Methods for treating congenital disorders of glycosylation

Abstract

Aspects of the present disclosure are directed to small molecules useful to treat congenital disorders of glycosylation.

Description

Docket No. BONO.P0076WOMETHODS FOR TREATING CONGENITAL DISORDERS OF GLYCOSYLATION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No.63 / 719,628, filed November 12, 2024, which is incorporated by reference herein in its entirety.BACKGROUNDI. Technical Field

[0002] Aspects of this disclosure relate to at least the fields of genetics, developmental disorders, and medicine.II. Background

[0003] Congenital disorders of glycosylation (CDG) include more than 200 inherited metabolic diseases that affect protein and lipid-linked glycosylation. CDGs may be broadly classified according to the glycosylation pathway(s) affected, including disorders of N-linked glycosylation, disorders of O-Linked protein glycosylation, disorders of lipid and glycosylphosphatidylinositol (GPI) anchor biosynthesis, disorders of multiple glycosylation pathways and disorders of deglycosylation. CDGs may also be classified as Types I (CDG-1) and II (CDG-II).

[0004] Phosphatidylinositol glycan anchor biosynthesis class N (PIGN) congenital disorder of glycosylation (PIGN-CDG), also referred to as multiple congenital anomalies-hypotonia-seizures syndrome type 1 (MCAHS1) or glycosylphosphatidylinositol biosynthesis defect 3 (GPIBD3), falls under the GPI anchor biosynthesis defects subclass of CDGs. It is an autosomal recessive disease caused by mutations in the PIGN gene, which encodes for phosphatidylinositol glycan anchor biosynthesis class N. The clinical presentation of PIGN-CDG varies among patients and is characterized by hypotonia, developmental delay, epilepsy, congenital and facial anomalies. Biallelic mutations in the PIGN gene are also associated with Fryns syndrome, an often-fatal condition characterized by congenital diaphragmatic hernia dysmorphic facial features, pulmonary hypoplasia and other various internal malformations, and a neurodevelopmental disorder, without congenital anomalies or dysmorphic features.

[0005] PIGN is involved in the first stage of GPI anchor protein (GPLAP) biosynthesis and catalyzes the attachment of ethanolamine phosphate (EtNP) to the first mannose residue in theDocket No. BONO.P0076WOGPI anchor. PIGN deficiency impairs GPI anchor biosynthesis, resulting in reduced expression of GPI anchor proteins on the cell surface. PIGN is also involved in regulating protein quality control within the endoplasmic reticulum. Over 150 proteins are anchored to the cell surface by GPI anchors (Kinoshita et al. 2020). GPI anchors are essential for localizing proteins into lipid rafts, to carry out their functions. Lipid rafts are specialized microdomains in cell membranes enriched in sphingolipids and cholesterol, and contribute to the organization and functionality of various cellular processes, including GPI anchored protein interactions and signaling events. GPI anchor proteins are are known to play important roles in various cellular processes, such as cell signaling and cell adhesion, and are critical during embryonic development and neurogenesis, further highlighted by the observation that the most prominent clinical symptoms of inherited GPI biosynthesis disorders are neurological (Bellai-Dussault et al. 2019).

[0006] Maintaining a balance of lipid raft components is critical for cellular homeostasis, and alterations in lipid raft components and structure is associated with various diseases (Grassi et al. 2020). There are two major mechanisms that regulate lipid raft dynamics are the availability of lipids that are critical for raft structure (i.e., cholesterol and sphingolipids) and changes in the cytoskeleton. Alterations in GPI anchor biosynthesis have been shown to impact sphingolipid levels (Mangold et al. 2012), and the synthesis and trafficking of sphingomyelins is regulated by cholesterol levels (Kim et al. 2023). Therefore, when GPI anchor biosynthesis and GPLAP expression are impaired, such as in PIGN-CDG and other GPI biosynthesis disorders, it also could disrupt the synthesis and balance of lipid raft components, such as cholesterol and sphingolipids, in turn disrupting lipid raft function.

[0007] There is no cure for PIGN-CDG or PIGN-related diseases, and treatment is mainly supportive and symptomatic, focusing on the management of individual symptoms as they arise. Restoring GPI anchor protein biosynthesis and increasing GPI anchor protein expression, rebalancing cholesterol and sphingolipid homeostasis, and restoring lipid raft function, may all or singularly contribute to a therapeutic effect in the disease.SUMMARY

[0008] Aspects of the present disclosure address needs in the art by providing methods and compositions for treating subjects with a CDG. Accordingly, disclosed herein are methods of treating a disease or disorder in a subject, methods of reversing a genetic disease, methods of treating a congenital disorders of glycosylation, methods of treating a disease associated withDocket No. BONO.P0076WOa PIGN deficiency, methods of restoring GPI anchor biosynthesis in a patient, and methods of treating any disorder disclosed herein In some aspects, the method comprises one or more steps including any of: administering, to a subject in need thereof, a therapeutically effective amount of one or more compounds selected from Compounds 1-48 listed in Tables A- J, administering, to a subject in need thereof, a pharmaceutical composition comprising one or more compounds selected from Compounds 1-48 listed in Tables A-J, administering, to a subject in need thereof, a second therapeutic composition, and determining whether the subject has a PIGN deficiency. In certain aspects, the method comprises administering, to a subject in need thereof, Compound 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 listed in Tables A-J, or any combination thereof. In certain aspects, the subject has, is suspected of having, is diagnosed with having, is genotyped to have, is haplotyped to have, or has symptoms of a congenital disorder of glycosylation (CDG)-related disease or disorder and / or phosphatidylinositol glycan class N (PIGN) -related disease or disorder.

[0009] In some aspects, the one or more compound comprises posaconazole, ziprasidone, voriconazole, and / or ascorbyl palmitate. In some aspects, the one or more compound comprises one or more azole antifungal compound selected from posaconazole, voriconazole, ketoconazole, climbazole, sertaconazole, clotrimazole, bifonazole, oxiconazole, terconazole, tioconazole, miconazole, butoconazole, and econazole. In some aspects, the one or more compound comprises one or more antifungal compound selected from clioquinol and broxyquinoline. In some aspects, the one or more compound comprises one or more antipsychotic compound selected from ziprasidone, haloperidol, and bromperidol. In some aspects, the one or more compound comprises one or more statin compound selected from cerivastatin and fluvastatin. In some aspects, the one or more compound comprises one or more plant-derived alkaloid compound selected from epiberberine and KukoaMine B. In some aspects, the one or more compound comprises one or more nutritional supplement compound selected from pantethine and tryptophan. In some aspects, the one or more compound comprises one or more PI3K inhibitor compound selected from PI- 103 and LY294002. In some aspects, the one or more compound comprises one or more compound selected from levocetirizine, tannic acid, docetaxel, D 4476, MK8745, BIM-46187 4 hydrochloride, PD0166285, Bobcat339, amuvatinib, AMD-070 hydrochloride, MLN0905, forchlorfenuron, uridine 5 ’-triphosphate tris salt (UTP), urapidil, acedapsone, D-(+)-trehalose dihydrate, isobutamben, pyrithione zinc, triciribine, amorolfine, and dyclonine. In some aspects, the one or more compound is capable of crossing the blood-brain barrier.Docket No. BONO.P0076WO

