WO2017040971A1

WO2017040971A1 - Methods of using inhibitors of pikfyve for the treatment of lysosomal storage disorders and neurodegenerative diseases

Info

Publication number: WO2017040971A1
Application number: PCT/US2016/050162
Authority: WO
Inventors: Shripad Bhagwat; Brett E. Crawford; Leonard Post
Original assignee: Biomarin Pharmaceutical Inc.
Priority date: 2015-09-03
Filing date: 2016-09-02
Publication date: 2017-03-09

Abstract

This disclosure features methods for treating lysosomal storage disorders (e.g. MPS) and/or neurodegenerative diseases (e.g., Alzheimers disease), which include administering to a subject (e.g., a human patient) in need of such treatment (e.g., identified as being in need of such treatment) an effective amount of one or more PIKfyve inhibitors, or a pharmaceutically acceptable salt thereof.

Description

METHODS OF USING INHIBITORS OF PIKFYVE FOR THE TREATMENT OF LYSOSOMAL STORAGE DISORDERS AND NEURODEGENERATIVE

DISEASE

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of United States Provisional Application No.

62/214,137, filed on September 3, 2015, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

This disclosure features methods for treating lysosomal storage disorders (e.g. Gangliosidosis, MPS, and other deficiencies in lysosomal enzymes) and/or

neurodegenerative diseases (e.g., Alzheimer's disease), which include administering to a subject (e.g., a human patient) in need of such treatment (e.g., identified as being in need of such treatment) an effective amount of one or more PIKfyve inhibitors, or a

pharmaceutically acceptable salt thereof. BACKGROUND

Lysosomal Storage Disorders

Lysosomes are organelles central to degradation and recycling processes in animal cells. Lysosomal storage disorders (LSDs) are inherited disorders that are thought to be caused by a deficiency of specific enzymes that are normally required for the breakdown of cellular metabolite substrates. If a specific lysosomal enzyme is not present in sufficient quantities, the normal breakdown of the substrate is incomplete or blocked. The cell is then unable to breakdown the material and it accumulates in the lysosomes of the cell. This accumulation disrupts the cell's normal functioning and gives rise to the clinical manifestations of LSDs. Nearly 50 types and subtypes of LSDs are known, and new types continue to be identified. Although the different types of LSDs are rare individually, when taken together they are estimated to affect about 1 in 7,700 births, making them a relatively common and significant health problem. Lysosomal storage disorders include diseases such as cholesteryl ester storage disease, gangliosidosis (e.g. GM1 gangliosidosis), Neimann-Pick disease (e.g. Neimann-Pick type C), and MPS disorders (e.g. MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS HID, MPS IVA, MPS IVB, MPS VI, MPS VII, or MPS IX). LSDs tend to be progressive, with the rate of progression, the severity of symptoms, and the organ systems affected varying between disorders and even within each disorder type. LSDs affect different body organs or systems including the skeleton and joints, eyes, heart, lungs, kidneys, skin, and frequently the central nervous system.

Lysosomal storage diseases, as well as certain neurodegenerative diseases, e.g., Alzheimer's and Parkinson's disease, share as a common feature the progressive accumulation of undigested macromolecules within the cell, either proteins that tend to form pathogenic aggregates, or intermediates of the cellular catabolism. This ultimately results in cellular dysfunction and clinical manifestations with variable association of visceral (hepatosplenomegaly), skeletal (joint limitation, bone disease and deformities), hematologic (anemia, lymphocyte vacuolization and inclusions), and neurological involvement, with often irreversible damage and invalidating or fatal consequences.

