Methods for treating bestrophin-related diseases

Abstract

The present disclosure provides, inter alia, methods for treating, ameliorating, or preventing the effects of bestrophin-related diseases, e.g, a bestrophinopathy or glaucoma in a subject in need thereof using e.g., one or more bestrophin activators. Also provided are the identification of bestrophin activators including glutamate analogs.

Description

Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 METHODS FOR TREATING BESTROPHIN-RELATED DISEASES

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 695,642, filed on September 17, 2024, which is incorporated herein by reference in its entirety.

[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosure of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described herein. FIELD OF DISCLOSURE

[0003] The present disclosure provides, inter alia, methods for treating, ameliorating, or preventing the effects of bestrophin-related diseases, e.g, a bestrophinopathy or glaucoma in a subject in need thereof using e.g., one or more bestrophin activators. GOVERNMENT SUPPORT

[0004] This invention was made with government support under TR001873, GM149252, and GM127652 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0005] The bestrophins are a family of Ca2+-activated anion channels consisting of four members in mammals1. They are widely distributed in various human organs including the airways, colon, kidney, pancreas and central nervous system, but best known for their physiological roles in the eye1. In particular, Best1 is predominantly expressed in retinal pigment epithelium (RPE) and genetically linked to a spectrum of retinal degenerative disorders collectively known as bestrophinopathies2,3. Over 350 different Best1 mutations, most of which result in loss-of-function (LOF), have been identified to cause bestrophinopathies1,2. The patients are susceptible to progressive vision loss that may eventually lead to blindness, and there is no treatment4. On the other hand, Best2 resides in ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 non-pigmented epithelium (NPE) regulating intra-ocular pressure (IOP), which must be properly maintained at all times as both hypertension and hypotension are deleterious conditions5-7. Therefore, bestrophins are potential drug targets, especially for the health of the eye.

[0006] However, this request is prohibited by the poor knowledge of small molecule activators or inhibitors specifically for the bestrophin channels. Ca2+was first identified as an essential activator of bestrophins, leading to the classification of this protein family as Ca2+- activated anion channels8,9. Later on, ATP was found to be required for the activation of a bacterial bestrophin homolog which lacks the Ca2+-binding site and for the full activation of mammalian bestrophins in the presence of Ca2+10. As Ca2+and ATP are both common physiological molecules involved in numerous biochemical activities, neither of them or their analogs is suited for pharmaceutically targeting bestrophins.

[0007] Besides Ca2+and ATP, it was recently reported that intracellular glutamate is permeable for Best2, strongly promotes Best2 channel function and relieves the inhibitory effect of glutamine synthetase (GS), which binds to Best2 on the intracellular side11. However, extracellular glutamate is barely permeable for Best2 and does not have these promotive effects11, suggesting the binding of glutamate to Best2 on the intracellular side.

[0008] Previous studies have revealed several key structural components on the pentameric assembly of bestrophins for controlling the channel function. These include: 1) two major permeation constrictions in the ion conducting pathway, namely the “neck” (I76 / F80 / F84 in Best1 and Best2) at the transmembrane pore and the “aperture” (I205 / Q208 / N212 in Best1 and S205 / K208 / E212 in Best2) at the cytosolic exit16-20; 2) a C- terminal autoinhibitory segment (AS, residues 346-378 / 379 in Best1 / Best2), which constricts the channel concentrically by wrapping around the channel periphery in an inter-protomer manner, and unleashes in concomitant with neck opening20-22; 3) a Ca2+clasp formed by an acidic cluster between S4a and S4b (E300 / D301 / D302 / D303 / D304 in Best1 / Best2) from one protomer and the N-terminal S1a-S1b helix-turn-helix element of an adjacent protomer16,20. One would expect that small molecule activators, 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 activator -bound bestrophin co-structures. 2 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 SUMMARY

[0009] Bestrophin-1 (Best1) and bestrophin-2 (Best2) are two members of the bestrophin family of anion channels critically involved in the prevention of retinal degeneration and maintenance of intraocular pressure, respectively. The present disclosure provides the structures of glutamate-bound Best2 by cryogenic electron microscopy, delineates a highly conserved intracellular glutamate binding site which could serve as a docking site, and identifies multiple glutamate analogs as Best2 activators. These findings provide the mechanism and lead compounds for the development of small molecule drugs to treat bestrophin-associated diseases / conditions.

[0010] Accordingly, in certain aspects, described herein is a method for treating, ameliorating, or preventing the effects of a disease in a subject in need thereof. This method comprises administering an effective amount of a Bestrophin (Best) activator to the subject. In some embodiments, the disease is a Bestrophin (Best)-related disease. In some embodiments, the Bestrophin-related disease is caused by one or more mutations on a gene selected from BEST1, BEST2, BEST3, and BEST4. In some embodiments, the Bestrophin-related disease is caused by one or more loss-of-function mutations on BEST1. In some embodiments, the Bestrophin-related disease is caused by one or more loss-of-function mutations on BEST2.

[0011] In some embodiments, the Bestrophin-related disease is a bestrophinopathy. In some embodiments, the bestrophinopathy is best vitelliform macular dystrophy (BVMD), autosomal recessive bestrophinopathy (ARB), adult-onset vitelliform dystrophy (AVMD), autosomal dominant vitreoretinoehoroidopathy (ADVIRC), autosomal dominant microcornea, rod-cone dystrophy, early-onset cataract, posterior staphyloma syndrome (MRCS syndrome), retinitis pigmentosa (RP), age-related macular degeneration (AMD), or combinations thereof. In some embodiments, the Bestrophin-related disease is associated with increased or decreased intraocular pressure (IOP) in the subject. In some embodiments, the subject has ocular hypertension or hypotension. In some embodiments, the IOP in the subject is above 21 mmHg. In some embodiments, the Bestrophin-related disease is glaucoma, myopia, pigment dispersion syndrome, pseudoexfoliation syndrome, age-related macular degeneration, or combinations thereof. In some embodiments, the Bestrophin-related disease is glaucoma.

[0012] In some embodiments, the disease is Alzheimer’s disease.

[0013] In some embodiments, the Best activator is a small molecule. In some embodiments, the Best activator is an activator for Best1. In some embodiments, the Best 3 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 activator is an activator for Best2. In some embodiments, the Best activator is Glutamate, 2- (1-amino-2,2,2-trifluoroethyl)pentanedioic acid (L:D = 1:1), Glutamine (L+D), γ- Carboxyglutamic acid, α-Aminoadipic acid, 1-Methyl L-glutamate, 1-Ethyl L-glutamate, (-)- Glutamic acid, 3-Aminopentanedioic acid, 4-Carboxybenzeneacetic acid, 2-Aminoterephthalic acid, Hydroxyterephthalic acid, 1,4-Cyclohexanedicarboxylic acid, 3-Phenylglutaric acid, L- Glutamine, D-Glutamine and combination thereof. In some embodiments, the Best activator is administered in a composition that comprises a pharmaceutically acceptable carrier and excipient. In some embodiments, the subject is a mammal. In some embodiments, the mammal is humans, veterinary animals, and agricultural animals. In some embodiments, the subject is a human.

[0014] In some embodiments, the pharmaceutical composition is administered via an ophthalmic route of delivery, an oral route of delivery, or a parenteral route of delivery. In some embodiments, the pharmaceutical composition is administered via an ophthalmic route of delivery, wherein the pharmaceutical composition is administered topically. In some embodiments, the pharmaceutical composition is administered via eye drops.

[0015] In certain aspects, described herein is a method for treating, ameliorating, or preventing the effects of a disease in a subject in need thereof, comprising administering an effective amount of a Best activator to the subject, wherein the Best activator is a compound of Formula I: or a pharmaceutically acceptableor solvate thereof; wherein: X is C(R1)3, OR2, N(R2)2, SR2, C6-C10aryl, or C5-C10heteroaryl; each occurrence of R1is independently H, halogen, N(Ra)2, ORa, SRa, -CORb, - COORa, -OCORa, -CON(Ra)2, -NRaCORb, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1- C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl; each occurrence of R2is independently H, -CORb, -CON(Ra)2, C1-C6alkyl, C1- C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6- C10aryl, or C5-C10heteroaryl; L is –(C(R3)2)n–, –(C(R3)2)nNR4–, –NR4(C(R3)2)nNR4–, –O(C(R3)2)nNR4–, – NR4(C(R3)2)nO–, –O(C(R3)2)nO–, –(C(R3)2)nO–, –(C(R3)2)nS–, C3-C7cycloalkyl, C3- C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl; n is an integer from 1 to 5; 4 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 each occurrence of R3is independently H, halogen, N(Ra)2, C(Ra)3, C(Ra)2N(Ra)2, C(Ra)2ORa, ORa, SRa, -CORb, -COORa, -OCORa, -CON(Ra)2, -NRaCORb, C1-C6alkyl, C1- C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6- C10aryl, or C5-C10heteroaryl; each occurrence of R4is independently H, -CORb, -CON(Ra)2, C1-C6alkyl, C1- C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6- C10aryl, or C5-C10heteroaryl; Z is H, halogen, -N(Rb)2, -ORb, -SRb, -SO2N(Rb)2, -SON(Rb)2, C(Rb)3, -CORb, - COORa, -OCORa, -CON(Ra)2, -NRaCORb, -C1-C6alkylCOORaC1-C6alkyl, C1-C6alkenyl, C1- C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6-C10aryl, or C5- C10heteroaryl; wherein when L is –O(C(R3)2)nNR4– or –O(C(R3)2)nO–, Z is not -ORb; each occurrence of Rais independently H, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl; each occurrence of Rbis independently H, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl; and the C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3- C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl in X, L, Z, R1, R2, R3, R4, Ra, and Rb, where applicable, are each optionally independently substituted by 1-4 substituents each independently selected from the group consisting of halogen, -OH, -OC1-C6alkyl, -NH2, C1- C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1-C6OH, C1-C6NH2, C3-C7cycloalkyl, oxo, -CN, - COOH, -CONH2, heterocyclyl, aryl, or heteroaryl.