[0010] In some aspects, the CDG- and / or PIGN -related disease or disorder involves deficient or defective glycosylation of proteins or lipids. In some aspects, the CDG- and / or PIGN-related disease or disorder involves one or more disorder of N-linked glycosylation, O-linked protein glycosylation, lipid and / or glycosylphosphatidylinositol (GPI) anchor protein biosynthesis, glycosylation pathways, and / or deglycosylation. In some aspects, the CDG-and / or PIGN-related disease or disorder involves one or more GPI anchor protein (GPI-AP) biosynthesis disorder. In some aspects, the GPI anchor biosynthesis disorder comprises phosphatidylinositol glycan class N (PIGN)-CDG (also known as MCAHS1 or GPIBD3). In some aspects, the CDG- and / or PIGN-related disease or disorder comprises Type I (CDG-1) or Type II (CDG-II). In some aspects, the CDG- and / or PIGN-related disease or disorder comprises a PIGN-related disease. In some aspects, the PIGN-related disease involves one or more biallelic PIGN mutation. In some aspects, the PIGN-related disease comprises Fryns syndrome and / or one or more neurological disorder.

[0011] In some aspects, the treating a type of CDG- and / or PIGN-related disease or disorder comprises one or more of restoring GPI anchor biosynthesis, increasing GPI anchor protein expression, rebalancing cholesterol and / or sphingolipid homeostasis, restoring lipid raft function, and / or treating congenital disorders of glycosylation. In some aspects, the treating a type of CDG- and / or PIGN-related disease or disorder comprises reducing or otherwise ameliorating one or more symptoms associated with hypotonia, developmental delays, various movement disorders, intellectual disability, encephalopathy, epilepsy, chorea, nystagmus, and / or congenital and / or facial anomalies, and / or reduced cell surface expression of GPI-anchored proteins in the subject.

[0012] In some aspects, the one or more compound is in an oral formulation. In some aspects, the one or more compound is administered in combination with one or more additional therapies such as an antiseizure therapy. In some aspects, the the one or more compound and the one or more additional therapies are administered together in one administration or composition. In some aspects, the one or more compound and the one or more additional therapies are administered separately in more than one administration or more than one composition. In some aspects, the subject is a pediatric subject. In some aspects, the subject is a human.

[0013] Also disclosed is a method of treating a human PIGN-CDG patient, the method comprising administering a composition comprising posaconazole, ziprasidone, voriconazole, and / or ascorbyl palmitate to the patient, wherein the patient is determined to have a mutation in a PIGN gene. Also disclosed is a method of treating a human PIGN-CDG patient, the methodDocket No. BONO.P0076WOcomprising administering a composition comprising posaconazole to the patient, wherein the patient is determined to have a mutation in a PIGN gene. Also disclosed is a method of treating a human PIGN-CDG patient, the method comprising administering a composition comprising ziprasidone to the patient, wherein the patient is determined to have a mutation in a PIGN gene. Also disclosed is a method of treating a human PIGN-CDG patient, the method comprising administering a composition comprising voriconazole to the patient, wherein the patient is determined to have a mutation in a PIGN gene. Also disclosed is a method of treating a human PIGN-CDG patient, the method comprising administering a composition comprising ascorbyl palmitate to the patient, wherein the patient is determined to have a mutation in a PIGN gene. Also disclosed is a compound selected from Compounds 1-48 listed in Tables A- J, or a pharmaceutical composition comprising said compound, for use in treating a congenital disorder of glycosylation (CDG)-related disease or disorder and / or a phosphatidylinositol glycan class N (PIGN) -related disease or disorder. Also disclosed is a use of a compound selected from Compounds 1-48 listed in Tables A-J, or a pharmaceutical composition comprising said compound, in treating a congenital disorder of glycosylation (CDG)-related disease or disorder and / or a phosphatidylinositol glycan class N (PIGN) -related disease or disorder.

[0014] Also disclosed is a method for screening a candidate compound for efficacy in treating a congenital disorder of glycosylation (CDG)-related disease or disorder and / or a phosphatidylinositol glycan class N (PIGN) -related disease or disorder, the method comprising one or more steps including any of:generating a model of PIGN-CDG comprising MCD4 mutant cells in culture; exposing the cells to one or more concentrations of one or more candidate compound; anddetermining one or more therapeutic effect of the candidate compound on the model; wherein a therapeutic effect of the candidate compound indicates efficacy in treating a CDG- and / or PIGN-related disease or disorder. In some aspects, the model comprises a MCD4 mutant yeast strain. In some aspects, the one or more effect of the compound comprises improved and / or increased cell growth. In some aspects, the candidate compound comprises an antifungal, antipsychotic, statin, nutritional supplement, plant-derived alkaloid, and / or PI3K inhibitor compound.

