 1 μM [Ca 2+ ] i compared to, p <0.05. (i) Compared to the case where GABA is the dominant anion in the external solution, when Cl... - When Best2 is the dominant anion (black) in both internal and external solutions, it is at 1 μM [Ca]. 2+ ] i IV relationship under; for each data point, n=7-9. (j) at 1 μM [Ca 2+ ] i In the presence of the reverse potential, the ratio of relative ionic conductivity (GABA / Cl, gray) is measured as the slope conductivity at which the reversal potential is increased by 50 mV (GABA / Cl, gray) or decreased by 50 mV (Cl / Cl, black). GABA / G Cl For each bar, n=7. (k) In the absence of (black) or in the presence of 100 μM external GABA, Best2 at 1 μM [Ca 2+ ] i The IV relationship below, where Cl - Glutamate and glutamate are the major anions in the internal and external solutions, respectively; for each point, n = 5-7. (l) G is measured as the slope conductivity at a reversal potential of 50 mV (Glu / Cl, gray) or 50 mV (Cl / Cl, black) in the presence or absence of 100 μM GABA. Glu / G Cl For each bar, n=5-7. All error bars in this figure represent sem.

[0040] Figure 3This study demonstrates the functional effect of small molecules on cantharidin channels. In HEK293 cells treated with 100 μM of the small molecule shown, the effect was observed at 1 μM [Ca]... 2+ ] i Current density of Best1 (top) or Best2 (bottom) expression at the next instant. n=5-6. *Compared to untreated cells, p <0.05.

[0041] Figure 4a-h The PABA-binding Best2 and Best1 structures are shown. (a) Side view of PABA-binding Best2, where the PABA molecule is shown as a blue rod. The black box indicates the focal region in Figure b. (b) Close-up of the PABA binding site, where hydrogen bonds are depicted as dashed lines and the interacting residue side chains are labeled. (c, d) Top views of Best2 (from the cell's outer membrane) for fully open (c) and closed (d) states, where the PABA molecule is shown as a blue sphere on the periphery, I76 as a brown sphere, and F80 / F84 as a pink sphere on the inner periphery (exemplary labeling provided for each case). In the fully closed state, from the top view, I76 is positioned above F80 / F84. (eh) PABA-binding Best1 structures of the same form as ad, except that h shows the intermediate state.

[0042] Figures 5a-g illustrate the functional rescue of mutations from Best1 patients by PABA. (af) In the absence (solid) or presence (hollow) of PABA treatment, at 1 μM [Ca 2+ ] i Below, IV relationships in HEK293 cells transiently expressing wild-type (WT) cells alone (black solid box, untreated) or co-expressing WT Best1 with patient-derived A10T (a), R218H (b), L234P (c), A243T (d), Q293K (e), and D302A (f) mutations in a 1:4 ratio (red); n=6–8; *Using two-tailed unpaired students t The test, compared with untreated WT cells, p <0.05. All error bars in this figure represent SEM values. (g) 1 μM [Ca] from donor-derived iPSC-RPE cells. 2+ ] i Current density under (black solid, untreated WT; red solid, untreated mutant; red hollow, mutant treated with PABA). n=5-6. *By two-tailed unpaired students t The test, compared with untreated WT cells, p<0.05.

[0043] Figure 6 PABA significantly increased Best2 function in NPE. 1 μM [Ca] from untreated (square) or human (round) NPE cells treated with 100 μM PABA was used. 2+ ] i IV relation under the following conditions. n=5-6. *By two-tailed unpaired students t The test, compared with untreated cells, p <0.05.

[0044] Figure 7 Further small molecule effects on the Best1 channel were demonstrated. Additional PABA / GABA analogs were screened using whole-cell patch-clamp and identified as Best1 activators. Bar graphs depict the effects of 1 μM [Ca] on the function of the Best1 channel in HEK293 cells treated with 100 μM of the small molecule shown. 2+ ] i The current density of Best1 expressed at the next instant. The small molecules are: 1: 4-amino-2-(hydroxymethyl)benzoic acid, 2: 4-isobutoxybenzoic acid, 3: 4-(aminomethyl)benzoic acid, 4: 2-amino-5-pyrimidinecarboxylic acid, 5: 2-(dimethylamino)-5-pyrimidinecarboxylic acid, 6: 4-[(2-hydroxyethyl)amino]benzoic acid, 7: 4-[(2-amino-2-oxoethyl)amino]benzoic acid, 8: 4-[(2-aminoethyl)amino]-3-nitrobenzoic acid, 9: 3,4-diaminobenzoic acid, 10: 4-amino-3-hydroxybenzoic acid. Acids, 11: 4-amino-N-(phenylmethyl)benzamide, 12: 4-amino-N-(2-amino-2-oxoethyl)benzamide, 13: 4-amino-N-(2-hydroxyethyl)benzamide, 14: 4-amino-N-(2-methylpropyl)benzamide, 15: lixocaine, 16: 4-amino-N-propylbenzamide, 17: 4-amino-1-naphthylcarboxylic acid, 18: 6-amino-4-quinolinecarboxylic acid, 19: 8-amino-4-quinolinecarboxylic acid, 20: 1H-indole-5-carboxylic acid. n=5-6. *By two-tailed unpaired students t The test, compared with untreated cells, p <0.05. All error bars in this figure represent SEM values.

[0045] Figures 8a-h illustrate the cryo-EM processing pathways for the Best1+GABA dataset. (a) Representative photomicrographs. (b) Representative 2D categories. (c) Refined consistency using a symmetrical mask with a covered hole liner spiral and GABA binding sites for 3D classification. (d) Results in the fully open state. Left sideThe top view of the density map. right side Orientation distribution map ( top ); FSC curve ( middle (Curves from bottom left to top right: no mask; loose; corrected; tight); and local resolution map ( bottom (e) Top view of the density map for the closed state. (fh) For the fully closed state (f) and intermediate states 1 (g) and 2 (h), it has the same form as d. illustration Color illustration.

[0046] Figures 9a-i illustrate the cryo-EM processing pathways for the Best2+GABA dataset. (a) Representative micrographs. (b) Representative 2D categories. (c) Consistent refinement using a symmetric mask with a covering hole liner spiral and GABA binding sites for 3D classification. (df) 3D classification results, depicting a top view of the density map and FSC curves (from bottom left to top right: no mask; loose; corrected; tight) for the fully open state. (d), local resolution map (e), and orientation distribution map (f). (gi) For the closed state, the same form as df. illustration Color illustration.

[0047] Figures 10a-d A comparison of the GABA-binding Best2 structure with the PABA-binding Best1 and Best2 structures is shown. (a) Left side A side view of the GABA-binding Best2 in its open state, where GABA molecules are depicted as blue rods. The black boxes indicate the focused areas in the middle and right images. middle and right side (b) Close-up of the GABA binding site and neck in the open (FFFFF) and closed (CCCCC) states, respectively, with local residues marked. Left side The image shows an open view of the GABA-binding Best2, where GABA molecules are depicted as blue spheres. The black boxes indicate the areas in focus in the middle and right images. middle and right side Model and density map of GABA binding sites in two views (rotated 45 degrees) (shown at sigma 7.5), with local residues marked. (cd) For PABA-binding types Best1 (c) or Best2 (d) in the open state, the same form as (b) is shown.

[0048] Figures 11a-dA comparison is shown between Best2 in its closed conformation and the small-molecule bound Best2 and Best1 in their fully open conformations. (ab) Side views of two opposing (144°) protopolymers of Best2 (a) in its closed unbound GABA state or Best2 (b) in its fully open GABA-bound state, where the ion permeation pathway is visualized and the pore size is depicted as colored dots: the closer the pore radii, the smaller and denser the dots. illustration Color illustration; GABA molecules are shown as blue spheres; the main narrow regions of the ion permeation pathway are marked with residues on the right; dashed lines indicate the approximate boundaries of the transmembrane domain (TM). (cd) Side views of two opposing (144°) protomers of PABA-bound Best1 (c) or Best2 (d) in a fully open state, identical to form a, where PABA molecules are shown as blue spheres.

[0049] Figures 12a-d This illustrates the effect of GABA on Best2 function in the presence of GS. When Cl - When the dominant anion in the internal and external solutions is (a) or glutamate is the dominant anion in the external solution (b), in the absence of (black) or presence of (red) 100 μM external GABA, in 1 μM [Ca 2+ ] i Below is the IV relationship of Best2 co-transfected with GS in HEK293 cells; for each point, n=6-8; *Using two-tailed unpaired students. t The test, compared to no GABA, p <0.05. (c, d) For endogenous currents from human NPE cells, the forms are the same as a and b, respectively; for each point, n = 5-6. All error bars in this figure represent sem.

[0050] Figure 13a-h The effects of small compounds on the function of the phytoretin channel are shown. (ad) Under conditions of absence (black) or presence (red solid), 100 µM PABA (a), PNBA (b), demethylarecanine (c), or PHBA (d), when Cl - When it is the dominant anion in both internal and external solutions, at 1 µM [Ca 2+ ] i Below, the IV relationship of Best2 in transiently transfected HEK293 cells; for each point, n=5-9; *using two-tailed unpaired students. t The test showed that, compared to untreated cells (black), p<0.05. (eh) For Best1, the same form as ad; for each point, n=6-7. The chemical structure of each compound is shown in the corresponding figure in eh. All error bars in this figure represent sem.

[0051] Figures 14a-i illustrate the cryo-EM processing pathways for the Best1+PABA dataset. (a) Representative micrographs. (b) Representative 2D categories. (c) Consistent refinement using a symmetric mask with a covering hole liner spiral and PABA binding sites for 3D classification. (df) 3D classification results, depicting a top view of the density map and FSC curves (from bottom left to top right: no mask; loose; corrected; tight) for the fully open state. (d), local resolution map (e), and orientation distribution map (f). (gi) For intermediate states, the same format as df. illustration Color illustration.

[0052] Figures 15a-i illustrate the cryo-EM processing pathways for the Best2+PABA dataset. (a) Representative micrographs. (b) Representative 2D categories. (c) Consistent refinement using a symmetric mask with a covering hole liner spiral and PABA binding sites for 3D classification. (df) 3D classification results, depicting a top view of the density map and FSC curves (from bottom left to top right: no mask; loose; corrected; tight) for the fully open state. (d), local resolution map (e), and orientation distribution map (f). (gi) For the closed state, the same form as df. illustration Color illustration.

[0053] Figures 16a-b GABA-bound Best1 in closed and fully open conformations are shown. (ab) Side views of two opposing (144°) Best1 protomers from the Best1+GABA dataset in either the GABA-unbound closed state (a) or the GABA-bound fully open state (b), where the ion permeation pathway is visualized and the pore size is depicted as colored dots: the closer the pore radius, the smaller and denser the dots. illustration Color legend. Major narrow regions of the ion permeation pathway are indicated by residues on the right. Dashed lines represent approximate boundaries of transmembrane domains (TM).

[0054] Figures 17a-c A comparison of the Best1 neck in different states is shown. (ac) Cryo-EM density map and top view of the model, showing the Best1 neck in fully open (a), partially open (b, from 8D1K) and closed (c) states.

[0055] Figures 18a-cThe effect of small compounds on the function of the phytoalveolar protein channel is shown in the (ab) bar graph, which shows the effect of Cl on the function of the phytoalveolar protein channel. - When the main internal and external anions are present, at 1 μM [Ca 2+ ] i Steady-state current density at +100 mV in HEK293 cells transiently expressing Best2 (a) or Best1 (b) treated with 100 μM of the small compound shown; n=5–9; *by two-tailed unpaired students t The test, compared to the current from untreated cells, p <0.05. (c) Normalized steady-state current density (Y-axis) of HEK293 cells instantaneously expressing Best1 at +100 mV versus different concentrations of the indicated small molecules in the external solution (X-axis), fitted to the Hill equation; n=6-7. All error bars in this figure represent SEM values. Detailed Implementation

[0056] In some aspects, this document describes a small molecule activator capable of binding to one or more paralogs of cantharidin, wherein the one or more paralogs of cantharidin include Best1, wherein the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best1 and the main-chain nitrogen atoms of V275 and F276 of Best1; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best1. In some embodiments, Best1 is human Best1.