Phosphoinositide Signaling Lipids

Phosphoinositide (PtdlnsP) signaling lipids form part of the intracellular signaling mechanisms present in eukaryotic cells. These lipids are believed to be involved with cellular functions including signal transduction, cytoskeletal dynamics and membrane trafficking (see, e.g., Ho, C.Y., Traffic 2012 73, 1). PtdlnsPs are phosphorylated derivatives of phosphatidylinositol (Ptdlns). Ptdlns can be phosphorylated on the inositol ring to generate seven distinct PtdlnsPs, each varying in subcellular distribution and levels. At least five of these - Ptdlns, PtdIns4P, PtdIns(4,5)P2, Ptdlns 3-phosphate (PtdIns3P), and Ptdlns 3,5-bisphosphate (PtdIns(3,5)P2) - are almost ubiquitous in eukaryotes, in which they have many widely conserved functions (see Michell, R.H., FEBS Journal 2013 280, 6241). Generally, each PtdlnsP interacts and recruits a diverse set of cognate soluble protein effectors to the host membrane, thus providing that membrane with a specific and distinct set of functions (Id.). Among the more widely studied PtdlnsP is phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2], commonly referred to as PIP2, which predominates in the plasma membrane where it regulates processes like clathrin-mediated endocytosis {Id.). More recently, PtdIns(3,5)P2, which contributes <0.1% of the total Ptdlns pool, has been shown to govern a wide variety of cellular functions including endolysosome morphology, trafficking and acidification, autophagy, stress-induced signaling and ion channel activity {Id.). PtdIns(3,5)P2 is predominantly found on the yeast vacuole and in endolysosomes of higher eukaryotes.

It is believed that PtdIns(3,5)P2 is largely synthesized from PtdIns3P by a PtdIns(3)P 5-kinase (PIP5K3), known as PIKfyve in mammals and as Fabl in

Saccharomyces cerevisiae {Id.). PIKfyve is a FYVE finger-containing phosphoinositide kinase. The principal enzymatic activity of PIKfyve is to phosphorylate PtdIns3P to PtdIns(3,5)P2, and studies have been reported showing that PIKfyve is responsible for all of the intracellular PtdIns(3,5)P2 pool and indirectly responsible for contributing to the PtdIns3P pool (see Zolov, S.N., Proc. Nat. Acad. Sci. USA 2012 109, 17472). PIKfyve activity is also responsible for the production of PtdIns(5)P via phosphorylation of Ptdlns.

Cellular Functions of Phosphoinositide Signaling Lipids/PIKfyve

It is recognized that the formation and metabolism of PtdIns(3,5)P2 can affect late endosomes and lysosomes (and the vacuole in yeast), and that the functions of

PtdIns(3,5)P2 are focused primarily on supporting cell functions in which these organelles are known to play one or more roles (see Michell, R.H., FEBS Journal 2013 280, 6241). However, there is also evidence that PtdIns(3,5)P2 functions in many other cellular processes that could impact lysosomal storage diseases.

Ion Transport

It has been reported that PtdIns(3,5)P2 regulates the mucolipin transient receptor potential channels (TRPML; see, Ho, C.Y., Traffic 2012 13, 1). TRPML1-3 are Ca²⁺ channels that localize to endolysosomes. Dong reported the use of biochemical binding assays to demonstrate that PI(3,5)P2 binds directly to the N terminus of TRPMLl (Dong et al., Nat. Commun. 2010 38, 1). Using whole-endolysosomal patch-clamp recordings, Dong demonstrated that PI(3,5)P2 robustly activates TRPMLl in the endolysosome and that the effect of PI(3,5)P2 was specific to this particular PtdlnsP. Using a spectrum of yeast mutant strains with variable degrees of PI(3,5)P2 deficiency, Dong established a high-degree of correlation between the amount of vacuolar Ca²⁺ release and the levels of PI(3,5)P2. For a discussion and overview of the link between Ca²⁺ transport and phospohoinositides, see Shen, D. Bioessays 2011, 33, 418.