[0015] Also disclosed are the following enumerated Aspects.Aspect 1 includes a method of treating a disease or disorder in a subject, the method comprising administering, to a subject in need thereof, a therapeutically effective amount of one or moreDocket No. BONO.P0076WOcompounds selected from Compounds 1-48 listed in Tables A-J, or a pharmaceutical composition comprising said compound, wherein the subject has a congenital disorder of glycosylation (CDG)-related disease or disorder and / or a phosphatidylinositol glycan class N (PIGN)-related disease or disorder.Aspect 2 depends upon Aspect 1, wherein the one or more compound comprises posaconazole, ziprasidone, voriconazole, and / or ascorbyl palmitate.Aspect 3 depends upon Aspect 1 or 2, wherein the one or more compound comprises one or more azole antifungal compound selected from posaconazole, voriconazole, ketoconazole, climbazole, sertaconazole, clotrimazole, bifonazole, oxiconazole, terconazole, tioconazole, miconazole, butoconazole, and econazole.Aspect 4 depends upon any one of Aspects 1 to 3, wherein the one or more compound comprises one or more antifungal compound selected from clioquinol and broxyquinoline. Aspect 5 depends upon any one of Aspects 1 to 4, wherein the one or more compound comprises one or more antipsychotic compound selected from ziprasidone, haloperidol, and bromperidol.Aspect 6 depends upon any one of Aspects 1 to 5, wherein the one or more compound comprises one or more statin compound selected from cerivastatin and fluvastatin.Aspect 7 depends upon any one of Aspects 1 to 6, wherein the one or more compound comprises one or more plant-derived alkaloid compound selected from epiberberine and KukoaMine B.Aspect 8 depends upon any one of Aspects 1 to 7, wherein the one or more compound comprises one or more nutritional supplement compound selected from pantethine and tryptophan.Aspect 9 depends upon any one of Aspects 1 to 8, wherein the one or more compound comprises one or more PI3K inhibitor compound selected from PI- 103 and LY294002.Aspect 10 depends upon any one of Aspects 1 to 9, wherein the one or more compound comprises one or more compound selected from levocetirizine, tannic acid, docetaxel, D 4476, MK8745, BIM-46187 4 hydrochloride, PD0166285, Bobcat339, amuvatinib, AMD-070 hydrochloride, MLN0905, forchlorfenuron, uridine 5 ’-triphosphate tris salt (UTP), urapidil, acedapsone, D-(+)-trehalose dihydrate, isobutamben, pyrithione zinc, triciribine, amorolfine, and dyclonine.Aspect 11 depends upon any one of Aspects 1 to 10, wherein the one or more compound is capable of crossing the blood-brain barrier.Docket No. BONO.P0076WOAspect 12 depends upon any one of Aspects 1 to 11, wherein the CDG and / or PIGN-related disease or disorder involves deficient or defective glycosylation of proteins or lipids.Aspect 13 depends upon any one of Aspects 1 to 12, wherein the CDG and / or PIGN-related disease or disorder involves one or more disorder of N-linked glycosylation, O-linked protein glycosylation, lipid and / or glycosylphosphatidylinositol (GPI) anchor protein biosynthesis, multiple glycosylation pathways, and / or deglycosylation.Aspect 14 depends upon any one of Aspects 1 to 13, wherein the CDG and / or PIGN-related disease or disorder involves one or more GPI anchor protein (GPI-AP) biosynthesis disorder.Aspect 15 depends upon Aspect 14, wherein the GPI anchor biosynthesis disorder comprises phosphatidylinositol glycan class N (PIGN)-CDG (also known as MCAHS1 or GPIBD3). Aspect 16 depends upon any one of Aspects 1 to 15, wherein the CDG and / or PIGN-related disease or disorder comprises Type I (CDG-I) or Type II (CDG-II).Aspect 17 depends upon any one of Aspects 1 to 16, wherein the CDG and / or PIGN-related disease or disorder comprises a PIGN-related disease.Aspect 18 depends upon Aspect 17, wherein the PIGN-related disease involves one or more biallelic PIGN mutation.Aspect 19 depends upon Aspect 17, wherein the PIGN-related disease comprises Fryns syndrome and / or one or more neurological disorder.Aspect 20 depends upon any one of Aspects 1 to 19, wherein treating a type of CDG and / or PIGN-related disease or disorder comprises one or more of restoring GPI anchor biosynthesis, increasing GPI anchor protein expression, rebalancing cholesterol and / or sphingolipid homeostasis, restoring lipid raft function, and / or treating congenital disorders of glycosylation.Aspect 21 depends upon any one of Aspects 1 to 20, wherein treating a type of CDG-related disease or disorder and / or PIGN-related disease or disorder comprises reducing or otherwise ameliorating one or more symptoms associated with hypotonia, developmental delays, various movement disorders, intellectual disability, encephalopathy, epilepsy, chorea, nystagmus, and / or congenital and / or facial anomalies, and / or reduced cell surface expression of GPI-anchored proteins in the subject.Aspect 22 depends upon any one of Aspects 1 to 21, wherein the one or more compound is in an oral formulation.Aspect 23 depends upon any one of Aspects 1 to 22, wherein the one or more compound is administered in combination with one or more additional therapies.Aspect 24 depends upon Aspect 23, wherein the one or more compound and the one or more additional therapies are administered together in one administration or composition.Docket No. BONO.P0076WOAspect 25 depends upon Aspect 23, wherein the one or more compound and the one or more additional therapies are administered separately in more than one administration or more than one composition.Aspect 26 depends upon any one of Aspects 1 to 25, wherein the subject is a pediatric subject.Aspect 27 depends upon any one of Aspects 1 to 26, wherein the subject is a human.Aspect 28 includes a method of treating a human PIGN-CDG patient, the method comprising administering a composition comprising posaconazole, ziprasidone, voriconazole, and / or ascorbyl palmitate to the patient, wherein the patient is determined to have a mutation in a PIGN gene.Aspect 29 includes a method of treating a human PIGN-CDG patient, the method comprising administering a composition comprising posaconazole to the patient, wherein the patient is determined to have a mutation in a PIGN gene.Aspect 30 includes a method of treating a human PIGN-CDG patient, the method comprising administering a composition comprising ziprasidone to the patient, wherein the patient is determined to have a mutation in a PIGN gene.Aspect 31 includes a method of treating a human PIGN-CDG patient, the method comprising administering a composition comprising voriconazole to the patient, wherein the patient is determined to have a mutation in a PIGN gene.Aspect 32 includes a method of treating a human PIGN-CDG patient, the method comprising administering a composition comprising ascorbyl palmitate to the patient, wherein the patient is determined to have a mutation in a PIGN gene.Aspect 33 includes a compound selected from Compounds 1-48 listed in Tables A-J, or a pharmaceutical composition comprising said compound, for use in treating a congenital disorder of glycosylation (CDG)-related disease or disorder and / or a phosphatidylinositol glycan class N (PIGN)-related disease or disorder.Aspect 34 includes the use of a compound selected from Compounds 1-48 listed in Tables A-J, or a pharmaceutical composition comprising said compound, in treating a congenital disorder of glycosylation (CDG)-related disease or disorder and / or a phosphatidylinositol glycan class N (PIGN) -related disease or disorder.Aspect 35 includes a method for screening a candidate compound for efficacy in treating a congenital disorder of glycosylation (CDG)-related disease or disorder and / or a phosphatidylinositol glycan class N (PIGN) -related disease or disorder, the method comprising: generating a model of PIGN-CDG comprising MCD4 mutant cells in culture; exposing the cells to one or more concentrations of one or more candidate compound; andDocket No. BONO.P0076WOdetermining one or more therapeutic effect of the candidate compound on the model; wherein a therapeutic effect of the candidate compound indicates efficacy in treating a CDG-related disease or disorder and / or a PIGN-related disease or disorder.Aspect 36 depends upon Aspect 35, wherein the model comprises a MCD4 mutant yeast strain.Aspect 37 depends upon Aspect 35 or 36, wherein the one or more effect of the compound comprises improved and / or increased cell growth.Aspect 38 depends upon any one of Aspects 35 to 37, wherein the candidate compound comprises an antifungal, antipsychotic, statin, nutritional supplement, plant-derived alkaloid, and / or PI3K inhibitor compound.

[0016] “Individual, “subject,” and “patient” are used interchangeably and can refer to either a human or non-human, such as primates, mammals, and vertebrates. In particular aspects, the subject is a human. The subject is of any age, gender, or race. The subject can be a patient, e.g., have or be suspected of having a disease or disorder disclosed herein. The subject may be undergoing or have undergone treatment. The subject may be asymptomatic. The subject may be a healthy individual desirous of prevention of a disease or condition.

[0017] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0018] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Any term used in singular form also comprise plural form and vice versa.

[0019] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” “(x and z) or y,” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an aspect or aspect.

[0020] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), “characterized by” (and any form of including, such as “characterized as”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.Docket No. BONO.P0076WO

[0021] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. The phrase “consisting of’ excludes any element, step, or ingredient not specified. The phrase “consisting essentially of’ limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments and aspects described in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”It is contemplated that any aspect discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0022] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0023] Use of the one or more sequences or compositions may be employed based on any of the methods described herein. Other aspects and embodiments are discussed throughout this application. Any embodiment or aspect discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa.

[0024] It is specifically contemplated that any limitation discussed with respect to one embodiment or aspect of the invention may apply to any other embodiment or aspect of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also aspects that may be implemented in the context of aspects discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary, Brief Description of the Drawings, Detailed Description, and / or Claims.