[0057] In some aspects, this document describes a small molecule activator capable of binding to one or more paralogs of cantharidin, wherein the one or more paralogs of cantharidin include Best2, wherein the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best2 and the main-chain nitrogen atoms of I275 and F276 of Best2; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best2. In some embodiments, Best2 is human Best2.

[0058] In some respects, this document describes a small molecule activator capable of binding to one or more paralogs of cantharidin, wherein the one or more paralogs include Best3, wherein the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best3 and the main-chain nitrogen atoms of I275 and F276 of Best3; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best3. In some embodiments, Best3 is human Best3.

[0059] In some respects, this document describes a small molecule activator capable of binding to one or more paralogs of cantharidin, wherein the one or more paralogs of cantharidin include Best4, wherein the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best4 and the main-chain nitrogen atoms of L290 and T291 of Best4; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of S72 of Best4. In some embodiments, Best4 is human Best4.

[0060] In some aspects, this document describes a small molecule activator capable of binding to one or more paralogs of cantharidin, wherein the small molecule activator activates the one or more paralogs of cantharidin by contacting a pocket formed at the N-terminal helical dipole of the helix S4a of the one or more paralogs of cantharidin. In some embodiments, the one or more paralogs of cantharidin include Best1, Best2, Best3, or Best4. In some embodiments, Best1, Best2, Best3, or Best4 is human Best1, human Best2, human Best3, or human Best4.

[0061] In some embodiments, the small molecule activator is an amino acid. In some embodiments, the amino acid is a non-proteinogenic amino acid. In some embodiments, the small molecule activator is GABA or a GABA analog. In some embodiments, the small molecule activator is GABA. In some embodiments, the small molecule activator is a GABA analog. In some embodiments, the GABA analog is desmethylarecanthine, pregabalin, gabapentin, enalacarbinol, acampalic acid, muscarinic acid, vigabatrin, gabapentin, or aminooxyacetic acid. In some embodiments, the small molecule activator is PABA or a PABA analog. In some embodiments, the small molecule activator is PABA. In some embodiments, the small molecule activator is a PABA analog. In some embodiments, the PABA analogues are 2-amino-5-methoxybenzoic acid, 3,4-diaminobenzenesulfonic acid, 3-amino-5-methoxybenzoic acid, 4-amino-2-fluorobenzoic acid, 4-hydroxybenzoic acid, 4-nitrobenzoic acid, 4-amino-2-(hydroxymethyl)benzoic acid, 4-isobutoxybenzoic acid, 4-(aminomethyl)benzoic acid, 2-amino-5-pyrimidinecarboxylic acid, 2-(dimethylamino)-5-pyrimidinecarboxylic acid, 4-[(2-hydroxyethyl)amino]benzoic acid, 4-[(2-amino-2-oxoethyl)amino]benzoic acid, 4 -[(2-aminoethyl)amino]-3-nitrobenzoic acid, 3,4-diaminobenzoic acid, 4-amino-3-hydroxybenzoic acid, 4-amino-N-(phenylmethyl)benzamide, 4-amino-N-(2-amino-2-oxoethyl)benzamide, 4-amino-N-(2-hydroxyethyl)benzamide, 4-amino-N-(2-methylpropyl)benzamide, lisocaine, 4-amino-N-propylbenzamide, 4-amino-1-naphthylcarboxylic acid, 6-amino-4-quinolinecarboxylic acid, 8-amino-4-quinolinecarboxylic acid, or 1H-indole-5-carboxylic acid. In some embodiments, the small molecule activator is not GABA, a GABA analog, PABA, or a PABA analog. In some embodiments, the small molecule activator is pantothenic acid or 5-hydroxyindole-2-carboxylic acid.

[0062] In some embodiments, the small molecule activator activates one or more paracellular homologs of cantharidin by binding to their extracellular space. In some embodiments, the small molecule activator promotes neck opening of one or more paracellular homologs of cantharidin. In some embodiments, neck opening results in the side chains of the gate-forming residues being located away from the channel pores of one or more paracellular homologs of cantharidin. In some embodiments, the gate-forming residues are I76, F80, and F84 of one or more paracellular homologs of cantharidin.

[0063] In some aspects, this document describes a method for activating one or more paralogs of cantharidin, the method comprising contacting the paralog with a composition comprising a small molecule activator of one or more paralogs of cantharidin. In some embodiments, the one or more paralogs of cantharidin include Best1, Best2, Best3, or Best4. In some embodiments, Best1, Best2, Best3, or Best4 is human Best1, human Best2, human Best3, or human Best4.

[0064] In some embodiments, the small molecule activator is an amino acid. In some embodiments, the amino acid is a non-proteinogenic amino acid. In some embodiments, the small molecule activator is GABA or a GABA analog. In some embodiments, the small molecule activator is GABA. In some embodiments, the small molecule activator is a GABA analog. In some embodiments, the GABA analog is desmethylarecanthine, pregabalin, gabapentin, enalacarbinol, acampalic acid, muscarinic acid, vigabatrin, gabapentin, or aminooxyacetic acid. In some embodiments, the small molecule activator is PABA or a PABA analog. In some embodiments, the small molecule activator is PABA. In some embodiments, the small molecule activator is a PABA analog. In some embodiments, the PABA analogues are 2-amino-5-methoxybenzoic acid, 3,4-diaminobenzenesulfonic acid, 3-amino-5-methoxybenzoic acid, 4-amino-2-fluorobenzoic acid, 4-hydroxybenzoic acid, 4-nitrobenzoic acid, 4-amino-2-(hydroxymethyl)benzoic acid, 4-isobutoxybenzoic acid, 4-(aminomethyl)benzoic acid, 2-amino-5-pyrimidinecarboxylic acid, 2-(dimethylamino)-5-pyrimidinecarboxylic acid, 4-[(2-hydroxyethyl)amino]benzoic acid, 4-[(2-amino-2-oxoethyl)amino]benzoic acid, 4 -[(2-aminoethyl)amino]-3-nitrobenzoic acid, 3,4-diaminobenzoic acid, 4-amino-3-hydroxybenzoic acid, 4-amino-N-(phenylmethyl)benzamide, 4-amino-N-(2-amino-2-oxoethyl)benzamide, 4-amino-N-(2-hydroxyethyl)benzamide, 4-amino-N-(2-methylpropyl)benzamide, lisocaine, 4-amino-N-propylbenzamide, 4-amino-1-naphthylcarboxylic acid, 6-amino-4-quinolinecarboxylic acid, 8-amino-4-quinolinecarboxylic acid, or 1H-indole-5-carboxylic acid. In some embodiments, the small molecule activator is not GABA, a GABA analog, PABA, or a PABA analog. In some embodiments, the small molecule activator is pantothenic acid or 5-hydroxyindole-2-carboxylic acid.

[0065] In some embodiments, the small molecule activator activates one or more paracellular homologs of cantharidin by binding to the extracellular side of the paracellular surface of the cantharidin. In some embodiments, the small molecule activator activates one or more paracellular homologs of cantharidin by contacting a pocket formed at the N-terminal helical dipole of the helix S4a of the paracellular homolog. In some embodiments, the small molecule activator promotes neck opening of one or more paracellular homologs of cantharidin. In some embodiments, neck opening results in the side chains of the gate-forming residues being displaced from the channel pores of one or more paracellular homologs of cantharidin. In some embodiments, the gate-forming residues are I76, F80, and F84 of one or more paracellular homologs of cantharidin.

[0066] In some embodiments, the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best1 and the main-chain nitrogen atoms of V275 and F276 of Best1; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best1. In some embodiments, the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best2 and the main-chain nitrogen atoms of I275 and F276 of Best2; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best2.

[0067] In some embodiments, the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best3 and the main-chain nitrogen atoms of I275 and F276 of Best3; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best3. In some embodiments, the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best4 and the main-chain nitrogen atoms of L290 and T291 of Best4; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of S72 of Best4.

[0068] In some aspects, this document describes a method for treating or preventing a disease or condition in a subject of need, the method comprising administering to the subject a pharmaceutical composition comprising a small molecule activator of one or more paralogs of cantharidin. In some embodiments, the subject is a human being. In some embodiments, the disease or condition is caused by dysregulation of one or more cantharidins. In some embodiments, the disease or condition is a cantharidin (Best)-related disease.

[0069] In some implementations, phytoacidosis-related diseases are caused by a selection of... BEST1 , BEST2 , BEST3 and BEST4 One or more mutations in the gene cause this. In some implementations, vitelline macular degeneration is known as vitrectomycosis. As used herein, “vitrectomycosis” refers to a clinically distinct group of inherited retinal dystrophys that typically affect the macular region, the area synonymous with central hypersensitivity vision. This set of disorders is believed to be caused by mutations in BEST1, which acts as a Ca2+ receptor agonist in the retinal pigment epithelium (RPE) of the eye. 2+ Activated Cl - Channel. Non-limiting examples of vitrectomyctic macular degeneration include Best vitrectomyctic dystrophy (BVMD), adult-onset vitrectomyctic dystrophy (AVMD), autosomal recessive vitrectomyctic macular degeneration (ARB), autosomal dominant vitreoretinal choroidal disease (ADVIRC), autosomal dominant microkeratosis, cone-rod dystrophy, early-onset cataract, posterior staphyloma syndrome (MRCS syndrome), age-related macular degeneration (AMD), retinitis pigmentosa (RP), or combinations thereof.

[0070] In some embodiments, plaque atrophy-related disease is associated with an increase or decrease in intraocular pressure (IOP) in the subject. As used herein, “intraocular pressure” or “IOP” refers to the fluid pressure inside the eye. It is maintained by the constant production and outflow of fluids such as aqueous humor, a watery fluid that fills the front of the eye. In a healthy eye, a small amount of new aqueous humor enters the eye while an equal amount drains. However, if the fluid abnormally accumulates or disperses, the IOP may increase or decrease, resulting in either high or low intraocular pressure, respectively. For example, in some embodiments of this disclosure, the subject has high intraocular pressure.

[0071] The normal range for intraocular pressure (IOP) is between 10 and 21 mmHg. Elevated IOP is a symptom and risk factor for various eye diseases such as glaucoma, and untreated high IOP can impair vision or lead to vision loss. In some embodiments of this disclosure, the subject's IOP is higher than 21 mmHg.

[0072] Therefore, in some embodiments of this disclosure, the diseases associated with macular degeneration are selected from the group consisting of: glaucoma, myopia, dispersive pigment syndrome, pseudoexfoliation syndrome, age-related macular degeneration, or combinations thereof.

[0073] In some implementations, the disease or condition is not a plaque atrophy protein-related disease. A non-limiting example of such a disease is Alzheimer's disease. Therefore, in some implementations, the disease or condition is Alzheimer's disease.

[0074] In some embodiments, one or more paralogs of the tartaric protein include Best1, Best2, Best3, or Best4. In some embodiments, Best1, Best2, Best3, or Best4 is human Best1, human Best2, human Best3, or human Best4.

[0075] In some embodiments, the small molecule activator is an amino acid. In some embodiments, the amino acid is a non-proteinogenic amino acid. In some embodiments, the small molecule activator is GABA or a GABA analog. In some embodiments, the small molecule activator is GABA. In some embodiments, the small molecule activator is a GABA analog. In some embodiments, the GABA analog is desmethylarecanthine, pregabalin, gabapentin, enalacarbinol, acampalic acid, muscarinic acid, vigabatrin, gabapentin, or aminooxyacetic acid. In some embodiments, the small molecule activator is PABA or a PABA analog. In some embodiments, the small molecule activator is PABA. In some embodiments, the small molecule activator is a PABA analog. In some embodiments, the PABA analogues are 2-amino-5-methoxybenzoic acid, 3,4-diaminobenzenesulfonic acid, 3-amino-5-methoxybenzoic acid, 4-amino-2-fluorobenzoic acid, 4-hydroxybenzoic acid, 4-nitrobenzoic acid, 4-amino-2-(hydroxymethyl)benzoic acid, 4-isobutoxybenzoic acid, 4-(aminomethyl)benzoic acid, 2-amino-5-pyrimidinecarboxylic acid, 2-(dimethylamino)-5-pyrimidinecarboxylic acid, 4-[(2-hydroxyethyl)amino]benzoic acid, 4-[(2-amino-2-oxoethyl)amino]benzoic acid, 4 -[(2-aminoethyl)amino]-3-nitrobenzoic acid, 3,4-diaminobenzoic acid, 4-amino-3-hydroxybenzoic acid, 4-amino-N-(phenylmethyl)benzamide, 4-amino-N-(2-amino-2-oxoethyl)benzamide, 4-amino-N-(2-hydroxyethyl)benzamide, 4-amino-N-(2-methylpropyl)benzamide, lisocaine, 4-amino-N-propylbenzamide, 4-amino-1-naphthylcarboxylic acid, 6-amino-4-quinolinecarboxylic acid, 8-amino-4-quinolinecarboxylic acid, or 1H-indole-5-carboxylic acid. In some embodiments, the small molecule activator is not GABA, a GABA analog, PABA, or a PABA analog. In some embodiments, the small molecule activator is pantothenic acid or 5-hydroxyindole-2-carboxylic acid.