[0016] In some embodiments, X is C(R1)3, OR2, or N(R2)2.

[0017] In some embodiments, X is CH2NH2, OH, or NH2.

[0018] In some embodiments, each occurrence of R1is independently H, halogen, N(Ra)2, ORa, or SRa.

[0019] In some embodiments, each occurrence of R1is independently H or N(Ra)2.

[0020] In some embodiments, each occurrence of R2is independently H, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C6-C10aryl, or C5-C10heteroaryl.

[0021] In some embodiments, each occurrence of R2is independently H, CH2, or CH3.

[0022] In some embodiments, L is –(C(R3)2)n–, –(C(R3)2)nNR4–, C3-C7cycloalkyl, or C6-C10aryl.

[0023] In some embodiments, n is an integer from 1 to 4. 5 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025

[0024] In some embodiments, n is 3 or 4.

[0025] In some embodiments, n is 1.

[0026] In some embodiments, each occurrence of R3is independently H, halogen, N(Ra)2, ORa, -COORa, C1-6alkyl, C1-6haloalkyl, or C6-C10aryl.

[0027] In some embodiments, each occurrence of R3is independently H, NH2, NHCH3, NHCH2CH3, COOH, CH(NH2)CF3, or phenyl.

[0028] In some embodiments, each occurrence of R4is independently H, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, or C6-C10aryl.

[0029] In some embodiments, each occurrence of R4is independently H.

[0030] In some embodiments, Z is H, halogen, -COORa, -CON(Ra)2, -C1- C6alkylCOORa, C1-C6alkyl, C1-C6haloalkyl, C1-C6heteroalkyl, or C6-C10aryl.

[0031] In some embodiments, Z is H, CH3, CF3, -COOH, CONH2, or CH2COOH.

[0032] In some embodiments, each occurrence of Rais independently H, C1-C6alkyl, C1-C6haloalkyl, or C6-C10aryl.

[0033] In some embodiments, each occurrence of Rais independently H, CH3, or CH2CH3.

[0034] In some embodiments, each occurrence of Rbis independently H or C1-C6alkyl.

[0035] In some embodiments, the Best activator is a compound of Formula I or a pharmaceutically acceptable enantiomer, salt, or solvate thereof, and wherein the compound of , ,6 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 , .BRIEF DESCRIPTION OF FIGURES

[0037] To facilitate further description of the embodiments of this disclosure, the following drawings are provided to illustrate and not to limit the scope of the disclosure.

[0038] The patent or application file contains at least one drawing originally in color.

[0039] Figs.1a-1f show glutamate-bound and -unbound Best2 structures. (a) Side view of glutamate-bound Best2 model with glutamate molecule shown as blue sticks, Ca2+as a green sphere. Regions of the AS are colored as follows: ACR1, yellow; Anchor, blue; ACR2, red. PM, plasma membrane. Black box indicates region of focus in Fig. 1b. (b) Close-up of glutamate binding site with hydrogen bonds depicted as dotted lines and interacting residue side chains labeled. (C) The model and map (shown at sigma 6) of the glutamate binding site with Q354 and the bound glutamate labeled. (d-f) The same format as a-c, respectively, for glutamate-unbound Best2 fully open model from the same data set.

[0040] Figs. 2a-2i show the cryo-EM processing pathway for the Best2 + glutamate dataset. (a) Representative micrograph. (b) Representative 2D classes. (c) Consensus refinement with symmetric mask covering the transmembrane domain used for 3D classification. (d-f) 3D classification results depicting top view of density and particle occupancy and FSC curves (d), local resolution maps (e) and orientation distribution plot (f) for the fully open state. (g-i) The same format as d-f, respectively, for the closed state; inset, color key.

[0041] Figs. 3a-3b show the comparison of glutamate-bound Best2 in the closed and fully open conformations. (a, b) Side views of two opposing (144°) Best2 protomers in the 7 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 glutamate-bound closed state (a) or glutamate-unbound fully open state (b) from the Best2 + glutamate dataset, with the ion permeation pathway visualized and pore diameter depicted as colored dots: the tighter the pore radius, the smaller and denser the dots; inset, color key; glutamate (Glu) molecules are shown as blue space-filling spheres; major constrictions to the ion permeation pathway are shown with residue labels on the right; dashed lines indicate approximate boundaries of the plasma membrane (PM).

[0042] Fig. 4 shows the functional influences of small molecules on Best2 channel. Current densities of transiently expressed Best2 at 1 μM [Ca2+]iin HEK293 cells treated with 50 mM of the indicated small molecules. Left-most bar, untreated cells. Glu, glutamate. 1: 2- (1-amino-2,2,2-trifluoroethyl)pentanedioic acid (L:D = 1:1), 2: Glutamine (L+D), 3: γ- Carboxyglutamic acid, 4: α-Aminoadipic acid, 5: 1-Methyl L-glutamate, 6: 1-Ethyl L- glutamate, 7: (-)-Glutamic acid, 8: 3-Aminopentanedioic acid, 9: 4-Carboxybenzeneacetic acid, 10: 2-Aminoterephthalic acid, 11: Hydroxyterephthalic acid, 12: 1,4- Cyclohexanedicarboxylic acid, 13: 3-Phenylglutaric acid, 2-L: L-Glutamine, and 2-D: D- Glutamine. n = 5-6. *p < 0.05 compared to untreated cells by two-tailed unpaired Student’s t test. All error bars in this figure represent s.e.m. DETAILED DESCRIPTION OF THE DISCLOSURE Definitions

[0043] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the invention can be practiced without these details. In other instances, well- known structures have not been shown or described in detail to avoid unnecessarily obscuring descriptions of the embodiments.

[0044] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0045] Reference throughout this specification to “one embodiment” or “an embodiment,” etc. means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the 8 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments. Also, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0046] “Alkyl” and “alk” refer to a straight or branched chain alkane (hydrocarbon) radical containing from 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms. Exemplary “alkyl” groups include methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, and the like. The term “C1-Cxalkyl,” “(C1-Cx)alkyl,” or “C1-xalkyl” refers to a straight or branched chain alkane (hydrocarbon) radical containing from 1 to x carbon atoms. For example, the term “C1-C4alkyl,” “(C1-C4)alkyl,” or “C1-4alkyl” refers to a straight or branched chain alkane (hydrocarbon) radical containing from 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, and isobutyl. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted.

[0047] The term “heteroalkyl” refers to an alkyl substituent as defined above wherein at least one carbon atom has been replaced by a heteroatom such as O, S, or N. For example, a heteroalkyl can be an alkyl group where one or more of its -CH2- groups are replaced by -O- , -S-, or -NRz-; and / or can be an alkyl group where one or more of its -CH- groups are replaced by -N-; wherein each occurrence of Rz is hydrogen, alkyl, cycloalkyl, heterocycle, or aryl. Non-limiting examples of heteroalkyl groups include , , ,, , , , ,9 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 ,and . Unless stated otherwise specifically inbromine, fluorine, or iodine.

[0049] “Cycloalkyl” refers to a fully saturated cyclic hydrocarbon radical group containing from 1 to 4 rings and 3 to 8 carbon atoms per ring. For example, “C3-C7cycloalkyl” or “C3-7cycloalkyl” refers to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl. Unless stated otherwise specifically in the specification, a cycloalkyl can be optionally substituted.

[0050] The term "heterocycloalkyl" or "cycloheteroalkyl" refers to cycloalkyl ring containing at least one heteroatom selected from the group consisting of nitrogen, sulfur, and oxygen, preferably from 1 to 3 heteroatoms in at least one ring. Each ring is preferably from 3 to 10 membered, more preferably 4 to 7 membered. Examples of suitable heterocycloalkyl substituents include, but are not limited to, azetidinyl, oxetanyl, pyrrolidyl, tetrahydrofuryl, tetrahydrothiofuranyl, piperidyl, piperazyl, tetrahydropyranyl, morpholino, 1,3-diazepanyl, 1,4-diazepanyl, 1,4-oxazepanyl, and 1,4-oxathiapanyl. Unless stated otherwise specifically in the specification, a heterocycloalkyl or cycloheteroalkyl can be optionally substituted.