[0025] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.Docket No. BONO.P0076WOBRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0027] FIG. 1 illustrates the growth defect of an MCD4 temperature-sensitive yeast strain at 37°C in a 24-hour luminescent-based growth assay. Wild Type (“WT”); MCD4 temperaturesensitive yeast (“MCD4 mutant”). A PIGN (MCD4 in yeast) temperature- sensitive yeast mutant displays a severe growth defect at 37C compared to wild-type yeast

[0028] FIGS. 2A-2B illustrates drug repurposing screens that identify compounds which rescue the growth defect of PIGN mutant yeast. Depicted are the Z-score distribution of MCD4 mutant cells treated with 20 pM of compounds from the Small Molecule Discovery Center Drug collection (FIG. 2A) and the L4000 Bioactive library (FIG. 2B) compared to DMSO controls. Mean Z-score for each group is shown. Compounds 1, 2, 3 and 4 are potent rescuers of MCD4 mutant yeast growth. Compound-treated MCD4 mutant (“MCD4 mutant (compounds)”; left group); DMSO MCD4 mutant (“MCD4 mutant (DMSO)”; middle group); DMSO wild type (“WT (DMSO)”; right group); Compound 1 (“posaconazole”); Compound 2 (“ziprasidone”); Compound 3 (“voriconazole”); Compound 4 (“ascorbyl palmitate”).

[0029] FIG. 3 illustrates the dose-response of Compounds 1-4 in MCD4 mutant yeast compared to DMSO control in a 24-hour luminescent-based growth assay. All compounds demonstrate dose-dependent rescue of MCD4 (PIGN) mutant yeast growth compared to the negative control. Compound 1-treated MCD4 mutant (“Compound 1”); Compound 2-treated MCD4 mutant (“Compound 2”); Compound 3-treated MCD4 mutant (“Compound 3”); Compound 4-treated MCD4 mutant (“Compound 4”); DSMO MCD4 mutant negative control (“DMSO”). Compound 1 (“posaconazole”); Compound 2 (“ziprasidone”); Compound 3 (“voriconazole”); Compound 4 (“ascorbyl palmitate”).

[0030] FIGS. 4A-4B illustrates the imbalance of GPI anchor proteins and lipid raft dynamics in PIGN-CDG and the mechanisms by which therapeutic compounds may restore this balance (FIG. 4A). (FIG. 4B) illustrates the reported effects of azole antifungal compounds and antipsychotics on the cholesterol biosynthesis pathway.

[0031] FIGS. 5A-5D illustrates reduced expression of surface glycosylphosphatidylinositol (GPI)-anchored proteins CD73, CD90, CD55, and CD59 inDocket No. BONO.P0076WOPIGN-CDG patient fibroblast lines relative to healthy control fibroblasts, as determined by median fluorescence intensity (MFI).

[0032] FIGS. 6A-6D illustrates the effect of Ascorbyl Palmitate (10 pM) on PIGN-CDG patient and control fibroblast lines relative to DMSO vehicle. (FIG. 6A) Change in surface expression (MFI) of GPI-anchored proteins relative to DMSO vehicle. (FIG. 6B) Change in the percentage of CD73-positive cells. (FIG. 6C) Change in the percentage of double-positive (CD73+ / partner+) cells. (FIG. 6D) Representative bivariate plot illustrating an increase in CD73+ / CD59+cells in patient line PIGN-4 following treatment with Ascorbyl Palmitate.

[0033] FIGS. 7A-7D illustrates the effect of Voriconazole (10 pM) on PIGN-CDG patient and control fibroblast lines relative to DMSO vehicle. (FIG. 7A) Change in surface expression (MFI) of GPI-anchored proteins relative to DMSO vehicle. (FIG. 7B) Change in the percentage of CD73-positive cells. (FIG. 7C) Change in the percentage of double-positive (CD73+ / partner+) cells. (FIG. 7D) Representative bivariate plot illustrating an increase in CD73+ / CD90+cells in patient line PIGN-3 following treatment with Voriconazole.

[0034] FIGS. 8A-8D illustrates the effect of Ziprasidone (1 pM) on PIGN-CDG patient and control fibroblast lines relative to DMSO vehicle. (FIG. 8A) Change in surface expression (MFI) of GPI-anchored proteins relative to DMSO vehicle. (FIG. 8B) Change in the percentage of CD73-positive cells. (FIG. 8C) Change in the percentage of double-positive (CD73+ / partner+) cells. (FIG. 8D) Representative bivariate plot illustrating an increase in CD73+ / CD90+cells in patient line PIGN-1 following treatment with Ziprasidone.DETAILED DESCRIPTION

[0035] A congenital disorder of glycosylation (CDG) is associated with deficient or defective glycosylation of proteins or lipids. PIGN deficiency or PIGN-CDG, also known as MCAHS1, is a rare disease with no cure. It is an autosomal recessive disease caused by mutations in the PIGN gene. The PIGN gene catalyzes the attachment of ethanolamine phosphate to the GPI anchor during GPI anchor biosynthesis, prior to its attachment to a protein and remodelling of the GPI anchor protein. Insufficient levels of PIGN lead to impaired GPI anchor biosynthesis and reduced expression of GPI anchor proteins on the cell surface. PIGN deficiency impairs GPI anchor protein expression, disrupting various cellular processes mediated by GPI anchor proteins. PIGN deficiency may also lead to impaired protein quality control, resulting in impaired secretion of proteins and aggregate formation. PIGN-CDG belongs to the subclass of CDG known as GPI anchor biosynthesis defects and biallelicDocket No. BGNO.P0076WGmutations in PIGN also cause PIGN-related diseases such as Fryns syndrome and a neurodevelopmental disorder. More than 75 patients with PIGN-related diseases have been reported in the scientific literature.

[0036] Provided herein are methods for restoring GPI anchor biosynthesis and increasing GPI anchor protein expression, rebalancing cholesterol and sphingolipid homeostasis, and restoring lipid raft function in a patient in need thereof, comprising administering a therapeutically effective amount of a compound as described herein, or a combination of the compounds described herein, or a composition as described herein.

[0037] Aspects herein include the use of any of the disclosed compounds, and various combinations of, to treat PIGN CDG, PIGN-related diseases, and any of the 200 identified other CDG types.I. Compounds

[0038] Aspects herein include compounds useful for the treatment of diseases and disorders disclosed herein. The compounds described herein include the compounds of Tables A- J.Table A: Certain Active Compounds" " ""Docket No. BONO.P0076WOTable B: Azole Antifungals<Docket No. BONO.P0076WODocket No. BONO.POQ76WOTable C: AntifungalsTable D: AntipsychoticsDocket No. BONO.POQ76WO""Table E: StatinsTable G: Plant-Derived AlkaloidsDocket No. BONO.POQ76WOTable H: Nutritional SupplementsTable I: PI3K InhibitorsDocket No. BONO.P0076WOTable J: Other Compounds< > << <><<"""Docket No. BONO.P0076WO">Docket No. BONO.P0076WODocket No. BONO.P0076WO<Docket No. BONO.P0076WO>" "II. Therapeutic Methods

[0039] Aspects of the present disclosure are directed to methods comprising treatment of a subject suffering from, or suspected of having, a disease disclosed herein including a congenital disorder of glycosylation, a disease or disorder associated with PIGN deficiency.