[0076] In some embodiments, the small molecule activator activates one or more paracellular homologs of cantharidin by binding to the extracellular side of the paracellular side of the cantharidin. In some embodiments, the small molecule activator activates one or more paracellular homologs of cantharidin by contacting a pocket formed at the N-terminal helical dipole of the helix S4a of the paracellular side of the cantharidin. In some embodiments, the small molecule activator binds to the extracellular side of the cantharidin. In some embodiments, the small molecule activator promotes neck opening of the cantharidin. In some embodiments, neck opening results in the side chains of the gate-forming residues being displaced from the channel pores of the cantharidin. In some embodiments, the gate-forming residues are I76, F80, and F84 of the cantharidin.

[0077] In some embodiments, the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best1 and the main-chain nitrogen atoms of V275 and F276 of Best1; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best1. In some embodiments, the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best2 and the main-chain nitrogen atoms of I275 and F276 of Best2; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best2.

[0078] In some embodiments, the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best3 and the main-chain nitrogen atoms of I275 and F276 of Best3; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best3. In some embodiments, the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best4 and the main-chain nitrogen atoms of L290 and T291 of Best4; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of S72 of Best4.

[0079] In some embodiments, the subject has a Best1 mutation. In some embodiments, the Best1 mutation is hereditary. In some embodiments, the Best1 mutation is a loss-of-function (LOF) mutation. In some embodiments, the composition is administered to salvage Best1 function.

[0080] Route of administration and dosage The route of administration and dosage of the effective amount of the pharmaceutical composition are also disclosed. The pharmaceutical agents of the present invention can be administered in combination with other pharmaceutical agents in multiple regimens for the effective treatment of diseases. In some embodiments, the pharmaceutical composition is administered topically via an ophthalmic delivery route, i.e., applied to the eye of the subject. Ophthalmic delivery routes include, but are not limited to, topically, periocularly (e.g., subconjunctival, subfascial, retro-orbital, and peri-orbital), intravitreal, and intra-anterior chamber. In some embodiments, the pharmaceutical composition is administered topically as eye drops. In some embodiments, the pharmaceutical composition is administered systemically, for example, via an oral or parenteral route.

[0081] The pharmaceutical composition is administered to the subject in a manner known in the art. The dosage administered will depend on the recipient's age, health and weight, the type of concurrent treatment (if any), the frequency of treatment, and the nature of the desired effect.

[0082] Pharmaceutical Composition Those skilled in the art will understand that the method of administering a pharmaceutically effective amount of a pharmaceutical composition to a patient in need can be determined empirically or by currently accepted standards in the medical field. The pharmaceutical composition may be administered to a patient as a combination of a pharmaceutical composition with one or more pharmaceutically acceptable excipients. It should be understood that when administered to human patients, the total daily dosage of the pharmaceutical composition will be determined by the attending physician within reasonable medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors: the type and extent of the cellular response to be achieved; the activity of the specific agent or composition used; the specific agent or composition used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the agent; the duration of treatment; drugs used in combination with or concurrently with the specific agent; and similar factors well known in the medical field. Those skilled in the art can certainly begin with a dose of the agent below the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0083] In some respects, this article describes a method for maintaining intraocular pressure (IOP) in subjects in need, comprising administering a pharmaceutical composition wherein the composition increases Best2-mediated Ca2+ in non-pigmented epithelial (NPE) cells. 2+ Dependence Cl -Electric current. In some implementations, the subject is a person. In some implementations, the subject has high or low intraocular pressure.

[0084] In some embodiments, the composition comprises a small molecule activator of Best2. In some embodiments, the small molecule activator is an amino acid. In some embodiments, the amino acid is a non-proteinogenic amino acid. In some embodiments, the small molecule activator is GABA or a GABA analog. In some embodiments, the small molecule activator is GABA. In some embodiments, the small molecule activator is a GABA analog. In some embodiments, the GABA analog is desmethylarecanthine, pregabalin, gabapentin, enalacarbinol, acampalic acid, muscarinic acid, vigabatrin, gabapentin, or aminooxyacetic acid. In some embodiments, the small molecule activator is PABA or a PABA analog. In some embodiments, the small molecule activator is PABA. In some embodiments, the small molecule activator is a PABA analog. In some embodiments, the PABA analogues are 2-amino-5-methoxybenzoic acid, 3,4-diaminobenzenesulfonic acid, 3-amino-5-methoxybenzoic acid, 4-amino-2-fluorobenzoic acid, 4-hydroxybenzoic acid, 4-nitrobenzoic acid, 4-amino-2-(hydroxymethyl)benzoic acid, 4-isobutoxybenzoic acid, 4-(aminomethyl)benzoic acid, 2-amino-5-pyrimidinecarboxylic acid, 2-(dimethylamino)-5-pyrimidinecarboxylic acid, 4-[(2-hydroxyethyl)amino]benzoic acid, 4-[(2-amino-2-oxoethyl)amino]benzoic acid, 4 -[(2-aminoethyl)amino]-3-nitrobenzoic acid, 3,4-diaminobenzoic acid, 4-amino-3-hydroxybenzoic acid, 4-amino-N-(phenylmethyl)benzamide, 4-amino-N-(2-amino-2-oxoethyl)benzamide, 4-amino-N-(2-hydroxyethyl)benzamide, 4-amino-N-(2-methylpropyl)benzamide, lisocaine, 4-amino-N-propylbenzamide, 4-amino-1-naphthylcarboxylic acid, 6-amino-4-quinolinecarboxylic acid, 8-amino-4-quinolinecarboxylic acid, or 1H-indole-5-carboxylic acid. In some embodiments, the small molecule activator is not GABA, a GABA analog, PABA, or a PABA analog. In some embodiments, the small molecule activator is pantothenic acid or 5-hydroxyindole-2-carboxylic acid.

[0085] In some embodiments, the small molecule activator activates Best2 by binding to the extracellular side of Best2. In some embodiments, the small molecule activator activates Best2 by contacting a pocket formed at the N-terminal helical dipole of helix S4a of Best2. In some embodiments, the small molecule activator promotes neck opening of Best2. In some embodiments, neck opening results in the side chains of the gate-forming residues moving away from the channel pores of Best2. In some embodiments, the gate-forming residues are I76, F80, and F84 of Best2. In some embodiments, the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best2 and the main-chain nitrogen atoms of I275 and F276 of Best2; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 and the side-chain hydroxyl group of T277; and the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best2. In some embodiments, the Best1 mutation is A243T, A10T, R218H, L234P, Q293K, or D302A. In some embodiments, the composition is applied to rescue Best1 function.

[0086] In some embodiments, the pharmaceutical composition is administered via ophthalmic delivery, oral delivery, or parenteral delivery. In some embodiments, the pharmaceutical composition is administered via ophthalmic delivery, wherein the pharmaceutical composition is applied topically. In some embodiments, the pharmaceutical composition is administered as eye drops.

[0087] Those skilled in the art will understand that the small molecule activators of the present invention are not limited to those specifically disclosed. Other small molecule activators (including GABA and PABA analogs) adapted to the binding pocket of the cantharidin channel and forming hydrogen bonds or other previously described interactions with the aforementioned residues of cantharidin can also be used in the present invention. Such small molecule activators are characterized by those having a molecular weight similar to the small molecule activators described above to adapt to the cantharidin binding pocket, and those containing a 5-membered or 6-membered ring to reach the aforementioned residues within the cantharidin binding pocket. Thus, in some embodiments, the small molecule activator contains a 5-membered or 6-membered ring. In some embodiments, the small molecule activator contains a 5-membered ring. In some embodiments, the small molecule activator contains a 6-membered ring.

[0088] In some embodiments, one or more paralogs of cantharidin include Best1, and the small molecule activator is GABA or a GABA analogue, wherein: The oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of Y68 of Best1 and the main chain nitrogen atoms of V275 and F276 of Best1; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main chain nitrogen atom of T277 and the side chain hydroxyl group of T277; the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side chain hydroxyl group of Y72 of Best1; the α carbon (C2) of the small molecule activator forms a van der Waals contact with the β carbon of P274 of Best1; the β carbon of the small molecule activator forms a van der Waals contact with the C3 carbon of the ring of the side chain of Y72 of Best1; and the nitrogen atom of the small molecule activator is adjacent to the hydroxyl group of the side chain of Y72 of Best1.

[0089] In some embodiments, one or more paralogs of taraxacin include Best1, and the small molecule activator is PABA or a PABA analogue, wherein: the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best1 and the main-chain nitrogen atoms of V275 and F276 of Best1; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 of Best1 and the side-chain hydroxyl group of T277 of Best1; the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best1; the amino group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of H267 of Best1; the aromatic ring of the small molecule activator forms a π-stacking interaction with the side chain of F257 of Best1; and the aromatic ring of the small molecule activator forms a side-to-side interaction with the side chain of Y72 of Best1.

[0090] In some embodiments, one or more paralogs of taraxacin include Best2, and the small molecule activator is PABA or a PABA analogue, wherein: the oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of Y68 of Best2 and the main-chain nitrogen atoms of I275 and F276 of Best2; another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main-chain nitrogen atom of T277 of Best2 and the side-chain hydroxyl group of T277; the nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side-chain hydroxyl group of Y72 of Best2; the amino group of the small molecule activator forms a hydrogen bond with the side-chain hydroxyl group of H267 of Best2; the aromatic ring of the small molecule activator forms a π-stacking interaction with the side chain of F257 of Best2; and the aromatic ring of the small molecule activator forms a side-to-side interaction with the side chain of Y72 of Best2.

[0091] Expression of paralogs of piracetam protein Four mammalian retinal pigment epithelial proteins play multiple roles in the body's physiology. Best1 is primarily expressed in the retinal pigment epithelium (RPE) of the human retina, which forms the external blood-retinal barrier and plays a crucial role in maintaining retinal physiology through transcellular transport of water, ions, metabolites, nutrients, and waste products. Best1 expression has also been found in the airways, colon, kidneys, central nervous system, colon cancer cells, human pancreatic duct cell lines, and testes of mice and / or humans. Among other regions, Best2 is expressed in the basolateral membrane of the non-pigmented epithelium (NPE) of the ciliary body and is important for aqueous humor secretion and maintenance of intraocular pressure (IOP). Among other functions, Best3 regulates vascular tone in smooth muscle of humans, mice, and rats. Best4 has been identified in different subsets of human intestinal absorptive epithelial cells. Previous structural analyses have shown that prokaryotic KpBest, avian cBest1, and mammalian bBest2 channels exhibit a variety of similarities, including the formation of homopentamer assemblies with cyclic symmetry (C5), four transmembrane helices per proton (20 per channel), and a flowing vase-shaped ion transport pathway traversing approximately 95 Å along the central axis of symmetry. Further background information on the bestrophin family of proteins can be found in Owji et al., Structure and Function of the Bestrophin family of calcium-activated chloride channels, Channels (Austin). 2021; 15(1): 604–623, the full text of which is incorporated herein by reference.