[0051] “Heterocyclyl,” “heterocyclic ring,” or “heterocycle” refers to a stable 3- to 20-membered non-aromatic ring radical which consists of two to twelve carbon atoms and from one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. Heterocyclyl or heterocyclic rings include heteroaryls as defined below. Unless stated otherwise specifically in the specification, the heterocyclyl radical can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical can be optionally oxidized; the nitrogen atom can be optionally quaternized; and the heterocyclyl radical can be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in the specification, a heterocyclyl group can be optionally substituted. 10 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025

[0052] “Aryl” refers to a hydrocarbon ring system radical comprising hydrogen, 5 to 18 carbon atoms and at least one aromatic ring. For purposes of this invention, the aryl radical can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems. Aryl radicals include, but are not limited to, aryl radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unless stated otherwise specifically in the specification, the term “aryl” is meant to include aryl radicals that are optionally substituted.

[0053] “Heteroaryl” refers to a 5- to 20-membered ring system radical comprising hydrogen atoms, one to thirteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and at least one aromatic ring. For purposes of this invention, the heteroaryl radical can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl radical can be optionally oxidized; the nitrogen atom can be optionally quaternized. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1- oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e. thienyl). Unless stated otherwise specifically in this disclosure, a heteroaryl group can be optionally substituted.

[0054] “Alkenyl” refers to a straight or branched chain hydrocarbon radical containing from 2 to 12 carbon atoms and at least one carbon-carbon double bond. Exemplary such groups include ethenyl or allyl. The term “C2-Cx alkenyl” or “C2-xalkenyl” refers to a straight or branched chain hydrocarbon radical containing from 2 to x carbon atoms and at least one 11 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 carbon-carbon double bond. For example, the term “C2-C6alkenyl” or “C2-6alkenyl” refers to a straight or branched chain hydrocarbon radical containing from 2 to 6 carbon atoms and at least one carbon-carbon double bond, such as ethylenyl, propenyl, 2-propenyl, (E)-but-2-enyl, (Z)-but-2-enyl, 2-methy(E)-but-2-enyl, 2-methy(Z)-but-2-enyl, 2,3-dimethy-but-2-enyl, (Z)- pent-2-enyl, (E)-pent-1-enyl, (Z)-hex-1-enyl, (E)-pent-2-enyl, (Z)-hex-2-enyl, (E)-hex-2-enyl, (Z)-hex-1-enyl, (E)-hex-1-enyl, (Z)-hex-3-enyl, (E)-hex-3-enyl, and (E)-hex-1,3-dienyl. Unless stated otherwise specifically in the specification, an alkenyl can be optionally substituted.

[0055] The term “substituted” used herein means any of the above groups (e.g., alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, alkoxy, alkylamino, alkylcarbonyl, thioalkyl, aryl, aralkyl, carbocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl) wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atoms such as, but not limited to: a halogen atom such as F, Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atom in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; a silicon atom in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups. “Substituted” also means any of the above groups in which one or more hydrogen atoms are replaced by a higher-order bond (e.g., a double- or triple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, “substituted” includes any of the above groups in which one or more hydrogen atoms are replaced with NRgC(=O)ORh, NRgSO2Rh, OC(=O)NRgRh, ORg, SRg, SORg, SO2Rg, OSO2Rg, SO2ORg, =NSO2Rg, and SO2NRgRh. “Substituted” also means any of the above groups in which one or more hydrogen atoms are replaced with C(=O)Rg, C(=O)ORg, C(=O)NRgRh, CH2SO2Rg, CH2SO2NRgRh. In the foregoing, Rg and Rh are the same or different and independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl. “Substituted” further means any of the above groups in which one or more hydrogen atoms are replaced by a bond to an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, 12 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl group. In addition, each of the foregoing substituents can also be optionally substituted with one or more of the above substituents.

[0056] “Optional” or “optionally” means that the subsequently described event of circumstances can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, “optionally substituted aryl” means that the aryl radical can or cannot be substituted and that the description includes both substituted aryl radicals and aryl radicals having no substitution.

[0057] The compounds of the invention, or their pharmaceutically acceptable salts can contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids. The present invention is meant to include all such possible isomers, as well as their racemic and optically pure forms whether or not they are specifically depicted herein. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0058] A “stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The present invention contemplates various stereoisomers and mixtures thereof and includes “enantiomers”, which refers to two stereoisomers whose molecules are nonsuperimposable mirror images of one another.

[0059] A “tautomer” refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present invention includes tautomers of any said compounds. 13 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025

[0060] Prodrugs and solvates of the compounds of the invention are also contemplated herein. The term “prodrug” as employed herein denotes a compound that, upon administration to a subject, undergoes chemical conversion by metabolic or chemical processes to yield a compound of the present invention, or a salt and / or solvate thereof. Solvates of the compounds of the present invention include, for example, hydrates.

[0061] “Pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0062] “Pharmaceutically acceptable salt” includes both acid and base addition salts.

[0063] “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4- acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

[0064] “Pharmaceutically acceptable base addition salt” refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, 14 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0065] As used herein, a “subject” is a mammal, preferably, a human. In addition to humans, categories of mammals within the scope of the present disclosure include, for example, agricultural animals, veterinary animals, laboratory animals, etc. Some examples of agricultural animals include cows, pigs, horses, goats, etc. Some examples of veterinary animals include dogs, cats, etc. Some examples of laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc. In some embodiments of the present disclosure, the phrase “a subject” means a subject having a Bestrophin-related disease such as, e.g., a bestrophinopathy.

[0066] A “pharmaceutical composition” refers to a formulation of a compound of the invention and a medium generally accepted in the art for the delivery of the biologically active compound to mammals, e.g., humans. Such a medium includes all pharmaceutically acceptable carriers, diluents or excipients therefor. A pharmaceutical composition may be suitable for any route of administration, including, for example, local or systemic administrations. Pharmaceutical compositions are administered to a subject in a manner known in the art. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0067] An “effective amount” refers to a therapeutically effective amount or a prophylactically effective amount. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a compound can vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. Dosage regimens can be adjusted to provide the optimum 15 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, increased life span, increased life expectancy or prevention of the progression of the disease or condition. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount can be less than a therapeutically effective amount. The pharmaceutical compositions of the inventions can be administered to any animal that can experience the beneficial effects of the agents of the invention. Such animals include humans and non-humans.

[0068] As used herein, the terms "treat," "treating," "treatment" and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient. In particular, the methods and compositions of the present disclosure may be used to slow the development of disease symptoms or delay the onset of the disease or condition, or halt the progression of disease development. However, because not every treated subject may respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population, e.g., patient population. Accordingly, a given subject or subject population, e.g., patient population, may fail to respond or respond inadequately to treatment.

[0069] As used herein, the terms “ameliorate”, "ameliorating" and grammatical variations thereof mean to decrease the severity of the symptoms of a disease in a subject.

[0070]

[0001] Following below are more detailed descriptions of various concepts related to, and embodiments of methods for the treatment of disease, including bestrophin- related disease. It should be appreciated that various concepts introduced above and discussed in greater detail below may be implemented in any of numerous ways, as the disclosed concepts are not limited to any particular manner of implementation. Examples of specific implementations and applications are provided primarily for illustrative purposes. Bestrophins and Bestrophin-related Diseases

[0071] The bestrophins are a family of Ca2+-activated anion channels consisting of four members in mammals. They are widely distributed in various human organs including the airways, colon, kidney, pancreas and central nervous system, but best known for their physiological roles in the eye. In particular, Best1 is predominantly expressed in retinal 16 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 pigment epithelium (RPE) and genetically linked to a spectrum of retinal degenerative disorders collectively known as bestrophinopathies. Over 350 different mutations in Best1 have been identified to cause bestrophinopathies. The patients are susceptible to progressive vision loss that may eventually lead to blindness, and there is no treatment. On the other hand, Best2 resides in non-pigmented epithelium (NPE) regulating intra-ocular pressure (IOP), which must be properly maintained at all times as both hypertension and hypotension are deleterious conditions. Therefore, bestrophins are potential drug targets for various human diseases, especially of the eye.

[0072] The present disclosure provides the cryogenic electron microscopy (cryoEM) structures of Best2 in complex with glutamate, an activator of the channel. A conserved glutamate binding site was discovered which is located on the intracellular side and highly conserved among all bestrophin channels (Best1-4), and multiple glutamate analogs were identified as Best2 activators. The results reveal a promising docking site for small molecule bestrophin activators (e.g., glutamate analogs) which could potentially be used as drugs for bestrophin-related diseases / conditions.