[0040] Also disclosed are compositions and methods for therapeutic use. The compositions of the disclosure may be used for in vivo, in vitro, or ex vivo administration. The route of administration of the composition may be, for example, intravenous, intramuscular, intraperitoneal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, through inhalation, or through a combination of two or more routes of administration.Docket No. BONO.P0076WO

[0041] As used herein, “treat,” “treating,” or “treatment” or equivalent terminology refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the growth, development, or spread of one or more symptoms or manifestation of a disease or condition. As an example, the disease or condition may be any disease disclosed herein and the one or more symptoms may be, for example, symptoms associated with the disease or disorder. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of symptoms, diminishment of extent of disease, stabilized (z.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented. The results of treatment can be determined by methods known in the art.

[0042] As used herein, “prevent,” and similar words such as “prevented,” “preventing,” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition including any disclosed herein. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, “prevention” and similar words also includes reducing the intensity, effect, symptoms and / or burden of a disease or condition prior to onset or recurrence of the disease or condition. Prevention may be considered complete when onset of a disease, disorder, or condition has been delayed for a predefined period of time.III. Administration of Therapeutic Compositions

[0043] Aspects herein concern the administration of a therapy, which can comprise one or more of the compounds or compositions disclosed herein, to a patient, including any patient disclosed herein. The therapy provided herein may comprise administration of a combination of therapeutic agents, such as one or more of the compounds of Tables A-J. It is also specifically contemplated that one or more of the compounds or compositions disclosed hereinDocket No. BONO.P0076WOare not administered to the patient. The therapy may be administered in any suitable manner known in the art.

[0044] Aspects of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed.

[0045] The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some aspects, the compound(s) is / are administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0046] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some aspects, a unit dose comprises a single administrable dose.

[0047] In some aspects, a single dose of a compound or combination of compounds is administered. In some aspects, multiple doses of the compound(s) is / are administered. In some aspects, the compound or combination of compounds is administered at a dose of between 1 mg / kg and 5000 mg / kg. In some aspects, the compound or combination of compounds is administered at a dose of at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,Docket No. BONO.P0076WO206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 mg / kg.

[0048] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. The term “therapeutic benefit” or “therapeutically effective” as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of the disease or disorder. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.

[0049] In the practice in certain aspects, it is contemplated that doses in the range from 10 mg / kg to 200 mg / kg can affect the protective capability of these agents. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 pg / kg, mg / kg, pg / day, orDocket No. BONO.P0076WOmg / day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and / or on multiple days, weeks, or months.

[0050] In certain aspects, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 pM to 150 pM. In another aspect, the effective dose provides a blood level of about 4 pM to 100 pM.; or about 1 pM to 100 pM; or about 1 pM to 50 pM; or about 1 pM to 40 pM; or about 1 pM to 30 pM; or about 1 pM to 20 pM; or about 1 pM to 10 pM; or about 10 pM to 150 pM; or about 10 pM to 100 pM; or about 10 pM to 50 pM; or about 25 pM to 150 pM; or about 25 pM to 100 pM; or about 25 pM to 50 pM; or about 50 pM to 150 pM; or about 50 pM to 100 pM (or any range derivable therein). In other aspects, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 pM or any range derivable therein. In certain aspects, the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0051] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0052] It will be understood by those skilled in the art and made aware that dosage units of pg / kg or mg / kg of body weight can be converted and expressed in comparable concentration units of pg / ml or mM (blood levels). It is also understood that uptake is species and organ / tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.Docket No. BONO.P0076WOIV. General Pharmaceutical Compositions

[0053] In some embodiments, pharmaceutical compositions, which can comprise one or more of the Compounds listed in Tables A- J, are contemplated. It is also contemplated that certain Compounds listed in Tables A-J may be absent from the pharmaceutical composition. Different aspects may involve administering an effective amount of a composition to a subject. In some embodiments, an antibody or antigen binding fragment capable of binding to an antigen may be administered to the subject to protect against or treat a condition (e.g., a CDG). Additionally, such compositions can be administered in combination with an additional therapeutic agent (e.g., an antiseizure drug). Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0054] The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate. The preparation of a pharmaceutical composition comprising an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.

[0055] As used herein, “pharmaceutically acceptable carrier” includes any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer’s dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0056] The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated.Docket No. BONO.P0076WOSupplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.

[0057] The active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0058] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0059] The proteinaceous compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0060] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0061] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumeratedDocket No. BONO.P0076WOabove, as required, followed by filtered sterilization or an equivalent procedure. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0062] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.Examples

[0063] The following examples are included to demonstrate aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.Example 1: General MethodsStrains and plasmids

[0064] All strains used herein are in the S288C background. Strains were grown in Yeast extract, Peptone and Dextrose (YPD) media (RPI Research Product International) at 30°C unless otherwise noted. A previously published MCD4 temperature-sensitive strain (mcd4-174) was procured from EuroScarf (Gaynor et al., 1999). Standard procedures were followed for yeast maintenance.Growth assay

[0065] Cells from overnight cultures were resuspended in YPD media to ODeoo = 0.4, then serial diluted into 100 pL YPD media in 96-well plates at 10’1, 10’2, 10’3, 10’4and 10’5. Plates were incubated at 37°C for 24 hours. 50 pL of yeast cell suspension was transferred to a 96-well plate into with 50 pL of BacTiter-Glo (Promega), briefly vortexed and incubated for 5Docket No. BONO.P0076WOminutes. Luminescence readings were measured by a plate reader (ThermoFisher Varioskan Lux Multimode Microplate Reader).Drug screens

[0066] Drug screens were performed using a 2,177-compound collection (Small Molecule Discovery Center (“SMDC”) Drug collection), and the 8,387 L4000 Bioactive Compound library (TargetMol). 50 nL of compounds the from SMDC Drug collection, 200 nL of compounds from the L4000 Bioactive Compound library, or DMSO, were dispensed into 384-well plates using the Echo acoustic dispenser (Beckman Coulter Labcyte Echo 650) to achieve a final concentration of 20 pM. Cells from overnight cultures were resuspended in YPD media to ODeoo = 0.0025 and 25 pL of yeast cell suspensions were dispensed into the 384-well plates containing compounds or DMSO with a EL406 automated dispenser (Biotek). Plates were covered and incubated at 37°C for 24 hours. 25 pL of BacTiter-Glo was dispensed into the plates. Plates were briefly vortexed and then incubated for 25-40 minutes prior to reading (Envision Plate reader).Dose response assays

[0067] Dose response assays were performed with select compounds identified through the drug screens. Each compound was dispensed at eight concentrations in triplicate, beginning with 40 pM and following a two-fold dilution (40 pM, 20 pM, 10 pM, 5 pM, etc.), while DMSO was administered at a single concentration of 0.72% to control wells. Compounds or DMSO were dispensed into 384-well plate using the Echo acoustic dispenser (Beckman Coulter Labcyte Echo 650). Within the plate, 16 wells were allocated for the negative control (MCD4 mutant DMSO) and 16 wells for the positive control (wild type yeast DMSO). Cells from overnight cultures were resuspended in YPD media to ODeoo = 0.0025 and 25 pL of yeast cell suspensions were dispensed into the 384-well plate containing compounds or DMSO with a EL406 automated dispenser (Biotek). Plates were covered and incubated at 37°C for 24 hours. 25 pL of BacTiter-Glo was dispensed into the plates. Plates were briefly vortexed and then incubated for 25-40 minutes prior to reading (Envision Plate reader).Example 2: Yeast Mutant Model of PIGN-CDGMCD4 temperature sensitive mutant displays a severe growth defect