[0092] Example Small molecule activators targeting cantharidin channels for disease treatment Ca2+ is composed of four members found in mammals. 2+ Activated anion channel family 1 They are widely distributed in various human organs, including the airways, colon, kidneys, pancreas, and central nervous system, but are best known for their physiological role in the eyes. 1 In particular, Best1 is primarily expressed in the retinal pigment epithelium (RPE) and is genetically associated with a range of retinal degenerative disorders collectively known as vitrectomycosis. 2,3 More than 350 different Best1 mutations have been identified that cause vitelline macular degeneration (most of which result in loss of function (LOF)). 1,2 Patients with this condition are prone to progressive vision loss, which can eventually lead to blindness, and there is no cure. 4On the other hand, Best2 resides in the non-pigmented epithelium (NPE) that regulates intraocular pressure (IOP). Since both high and low blood pressure are harmful conditions, it is essential to maintain appropriate intraocular pressure at all times. 5-7 Therefore, laminarin is a potential drug target, especially for eye health.

[0093] However, this request was prohibited due to a lack of knowledge about small molecule activators or inhibitors specifically targeting the cantharidin channel. 2+ It was first identified as an essential activator of cantharidin, leading to the classification of this protein family as Ca. 2+ Activated anion channels 8,9 Later, it was discovered that ATP activates calcium-deficient cells. 2+ Bacterial scab protein homologs at binding sites and in Ca 2+ The presence of these proteins is necessary for the complete activation of mammalian frenulum proteins. 10 Due to Ca 2+ Both ATP and β-hydroxypropyl methylphenidate (Best1) are common physiological molecules involved in numerous biochemical activities, therefore they or their analogues are not suitable for drug targeting of cantharidin. Interestingly, Best1 has been shown to be permeable to the major inhibitory neurotransmitter γ-aminobutyric acid (GABA). 11-14 However, the penetration and impact of GABA on Best2 remain unknown.

[0094] Previous studies have revealed several key structural components on the pentamer assembly of cantharidin, which controls channel function. These include: 1) two major permeational narrowing regions in the ion transport pathway, namely the “neck” at the transmembrane pore (I76 / F80 / F84 in Best1 and Best2) and the “pore” at the cytoplasmic exit (I205 / Q208 / N212 in Best1 and S205 / K208 / E212 in Best2). 15-19 ;2) The C-terminal self-inhibitory region (residues 346-378 / 379 in AS, Best1 / Best2) causes the channel to concentrically contract by wrapping around the channel periphery in an interpromeric manner, and is released as the neck opens. 19,20 ;3) Ca formed by acidic clusters (E300 / D301 / D302 / D303 / D304 in Best1 / Best2) between S4a and S4b of a protopolymer and N-terminal S1a-S1b helical-turn-helical elements of adjacent protopolymers. 2+ buckle 15,19 It is expected that small molecule activators / inhibitors (such as glutamate and ATP) affect the conformation of one or more of these components, but the specific mechanisms remain unclear due to the lack of co-structures of laminarin that bind to activators / inhibitors.

[0095] Here, we resolved the structures of GABA-binding Best1 and Best2, depicting the extracellular GABA binding sites on the channels, and identified extracellular GABA as an activator of both Best1 and Best2. We further identified various small molecules, including multiple GABA analogs, as activators of cantharidin channels. Extensive analysis was performed on the potent activator 4-aminobenzoic acid (PABA): the structures of PABA-binding Best1 and Best2 were resolved and showed the same binding sites as GABA; PABA treatment rescued the functional deficits of patient-derived Best1 mutations. In conclusion, our results demonstrate the mechanisms and potential of various small molecule candidates as clinically applicable medicines for cantharidin-related diseases / conditions.

[0096] GABA-binding Best1 structure We incubated purified Best1 with 20 mM GABA before preparing the cryo-EM mesh and resolved the GABA-bound Best1 structure at a resolution of 2.4–2.5 Å (Figs. 1a and 8a–h). 20 The GABA binding site on Best1 is extracellular (Fig. 1a) and was previously identified as Cl by X-ray anomalous diffraction studies using chicken Best1. - binding site 15 Several contact points exist between GABA and Best1 (Fig. 1b): one oxygen atom of the GABA carboxyl group forms a hydrogen bond with the side chain hydroxyl group of Y68 (2.8 Å) and the main chain nitrogen atoms of V275 (3.1 Å) and F276 (3.1 Å); another carboxyl oxygen atom forms a hydrogen bond with the side chain hydroxyl group of T277 (3.2 Å) and is adjacent to the main chain nitrogen of T277 (3.5 Å); the α carbon (C2) forms a van der Waals contact with the β carbon of P274 (3.5 Å), while the GABA β carbon forms a van der Waals contact with the C3 carbon of the ring of the Y72 side chain (3.4 Å); the nitrogen atom of the GABA molecule is adjacent to the hydroxyl group of the Y72 side chain (3.4 Å); the nitrogen atom of the GABA molecule is adjacent to the hydroxyl group of the Y72 side chain (3.6 Å, Fig. 1b). Notably, 18% of the particles in the Best1+GABA dataset exhibited a fully open conformation at the "neck" of the channel (FFFFF, Fig. 1c), which was previously only obtained using truncated or mutant Best1. This demonstrates a similarity to Ca... 2+ The bound, GABA-free Best1 (PPPPP, Fig. 1c) showed a significantly larger neck opening and lacked the C-terminal autoinhibitory segment (AS) in our previous study of the truncated Best1. 1-345The observed conformations are very similar (root mean square deviation, rmsd 0.41, relative to 8D1O). 19 In this GABA-bound open state, a GABA molecule resides within a pocket formed at the N-terminal helical dipole of the helix S4a of each Best1 proton (FFFFF, Fig. 1c). In stark contrast, 10% of the Best1 particles from the same sample are in a completely closed state (CCCCC, Fig. 1c). In addition to the completely open (FFFFF) and closed (CCCCC) states, we also identified two intermediate states: one with four closed protons plus one partially open proton (PCCCC, 32%), and one with three closed protons plus two partially open protons (PCPCC, 31%) (Fig. 1c). Notably, in these intermediate states, only four of the five Best1 protons in each pentamer assembly are related to the GABA-like density (therefore four GABA molecules per channel), which is the opposite of all five protons bound to GABA in the completely open state (Fig. 1c). In summary, our results indicate that extracellular GABA binding strongly promotes Best1 neck opening, and complete GABA occupancy of all five protomers is necessary for complete Best1 neck opening.

[0097] The pores of GABA-bonded Best1 are indistinguishable from those of Best1 without GABA (Fig. 16), indicating that the bonding of GABA to Best1 does not affect the structural conformation of the pores.

[0098] Extracellular GABA stimulation of Best1-mediated Cl - Current The identification of the extracellular GABA binding site on Best1 prompted us to further examine the effect of GABA on Best1 channel function using whole-cell patch-clamp. Consistent with the structural results, when 20 mM GABA was added to the external rather than the internal patch solution, the inward and outward Ca2+ expression from Best1-expressing HEK293 cells increased. 2+ Activated Cl - The currents all increased significantly (Figure 1d, e). 20 To evaluate the dose dependence of GABA, we performed patch-clamp analyses with different concentrations of GABA in an external solution. The outward current density (Cl...) was... - The relationship between inflow and extracellular GABA concentration was fitted to the Hill equation, and the EC50 of extracellular GABA required to activate Best1 was calculated. 50 The concentration was calculated to be 371 nM (Figure 1f). 20 .

[0099] Our results indicate that GABA binds to Best1 on the extracellular side and promotes Best1-mediated Cl- at nanomolar concentrations. - Electric current.

[0100] GABA-binding Best2 structure To test whether GABA had any effect on the conformation of Best2, we incubated the purified Best2 protein with GABA before freeze-EM mesh preparation and found that GABA also bound to Best2. The GABA-bound Best2 structure was resolved at 2.3 Å resolution (Figs. 2a-b and 9a-i), revealing an extracellular GABA binding site similar to that in Best1, and previously identified as Cl by X-ray anomalous diffraction using chicken Best1. - binding site 15,20 There are several contact points between GABA and Best2 (Figure 2b): one oxygen atom of the GABA carboxyl group forms a hydrogen bond with the side chain hydroxyl group of Y68 (2.4 Å) and the main chain nitrogen atoms of I275 (3.4 Å) and F276 (2.9 Å), while the other oxygen atom of the carboxyl group forms a hydrogen bond with the main chain nitrogen atom of T277 (2.9 Å) and the side chain hydroxyl group of T277 (2.4 Å); the nitrogen atom of the GABA amino group forms a hydrogen bond (3.4 Å) with the oxygen atom of the side chain hydroxyl group of Y72.

[0101] Notably, 65% of the particles in the Best2+GABA dataset were GABA-bound (one GABA molecule per Best2 proton, and therefore five per pentamer) and exhibited fully open necks (Fig. 2c). In contrast, the remaining 35% of the particles were not GABA-bound and exhibited fully closed necks (Fig. 2d). In summary, our results suggest that GABA binding to the extracellular pocket on Best2 strongly promotes neck opening, resulting in the I76 / F80 / F84 side chains being displaced from the channel pores.

[0102] Notably, GABA binding induces a conformational change in the pore-lined helix by recruiting Y68 to form hydrogen bonds with the GABA carboxyl group, resulting in upward and slightly off-center shifts of the Y72 and Y68 α-carbons by 1.2 Å and 1.4 Å, respectively (Fig. 10b). Multiple hydrogen bonds between the GABA carboxyl group and the electronegative N-terminal helical dipole anchor the GABA molecule within the pocket, while another hydrogen bond anchors Y68 in an open position and spatially closes it from the open-to-close conformational transition (Figs. 2b and 10). These interactions anchor the pore-lined helix in a slightly expanded position with symmetrical five-fold occupancy, which is energy-friendly for the helix to unwind at P77, resulting in full opening of the neck.

[0103] GABA is a Best2 activator and promotes the influx of extracellular glutamate.

[0104] The identification of extracellular GABA binding sites on Best2 cells prompted us to add GABA to standard Cl during patch-clamping. - The effect of GABA on Best2 channel function was further examined in a patch solution. Consistent with the structural results, when 100 μM GABA was added to the external solution, the inward and outward Cl-channel activity from HEK293 cells expressing Best2 was significantly improved. - The currents all increased significantly (Figure 2e). To evaluate the dose dependence of GABA, we performed patch-clamp analyses with different concentrations of GABA in the external solution. The outward current density (Cl... - The relationship between inflow and extracellular GABA concentration was fitted to the Hill equation, and the EC50 of extracellular GABA required to promote Best2 was also calculated. 50 The measurement was 191 nM (Figure 2f). Notably, both GABA and glutamate were expressed as Ca²⁺. 2+ The dependency mechanism promotes Best2, and at 1 μM [Ca 2+ ] i The peak current is reached at this time (Figure 2g, h).

[0105] To test whether Best2 is permeable to GABA, we used GABA as the main permeable ion in the external solution, Cl... - As the only anion in the internal solution. The reversal potential shifts to the right (E rev = 16.6 ± 5.3 mV (Figure 2i), corresponding to GABA to Cl in Best2 - relative penetration rate (P) GABA / P Cl The value was 0.56. Furthermore, GABA was used to replace Cl in the external solution. -This not only leads to a strong increase in outward current (GABA inflow), but also to an increase in inward current (Cl). - A strong increase in outflow (Fig. 2j). These results indicate that: 1) Best2 is permeable to extracellular GABA; 2) GABA on the extracellular side of the channel trans-promotes Cl. - They move from the inside of the cell outwards, which is consistent with the structural results.

[0106] Previously, we found that in the external glutamate and internal Cl... - In this case, the reversal potential of Best2 shifts significantly to the right (E rev = 46.2 ± 2.5 mV (Figure 2k, black), and glutamate relative to Cl - The penetration ratio (P) Glu / P Cl Only 0.1 21 To test whether GABA affects glutamate permeability, 100 μg GABA was added to the external glutamate solution. Under these conditions, the reversal potential shifted significantly to the left (E0). rev = 18.0 ± 5.5 mV (Figure 2k, red), and (P Glu / P Cl The concentration of glutamate from Best2 increased to 0.53. Furthermore, after adding 100 μM GABA to the external solution, the concentrations of glutamate from Best2 and introverted Cl- from Best2 increased. - The currents all increased significantly (Figure 2l).

[0107] In summary, our results indicate that GABA binds to Best2 on the extracellular surface and promotes Best2-mediated Cl- at nanomolar concentrations. - The current significantly increases the permeability of extracellular glutamate.

[0108] GABA releases GS-mediated inhibition of Best2 in vivo.