[0073] Accordingly, one embodiment of the present disclosure is directed to a method for treating, ameliorating, or preventing the effects of a disease in a subject in need thereof. This method comprises administering an effective amount of a Best activator to the subject. In some embodiments, the disease is a Bestrophin (Best)-related disease.

[0074] In some embodiments, the Bestrophin-related disease is caused by one or more mutations on a gene selected from BEST1, BEST2, BEST3, and BEST4. In some embodiments, the Bestrophin-related disease is caused by one or more loss-of-function mutations on BEST1. In some embodiments, the Bestrophin-related disease is caused by one or more loss-of-function mutations on BEST2.

[0075] In some embodiments, the Bestrophin-related disease is a bestrophinopathy. As used herein, “bestrophinopathies” refer to are a group of clinically distinct inherited retinal dystrophies that typically affect the macular region, an area synonymous with central high acuity vision. This spectrum of disorders is believed to be caused by mutations in BEST1, which acts as a Ca2+-activated Cl- channel in the retinal pigment epithelium (RPE) of the eye. Non-limiting examples of a bestrophinopathy include best vitelliform macular dystrophy (BVMD), autosomal recessive bestrophinopathy (ARB), adult-onset vitelliform dystrophy (AVMD), autosomal dominant vitreoretinoehoroidopathy (ADVIRC), autosomal dominant microcornea, rod-cone dystrophy, early-onset cataract, posterior staphyloma syndrome (MRCS 17 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 syndrome), retinitis pigmentosa (RP), age-related macular degeneration (AMD), or combinations thereof.

[0076] In some embodiments, the Bestrophin-related disease is associated with increased or decreased 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 the aqueous humor, a watery liquid that fills the front part of the eye. In a healthy eye, a small amount of new aqueous humor enters the eye while an equal amount drains out. However, if the fluid abnormally accumulates or disperses, IOP can build up or decrease, resulting in ocular hypertension or hypotension, respectively. For example, in some embodiments of the present dislcosure, the subject has ocular hypertension.

[0077] A normal range for IOP is between 10 and 21 millimeters of mercury (mmHg). Elevated IOP is a symptom and risk factor for various eye diseases such as glaucoma, and untreated high eye pressure can harm vision, or lead to vision loss. In some embodiments of the present disclosure, the IOP in the subject is above 21 mmHg.

[0078] Accordingly, in some embodiments of the present disclosure, the Bestrophin- related disease is glaucoma, myopia, pigment dispersion syndrome, pseudoexfoliation syndrome, age-related macular degeneration, or combinations thereof. In some embodiments the Bestrophin-related disease is glaucoma.

[0079] In some embodiments, the disease is not a Bestrophin-related disease. One non- limiting example of such a disease is Alzheimer’s disease. Thus, in some embodiments, the disease is Alzheimer’s disease.

[0080] In some embodiments, the Best activator is a small molecule.

[0081] In some embodiments, the Best activator is an activator for Best1.

[0082] In some embodiments, the Best activator is an activator for Best2.

[0083] In some embodiments, the Best activator is Glutamate, 2-(1-amino-2,2,2- trifluoroethyl)pentanedioic acid (L:D = 1:1), Glutamine (L+D), γ-Carboxyglutamic acid, α- Aminoadipic acid, 1-Methyl L-glutamate, 1-Ethyl L-glutamate, (-)-Glutamic acid, 3- Aminopentanedioic acid, 4-Carboxybenzeneacetic acid, 2-Aminoterephthalic acid, Hydroxyterephthalic acid, 1,4-Cyclohexanedicarboxylic acid, 3-Phenylglutaric acid, L- Glutamine, D-Glutamine, and combinations thereof. In some embodiments, the Best activator is 1-Ethyl L-glutamate or Hydroxyterephthalic acid.

[0084] As used herein, “Loss-of-function mutations” refers to mutations in the bestrophin gene that result in reduced anion currents mediated by the mutant bestrophin protein 18 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 and reduced channel activity compared to a wild-type protein. Exemplary BEST1 loss-of- function mutations include, but are not limited to, A10T, R218H, L234P, A243T, Q293K, and D302A.

[0085] In some embodiments, the Best activator is administered in a composition that comprises a pharmaceutically acceptable carrier and excipient. The small molecule activators of the present invention can be administered in combination with other pharmaceutical agents in a variety of protocols and at effective doses for treating, ameliorating, or preventing disease.

[0086] Pharmaceutical compositions are administered to a subject in a manner known in the art. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. Bestrophin Activators

[0087] In one aspect, described herein is a method for treating, ameliorating, or preventing the effects of a disease in a subject in need thereof wherein the method includes administering an effective amount of a Best activator to the subject, wherein the Best activator is a compound of Formula I: or a pharmaceutically acceptableor solvate thereof; wherein: X is C(R1)3, OR2, N(R2)2, SR2, C6-C10aryl, or C5-C10heteroaryl; each occurrence of R1is independently H, halogen, N(Ra)2, ORa, SRa, -CORb, - COORa, -OCORa, -CON(Ra)2, -NRaCORb, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1- C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl; each occurrence of R2is independently H, -CORb, -CON(Ra)2, C1-C6alkyl, C1- C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6- C10aryl, or C5-C10heteroaryl; L is –(C(R3)2)n–, –(C(R3)2)nNR4–, –NR4(C(R3)2)nNR4–, –O(C(R3)2)nNR4–, – NR4(C(R3)2)nO–, –O(C(R3)2)nO–, –(C(R3)2)nO–, –(C(R3)2)nS–, C3-C7cycloalkyl, C3- C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl; n is an integer from 1 to 5; each occurrence of R3is independently H, halogen, N(Ra)2, C(Ra)3, C(Ra)2N(Ra)2, C(Ra)2ORa, ORa, SRa, -CORb, -COORa, -OCORa, -CON(Ra)2, -NRaCORb, C1-C6alkyl, C1- 19 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6- C10aryl, or C5-C10heteroaryl; each occurrence of R4is independently H, -CORb, -CON(Ra)2, C1-C6alkyl, C1- C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6- C10aryl, or C5-C10heteroaryl; and Z is H, halogen, -N(Rb)2, -ORb, -SRb, -SO2N(Rb)2, -SON(Rb)2, C(Rb)3, -CORb, - COORa, -OCORa, -CON(Ra)2, -NRaCORb, -C1-C6alkylCOORaC1-C6alkyl, C1-C6alkenyl, C1- C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6-C10aryl, or C5- C10heteroaryl; wherein when L is –O(C(R3)2)nNR4– or –O(C(R3)2)nO–, Z is not -ORb; each occurrence of Rais independently H, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl; each occurrence of Rbis independently H, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl; and the C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3- C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl in X, L, Z, R1, R2, R3, R4, Ra, and Rb, where applicable, are each optionally independently substituted by 1-4 substituents each independently selected from the group consisting of halogen, -OH, -OC1-C6alkyl, -NH2, C1- C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1-C6OH, C1-C6NH2, C3-C7cycloalkyl, oxo, -CN, - COOH, -CONH2, heterocyclyl, aryl, or heteroaryl.

[0088] In some embodiments, X is C(R1)3, OR2, N(R2)2, SR2, C6-C10aryl, or C5- C10heteroaryl. In some embodiments, X is C(R1)3, OR2, or N(R2)2. In some embodiments, X is C(R1)3. In some embodiments, X is OR2. In some embodiments, X is N(R2)2. In some embodiments, X is CH2NH2, OH, or NH2.

[0089] In some embodiments, each occurrence of R1is independently H, halogen, N(Ra)2, ORa, SRa, -CORb, -COORa, -OCORa, -CON(Ra)2, -NRaCORb, C1-C6alkyl, C1- C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6- C10aryl, or C5-C10heteroaryl. In some embodiments, each occurrence of R1is independently H, halogen, N(Ra)2, ORa, or SRa. In some embodiments, each occurrence of R1is independently H or N(Ra)2.

[0090] In some embodiments, each occurrence of R2is independently H, -CORb, - CON(Ra)2, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3- C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl. In some embodiments, each occurrence 20 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 of R2is independently H, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C6-C10aryl, or C5- C10heteroaryl. In some embodiments, each occurrence of R2is independently H, C1-C6alkyl, C1-C6haloalkyl, or C6-C10aryl. In some embodiments, each occurrence of R2is independently H, CH2, or CH3.

[0091] In some embodiments, L is –(C(R3)2)n–, –(C(R3)2)nNR4–, –NR4(C(R3)2)nNR4–, –O(C(R3)2)nNR4–, –NR4(C(R3)2)nO–, –O(C(R3)2)nO–, –(C(R3)2)nO–, –(C(R3)2)nS–, C3- C7cycloalkyl, C3-C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl. In some embodiments, L is –(C(R3)2)n–, –(C(R3)2)nNR4–, C3-C7cycloalkyl, or C6-C10aryl.

[0092] In some embodiments, n is an integer from 1 to 5. In some embodiments, n is an integer from 1 to 4. In some embodiments, n is 3 or 4. In some embodiments, n is 1.