[0068] PIGN is involved in the addition of ethanolamine phosphate (EtNP) to the first mannose (Mani) in GPI biosynthesis. Mammalian PIGN and the yeast homolog, Mcd4 share 38% identity at the amino-acid level. MCD4 is an essential gene in yeast because the presenceDocket No. BONO.P0076WOof EtNP on Mani in the GPI intermediate is critical for the subsequent attachment of the third mannose residue by GpilO (Imhof et al. 2004). In yeast, the EtNP attached to Mani is removed by Cdcl which may facilitate the integration of GPI-APs in the yeast cell wall (Vazquez et al.2014). In mammalian cells, Manl-linked EtNP is a conserved side chain in mature GPI-Aps (Kinoshita et al. 2020), however, its function remains unknown. It is plausible that it may interact electrostatically with the hydroxyl group of cholesterol, potentially facilitating GPI-AP association within the cell membrane.

[0069] To model PIGN-CDG in yeast, a temperature- sensitive MCD4 allele (mcd4-174) harboring a single mutation in a conserved motif, G227E, previously identified and characterized (Gaynor et al. 1999), was used. At a non-permissive temperature of 38°C, the MCD4 mutant is defective in ER-to-Golgi transport of GPI anchor proteins, is required for GPI anchoring, secretes proteins into the medium, and accumulates GPI anchor precursors (Gaynor et al. 1999). We observed that the MCD4 mutant exhibits a severe growth defect compared to wild-type yeast at 37°C in a 24-hour luminescent-based growth assay (Fig. 1).

[0070] Biallelic truncating genotypes, predicted to result in a total loss of function of, and biallelic missense and mixed genotypes, which may retain some protein function, have been reported in patients with PIGN-CDG and related diseases (Bayat et al. 2022, Loong et al.2022). PIGN genotypes predicted to result in a total loss-of-function, representative of the MCD4 yeast mutant, appear to be significantly more strongly associated with congenital anomalies than the genotypes that may retain some functional protein (Loong et al. 2022).Example 3: Compounds for PIGNDrug screen in a yeast model of PIGN-CDG identifies chemical modifiers

[0071] Two high-throughput drug screens were conducted, utilizing both the SDMC Drug collection and the L4000 Bioactive Compound library, to screen the MCD4 mutant for chemical modifiers. The SDMC library consisted of 2,177 compounds, including the Pharmakon-1600 drug library (Microsource Discovery Systems), containing US and international drugs at clinical stage evaluation or approval, along with 577 drugs selected by the SMDC. The 8,387 compound L4000 Compound library consisted of FDA approved drugs, drugs in various stages of clinical development, bioactive tool compounds, and natural products. In each screen, the MCD4 mutant strain was screened in singlicate and in 384-well plates, with each plate containing 32 wells of the negative control (MCD4 mutant DMSO) and 32 wells of the positive controls (wild type yeast DMSO).Docket No. BONO.P0076WO

[0072] In each screen, the positive and negative controls exhibited a distinct separation of Z-scores, enabling the identification of compounds which rescued growth of the mutant strain (Fig. 2). Using a Z-score cut-off of -0.137 in the SMDC Drug screen (Fig. 2A), and -0.243 in the L4000 Bioactive screen (Fig. 2B), excluding pan-assay interference compounds, we identified 48 compounds that rescued the growth of the MCD4 mutant strain. It is hypothesized that these compounds may exert their therapeutic effects in PIGN-CDG by restoring GPI anchor biosynthesis and increasing GPI anchor protein expression, rebalancing cholesterol and / or sphingolipid homeostasis, and / or restoring of lipid raft function (Fig. 4A).

[0073] Compound 1 (Z-score = 0.023) and Compound 3 (Z-score =-0.034), are the triazole antifungal compounds, posaconazole and voriconazole, respectively (Fig. 2A). Azole antifungal compounds emerged as a prominent rescue class, with 11 additional compounds exceeding the Z-score thresholds identified as chemical modifiers of MCD4 mutant yeast. Azole antifungals are a first- line treatment for invasive fungal infections. They act by inhibiting the cytochrome P450-dependent enzyme 14a-lanosterol demethylase (CYP51) which catalyzes the conversion of lanosterol to ergosterol. Ergosterol, a sterol homologous to cholesterol, is a key component of fungal cell membranes. Azoles can inhibit human CYP51, which is involved in the cholesterol biosynthesis pathway (Strushkevich et al. 2010) (Fig. 4B). Some azoles, such as voriconazole, have been shown to modulate cholesterol metabolism in in vitro neuronal models (Hennegen et al. 2024) and impact lipid profiles in patients (Wu et al.2021).

[0074] Compound 2 (Z-score = -0.033) is the atypical antipsychotic, ziprasidone (Fig. 2A). Two additional compounds belonging to the drug class of typical antipsychotics, haloperidol and bromperidol, exceed the Z-score thresholds and were identified as chemical modifiers of MCD4 mutant yeast. Antipsychotics are a class of drugs used to treat psychiatric conditions such as schizophrenia, mania and irritability by modulating neurotransmitter activity in the brain. Antipsychotics frequently cause metabolic disorders in patients through effects that are independent of their receptor binding activity in the brain (Vantaggiato et al. 2019). Antipsychotics can disrupt lysosomal function, affect cholesterol trafficking and inhibit cholesterol biosynthesis (Fig. 4B). Ziprasidone has been shown to inhibit reactions in the cholesterol biosynthesis pathway catalyzed by A14-reductase and A7-reductase in vitro, leading to an increase of sterol intermediates and decrease of intracellular cholesterol (Canfran-Duque et al. 2013). Haloperiodol has been shown to impact lipid rafts and insulin signalling in neuronal cell lines (Sanchez-Wandelmer et al. 2010). By inhibiting cholesterol biosynthesis,Docket No. BONO.P0076WOazoles and antipsychotics may rebalance cholesterol homeostasis and restore lipid raft function in PIGN-CDG.

[0075] Compound 4 (Z-score = -0.106), is the compound ascorbyl palmitate, a fat-soluble form of Vitamin C. Vitamin C intake may improve metabolic function and reduce cholesterol levels in patients with diabetes or metabolic syndrome (Dludla et al. 2020), however little is known about how it may be regulating cholesterol metabolism.Chemical modifiers demonstrate a dose-dependent growth rescue effect in a yeast model ofPIGN-CDG

[0076] Dose response assays were performed to evaluate the effect of Compound 1, Compound 2, Compound 3 and Compound 4 on MCD4 mutant yeast growth. All compounds exhibited a dose- dependent rescue effect on MCD4 mutant yeast compared to the negative control. It is contemplated that compounds restore GPI anchor biosynthesis and increase GPI anchor protein expression, rebalance cholesterol and / or sphingolipid homeostasis and / or restore lipid raft function in PIGN-CDG and related diseases (Fig. 4A).Example 4: Validation of Chemical ModifiersFlow Cytometry Assay Methods

[0077] Patient-derived fibroblast cell lines from individuals with PIGN-CDG (PIGN-1, PIGN-2, PIGN-3, PIGN-4) and control fibroblast cell lines were analyzed for cell surface expression of the glycosylphosphatidylinositol-anchored proteins (GPI-APs) CD55, CD59, CD73, and CD90 by flow cytometry, both under basal conditions and following drug treatment.