[0109] Previously, we discovered that GS combines Best2 and in NPE 21 In the absence of intracellular glutamate, channel function is inhibited. To test whether extracellular GABA alleviates the inhibitory effect of GS, we examined the effect of GABA on currents from HEK293 cells co-expressing Best2 and GS. Consistent with previous studies, when Cl... - When it is the only anion in the internal solution, regardless of whether the dominant anion in the external solution is Cl... - Both glutamate and GS significantly reduced the current (Fig. 12a-b, black). In contrast, when 100 μM GABA was added to the external solution, Cl... -Both glutamate and GS currents increased significantly to levels similar to those in the absence of GS (Fig. 12a-b, red), and both external glutamate and internal Cl- currents increased significantly. - Under these conditions, the reversal potential also shifts to a position similar to that without GS (E rev = 16.8 ± 4.6 mV (Figure 12b, red), where P Glu / P Cl It is 0.54.

[0110] We then examined the effect of GABA on the innate Best2-mediated current in NPE cells using patch clamp under the same conditions. The results were similar to those from transiently transfected HEK293 cells (Fig. 12c-d), strongly suggesting that GABA promotes Best2-mediated Cl- in NPE cells. - Physiological effects on electrical current and glutamate inflow, despite the presence of GS 21 .

[0111] Best1 / Best2 small molecule activators Since GABA acts as a potent activator of Best2 and its binding site is extracellular, our findings suggest the potential for clinical applications of small molecules stimulating channel function via this binding site. Through electrophysiological analysis, we identified four additional small-molecule activators of Best2, including the GABA analogue desmethylarecanthine and three benzoic acid derivatives with different chemical groups at the para position: 4-aminobenzoic acid (PABA), 4-nitrobenzoic acid (PNBA), and 4-hydroxybenzoic acid (PHBA). During patch-clamp recording, Best2-mediated Cl... - The current was significantly increased by each substance in the external solution at 100 µM (Figs. 13a-d and 18a-c).

[0112] Since the residues that form GABA binding sites on Best2 are highly conserved in cantharidin, we inferred that small molecule activators could also stimulate Best1. Indeed, Best1-mediated Cl- activating agents were observed in transiently transfected HEK293 cells. - The current was significantly enhanced by each small molecule at 100 µM in the external solution (Fig. 13e-h and 18a-c).

[0113] To further investigate the dose-dependent effect of small molecule activators on Best1 (whose loss of function leads to macular degeneration), we performed patch-clamp analyses in external solutions using a series of concentrations of PABA, PNBA, demethylarecanine, and PHBA. The outward current density (Cl...) - The relationship between inflow and extracellular small molecule concentration was fitted to the Hill equation. EC20 required to activate Best1 50The measured values ​​were: 192 nM for PABA, 261 nM for PNBA, 258 nM for demethylarecanthine, and 180 nM for PHBA. Figures 18a-c Furthermore, for Best1 and Best2 with 100 µM PABA, in the absence of Ca... 2+ Under these conditions, tiny currents were recorded (Figures 4a and 4e), indicating that PABA-mediated activation is Ca 2+ Dependency-dependent.

[0114] PABA-binding Best1 and Best2 structures To verify the role of the newly identified GABA binding site in accommodating other small molecule activators of the cantharidin channel, we resolved the PABA-binding Best1 and Best2 structures at a resolution of 2.4–2.7 Å (Fig. 10, see also Figs. 4, 11, 14, and 15). The PABA-binding costructures show that PABA binds to both channels at the same site where GABA binds to Best2, thus preserving all the contact points observed in the GABA-Best2 costructure, plus the hydrogen bond with H267 (Figs. 4b, f, and 10c–d). Furthermore, the aromatic ring of PABA exhibits π-stacking and side-to-side interactions with the side chains of F257 and Y72, respectively (Figs. 4b, f, and 10c–d).

[0115] In the Best2+PABA dataset, fully open necks were observed in 57% of the particles (Figs. 4c and 15), and closed necks were present in 43% of the particles (Figs. 4d and 15). In the Best1+PABA dataset, fully open necks were observed in 18% of the particles (Figs. 4g and 14d), while fully closed necks were not present at all, and a novel intermediate state consisting of two closed and three partially open protopolymers (PPCPC) was identified (Figs. 4h and 14g). For comparison, most Best1 particles (89%) from the PABA-free condition were in a fully closed state, while fully open necks were not present at all. 19 In summary, our results indicate that, similar to GABA, PABA binds to the extracellular side of laminarin and strongly promotes laminarin neck opening.

[0116] PABA rescues patient-derived mutations in Best1 in HEK293 cells.

[0117] Since most disease-causing Best1 mutations are autosomal dominant LOF mutations, the coexistence of WT and LOF mutant Best1 proteins in patients forms the basis for a potential therapeutic strategy for vitrectomycosis using small molecule activators of Best1. 4To verify this idea, we co-transfected six patient-derived dominant LOF mutants (A10T, R218H, L234P, A243T, Q293K, and D302A) with WT Best1 in HEK293 cells at a ratio of 4:1 to simulate the endogenous allele expression ratio in patient RPE cells. 22 And by patch-clamping, PABA was examined in these cells for its effects on Ca2+. 2+ Dependence Cl - The effect of current. Consistent with our previous results. 22 All six mutations caused a decrease in Best1-mediated current, which was completely rescued by different concentrations of PABA. Figures 5a-f Notably, A243T exhibited the fewest defects among the six mutants and required the lowest concentration of PABA (10 nM) for its functional rescue, while the other mutants A10T, R218H, L234P, Q293K, and D302A required 100 nM, 10 μM, 10 μM, 1 μM, and 1 μM PABA, respectively. Furthermore, PABA treatment restored Ca2+ in patient-derived iPSC-RPE cells carrying Best1 mutations in A10T, R218H, L234P, A243T, Q293K, or D302A. 2+ Dependence Cl - Current ( Figure 5g These results demonstrate the potential of PABA as a drug treatment for vitelloid macular degeneration caused by Best1 dominant LOF mutations.

[0118] Other small molecule activators of Best1 / Best2 Using patch-clamp techniques, we screened a range of small molecule candidates for their effects on Best1 / Best2 function using whole-cell patch-clamp therapy and identified several compounds, including some FDA-approved drugs, as activators of both Best1 and Best2. Figure 3 and 7Those include: 1) GABA analogs such as pregabalin, gabapentin, ennacarbi, acanolic acid, muscarinic acid, vigabatrin, gabapentin, and aminooxyacetic acid; 2) PABA analogs such as 2-amino-5-methoxybenzoic acid, 3,4-diaminobenzoic acid, 3,4-diaminobenzenesulfonic acid, 3-amino-5-methoxybenzoic acid, 4-amino-2-fluorobenzoic acid, 4-amino-2-(hydroxymethyl)benzoic acid, 4-isobutoxybenzoic acid, 4-(aminomethyl)benzoic acid, 2-amino-5-pyrimidinecarboxylic acid, 2-(dimethylamino)-5-pyrimidinecarboxylic acid, 4-[(2-hydroxyethyl)amino]benzoic acid, 4-[(2-amino-2-oxoethyl)amino]benzoic acid, 4-[(2-aminoethyl)amino]-3-nitrobenzoic acid, 4-Amino-3-hydroxybenzoic acid, 4-amino-N-(phenylmethyl)benzamide, 4-amino-N-(2-amino-2-oxoethyl)benzamide, 4-amino-N-(2-hydroxyethyl)benzamide, 4-amino-N-(2-methylpropyl)benzamide, lisocaine, 4-amino-N-propylbenzamide, 4-amino-1-naphthylcarboxylic acid, 6-amino-4-quinolinecarboxylic acid, 8-amino-4-quinolinecarboxylic acid, or 1H-indole-5-carboxylic acid; 3) pantothenic acid and 5-hydroxyindole-2-carboxylic acid, which are neither GABA analogs nor PABA analogs.

[0119] In summary, our results demonstrate the mechanism of GABA-mediated cantharidin channel activation and the mechanism and potential of various candidate compounds as clinically applicable drugs for cantharidin-related diseases / conditions.

[0120] Retinal degeneration, specifically vitrectomycosis (Vitamin B1) macular degeneration, is caused by Best1 mutations, most of which are autosomal dominant LOF (loose-of-function) mutations. This results in the presence of the WT Best1 protein in the patient's cells, which can be activated upon binding with small molecule activators to compensate for the LOF caused by the mutation. On the other hand, the response of Best1 recessive mutations to each small molecule should be examined individually, as these cells lack the WT Best1 protein, and the mutant Best1 may or may not be bound / activated by small molecule activators. Although rare Best1 gain-of-function mutations are not suitable for this treatment, autosomal dominant LOF accounts for >95% of all vitrectomycosis cases, thus underpinning the enormous pharmaceutical potential of small molecule Best1 activators. This provides an alternative treatment strategy for the cure of vitrectomycosis, in addition to gene therapy.

[0121] Best2 knockout mice exhibited reduced IOP. 5,6This indicates a positive contribution of Best2 channel function to IOP. GABA activation of Best2 suggests that increased GABA levels in the ciliary body may be a pathogenic factor in high intraocular pressure. Notably, experimentally induced high intraocular pressure has been shown to cause a decrease in GABAergic activity in the eye. 23,24 This indicates a negative feedback loop in the GABAergic system following an increase in IOP. On the other hand, small molecule activators of Best2 may have potential applications in the treatment of hypotony.

[0122] method cell lines HEK293 and HEK293F cells were purchased from ATCC (catalog number #CRL-1573) and Thermo Fisher Scientific (catalog number #R79007), respectively. Cells used in this study were validated by short tandem repeat (STR) DNA profiling and tested negative for mycoplasma contamination. HEK293 cells were cultured in DMEM (Corning, catalog number 10013CV) supplemented with 100 µg / mL penicillin-streptomycin and 10% fetal bovine serum, while HEK293F cells were cultured in FreeStyle™ 293 expression medium (Thermo Fisher Scientific, catalog number #12338026). Human NPE cells were purchased from ScienCell Research Laboratories (catalog number #6580), cultured in Epithelial Cell Medium (EpiCM, catalog number #4101), and morphologically validated. No mycoplasma contamination was detected by DAPI staining.

[0123] transfection 20–24 h before transfection, cells were aliquoted into new 3.5 cm culture dishes at 50% confluence. Plasmid and siRNA transfections were performed using PolyJet transfection reagent (SignaGen, catalog number #SL100688) and Lipofectamine RNAiMAX reagent (Invitrogen, catalog number #13778-030), respectively. After 4–8 h, the transfection mixture was removed, cells were washed with PBS, and fresh culture medium was added until downstream analysis or harvesting.

[0124] Electrophysiology Whole-cell patch-clamp recordings were performed 48–96 h after NPE, iPSC-RPE division, or transfection of HEK293 cells using an EPC10 patch-clamp amplifier (HEKA Electronics) controlled by Patchmaster v2x90.5 (HEKA). 22,25The micropipette is fabricated by pulling it out of a 1.5 mm thin-walled glass filament (WPI Instruments). The series resistance is typically 1.5–2.5 MΩ. No electronic series resistance compensation was performed. The experiment was conducted at room temperature (23 ± 2℃). The liquid junction potential was measured and corrected using the HEKA built-in function. Standard zero Ca 2+ The pipette solution contained (mM): 146 CsCl, 2 MgCl2, 5 EGTA, 2 MgATP (freshly added), 10 HEPES, and was adjusted to pH 7.3 with NMDG. As calculated using the MaxChelator program, various free Ca2+ compounds were prepared by mixing CaCl2 with EGTA. 2+ A solution of a certain concentration, and using Ca 2+ Ion-selective electrode verification of free Ca 2+ Concentration. The standard extracellular solution contains (mM): 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 15 mM glucose, 10 mM HEPES, pH 7.4, and NMDG. In solutions containing GABA / small molecules, 100 mM, 10 mM, 1 mM, 100 nM, 10 nM, or 1 nM GABA / GABA / small molecules are added to the external solution, and the pH is adjusted to 7.4 with NMDG. In GABA / glutamate external solutions, 140 mM GABA / glutamate replaces Cl2. - The pH was adjusted to 7.4 using NMDG. In the glutamate internal solution, 146 mM Cs-glutamate replaced CsCl, and the pH was adjusted to 7.4 using NMDG. The glucose solution had an osmotic pressure of 290–310 mOsm / L, and was approximately 5 mOsm lower in the internal solution than in the external solution in the same experiment. Low and high Ca2+ solutions from the same group of experiments were compared. 2+ Adjust the solution to have the exact same osmotic pressure. Replace the solution manually.