[0093] In some embodiments, L is –(C(R3)2)n– and n is an integer from 1 to 5. In some embodiments, L is –(C(R3)2)n– and n is an integer from 3 to 4. In some embodiments, L is – (C(R3)2)nNR4–, and n is an integer from 1 to 5. In some embodiments, L is –(C(R3)2)nNR4–, and n is 1.

[0094] In some embodiments, each occurrence of R3is independently H, halogen, N(Ra)2, C(Ra)3, C(Ra)2N(Ra)2, C(Ra)2ORa, ORa, SRa, -CORb, -COORa, -OCORa, -CON(Ra)2, - NRaCORb, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3- C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl. In some embodiments, each occurrence of R3is independently H, halogen, N(Ra)2, ORa, -COORa, C1-6alkyl, C1-6haloalkyl, or C6- C10aryl. In some embodiments, each occurrence of R3is independently H, NH2, NHCH3, NHCH2CH3, COOH, CH(NH2)CF3, or phenyl.

[0095] In some embodiments, each occurrence of R4is independently H, -CORb, - CON(Ra)2, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3- C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl. In some embodiments, each occurrence of R4is independently H, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, or C6-C10aryl. In some embodiments, each occurrence of R4is independently H.

[0096] In some embodiments, Z is H, halogen, -N(Rb)2, -ORb, -SRb, -SO2N(Rb)2, - SON(Rb)2, C(Rb)3, -CORb, -COORa, -OCORa, -CON(Ra)2, -NRaCORb, -C1-C6alkylCOORaC1- C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3- C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl; wherein when L is –O(C(R3)2)nNR4– or – O(C(R3)2)nO–, Z is not -ORb. In some embodiments, Z is H, halogen, -COORa, -CON(Ra)2, - C1-C6alkylCOORa, C1-C6alkyl, C1-C6haloalkyl, C1-C6heteroalkyl, or C6-C10aryl. In some embodiments, Z is H, CH3, CF3, -COOH, CONH2, or CH2COOH. 21 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025

[0097] In some embodiments, each occurrence of Rais independently H, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6- C10aryl, or C5-C10heteroaryl. In some embodiments, each occurrence of Rais independently H, C1-C6alkyl, C1-C6haloalkyl, or C6-C10aryl. In some embodiments, each occurrence of Rais independently H, CH3, or CH2CH3.

[0098] In some embodiments, each occurrence of Rbis independently H, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6- C10aryl, or C5-C10heteroaryl. In some embodiments, each occurrence of Rbis independently H or C1-C6alkyl.