[0078] Fibroblasts were cultured in Dulbecco’s Modified Eagle Medium (DMEM, low glucose, 1 g / L, with pyruvate) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% GlutaMAX, and 1% penicillin- streptomycin, and maintained at 37 °C in a humidified 5% CO2incubator. For baseline expression studies, patient and control fibroblast lines were grown until confluent and processed directly for surface staining. For drug treatment experiments, fibroblasts were seeded at 13.000 cells / cm2in T25 flask or 1.3 x 105cells per well into 6-well plates and allowed to adhere for 24 hours. Medium was then replaced with fresh medium containing 10 pM voriconazole, 10 pM ascorbyl palmitate, 1 - 10 pM ziprasidone, or with the equivalent final concentration of DMSO (0.1 % v / v). Drug- or vehiclecontaining media were refreshed daily for 48 hours.

[0079] At harvest, untreated and treated cells were detached using trypsin-EDTA, washed with phosphate-buffered saline (PBS), and stained for viability using Zombie NIR dye (1:2000Docket No. BONO.P0076WOdilution) for 10 minutes at room temperature in the dark. Following centrifugation, cells were stained in FACS buffer (PBS + 2.5% FBS + 0.1% sodium azide) with the following antibody panel: CD55-PE (1:16,000), CD59-RB780 (1:400), CD73-NovaFluor Yellow 700 (1:100), and CD90-APC (1:400). Cells were incubated for 10 minutes at room temperature in the dark, washed twice and fixed in 1% paraformaldehyde in FACS buffer.

[0080] Flow cytometric data (>10,000 events / s ample) were acquired on a Cytek Aurora 5L spectral flow cytometer and analyzed using FlowJo vlO.10.0. Data were gated to exclude debris and doublets (forward / side scatter, (FSC-A vs FSC-H, SSC-H vs SSC-A). Live cells were identified by exclusion of Zombie NIR-positive events. Bivariate plots comparing CD73 fluorescence intensity with CD55, CD59, and CD90 fluorescence were generated to assess coexpression patterns. Gates were set so that < 1% of events in the fluorescence-minus-one (FMO) control fell within the positive quadrants, and these positions were then applied to fully stained samples to ensure consistent and objective definition of positive and negative populations across all samples.Analysis

[0081] Baseline expression of GPLAPs in PIGN-CDG patient fibroblasts was determined as median fluorescence intensity (MFI) normalized to the mean of control lines for CD73, CD55, CD59, and CD90 (FIG. 5A-D). Rescue of GPLAP expression following compound treatment was evaluated by (i) calculating the percent change in MFI for CD73, CD55, CD59, and CD90 relative to the vehicle-treated baseline for each cell line (FIGS. 6A-8A); (ii) quantifying the change in percentage of CD73-positive cells relative to vehicle-treated baseline, representing expansion of the CD73+population (FIGS. 6B-8B); and (iii) by determining the change in percentage of double-positive (CD73+ / partner+) cells for each comarker (CD55, CD59, CD90) relative to the vehicle-treated baseline, representing coordinated restoration of multiple GPI-anchored proteins (FIGS. 6C-8C). Representative bivariate plots (FIGS. 6D-8D) illustrate treatment-dependent co-expression shifts, showing contraction of the CD73 / partner quadrant and expansion of CD73+ / partner+populations following compound treatment. Ziprasidone was evaluated at concentrations between 1 pM and 10 pM; data shown correspond to 1 pM, which represents the evaluated range (FIG. 8). Higher concentrations (> 5 pM) showed variable and negative effects on GPLAP expression across cell lines.

[0082] Given that the drug-response assay was performed as a single experimental run without biological replicates, changes were interpreted against published reproducibility benchmarks for flow cytometry Kalina et al., Cytometry A 97:137-147, 2020; Cossarizza etDocket No. BONO.P0076WOal., Eur J Immunol. 51:2708-3145, 2021). Accordingly, a treatment was defined as rescuing GPI- anchored-protein expression when it produced (i) a > 20 % increase in MFI over the vehicle-treated baseline and / or (ii) a > 5 percentage-point increase in the frequency of CD73+or CD73+ / partner+cells. These thresholds exceed commonly reported technical variability for intensity and population measurements and provide a conservative criterion for identifying treatment- associated improvements in surface GPI-AP expression.Results

[0083] PIGN-CDG fibroblasts exhibited reduced surface levels of CD73, CD55, CD59, and CD90 compared with control fibroblasts, consistent with impaired GPI-anchor biosynthesis and prior reports (Tiffault, I et al. 2017; Sidpra, J et al. 2024) (FIG 5).

[0084] Treatment with 10 pM ascorbyl palmitate produced increases in MFI for multiple GPI-APs and in positive-cell frequencies relative to vehicle-treated baselines, meeting predefined rescue thresholds in several patient fibroblast lines and showing generally concordant increases across markers (FIG. 6), consistent with a rescue of GPI-AP expression.

[0085] Treatment with 10 pM voriconazole produced patient- line-dependent effects on GPI-AP expression. Increases or stable MFI values were observed in PIGN-3, while other patient fibroblast lines showed mixed changes across individual GPI-APs relative to vehicle-treated baselines. CD73+cell frequencies increased in three lines, meeting predefined rescue thresholds in PIGN-2 and PIGN-3 but decreasing in PIGN-4. Changes in double-positive (CD73+ / partner+) populations were similarly variable, with PIGN-3 showing increases across all partners, consistent with a rescue of GPI-AP expression, whereas other lines exhibited mixed response patterns (FIG. 7).

[0086] Treatment with IpM ziprasidone produced patient- line-dependent effects on GPI-AP expression. The PIGN-1 line exhibited coordinated increases in MFI and positive-cell frequencies for multiple GPI-APs relative to vehicle-treated baselines, meeting predefined rescue thresholds across multiple analytical metrics, and consistent with a rescue of GPI-AP expression. Other patient lines exhibited reduced CD73 MFI or decreases in CD73+cell frequencies, and higher concentrations (> 5 pM) were associated with further down-modulation of several markers (FIG. 8).* * *Docket No. BONO.POQ76WO

[0087] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of certain aspects, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

Docket No. BGNO.P0076WGCLAIMSWhat is claimed is:

1. A method of treating a disease or disorder in a subject, the method comprising administering, to a subject in need thereof, a therapeutically effective amount of one or more compounds selected from Compounds 1-48 listed in Tables A- J, or a pharmaceutical composition comprising said compound, wherein the subject has a congenital disorder of glycosylation (CDG)-related disease or disorder and / or a phosphatidylinositol glycan class N (PIGN)-related disease or disorder.