It is known that PtdIns(3,5)P2 is required for vacuolar acidification because fablA, vac7A, and vacl4A cells all have neutral vacuoles, a defect that reportedly appears to occur independently of vacuole enlargement (see Dove, et al., Biochem J. 2009 419, 1). Dove also reports that the levels of PtdIns(3,5)P2 in yeast, plant and some animal cells increase in response to various stresses, such as heat, hyperosmotic and alkaline stresses, and do so in a manner that suggests that this lipid may be a regulator of stress- driven cell responses {Id.). Dove further indicates that rapid Fablp/PtdIns(3,5)P2- dependent fragmentation/fission of the vacuole occurs as a result of transient

PtdIns(3,5)P2 accumulation in response to the stress {Id.). Doves also describes work that indicates an apparent link between PtdIns(3,5)P2 and the regulation of exocytosis. AMPAR Trafficking

McCartney reports that the levels of PtdIns(3,5)P2 in neurons increased during two distinct forms of synaptic depression, and inhibition of PIKfyve activity prevented or reversed induction of synaptic weakening; moreover, altering neuronal PtdIns(3,5)P2 levels was sufficient to regulate synaptic strength bidirectionally, with enhanced synaptic function accompanying loss of PtdIns(3,5)P2 and reduced synaptic strength following increased PtdIns(3,5)P2 levels. Inhibiting PtdIns(3,5)P2 synthesis was also found to alter endocytosis and recycling of AMPA-type glutamate receptors (AMPARs), implicating PtdIns(3,5)P2 dynamics in AMPAR trafficking. See McCartney et al., Proc Natl Acad Sci USA. 2014 111 4896. TFEB Activation

Most lysosomal genes are believed to be regulated by the transcription factor EB (TFEB), a master gene associated with lysosomal biogenesis. It encodes a transcription factor that coordinates expression of lysosomal hydrolases, membrane proteins and genes involved in autophagy. Under aberrant lysosomal storage conditions such as in lysosomal storage diseases, TFEB is translocated from the cytoplasm to the nucleus, resulting in the activation of its target genes. TFEB overexpression in cultured cells induced lysosomal biogenesis and increased the degradation of complex molecules, such as

glycosaminoglycans and the pathogenic protein that causes Huntington disease. See, e.g., Sardiello M, et al. Science 2009 325, 473-7.

Wang describes investigations aimed at understanding the role of PtdIns(3,5)P2 in TFEB activation (Wang et al., 2015). These investigations were carried using two different inhibitors of the PtdIns(3,5)P2-synthesizing enzyme, PIKfyve: YM201636 (38) and Apilimod. HEK293 cells that stably expressed TFEB were treated with YM201636 or Apilimod, and TFEB nuclear translocation was observed in both experiments.

Moreover, the extent of translocation in each case was comparable to that observed with Torin-1 treatment. Correspondingly, the treatment of Cos-1 cells with Apilimod, but not vacuolin-1, increased MLl -mediated lysosomal Ca²⁺ release. Previously, lysosome inhibitors have been found to induce TFEB nuclear translocation by reducing mTOR activity (Id). However, treatment with Apilimod did not cause an obvious inhibition of lysosomes or lysosomal membrane damage. In addition, p-S6K levels were only slightly reduced following YM201636 treatment and remained unchanged following Apilimod treatment. Similar results were observed for p-4E-BPl, another mTORC l effector. In contrast, rapamycin, which was unable to induce TFEB nuclear translocation, completely suppressed the level of p-S6K. Because PtdIns(3,5)P2 levels are potently reduced by both compounds, these results suggest that regulation of TFEB nuclear translocation during starvation may use a PtdIns(3,5)P2-dependent mechanism that is independent of mTOR. Because PtdIns(3,5)P2 is an endogenous agonist of MLl (Id.), starvation-induced MLl upregulation may occur as the result of a compensatory mechanism caused by a reduction in the levels of PtdIns(3,5)P2 (Id.). Golgi Apparatus Function

Phosphoinositides are also known to play important roles in Golgi traffic and structural integrity. See, e.g., Mayinger, et al., Seminars in Cell and Developmental Biology 2009 20, 793 and Dippold, et. al., Cell 2009 139, 337. Alterations in the highly regulated trafficking of the Golgi would likely alter the biosynthesis of many substrates that can accumulate in lysosomal diseases. Alternatively, altered Golgi or ER function could reduce the secretory flow of factors that ultimately transit to the lysosome for degredation and therefore alleviate the demand on a compromised lysosomal system.