[0125] Electrophysiological data collection and analysis Acquire traces at 4-second repetition intervals. 28 The current was sampled at 25 kHz and filtered at 5 or 10 kHz. IV curves were generated from a set of step potentials (from a holding potential of 0 mV, -100 to +100 mV). Data were processed offline in Patchmaster. Statistical analysis was performed using built-in functions in OriginPro 8.5. Relative permeability was calculated according to the Goldman-Hodgkin-Katz equation. The relative X / Cl... - (X = GABA or glutamate, where X or Cl) -In the external solution, the inward movement (outward current) conductivity (G) X，具有ex-X / G Cl，具有ex-Cl The slope conductivity is measured at a reversal potential of 50 mV. This represents the relative Cl... - (In the internal solution) Outward movement (inward current) conductivity (G) Cl，具有ex-X / G Cl，具有ex-Cl The inverse effect of ) is measured as the slope conductivity at the inversion potential minus 50 mV. The patch-clamp recording is shown in the legend. n The value represents the total number of individual cells.

[0126] Cryo-EM Sample Preparation Best1 and Best2 proteins were purified in GDN. 19, 32 Following nickel affinity chromatography and size exclusion chromatography, the protein was concentrated to 5 mg / mL and incubated with 20 mM GABA / PABA for 1 h prior to grid generation for GABA-binding Best2 and PABA-binding Best1. The protein was then concentrated to 5 mg / mL for grid generation.

[0127] 2.8 µL of protein was incorporated into 5 mM CaCl2 and immediately applied to a plasma-treated UltrAuFoil R0.6 / 1 on a Vitrobot Mark IV. The mixture was incubated at 100% humidity and 10°C for 30 s, transferred under force 4 for 5–7 s, and immediately immersed in liquid ethane cooled by liquid nitrogen. A grid was screened on a Glacios prior to data collection.

[0128] Data collection and image processing Data were collected using Leginon 3.5 on Krios. For the Best1+PABA dataset, 1,634 photomicrographs were collected in counting mode using a K3 direct electron detector at a magnification of 105,000x, corresponding to a total dose of 59 e. - / Å 2 0.83 Å 2 The physical pixel size per pixel, graded across 50 frames, corresponds to 1.18 e. - / Å 2The dose rate was set at / frame, with a defocus range of 0.8–1.8 μm. The film was aligned with MotionCorr2 via Relion-3.1 and imported into cryoSPARCv4 for further processing via PatchCTF estimation, template selection and extraction, and initial 2D classification, resulting in 46,097 final particles. These particles underwent homogenization refinement (C5), symmetry expansion (generating 230,485 particles), and 3D classification into 6 categories using a mask covering the neck and ligand binding sites, with a target resolution of 3.2 Å. One category (41,771 particles, 18.1%) was completely open and underwent local refinement (C1) to 2.60 Å. At least three categories represented intermediate states. All closed particles were combined and symmetry expansion was de-exposed (to remove duplicates), leaving 40,311 particles, and refinement was performed on the intermediate state volume, which was obtained by reconstructing an intermediate state from the symmetry expansion-based 3D classification. Homogeneous refinement with symmetric relaxation (marginalization method) using a 4 Å low-pass filter is employed to force proto-cluster classification based on high-resolution features. The resulting density is almost identical to the intermediate state obtained by reconstructing the closed symmetric extended particles from the 3D classification, except that the density of the intermediate neck state becomes enhanced, possibly due to the less-than-ideal classification of the unwinding helices during the 3D classification. Following homogeneous refinement with symmetric relaxation (C5), the particles undergo a final round of homogeneous refinement without applying symmetry (C1) and a 4 Å low-pass filter to maintain the original alignment from the symmetric relaxation refinement.

[0129] For the Best1+GABA dataset, 713 photomicrographs were collected in counting mode using a K3 direct electron detector at a magnification of 105,000x, corresponding to a total dose of 58 e. - / A 2 0.83 Å 2 The physical pixel size per pixel, graded across 50 frames, corresponds to 1.16 e. - / A 2The dose rate was set at / frame, with a defocus range of -0.8 to 1.5 micrometers. The film was aligned with MotionCorr2 via the Relion-3.1 GUI and imported into cryoSPARCv4 for further processing via PatchCTF correction, template selection, extraction, and initial 2D classification of 245,860 selected particles. 109,930 particles were selected for homogenization refinement with C5 symmetry (2.36 Å) and polished in Relion-3.1. The polished particles underwent homogenization refinement with C5 symmetry (2.14 Å) and then symmetric expansion to generate 549,650 particles, which underwent 3D classification at a target resolution of 3.2 Å using a mask covering the neck and GABA binding sites, into 6 categories. One category (Category 0) was in a fully open state, while the other 5 categories were in a closed state. Particles in the open state were stripped of duplicates (leaving 30,219 particles), then symmetrically expanded (151,395 particles), and locally refined using C1 symmetry with a global mask to obtain a final map of 2.41 Å. Particles in the closed state were stripped of duplicates, re-expanded, and subjected to another round of 3D classification using the same mask, resulting in eight categories (target resolution of 3.2 Å) to separate intermediate closed states. Three categories were in the intermediate 1 conformation (PCCCC, 32% of particles), three categories were in the intermediate 2 conformation (PCPCC, 31%), and one category was in the closed conformation (CCCCC, 10%). The last category, exhibiting poor characteristics due to excessive heterogeneity, was discarded as garbage. For each detected state, a single category was selected for local refinement utilizing C1 symmetry with a global mask. For intermediate 1 (PCCCC), 59,401 particles were refined to 2.42 Å. For intermediate 2 (PCPCC), one category was refined to 2.45 Å, and a single fully closed category containing 52,529 particles was refined to 2.50 Å.

[0130] For the Best2+GABA dataset, 1,130 K3 films were processed using MotionCorr2 via Relion-3.1 GUI, and dose-weighted micrographs were imported into cryoSPARCv4 for further processing via PatchCTF correction, template selection, extraction, and initial 2D classification, resulting in 161,366 final particles. These particles underwent homogenization refinement (C5) and 3D classification into 6 classes using a mask covering the neck and ligand binding sites, with a target resolution of 3.4 Å. 3D classification was performed without symmetry expansion. Four classes were in the fully open neck conformation, accounting for 65.4% (105,576 particles) of the total particles, while two classes were fully closed. The fully open particles were refined and polished in Relion. The final 103,839 particles were homogenized (C5) to 2.31 Å. Particles representing the closed state were similarly refined, polished in Relon, and the final 54,947 polished particles were uniformly refined to 2.27 Å. 3D classification was performed, and no heterogeneity within the neck was found in either the open or closed state.

[0131] For the Best2+PABA dataset, at 0.8215 Å 2 Data was collected at a nominal magnification of 105,000x, representing the physical pixel size per pixel. The energy filter width was 20 e. - On the K3 detector of V, 50.52 e - / Å 2 The total dose was divided into 40 frames, with a total exposure time of 1,200 ms (30 ms per frame). 1,051 K3 films were motion-corrected via Relion-3.1 GUI using MotionCorr2, and the dose-weighted micrographs were imported into cryoSPARCv4 for further processing through PatchCTF correction, template selection, extraction, and initial 2D classification, resulting in 47,438 final particles. These particles underwent homogenization refinement (C5), Bayesian polishing in Relion, symmetry expansion, and single-round focusing 3D classification (5 categories, target resolution 3.4 Å) using a mask covering the transmembrane neck and PABA binding sites. The two categories representing fully closed states were combined, symmetry expansion was removed (to remove duplicates), leaving 26,692 particles, which were then homogenized (C5) to 2.6 Å. The three categories representing fully open states were combined and symmetric expansion was removed (to eliminate repetition), leaving 33,154 particles, which were then uniformly refined (C5) to 2.4 Å. Despite extensive 3D classification, no intermediate states with partially open necks were identified. Each final state underwent another round of focused 3D classification to ensure conformational homogeneity.

[0132] Model refinement and validation The spectra used for model building and refinement were obtained by sharpening to a b-factor, which was determined by a Guinier plot as implemented in cryoSPARC. PDB 8D1I (for Best1) and PDB 8D1E and 8D1G (for Best2) rigid bodies were fitted to their respective cryo-EM spectra and refined iteratively multiple times using coot, phenix real space refinement, and REFMAC5 (Servalcat). 26-30 Validation was performed using the comprehensive cryo-EM validation tools in Phenix (including MolProbity). 31 .

[0133] Statistics and Reproducibility Based on the specific methods used in this experiment, a sufficient number of samples were examined to draw statistical conclusions. This involved examining two-tailed unpaired students. t The test determines the statistical significance of the difference between the means of the two groups. p <0.05). Data are presented as mean + / - SEM. Immunoblotting and pull-down assays were biologically replicated three times with similar results.

[0134] References

Claims

1. A small molecule activator capable of binding to one or more paralogs of cantharidin, wherein the one or more paralogs of cantharidin include Best1, wherein: The oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of Y68 of Best1 and the main chain nitrogen atoms of V275 and F276 of Best1. The other oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main chain nitrogen atom of T277 and the side chain hydroxyl group of T277; and The nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the hydroxyl group on the side chain of Best1's Y72.

2. The small molecule activator of claim 1, wherein Best1 is human Best1.

3. A small molecule activator capable of binding to one or more paralogs of cantharidin, wherein the one or more paralogs of cantharidin include Best2, wherein: The oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of Y68 of Best2 and the main chain nitrogen atoms of I275 and F276 of Best2. The other oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main chain nitrogen atom of T277 and the side chain hydroxyl group of T277; and The nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the hydroxyl group on the side chain of Best2's Y72.

4. The small molecule activator of claim 3, wherein Best2 is human Best2.

5. A small molecule activator capable of binding to one or more paralogs of cantharidin, wherein the one or more paralogs of cantharidin include Best3, wherein: The oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of Y68 of Best3 and the main chain nitrogen atoms of I275 and F276 of Best3. The other oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main chain nitrogen atom of T277 and the side chain hydroxyl group of T277; and The nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the hydroxyl group on the side chain of Best3's Y72.

6. The small molecule activator of claim 5, wherein Best3 is human Best3.

7. A small molecule activator capable of binding to one or more paralogs of cantharidin, wherein the one or more paralogs of cantharidin comprise Best4, wherein: The oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of Y68 of Best4 and the main chain nitrogen atoms of L290 and T291 of Best4. The other oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main chain nitrogen atom of T277 and the side chain hydroxyl group of T277; and The nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the hydroxyl group on the side chain of Best4's S72.

8. The small molecule activator of claim 7, wherein Best4 is human Best4.

9. A small molecule activator capable of binding one or more paralogs of cantharidin, wherein the small molecule activator activates the one or more paralogs of cantharidin by contacting a pocket formed at the N-terminal helical dipole of the helix S4a of the one or more paralogs of cantharidin.

10. The small molecule activator of claim 9, wherein one or more paralogs of the tartaric protein include Best1, Best2, Best3 or Best4.

11. The small molecule activator of claim 10, wherein Best1, Best2, Best3 or Best4 is human Best1, human Best2, human Best3 or human Best4.

12. The small molecule activator according to any one of claims 1-11, wherein the small molecule activator is an amino acid.

13. The small molecule activator of claim 12, wherein the amino acid is a non-proteinogenic amino acid.

14. The small molecule activator according to any one of claims 1-13, wherein the small molecule activator is GABA or a GABA analogue.

15. The small molecule activator of claim 14, wherein the small molecule activator is GABA.

16. The small molecule activator of claim 14, wherein the small molecule activator is a GABA analog.

17. The small molecule activator of claim 16, wherein the GABA analogue is desmethylarecanine, pregabalin, gabapentin, ennacarpi, acampolic acid, muscarinic acid, aminohexenoic acid, gabapentin, or aminooxyacetic acid.