2. The method of claim 1, wherein the one or more compound comprises posaconazole, ziprasidone, voriconazole, and / or ascorbyl palmitate.

3. The method of any one of claims 1 or 2, wherein the one or more compound comprises one or more azole antifungal compound selected from posaconazole, voriconazole, ketoconazole, climbazole, sertaconazole, clotrimazole, bifonazole, oxiconazole, terconazole, tioconazole, miconazole, butoconazole, and econazole.

4. The method of any one of claims 1 to 3, wherein the one or more compound comprises one or more antifungal compound selected from clioquinol and broxyquinoline.

5. The method of any one of claims 1 to 4, wherein the one or more compound comprises one or more antipsychotic compound selected from ziprasidone, haloperidol, and bromperidol.

6. The method of any one of claims 1 to 5, wherein the one or more compound comprises one or more statin compound selected from cerivastatin and fluvastatin.

7. The method of any one of claims 1 to 6, wherein the one or more compound comprises one or more plant-derived alkaloid compound selected from epiberberine and KukoaMine B.

8. The method of any one of claims 1 to 7, wherein the one or more compound comprises one or more nutritional supplement compound selected from pantethine and tryptophan.

9. The method of any one of claims 1 to 8, wherein the one or more compound comprises one or more PI3K inhibitor compound selected from PL103 and LY294002.

10. The method of any one of claims 1 to 9, wherein the one or more compound comprises one or more compound selected from levocetirizine, tannic acid, docetaxel, D 4476, MK8745, BIM-46187 4 hydrochloride, PD0166285, Bobcat339, amuvatinib, AMD-070 hydrochloride, MLN0905, forchlorfenuron, uridine 5 ’-triphosphate tris salt (UTP), urapidil, acedapsone, D-(+)-trehalose dihydrate, isobutamben, pyrithione zinc, triciribine, amorolfine, and dyclonine.Docket No. BGNO.P0076WG11. The method of any one of claims 1 to 10, wherein the one or more compound is capable of crossing the blood-brain barrier.

12. The method of any one of claims 1 to 11, wherein the CDG- and / or PIGN-related disease or disorder involves deficient or defective glycosylation of proteins or lipids.

13. The method of any one of claims 1 to 12, wherein the CDG- and / or PIGN-related disease or disorder involves one or more disorder of N-linked glycosylation, O-linked protein glycosylation, lipid and / or glycosylphosphatidylinositol (GPI) anchor protein biosynthesis, multiple glycosylation pathways, and / or deglycosylation.

14. The method of claim any one of claims 1 to 13, wherein the CDG- and / or PIGN-related disease or disorder involves one or more GPI anchor protein (GPI-AP) biosynthesis disorder.

15. The method of claim 14, wherein the GPI anchor biosynthesis disorder comprises phosphatidylinositol glycan class N (PIGN)-CDG (also known as MCAHS1 or GPIBD3).

16. The method of any one of claims 1 to 16, wherein the CDG- and / or PIGN-related disease or disorder comprises Type I (CDG-1) or Type II (CDG-II).

17. The method of any one of claims 1 to 17, wherein the CDG- and / or PIGN-related disease or disorder comprises a PIGN-related disease.

18. The method of claims 17, wherein the PIGN-related disease involves one or more biallelic PIGN mutation.

19. The method of claim 17, wherein the PIGN-related disease comprises Fryns syndrome and / or one or more neurological disorder.

20. The method of any one of claims 1 to 19, wherein treating a type of CDG- and / or PIGN-related disease or disorder comprises one or more of restoring GPI anchor biosynthesis, increasing GPI anchor protein expression, rebalancing cholesterol and / or sphingolipid homeostasis, restoring lipid raft function, and / or treating congenital disorders of glycosylation.

21. The method of any one of claims 1 to 20, wherein treating a type of CDG- and / or PIGN-related disease or disorder comprises reducing or otherwise ameliorating one or more symptoms associated with hypotonia, developmental delays, various movement disorders, intellectual disability, encephalopathy, epilepsy, chorea, nystagmus, and / or congenital and / or facial anomalies, and / or reduced cell surface expression of GPI-anchored proteins in the subject.

22. The method of any one of claims 1 to 21, wherein the one or more compound is in an oral formulation.

23. The method of any one of claims 1 to 22, wherein the one or more compound is administered in combination with one or more additional therapies.Docket No. BGNO.P0076WG24. The method of any one of claims 1 to 23, wherein the one or more compound and the one or more additional therapies are administered together in one administration or composition.

25. The method of any one of claims 1 to 24, wherein the one or more compound and the one or more additional therapies are administered separately in more than one administration or more than one composition.

26. The method of any one of claims 1 to 25, wherein the subject is a pediatric subject.

27. The method of any one of claims 1 to 26, wherein the subject is a human.

28. A method of treating a human PIGN-CDG patient, the method comprising administering a composition comprising posaconazole, ziprasidone, voriconazole, and / or ascorbyl palmitate to the patient, wherein the patient is determined to have a mutation in a PIGN gene.

29. A method of treating a human PIGN-CDG patient, the method comprising administering a composition comprising posaconazole to the patient, wherein the patient is determined to have a mutation in a PIGN gene.

30. A method of treating a human PIGN-CDG patient, the method comprising administering a composition comprising ziprasidone to the patient, wherein the patient is determined to have a mutation in a PIGN gene.

31. A method of treating a human PIGN-CDG patient, the method comprising administering a composition comprising voriconazole to the patient, wherein the patient is determined to have a mutation in a PIGN gene.

32. A method of treating a human PIGN-CDG patient, the method comprising administering a composition comprising ascorbyl palmitate to the patient, wherein the patient is determined to have a mutation in a PIGN gene.

33. A compound selected from Compounds 1-48 listed in Tables A- J, or a pharmaceutical composition comprising said compound, for use in treating a congenital disorder of glycosylation (CDG)-related disease or disorder and / or a phosphatidylinositol glycan class N (PIGN)-related disease or disorder.

34. Use of a compound selected from Compounds 1-48 listed in Tables A- J, or a pharmaceutical composition comprising said compound, in treating a congenital disorder of glycosylation (CDG)-related disease or disorder and / or a phosphatidylinositol glycan class N (PIGN)-related disease or disorder.Docket No. BGNO.P0076WG35. A method for screening a candidate compound for efficacy in treating a congenital disorder of glycosylation (CDG)-related disease or disorder and / or a phosphatidylinositol glycan class N (PIGN)-related disease or disorder, the method comprising:generating a model of PIGN-CDG comprising MCD4 mutant cells in culture; exposing the cells to one or more concentrations of one or more candidate compound; anddetermining one or more therapeutic effect of the candidate compound on the model; wherein a therapeutic effect of the candidate compound indicates efficacy in treating a CDG- and / or PIGN-related disease or disorder.

36. The method of claim 35, wherein the model comprises a MCD4 mutant yeast strain.

37. The method of claim 35 or 36, wherein the one or more effect of the compound comprises improved and / or increased cell growth.

38. The method of any one of claims 35 to 37, wherein the candidate compound comprises an antifungal, antipsychotic, statin, nutritional supplement, plant-derived alkaloid, and / or PI3K inhibitor compound.

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