[0099] In some embodiments, the C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1- C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl in X, L, Z, R1, R2, R3, R4, Ra, and Rb, where applicable, are each optionally independently substituted by 1-4 substituents each independently selected from the group consisting of halogen, -OH, - OC1-C6alkyl, -NH2, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1-C6OH, C1-C6NH2, C3- C7cycloalkyl, oxo, -CN, -COOH, -CONH2, heterocyclyl, aryl, or heteroaryl. [000100] In some embodiments, the Best activator is a compound of Formula I or a pharmaceutically acceptable enantiomer, salt, or solvate thereof, wherein the compound of , , ,22 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 [000101] Routes of administration and dosages of effective amounts of the pharmaceutical compositions are also disclosed. These pharmaceutical compositions or formulations may be designed for various routes of administration, including but not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, intranasal, intraperitoneal, intradermal, or topical delivery. The agents of the present invention can be administered in combination with other pharmaceutical agents in a variety of protocols for effective treatment of disease. In some embodiments, the pharmaceutical composition is administered locally via an ophthalmic route of delivery, i.e., administered to a subject’s eye. Ophthalmic routes of delivery include, but are not limited to, topical, periocular (e.g., subconjunctival, sub-Tenon, retrobulbar, and peribulbar), intravitreal and intracameral. In some embodiments, the pharmaceutical composition is administered via an ophthalmic route of delivery, wherein the pharmaceutical composition is administered topically. In some embodiments, the pharmaceutical composition is administered topically via eye drops. In some embodiments, the pharmaceutical composition is administered systemically, e.g., via an oral route or a parenteral route. Oral routes include, but are not limited to, administration by pills, tablets, or liquid formulations. Parenteral routes include, but are not limited to, intravenous, intramuscular, subcutaneous, intranasal, intraperitoneal, intradermal injection. [000102] Pharmaceutical compositions are administered to a subject in a manner known in the art. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. Pharmaceutical Compositions [000103] One of ordinary skill in the art will appreciate that a method of administering pharmaceutically effective amounts of the pharmaceutical compositions to a patient in need thereof, can be determined empirically, or by standards currently recognized in the medical arts. The pharmaceutical compositions can be administered to a patient as pharmaceutical compositions in combination with one or more pharmaceutically acceptable excipients. Suitable excipients include, but are not limited to, stabilizers, preservatives, solubilizers, emulsifiers, buffers, and agents that enhance bioavailability or control release kinetics. Other examples of suitable excipients are known in the art and are described in e.g., Remington’s: The Science and Practice of Pharmacy, 22ndEd. (Allen, Loyd V., Jr ed., Pharmaceutical Press (2012)), the disclosure of which is incorporated herein by reference in its entirety. Such pharmaceutical compositions or formulations may be tailored to improve stability, shelf-life, 23 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 patient compliance, and therapeutic performance. The pharmaceutical compositions or formulations may be provided in dosage forms such as tablets, pills, capsules, solutions, suspensions, emulsions, injectables, topicals, eye drops, or transdermal patches, depending on the intended use and delivery method. It will be understood that, when administered to a human patient, the total daily usage of the agents of the pharmaceutical compositions will be decided within the scope of sound medical judgment by the attending physician. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors: the type and degree of the cellular response to be achieved; activity of the specific agent or composition employed; the specific agents or composition employed; the age, body weight, general health, gender and diet of the patient; the time of administration, route of administration, and rate of excretion of the agent; the duration of the treatment; drugs used in combination or coincidental with the specific agent; and like factors well known in the medical arts. It is well within the skill of the art to start doses of the agents at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosages until the desired effect is achieved. [000104] In certain aspects, described herein is a method of maintaining intraocular pressure (IOP) in a subject in need thereof, the method comprising administering a pharmaceutical composition, wherein the composition increases Best2-mediated Ca2+- dependent Cl- currents in non-pigmented epithelium (NPE) cells. In some embodiments, the subject is a human. In some embodiments, the subject suffers from ocular hypertension or ocular hypotension.In some embodiments, the pharmaceutical composition is administered via an ophthalmic route of delivery, an oral route of delivery, or a parenteral route of delivery. In some embodiments, the pharmaceutical composition is administered via an ophthalmic route of delivery, wherein the pharmaceutical composition is administered topically. In some embodiments, the pharmaceutical composition is administered via eye drops. Prodrugs [000105] A "pro-drug" or "pro-drug" refers to an agent which is converted into the active drug in vivo. Pro-drugs are often useful because, in some situations, they are easier to administer than the parent drug. They are bioavailable, for instance, by oral administration whereas the parent drug is either less bioavailable or not bioavailable. In some embodiments, the pro-drug has improved solubility in pharmaceutical compositions over the parent drug. For example, the compound carries protective groups that are removed in vivo, thus releasing active compound. The term "pro-drug" may apply to such functionalities as, for example, the acid functionalities of the 24 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 compounds of Formula (I). Pro-drugs may be comprised of structures wherein an acid group is masked, for example, as an ester or amide. Further examples of pro-drugs are discussed herein and, for example, by Alexander et al., J Med Chem.1988, 31, 318 (hereby incorporated by reference in its entirety). In some embodiments, a prodrug form of a compound of Formula (I) can be used to improve pharmacokinetic or penetration of cell membrane. In some embodiments, a prodrug form of a compound of Formula (I) includes but is not limited to an ester of a carboxylic acid. [000106] In one embodiment, the present invention also encompasses methods comprising pro-drugs of compounds of Formula (I) and / or pharmaceutical compositions thereof. Prodrugs include derivatives of compounds that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide an active compound of the invention. Examples of pro-drugs include, but are not limited to, derivatives and metabolites of a compound of the invention that include biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, and biohydrolyzable phosphate analogues. Pro-drugs are often useful because, in some situations, they are easier to administer than the parent drug. They are bioavailable, for instance, by oral administration whereas the parent drug is either less bioavailable or not bioavailable. In some embodiments, the pro-drug has improved solubility in pharmaceutical compositions over the parent drug. For example, the compound carries protective groups that are removed in vivo, thus releasing active compound. [000107] The following examples are provided to further illustrate the methods of the present disclosure. These examples are illustrative only and are not intended to limit the scope of the disclosure in any way. EXAMPLES Example 1 [000108] Methods and Materials [000109] Cryo-EM Sample Preparation [000110] Human Bestrophin proteins were purified in GDN2. After nickel affinity and size exclusion chromatography, the protein was concentrated to 5 mg / mL for glutamate-bound Best2, the concentrated protein was buffer exchanged into 50 mM Na-glutamate, 25 mM HEPES pH 7.8 supplemented with 0.008% GDN on a Sartorius 100 kDa MWCO centrifugal concentrator. Protein was concentrated to 5mg / mL for grid production. 25 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 [000111] 2.8 µL of protein was spiked with 5 mM CaCl2 and immediately applied to a plasma treated UltrAuFoil R0.6 / 1 on a vitrobot Mark IV, incubated for 30 s at 100% humidity and 10 ̊C, blotted for 5-7 s at force 4 and immediately plunged into liquid ethane cooled by liquid nitrogen. Grids were screened on a glacios prior to data collection on a Krios. [000112] Data Collection and Image Processing [000113] For the Best2 + glutamate dataset, 6,625 micrographs were collected with a K3 direct electron detector in counting mode at a magnification of 105,000x, corresponding to a physical pixel size of 0.825 Å2 / pix at a total dose of 59 e- / A2, fractionated over 50 frames, corresponding to a dose rate of 1.18 e- / A2 / frame with a defocus range of -1-2.5 microns. Movies were aligned with MotionCorr2 via Relion3.1 and imported to cryoSPARCv4 for further processing by PatchCTF estimation, template picking, and extraction. After initial 2D classification of 3,529,672 picked particles, 189,723 particles were selected for ab initio reconstruction and local refinement using a global mask with C5 symmetry (3.1 Å). Particles underwent symmetry expansion (C5) to generate 948,615 particles, which underwent 3D classification into 3 classes using a mask encompassing the transmembrane domain. One class (class 2) was in the fully open state and the 2 other classes were closed. The open state particles were local refined with C1 symmetry using a global mask, resulting in a final map at 3.15 Å. The 635,068 closed state particles were local refined to 3.03 Å with a global mask. [000114] Model Refinement and Validation [000115] Maps used for model building and refinement were obtained by sharpening to a b-factor determined by Guinier plot as implemented in cryoSPARC. PDB 8D1G (for Best2) was rigid body fit into cryo-EM maps and subjected to multiple iterations of refinement in coot, phenix real space refinement, and REFMAC5 (Servalcat). Validation was performed with comprehensive cryo-EM validation tools in phenix, including MolProbity. [000116] Cell Lines [000117] HEK293 and HEK293F cells were purchased from ATCC (Cat. #CRL-1573) and Thermo Fisher Scientific (Cat. #R79007), respectively. The cells used in this study were authenticated by short tandem repeat (STR) DNA profiling and tested negative for mycoplasma contamination. HEK293 cells were cultured in DMEM (Corning, Cat. #10013CV) supplemented with 100 µg / mL penicillin-streptomycin and 10% fetal bovine serum, and HEK293F cells were cultured in FreeStyle™ 293 Expression Medium (Thermo Fisher Scientific, Cat. #12338026). [000118] Transfection 26 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 [000119] 20-24 h before transfection, cells were split into new 3.5 cm culture dishes at 50% confluency. Plasmid and siRNA transfections were conducted using the PolyJet Transfection Reagent (SignaGen, Cat. #SL100688) and Lipofectamine RNAiMAX Reagent (Invitrogen, Cat. #13778-030), respectively. The transfection mix was removed after 4-8 h, and cells were washed with PBS and fed with fresh media until downstream analysis or harvest. [000120] Electrophysiology [000121] Whole-cell patch clamp recording was conducted 48-96 h after transfection of HEK293 cells with EPC10 patch clamp amplifier (HEKA Electronics) controlled by Patchmaster v2x90.5 (HEKA). Micropipettes were pulled and fashioned from filamented 1.5 mm thin-walled glass (WPI Instruments). Series resistance was typically 1.5-2.5 MΩ, with no electronic series resistance compensation. Experiments were conducted at room temperature (23 ± 2 °C). Liquid junction potentials were measured and corrected using HEKA built-in functions. The standard zero Ca2+pipette solution contained (mM): 146 CsCl, 2 MgCl2, 5 EGTA, 2 MgATP (added fresh), 10 HEPES, pH 7.3 adjusted with NMDG. Solutions with various free Ca2+concentrations were made by mixing CaCl2 with EGTA as calculated by the MaxChelator Program, and the free Ca2+concentration was verified using a Ca2+ion-selective electrode. The standard extracellular solution contained (mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 15 glucose, 10 HEPES, pH 7.4 with NMDG. In the glutamate (analog) internal solution, 50 mM Cs-glutamate (analog) replaced CsCl, pH 7.4 adjusted with NMDG. Solution osmolarity was 290~310 mOsm / L with glucose, and ~5 mOsm lower in the internal solutions than the external solutions of the same experiment. The low and high Ca2+solutions in the same set of experiments were adjusted to have the exact same osmolarity. Solution changes were performed manually. [000122] Electrophysiological Data Collection and Analyses [000123] Traces were acquired at a repetition interval of 4 s. Currents were sampled at 25 kHz and filtered at 5 or 10 kHz. I-V curves were generated from a group of step potentials (- 100 to +100 mV from a holding potential of 0 mV). Data were processed off-line in Patchmaster. Statistical analyses were performed using built-in functions in OriginPro 8.5. The “n” value in patch clamp recording figure legends indicates the total number of individual cells. [000124] Statistics and Reproducibility [000125] A sufficient number of samples were examined to reach statistical conclusion according to the specific method utilized in that experiment. Statistically significant differences 27 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 (p < 0.05) between means of two groups were determined by two-tailed unpaired Student’s t test. Data are presented as means values + / - SEM. [000126] Glutamate-bound Best2 structure [000127] We recently reported that glutamate promotes Best2 function specifically from the intracellular / cytosolic side of the channel with a half maximal effective concentration (EC50) at 46 mM11. [000128] To elucidate the structural basis of this stimulating effect of intracellular glutamate, we incubated purified Best2 with 50 mM glutamate prior to cryo-EM grids preparation, and solved the glutamate-bound and -unbound Best2 structures under this condition at 3.0-3.2 Å (Figs.1a-1c, Figs.2a-2i, Figs.3a-3b and Table 1). Consistent with the functional results, the Best2 + glutamate data set shows a significantly higher percentage of particles with a fully open neck compared to that in the glutamate-free data set from our previous study (33% vs 12%, Fig. 2d)11,20. Glutamate binds to Best2 in a hydrophilic pocket just below the Ca2+-clasp, making hydrogen bonds with the backbone of the Ca2+-clasp, the N- terminal segment, and the C-terminal AS, all of which are critical for channel gating20: 1) the side chain carboxyl group of the glutamate molecule makes hydrogen bonds with the N- terminus of the N-terminal segment, the side chain carboxyl of E306, and the backbone carbonyls of D301, D302 and D304; 2) the carboxyl group of the main chain makes hydrogen bonds with the backbone carbonyls of F305, G299, and the side chain hydroxyl group of T307; 3) the amino group makes a hydrogen bond with the side chain of Q354 within the AS (Fig. 1b). Table 1. Cryo-EM data collection and refinement statistics for Best2 + glutamate structures. State Best2 + glutamate Open Best2 + glutamate Closed8 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 Final symmetry imposed C1 C1 Micrographs (#) 6,625 6,625region, including both AS Cooperativity Regions (ACR1 and ACR2, corresponding to residues 346-356, which contains the critical residue Q354 and 369-379, respectively), are simultaneously abolished in the open state structure compared to the closed state (Fig.1c and 29 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 1f), suggesting dissociation of the AS from the channel upon glutamate binding. As unleashing AS is required for opening the neck20, these results suggest that: 1) glutamate binds to the closed channel, and comes off upon channel opening; 2) Q354 is critical for glutamate binding. The binding of glutamate to Best2 may sterically occlude AS from association with the channel core, thus unleashing the restriction of the neck gate20. [000130] Small molecule activators of Best2 [000131] As glutamate acts as a potent activator of Best2 (Fig. 4), our findings suggest the potential of small molecules to stimulate the channel function via this binding site for clinical applications. By electrophysiological analysis, we identified another 15 small molecule activators of Best2, namely 2-(1-amino-2,2,2-trifluoroethyl)pentanedioic acid (L:D = 1:1), Glutamine (L+D), γ-Carboxyglutamic acid, α-Aminoadipic acid, 1-Methyl L-glutamate, 1- Ethyl L-glutamate, (-)-Glutamic acid, 3-Aminopentanedioic acid, 4-Carboxybenzeneacetic acid, 2-Aminoterephthalic acid, Hydroxyterephthalic acid, 1,4-Cyclohexanedicarboxylic acid, 3-Phenylglutaric acid, L-Glutamine, and D-Glutamine. Best2-mediated Cl- currents were significantly elevated by 50 mM of each substance in the internal solution during patch clamp recording (Fig.4). [000132] As the residues forming the glutamate-binding site on Best2 is highly conserved among bestrophins, the small molecule activators may also stimulate Best1, Best3 and Best4. [000133] In summary, the glutamate-bound Best2 structure suggests that the binding of glutamate to Best2 sterically occludes AS from association with the channel core, thus unleashing the restriction of the neck gate2. The residues making contact with glutamate are highly conserved in all bestrophins (Best1-4), underlining a promising docking site for small molecule activators of the channels. [000134] Further information regarding this disclosure may be found in Owji, A. P.; et al., “Neurotransmitter-bound Bestrophin channel structures reveal small molecule drug targeting sites for disease treatment” Nature Communications 15, 10766, doi: 10.1038 / s41467- 024-54938-z (2024), the full disclosure of which is incorporated herein by reference in its entirety. 30 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 CITED DOCUMENTS 1 Owji, A. P., Kittredge, A., Zhang, Y. & Yang, T. Structure and Function of the Bestrophin family of calcium-activated chloride channels. Channels (Austin) 15, 604- 623, doi:10.1080 / 19336950.2021.1981625 (2021). 2 Johnson, A. A. et al. Bestrophin 1 and retinal disease. Progress in retinal and eye research, doi:10.1016 / j.preteyeres.2017.01.006 (2017). 3 Petrukhin, K. et al. Identification of the gene responsible for Best macular dystrophy. Nat Genet 19, 241-247, doi:10.1038 / 915 (1998). 4 Yang, T., Justus, S., Li, Y. & Tsang, S. H. BEST1: the Best Target for Gene and Cell Therapies. Molecular therapy : the journal of the American Society of Gene Therapy 23, 1805-1809, doi:10.1038 / mt.2015.177 (2015). 5 Bakall, B. et al. Bestrophin-2 is involved in the generation of intraocular pressure. Investigative ophthalmology & visual science 49, 1563-1570, doi:49 / 4 / 1563 [pii] 10.1167 / iovs.07-1338 (2008). 6 Zhang, Y. et al. Enhanced inflow and outflow rates despite lower IOP in bestrophin-2- deficient mice. Investigative ophthalmology & visual science 50, 765-770, doi:iovs.08- 2501 [pii] 10.1167 / iovs.08-2501 (2009). 7 Zhang, Y., Patil, R. V. & Marmorstein, A. D. Bestrophin 2 is expressed in human non- pigmented ciliary epithelium but not retinal pigment epithelium. Molecular vision 16, 200-206, doi:25 [pii] (2010). 8 Sun, H., Tsunenari, T., Yau, K. W. & Nathans, J. The vitelliform macular dystrophy protein defines a new family of chloride channels. Proceedings of the National Academy of Sciences of the United States of America 99, 4008-4013, doi:10.1073 / pnas.05269299999 / 6 / 4008 [pii] (2002). 9 Tsunenari, T. et al. Structure-function analysis of the bestrophin family of anion channels. The Journal of biological chemistry 278, 41114-41125, doi:10.1074 / jbc.M306150200 M306150200 [pii] (2003). 10 Zhang, Y. et al. ATP activates bestrophin ion channels through direct interaction. Nature communications 9, 3126, doi:10.1038 / s41467-018-05616-410.1038 / s41467- 018-05616-4 [pii] (2018). 11 Owji, A. P. et al. Bestrophin-2 and glutamine synthetase form a complex for glutamate release. Nature 611, 180-187, doi:10.1038 / s41586-022-05373-x (2022). 31 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 12 Lee, S. et al. Channel-mediated tonic GABA release from glia. Science 330, 790-796, doi:10.1126 / science.1184334 (2010). 13 Woo, D. H. et al. TREK-1 and Best1 channels mediate fast and slow glutamate release in astrocytes upon GPCR activation. Cell 151, 25-40, doi:10.1016 / j.cell.2012.09.005 (2012). 14 Jo, S. et al. GABA from reactive astrocytes impairs memory in mouse models of Alzheimer's disease. Nature medicine 20, 886-896, doi:10.1038 / nm.3639 (2014). 15 Park, H. et al. High glutamate permeability and distal localization of Best1 channel in CA1 hippocampal astrocyte. Molecular brain 6, 54, doi:10.1186 / 1756-6606-6-54 (2013). 16 Kane Dickson, V., Pedi, L. & Long, S. B. Structure and insights into the function of a Ca(2+)-activated Cl(-) channel. Nature 516, 213-218, doi:10.1038 / nature13913 (2014). 17 Yang, T. et al. Structure and selectivity in bestrophin ion channels. Science 346, 355- 359, doi:10.1126 / science.1259723 (2014). 18 Ji, C. et al. Dual Ca(2+)-dependent gates in human Bestrophin1 underlie disease- causing mechanisms of gain-of-function mutations. Commun Biol 2, 240, doi:10.1038 / s42003-019-0433-3433 [pii] (2019). 19 Owji, A. P. et al. Structural and functional characterization of the bestrophin-2 anion channel. Nature structural & molecular biology 27, 382-391, doi:10.1038 / s41594-020- 0402-z (2020). 20 Owji, A. P. et al. Structures and gating mechanisms of human bestrophin anion channels. Nature communications 13, 3836, doi:10.1038 / s41467-022-31437-7 (2022). 21 Qu, Z., Cui, Y. & Hartzell, C. A short motif in the C-terminus of mouse bestrophin 3 [corrected] inhibits its activation as a Cl channel. FEBS Lett 580, 2141-2146, doi:S0014-5793(06)00327-9 [pii] 10.1016 / j.febslet.2006.03.025 (2006). 22 Qu, Z. Q., Yu, K., Cui, Y. Y., Ying, C. & Hartzell, C. Activation of bestrophin Cl- channels is regulated by C-terminal domains. The Journal of biological chemistry 282, 17460-17467, doi:M701043200 [pii] 10.1074 / jbc.M701043200 (2007). [000135] All documents cited in this application are hereby incorporated by reference as if recited in full herein. In the event of a conflict between the teachings of this application and those of the incorporated documents, the teachings of this application control. 32 ActiveUS 211472149Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 [000136] Although illustrative embodiments of the present disclosure have been described herein, it should be understood that the disclosure is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the disclosure. 33 ActiveUS 211472149