18. The small molecule activator according to any one of claims 1-13, wherein the small molecule activator is PABA or a PABA analogue.

19. The small molecule activator of claim 18, wherein the small molecule activator is PABA.

20. The small molecule activator of claim 18, wherein the small molecule activator is a PABA analog.

21. The small molecule activator of claim 20, wherein the PABA analogue is 2-amino-5-methoxybenzoic acid, 3,4-diaminobenzenesulfonic acid, 3-amino-5-methoxybenzoic acid, 4-amino-2-fluorobenzoic acid, 4-hydroxybenzoic acid, 4-nitrobenzoic acid, 4-amino-2-(hydroxymethyl)benzoic acid, 4-isobutoxybenzoic acid, 4-(aminomethyl)benzoic acid, 2-amino-5-pyrimidinecarboxylic acid, 2-(dimethylamino)-5-pyrimidinecarboxylic acid, 4-[(2-hydroxyethyl)amino]benzoic acid, 4-[(2-amino-2-oxoethyl)amino]amino [Bento] benzoic acid, 4-[(2-aminoethyl)amino]-3-nitrobenzoic acid, 3,4-diaminobenzoic acid, 4-amino-3-hydroxybenzoic acid, 4-amino-N-(phenylmethyl)benzamide, 4-amino-N-(2-amino-2-oxoethyl)benzamide, 4-amino-N-(2-hydroxyethyl)benzamide, 4-amino-N-(2-methylpropyl)benzamide, lisocaine, 4-amino-N-propylbenzamide, 4-amino-1-naphthylcarboxylic acid, 6-amino-4-quinolinecarboxylic acid, 8-amino-4-quinolinecarboxylic acid, or 1H-indole-5-carboxylic acid.

22. The small molecule activator according to any one of claims 1-13, wherein the small molecule activator is not GABA, a GABA analog, PABA, or a PABA analog.

23. The small molecule activator of claim 22, wherein the small molecule activator is pantothenic acid or 5-hydroxyindole-2-carboxylic acid.

24. The small molecule activator of any one of claims 1-23, wherein the small molecule activator activates the one or more paracellular homologs of the ...

25. The small molecule activator of any one of claims 1-24, wherein the small molecule activator promotes neck opening of the one or more paralogs of the phytohemagglutinin protein.

26. The small molecule activator of claim 25, wherein the neck opening causes the side chain of the gate-forming residue to be away from the channel pore of the one or more paralogs of the canthalin.

27. The small molecule activator of claim 26, wherein the gate-forming residues are I76, F80, and F84 of one or more paralogs of the cantharidin.

28. A method for activating one or more paralogs of tartaric acid, the method comprising contacting the paralogs of tartaric acid with a composition comprising a small molecule activator of one or more paralogs of tartaric acid.

29. The method of claim 28, wherein the small molecule activator is an amino acid.

30. The method of claim 29, wherein the amino acid is a non-proteinogenic amino acid.

31. The method of any one of claims 28-30, wherein the small molecule activator is GABA or a GABA analogue.

32. The method of claim 31, wherein the small molecule activator is GABA.

33. The method of claim 32, wherein the small molecule activator is a GABA analog.

34. The method of claim 33, wherein the GABA analogue is desmethylarecanine, pregabalin, gabapentin, ennacarbi, acanolic acid, muscarinic acid, aminohexenoic acid, gabapentin, or aminooxyacetic acid.

35. The method of any one of claims 28-30, wherein the small molecule activator is PABA or a PABA analogue.

36. The method of claim 35, wherein the small molecule activator is PABA.

37. The method of claim 35, wherein the small molecule activator is a PABA analog.

38. The method of claim 37, wherein the PABA analog is 2-amino-5-methoxybenzoic acid, 3,4-diaminobenzenesulfonic acid, 3-amino-5-methoxybenzoic acid, 4-amino-2-fluorobenzoic acid, 4-hydroxybenzoic acid, 4-nitrobenzoic acid, 4-amino-2-(hydroxymethyl)benzoic acid, 4-isobutoxybenzoic acid, 4-(aminomethyl)benzoic acid, 2-amino-5-pyrimidinecarboxylic acid, 2-(dimethylamino)-5-pyrimidinecarboxylic acid, 4-[(2-hydroxyethyl)amino]benzoic acid, 4-[(2-amino-2-oxoethyl)amino]amino] Benzoic acid, 4-[(2-aminoethyl)amino]-3-nitrobenzoic acid, 3,4-diaminobenzoic acid, 4-amino-3-hydroxybenzoic acid, 4-amino-N-(phenylmethyl)benzamide, 4-amino-N-(2-amino-2-oxoethyl)benzamide, 4-amino-N-(2-hydroxyethyl)benzamide, 4-amino-N-(2-methylpropyl)benzamide, lisocaine, 4-amino-N-propylbenzamide, 4-amino-1-naphthylcarboxylic acid, 6-amino-4-quinolinecarboxylic acid, 8-amino-4-quinolinecarboxylic acid, or 1H-indole-5-carboxylic acid.

39. The method of any one of claims 28-30, wherein the small molecule activator is not GABA, a GABA analog, PABA, or a PABA analog.

40. The method of claim 39, wherein the small molecule activator is pantothenic acid or 5-hydroxyindole-2-carboxylic acid.

41. The method of any one of claims 29-40, wherein the small molecule activator activates the one or more paracellular homologs of the ...

42. The method of any one of claims 29-41, wherein the small molecule activator activates the one or more paralogs of the paralog by contacting a pocket formed at the N-terminal helical dipole of the helix S4a of the one or more paralogs of the paralog.

43. The method of any one of claims 29-42, wherein the small molecule activator promotes neck opening of the one or more paralogs of the phytoretin.

44. The method of claim 43, wherein the neck opening causes the side chain of the gate-forming residue to be away from the channel pore of the one or more paralogs of the phytokeratin.

45. The method of claim 44, wherein the gate-forming residues are I76, F80, and F84 of one or more paralogs of the tartaric protein.

46. ​​The method of any one of claims 29-45, wherein the one or more paralogs of cantharidin comprise Best1, wherein: The oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of Y68 of Best1 and the main chain nitrogen atoms of V275 and F276 of Best1. The other oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main chain nitrogen atom of T277 and the side chain hydroxyl group of T277; and The nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the hydroxyl group on the side chain of Best1's Y72.

47. The method of claim 46, wherein Best1 is a person Best1.

48. The method of any one of claims 29-47, wherein the one or more paralogs of cantharidin comprise Best2, wherein: The oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of Y68 of Best2 and the main chain nitrogen atoms of I275 and F276 of Best2. The other oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main chain nitrogen atom of T277 and the side chain hydroxyl group of T277; and The nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the hydroxyl group on the side chain of Best2's Y72.

49. The method of claim 48, wherein Best2 is person Best2.

50. The method of any one of claims 29-49, wherein the one or more paralogs of cantharidin comprise Best3, wherein: The oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of Y68 of Best3 and the main chain nitrogen atoms of I275 and F276 of Best3. The other oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main chain nitrogen atom of T277 and the side chain hydroxyl group of T277; and The nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the hydroxyl group on the side chain of Best3's Y72.

51. The method of claim 50, wherein Best3 is a person Best3.

52. The method of any one of claims 29-51, wherein the one or more paralogs of cantharidin comprise Best4, wherein: The oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of Y68 of Best4 and the main chain nitrogen atoms of L290 and T291 of Best4. The other oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main chain nitrogen atom of T277 and the side chain hydroxyl group of T277; and The nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the hydroxyl group on the side chain of Best4's S72.

53. The method of claim 52, wherein Best4 is a person Best4.

54. A method for treating or preventing a disease or condition in a subject in need, the method comprising administering a pharmaceutical composition to the subject, wherein the composition comprises a small molecule activator of one or more paralogs of pachyphytoprotein.

55. The method of claim 54, wherein the subject is a human being.

56. The method of any one of claims 54-55, wherein the disease or symptom is caused by an imbalance of one or more phytoalveolar proteins.

57. The method of any one of claims 54-56, wherein the disease or condition is a disease related to the phytohemagglutinin protein (Best).

58. The method of claim 57, wherein the plaque atrophy-associated disease is selected from... BEST1 , BEST2 , BEST3 and BEST4 It is caused by one or more mutations in the gene.

59. The method of any one of claims 57-58, wherein the macular degeneration-related disease is yolk-like macular degeneration.

60. The method of claim 59, wherein the vitrectomyctic lesion is Best vitrectomyctic dystrophy (BVMD), adult-onset vitrectomyctic dystrophy (AVMD), autosomal recessive vitrectomyctic dystrophy (ARB), autosomal dominant vitreoretinal choroidal dysplasia (ADVIRC), autosomal dominant microkeratosis, cone-rod dystrophy, early-onset cataract, posterior staphyloma syndrome (MRCS syndrome), age-related macular degeneration (AMD), retinitis pigmentosa (RP), or a combination thereof.

61. The method of claim 57, wherein the plaque atrophy-related disease is associated with an increase or decrease in the intraocular pressure (IOP) of the subject.

62. The method of claim 61, wherein the subject's IOP is greater than 21 mmHg.

63. The method of claim 62, wherein the macular degeneration-related disease is glaucoma, myopia, dispersive pigment syndrome, pseudoexfoliation syndrome, age-related macular degeneration, or a combination thereof.

64. The method of any one of claims 54-55, wherein the disease or condition is Alzheimer's disease.

65. The method of any one of claims 54-64, wherein the small molecule activator is an amino acid.

66. The method of claim 65, wherein the amino acid is a non-proteinogenic amino acid.

67. The method of any one of claims 54-66, wherein the small molecule activator is GABA or a GABA analogue.

68. The method of claim 67, wherein the small molecule activator is GABA.

69. The method of claim 67, wherein the small molecule activator is a GABA analog.

70. The method of claim 69, wherein the GABA analogue is desmethylarecanine, pregabalin, gabapentin, ennacarbi, acanolic acid, muscarinic acid, aminohexenoic acid, gabapentin, or aminooxyacetic acid.

71. The method of any one of claims 54-66, wherein the small molecule activator is PABA or a PABA analogue.

72. The method of claim 71, wherein the small molecule activator is PABA.

73. The method of claim 71, wherein the small molecule activator is a PABA analog.

74. The method of claim 73, wherein the PABA analog is 2-amino-5-methoxybenzoic acid, 3,4-diaminobenzenesulfonic acid, 3-amino-5-methoxybenzoic acid, 4-amino-2-fluorobenzoic acid, 4-hydroxybenzoic acid, 4-nitrobenzoic acid, 4-amino-2-(hydroxymethyl)benzoic acid, 4-isobutoxybenzoic acid, 4-(aminomethyl)benzoic acid, 2-amino-5-pyrimidinecarboxylic acid, 2-(dimethylamino)-5-pyrimidinecarboxylic acid, 4-[(2-hydroxyethyl)amino]benzoic acid, 4-[(2-amino-2-oxoethyl)amino]amino] Benzoic acid, 4-[(2-aminoethyl)amino]-3-nitrobenzoic acid, 3,4-diaminobenzoic acid, 4-amino-3-hydroxybenzoic acid, 4-amino-N-(phenylmethyl)benzamide, 4-amino-N-(2-amino-2-oxoethyl)benzamide, 4-amino-N-(2-hydroxyethyl)benzamide, 4-amino-N-(2-methylpropyl)benzamide, lisocaine, 4-amino-N-propylbenzamide, 4-amino-1-naphthylcarboxylic acid, 6-amino-4-quinolinecarboxylic acid, 8-amino-4-quinolinecarboxylic acid, or 1H-indole-5-carboxylic acid.

75. The method of any one of claims 54-66, wherein the small molecule activator is not GABA, a GABA analog, PABA, or a PABA analog.

76. The method of claim 75, wherein the small molecule activator is pantothenic acid or 5-hydroxyindole-2-carboxylic acid.

77. The method of any one of claims 54-76, wherein the small molecule activator activates the one or more paracellular homologs of the ...

78. The method of any one of claims 54-77, wherein the small molecule activator activates the one or more paralogs of the platypophyte protein by contacting a pocket formed at the N-terminal helical dipole of the helix S4a of the one or more paralogs of the platypophyte protein.

79. The method of any one of claims 54-78, wherein the small molecule activator binds to the extracellular side of one or more paralogs of the phytohemagglutinin protein.

80. The method of any one of claims 54-79, wherein the small molecule activator promotes neck opening of the one or more paralogs of the phytoretin.

81. The method of claim 80, wherein the neck opening causes the side chain of the gate-forming residue to be away from the channel pore of the one or more paralogs of the tarsalin.

82. The method of claim 81, wherein the gate-forming residues are I76, F80, and F84 of one or more paralogs of the tartaric protein.

83. The method of any one of claims 54-82, wherein the one or more paralogs of cantharidin comprise Best1, wherein: The oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of Y68 of Best1 and the main chain nitrogen atoms of V275 and F276 of Best1. The other oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main chain nitrogen atom of T277 and the side chain hydroxyl group of T277; and The nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the hydroxyl group on the side chain of Best1's Y72.

84. The method of claim 83, wherein Best1 is person Best1.

85. The method of any one of claims 54-84, wherein the one or more paralogs of cantharidin comprise Best2, wherein: The oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of Y68 of Best2 and the main chain nitrogen atoms of I275 and F276 of Best2. The other oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main chain nitrogen atom of T277 and the side chain hydroxyl group of T277; and The nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the hydroxyl group on the side chain of Best2's Y72.

86. The method of claim 85, wherein Best2 is person Best2.

87. The method of any one of claims 54-86, wherein the one or more paralogs of cantharidin comprise Best3, wherein: The oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of Y68 of Best3 and the main chain nitrogen atoms of I275 and F276 of Best3. The other oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main chain nitrogen atom of T277 and the side chain hydroxyl group of T277; and The nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the hydroxyl group on the side chain of Best3's Y72.

88. The method of claim 87, wherein Best3 is person Best3.

89. The method of any one of claims 54-88, wherein the one or more paralogs of cantharidin comprise Best4, wherein: The oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of Y68 of Best4 and the main chain nitrogen atoms of L290 and T291 of Best4. The other oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main chain nitrogen atom of T277 and the side chain hydroxyl group of T277; and The nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the hydroxyl group on the side chain of Best4's S72.

90. The method of any one of claims 54-89, wherein the subject suffers from a disease caused by an imbalance of one or more phytoalveolar proteins.

91. The method of any one of claims 54-90, wherein the subject has the Best1 mutation.

92. The method of claim 91, wherein the Best1 mutation is hereditary.

93. The method of any one of claims 91-92, wherein the Best1 mutation is a loss-of-function (LOF) mutation.

94. The method of any one of claims 91-93, wherein the composition is applied to salvage the Best1 function.

95. The method of any one of claims 54-94, wherein the pharmaceutical composition is administered via an ophthalmic delivery route, an oral delivery route, or a parenteral delivery route.

96. The method of claim 95, wherein the pharmaceutical composition is administered via an ophthalmic delivery route, wherein the pharmaceutical composition is administered topically.

97. The method of claim 96, wherein the pharmaceutical composition is administered via eye drops.

98. A method for maintaining intraocular pressure (IOP) in a subject in need, the method comprising administering a pharmaceutical composition, wherein the composition increases Best2-mediated Ca2+ in non-pigmented epithelial (NPE) cells. 2+ Dependence Cl - Electric current.

99. The method of claim 98, wherein the subject is a human being.

100. The method of any one of claims 98-99, wherein the disease is caused by an imbalance of one or more phytoalveolar proteins.

101. The method of any one of claims 98-99, wherein the subject suffers from atrophic protein-related disease.

102. The method of claim 101, wherein the plaque atrophy-related disease is associated with an increase or decrease in the intraocular pressure (IOP) of the subject.

103. The method of any one of claims 98-102, wherein the subject has high or low intraocular pressure.

104. The method of any one of claims 98-103, wherein the subject's IOP is greater than 21 mmHg.

105. The method of claim 101, wherein the macular degeneration-related disease is glaucoma, myopia, dispersive pigment syndrome, pseudoexfoliation syndrome, age-related macular degeneration, or a combination thereof.

106. The method of any one of claims 98-105, wherein the composition comprises a small molecule activator of Best2.

107. The method of any one of claims 98-106, wherein the small molecule activator is an amino acid.

108. The method of claim 107, wherein the amino acid is a non-proteinogenic amino acid.

109. The method of any one of claims 98-108, wherein the small molecule activator is GABA or a GABA analogue.

110. The method of claim 109, wherein the small molecule activator is GABA.

111. The method of claim 109, wherein the small molecule activator is a GABA analog.

112. The method of claim 111, wherein the GABA analogue is desmethylarecanine, pregabalin, gabapentin, ennacarbi, acanolic acid, muscarinic acid, aminohexenoic acid, gabapentin, or aminooxyacetic acid.

113. The method of any one of claims 98-108, wherein the small molecule activator is PABA or a PABA analogue.

114. The method of claim 113, wherein the small molecule activator is PABA.

115. The method of claim 113, wherein the small molecule activator is a PABA analog.

116. The method of claim 115, wherein the PABA analog is 2-amino-5-methoxybenzoic acid, 3,4-diaminobenzenesulfonic acid, 3-amino-5-methoxybenzoic acid, 4-amino-2-fluorobenzoic acid, 4-hydroxybenzoic acid, 4-nitrobenzoic acid, 4-amino-2-(hydroxymethyl)benzoic acid, 4-isobutoxybenzoic acid, 4-(aminomethyl)benzoic acid, 2-amino-5-pyrimidinecarboxylic acid, 2-(dimethylamino)-5-pyrimidinecarboxylic acid, 4-[(2-hydroxyethyl)amino]benzoic acid, 4-[(2-amino-2-oxoethyl)amino]amino Benzoic acid, 4-[(2-aminoethyl)amino]-3-nitrobenzoic acid, 3,4-diaminobenzoic acid, 4-amino-3-hydroxybenzoic acid, 4-amino-N-(phenylmethyl)benzamide, 4-amino-N-(2-amino-2-oxoethyl)benzamide, 4-amino-N-(2-hydroxyethyl)benzamide, 4-amino-N-(2-methylpropyl)benzamide, lisocaine, 4-amino-N-propylbenzamide, 4-amino-1-naphthylcarboxylic acid, 6-amino-4-quinolinecarboxylic acid, 8-amino-4-quinolinecarboxylic acid, or 1H-indole-5-carboxylic acid.

117. The method of any one of claims 98-108, wherein the small molecule activator is not GABA, a GABA analog, PABA, or a PABA analog.

118. The method of claim 117, wherein the small molecule activator is pantothenic acid or 5-hydroxyindole-2-carboxylic acid.

119. The method of any one of claims 98-118, wherein the small molecule activator activates Best2 by binding to the extracellular surface of Best2.

120. The method of any one of claims 98-119, wherein the small molecule activator activates Best2 by contacting a pocket formed at the N-terminal helical dipole of the helix S4a of Best2.

121. The method of any one of claims 98-120, wherein the small molecule activator promotes neck opening of Best2.

122. The method of claim 121, wherein the neck opening causes the side chain of the gate-forming residue to be away from the channel pore of Best2.

123. The method of claim 122, wherein the gate-forming residues are I76, F80, and F84 of Best2.

124. The method according to any one of claims 98-123, wherein: The oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of Y68 of Best2 and the main chain nitrogen atoms of I275 and F276 of Best2. The other oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main chain nitrogen atom of T277 and the side chain hydroxyl group of T277; and The nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the hydroxyl group on the side chain of Best2's Y72.

125. The method of any one of claims 91-92, wherein the Best1 mutation is A243T, A10T, R218H, L234P, Q293K or D302A.

126. The method of claim 125, wherein the composition is applied to salvage the Best1 function.

127. The method of any one of claims 98-126, wherein the pharmaceutical composition is administered via an ophthalmic delivery route, an oral delivery route, or a parenteral delivery route.

128. The method of claim 127, wherein the pharmaceutical composition is administered via an ophthalmic delivery route, wherein the pharmaceutical composition is administered topically.

129. The method of claim 128, wherein the pharmaceutical composition is administered via eye drops.

130. The small molecule activator according to any one of claims 1-27, wherein the small molecule activator comprises a 5-membered ring or a 6-membered ring.

131. The small molecule activator of claim 130, wherein the small molecule activator comprises a 5-membered ring.

132. The small molecule activator of claim 130, wherein the small molecule activator comprises a 6-membered ring.

133. The method of any one of claims 28-97, wherein the small molecule activator comprises a 5-membered ring or a 6-membered ring.

134. The method of claim 133, wherein the small molecule activator comprises a 5-membered ring.

135. The method of claim 133, wherein the small molecule activator comprises a 6-membered ring.

136. The method of any one of claims 98-129, wherein the small molecule activator comprises a 5-membered ring or a 6-membered ring.

137. The method of claim 136, wherein the small molecule activator comprises a 5-membered ring.

138. The method of claim 136, wherein the small molecule activator comprises a 6-membered ring.

139. The small molecule activator according to any one of claims 14-17, wherein the paralog of the cantharidin comprises Best1, wherein: The α-carbon (C2) of the small molecule activator forms a van der Waals contact with the β-carbon of P274 of Best1. The β carbon of the small molecule activator forms a van der Waals contact with the C3 carbon of the ring of the Y72 side chain of Best1, and The nitrogen atom of the small molecule activator is adjacent to the hydroxyl group of the side chain of Best1's Y72.

140. The small molecule activator according to any one of claims 18-21, wherein the paralog of the cantharidin comprises Best1, wherein: The amino group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of H267 of Best1; The aromatic ring of the small molecule activator forms a π-stacking interaction with the side chain of Best1's F257; and The aromatic ring of the small molecule activator forms a side-to-side interaction with the Y72 side chain of Best1.

141. The small molecule activator according to any one of claims 18-21, wherein the paralog of the cantharidin comprises Best2, wherein: The amino group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of H267 of Best2; The aromatic ring of the small molecule activator forms a π-stacking interaction with the side chain of Best2's F257; and The aromatic ring of the small molecule activator forms a side-to-side interaction with the Y72 side chain of Best2.

142. The method of any one of claims 31-34, 67-70, and 109-112, wherein the one or more paralogs of cantharidin comprise Best1, wherein: The oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of Y68 of Best1 and the main chain nitrogen atoms of V275 and F276 of Best1. Another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main chain nitrogen atom of T277 and the side chain hydroxyl group of T277; The nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side chain hydroxyl group of Best1's Y72; the α carbon (C2) of the small molecule activator forms a van der Waals contact with the β carbon of Best1's P274. The β-carbon of the small molecule activator forms a van der Waals contact with the C3 carbon of the ring of the Y72 side chain of Best1; and The nitrogen atom of the small molecule activator is adjacent to the hydroxyl group of the side chain of Best1's Y72.

143. The method of any one of claims 35-38, 71-74, and 113-116, wherein the one or more paralogs of cantharidin comprise Best1, wherein: The oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of Y68 of Best1 and the main chain nitrogen atoms of V275 and F276 of Best1. Another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main chain nitrogen atom of Best1's T277 and the side chain hydroxyl group of Best1's T277. The nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side chain hydroxyl group of Best1's Y72; The amino group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of H267 of Best1; The aromatic ring of the small molecule activator forms a π-stacking interaction with the side chain of Best1's F257; and The aromatic ring of the small molecule activator forms a side-to-side interaction with the Y72 side chain of Best1.

144. The method of any one of claims 35-38, 71-74, and 113-116, wherein the one or more paralogs of cantharidin comprise Best2, wherein: The oxygen atom of the carboxyl group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of Y68 of Best2 and the main chain nitrogen atoms of I275 and F276 of Best2. Another oxygen atom of the same carboxyl group of the small molecule activator forms a hydrogen bond with the main chain nitrogen atom of Best2's T277 and the side chain hydroxyl group of T277; The nitrogen atom of the amino group of the small molecule activator forms a hydrogen bond with the oxygen atom of the side chain hydroxyl group of Best2's Y72; The amino group of the small molecule activator forms a hydrogen bond with the side chain hydroxyl group of H267 of Best2; The aromatic ring of the small molecule activator forms a π-stacking interaction with the side chain of Best2's F257; and The aromatic ring of the small molecule activator forms a side-to-side interaction with the Y72 side chain of Best2.

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