Claims

1. Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 CLAIMS What is claimed is:

1. A method for treating, ameliorating, or preventing the effects of a disease in a subject in need thereof, comprising administering an effective amount of a Best activator to the subject.

2. The method of claim 1, wherein the disease is a Bestrophin (Best)-related disease.

3. The method of claim 2, wherein the Bestrophin-related disease is caused by one or more mutations on a gene selected from BEST1, BEST2, BEST3, and BEST4.

4. The method of claim 3, wherein the Bestrophin-related disease is caused by one or more loss-of-function mutations on BEST1.

5. The method of claim 3, wherein the Bestrophin-related disease is caused by one or more loss-of-function mutations on BEST2.

6. The method of claim 2, wherein the Bestrophin-related disease is a bestrophinopathy.

7. The method of claim 6, wherein the bestrophinopathy is best vitelliform macular dystrophy (BVMD), autosomal recessive bestrophinopathy (ARB), adult-onset vitelliform dystrophy (AVMD), autosomal dominant vitreoretinoehoroidopathy (ADVIRC), autosomal dominant microcornea, rod-cone dystrophy, early-onset cataract, posterior staphyloma syndrome (MRCS syndrome), retinitis pigmentosa (RP), age-related macular degeneration (AMD), or combinations thereof.

8. The method of claim 2, wherein the Bestrophin-related disease is associated with increased or decreased intraocular pressure (IOP) in the subject.

9. The method of claim 8, wherein the subject has ocular hypertension or hypotension.

10. The method of claim 8, wherein the IOP in the subject is above 21 mmHg.

11. The method of claim 8, wherein the Bestrophin-related disease is glaucoma, myopia, pigment dispersion syndrome, pseudoexfoliation syndrome, age-related macular degeneration, or combinations thereof.

12. The method of claim 2, wherein the Bestrophin-related disease is glaucoma.

13. The method of claim 1, wherein the disease is Alzheimer’s disease.

14. The method of any of claims 1-13, wherein the Best activator is a small molecule.

15. The method of any of claims 1-14, wherein the Best activator is an activator for Best1.

16. The method of any of claims 1-14, wherein the Best activator is an activator for Best2. 34 ActiveUS 211472149 Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 17. The method of any of claims 1-16, wherein the Best activator is Glutamate, 2-(1- amino-2,2,2-trifluoroethyl)pentanedioic acid (L:D = 1:1), Glutamine (L+D), γ- Carboxyglutamic acid, α-Aminoadipic acid, 1-Methyl L-glutamate, 1-Ethyl L- glutamate, (-)-Glutamic acid, 3-Aminopentanedioic acid, 4-Carboxybenzeneacetic acid, 2-Aminoterephthalic acid, Hydroxyterephthalic acid, 1,4- Cyclohexanedicarboxylic acid, 3-Phenylglutaric acid, L-Glutamine, and D-Glutamine and combination thereof.

18. The method of any of claims 1-17, wherein the Best activator is administered in a composition that comprises a pharmaceutically acceptable carrier and excipient.

19. The method of any of claims 1-18, wherein the subject is a mammal.

20. The method of claim 19, wherein the mammal is humans, veterinary animals, and agricultural animals.

21. The method of any of claims 1-20, wherein the subject is a human.

22. The method of any of claims 18-21, wherein the pharmaceutical composition is administered via an ophthalmic route of delivery, an oral route of delivery, or a parenteral route of delivery.

23. The method of claim 22, wherein the pharmaceutical composition is administered via an ophthalmic route of delivery, wherein the pharmaceutical composition is administered topically.

24. The method of claim 23, wherein the pharmaceutical composition is administered via eye drops.

25. A method for treating, ameliorating, or preventing the effects of a disease in a subject in need thereof, comprising administering an effective amount of a Best activator to the subject, wherein the Best activator is a compound of Formula I: or a pharmaceutically acceptable solvate thereof; wherein: X is C(R1)3, OR2, N(R2)2, SR2, C6-C10aryl, or C5-C10heteroaryl; each occurrence of R1is independently H, halogen, N(Ra)2, ORa, SRa, -CORb, - COORa, -OCORa, -CON(Ra)2, -NRaCORb, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1- C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl; 35 ActiveUS 211472149 Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 each occurrence of R2is independently H, -CORb, -CON(Ra)2, C1-C6alkyl, C1- C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6- C10aryl, or C5-C10heteroaryl; L is –(C(R3)2)n–, –(C(R3)2)nNR4–, –NR4(C(R3)2)nNR4–, –O(C(R3)2)nNR4–, – NR4(C(R3)2)nO–, –O(C(R3)2)nO–, –(C(R3)2)nO–, –(C(R3)2)nS–, C3-C7cycloalkyl, C3- C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl; n is an integer from 1 to 5; each occurrence of R3is independently H, halogen, N(Ra)2, C(Ra)3, C(Ra)2N(Ra)2, C(Ra)2ORa, ORa, SRa, -CORb, -COORa, -OCORa, -CON(Ra)2, -NRaCORb, C1-C6alkyl, C1- C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6- C10aryl, or C5-C10heteroaryl; each occurrence of R4is independently H, -CORb, -CON(Ra)2, C1-C6alkyl, C1- C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6- C10aryl, or C5-C10heteroaryl; Z is H, halogen, -N(Rb)2, -ORb, -SRb, -SO2N(Rb)2, -SON(Rb)2, C(Rb)3, -CORb, - COORa, -OCORa, -CON(Ra)2, -NRaCORb, -C1-C6alkylCOORaC1-C6alkyl, C1-C6alkenyl, C1- C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6-C10aryl, or C5- C10heteroaryl; wherein when L is –O(C(R3)2)nNR4– or –O(C(R3)2)nO–, Z is not -ORb; each occurrence of Rais independently H, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl; each occurrence of Rbis independently H, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3-C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl; and the C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1-C6heteroalkyl, C3-C7cycloalkyl, C3- C7heterocycloalkyl, C6-C10aryl, or C5-C10heteroaryl in X, L, Z, R1, R2, R3, R4, Ra, and Rb, where applicable, are each optionally independently substituted by 1-4 substituents each independently selected from the group consisting of halogen, -OH, -OC1-C6alkyl, -NH2, C1- C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C1-C6OH, C1-C6NH2, C3-C7cycloalkyl, oxo, -CN, - COOH, -CONH2, heterocyclyl, aryl, or heteroaryl.

26. The method of claim 25, wherein X is C(R1)3, OR2, or N(R2)2.

27. The method of claim 25 or claim 26, wherein X is CH2NH2, OH, or NH2.

28. The method of claim 25 or 26, wherein each occurrence of R1is independently H, halogen, N(Ra)2, ORa, or SRa. 36 ActiveUS 211472149 Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 29. The method of claim 28, wherein each occurrence of R1is independently H or N(Ra)2.

30. The method of claim 25 or 26, wherein each occurrence of R2is independently H, C1- C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, C6-C10aryl, or C5-C10heteroaryl.

31. The method of any one of claims 25-26 and 30, wherein each occurrence of R2is independently H, CH2, or CH3.

32. The method of any one of claims 25-31, wherein L is –(C(R3)2)n–, –(C(R3)2)nNR4–, C3-C7cycloalkyl, or C6-C10aryl.

33. The method of any one of claims 25-32, wherein n is an integer from 1 to 4.

34. The method of any one of claims 25-33, wherein n is 3 or 4.

35. The, method of any one of claims 25-33, wherein n is 1.

36. The method of any one of claims 25-35, wherein each occurrence of R3is independently H, halogen, N(Ra)2, ORa, -COORa, C1-6alkyl, C1-6haloalkyl, or C6-C10aryl.

37. The method of any one of claims 23-36, wherein each occurrence of R3is independently H, NH2, NHCH3, NHCH2CH3, COOH, CH(NH2)CF3, or phenyl.

38. The method of any one of claims 25-37, wherein each occurrence of R4is independently H, C1-C6alkyl, C1-C6alkenyl, C1-C6haloalkyl, or C6-C10aryl.

39. The method of any one of claims 25-38, wherein each occurrence of R4is independently H.

40. The method of any one of claims 25-39, wherein Z is H, halogen, -COORa, - CON(Ra)2, -C1-C6alkylCOORa, C1-C6alkyl, C1-C6haloalkyl, C1-C6heteroalkyl, or C6-C10aryl.

41. The method of any one of claims 25-40, wherein Z is H, CH3, CF3, -COOH, CONH2, or CH2COOH.

42. The method of any one of claims 25-41, wherein each occurrence of Rais independently H, C1-C6alkyl, C1-C6haloalkyl, or C6-C10aryl.

43. The method of any one of claims 25-42, wherein each occurrence of Rais independently H, CH3, or CH2CH3.

44. The method of any one of claims 25-43, wherein each occurrence of Rbis independently H or C1-C6alkyl.

45. The method of claim 25, wherein the Best activator is a compound of Formula I or a pharmaceutically acceptable enantiomer, salt, or solvate thereof, and wherein the compound of Formula I is , 37 ActiveUS 211472149 Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 , , 46. - related disease.

47. The method of claim 46, wherein the Bestrophin-related disease is caused by one or more mutations on a gene selected from BEST1, BEST2, BEST3, and BEST4.

48. The method of claim 47, wherein the Bestrophin-related disease is caused by one or more loss-of-function mutations on BEST1.

49. The method of claim 47, wherein the Bestrophin-related disease is caused by one or more loss-of-function mutations on BEST2.

50. The method of claim 46, wherein the Bestrophin-related disease is a bestrophinopathy.

51. The method of claim 50, wherein the bestrophinopathy is best vitelliform macular dystrophy (BVMD), autosomal recessive bestrophinopathy (ARB), adult-onset vitelliform dystrophy (AVMD), autosomal dominant vitreoretinoehoroidopathy (ADVIRC), autosomal dominant microcornea, rod-cone dystrophy, early-onset cataract, posterior staphyloma syndrome (MRCS syndrome), retinitis pigmentosa (RP), age-related macular degeneration (AMD), or combinations thereof.

52. The method of claim 46, wherein the Bestrophin-related disease is associated with increased or decreased intraocular pressure (IOP) in the subject. 38 ActiveUS 211472149 Attorney Docket No.: 19240.01332WO4 Date of Electronic Filing: September 17, 2025 53. The method of claim 52, wherein the subject has ocular hypertension or hypotension.

54. The method of claim 52, wherein the IOP in the subject is above 21 mmHg.

55. The method of claim 52, wherein the Bestrophin-related disease is glaucoma, myopia, pigment dispersion syndrome, pseudoexfoliation syndrome, age-related macular degeneration, or combinations thereof.

56. The method of claim 46, wherein the Bestrophin-related disease is glaucoma.

57. The method of claim any one of claims 25-45, wherein the disease is Alzheimer’s disease.

58. The method of any of claims 25-57, wherein the Best activator is an activator for Best1.

59. The method of any of claims 25-57, wherein the Best activator is an activator for Best2.

60. The method of any of claims 25-59, wherein the Best activator is administered in a composition that comprises a pharmaceutically acceptable carrier and excipient.

61. The method of any of claims 25-60, wherein the subject is a mammal.

62. The method of claim 61, wherein the mammal is humans, veterinary animals, or agricultural animals.

63. The method of any of claims 25-62, wherein the subject is a human.

64. The method of any of claims 60-63, wherein the pharmaceutical composition is administered via an ophthalmic route of delivery, an oral route of delivery, or a parenteral route of delivery.

65. The method of claim 64, wherein the pharmaceutical composition is administered via an ophthalmic route of delivery, wherein the pharmaceutical composition is administered topically.

66. The method of claim 65, wherein the pharmaceutical composition is administered via eye drops 67. A composition or process as disclosed or depicted herein. 39 ActiveUS 211472149

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