Modulation of Endocytosis

Phosphoinositides are also known to play important roles in the regulation of endocytosis. By modulating the rate of endocytosis of lysosomal substrates, it is possible to alter the rate of accumulation of substrates in lysosomal diseases.

Regulation of Autophagy

Autophagy is a critical cellular function required for the turnover of damaged organelles and the turnover of long lived proteins. Modulation of autophagy could be useful in treatment of lysosomal storage diseases. Phosphoinositides play essential roles in the regulation of autophagy. Devereaux and others have shown that phosphoinositides are critical regulators of autophagosome biogenesis (Devereaux, PLOS One 2013)(Petiot, 2000, JBC). A critical role for PI(5)P in the regulation of autophagosomes has been demonstrated (Vicinanza, Molecular Cell 2015). Due to the complex biosynthetic pathway of phosphoinositides, inhibition of PIKfyve or other lipid kinases would be expected to alter the abundance or cellular localization of PI(5)P and therefore effect autophagosome biogenesis.

Biological Effects of modulation of PIKfyve in lysosomal disease

Through the diverse biological roles that phosphoinositides play in cellular biology, the modulation of the abundance of phosphoinositides through inhibition of PIKfyve can alter the biochemical progression of lysosomal storage diseases. This effect could be mediated through direct effects on PtdIns(3,5)P2 or through indirect effects on the synthesis of other Ptdlns such as PtdIns4P, PtdIns(4,5)P2, or Ptdlns 3-phosphate (PtdIns3P) that have interconnected biosynthetic and degradative pathways.

Approaches to modulate phosphoinositides

While inhibition of the kinases required for the synthesis of phosphoinositides would be the most direct therapeutic approach to modulating phosphoinositide levels. This could also be accomplished by inhibition of other members of protein complexes that are required for the function of the biosynthetic pathway. Many of the lipid kinases exist in complexes with other proteins that are required for function. For example, PIKfyve is known to exist in a complex with FIG4 (a phosphoinositide phosphatase) and Vacl4 (a scaffolding protein). Alteration of the function of either of the binding partners can alter the biological function of PIKfyve. Paradoxically, reduction in the phosphatase can reduce the abundance of PtdIns(3,5)P2.

Due to the complex and interconnected biosynthetic pathways of

phosphoinositide biosynthesis, there are likely multiple points of inhibition that will yield an alteration in phosphoinositides that would generate a therapeutic effect. Inhibition of PIKfyve and other lipid kinases could also be used to generate a similar effect.

Pathologies

PtdIns(3,5)P2 deficiency is typically manifested by the presence of massively enlarged, single-lobed vacuole in yeast and dilated endolysosomes in higher eukaryotes (see, Ho, C.Y., Traffic 2012 73, 1). PtdIns(3,5)P2 dysfunction is an underlying cause in certain forms of the human neuropathologies of Charcot-Marie-Tooth Type 4J (CMT4J) disease and amyotrophic lateral sclerosis {Id.). PtdIns(3,5)P2 signaling malfunction could also be potentially linked to myotubular dystrophy, mucolipidosis type IV, cardiac illness, diabetes, cancer and immunity {Id.). It has been reported that mice and humans that are unable to make PtdIns(3,5)P2 (or only able to make limited amounts) can sometimes lead to death during embryonic development in utero; or suffer from severely defective nervous systems, skeletal deformities and a rapidly increasing number of other developmental faults (see Michell, R.H., FEBS Journal 2013 280, 6241).

SUMMARY

This disclosure is based, in part, on the discovery that manipulation of

phosphatidylinositol metabolism can reduce progressive accumulation of undigested macromolecules within the cell. In particular, various classes of inhibitors of PIKfyve (PIP5K3) have been found to reduce lysosomal accumulation in cellular models of lysosomal storage diseases. To the best of our knowledge, the use of PIKfyve inhibitors for treatment of lysosomal storage disorders is not known in the art, and in fact the current knowledge in the field suggests that inhibition of PIKfyve could compromise TRPMLl function which would not be therapeutically beneficial for lysosomal storage disorders. Other methods for reduction of the progressive accumulation of undigested macromolecules within the cell by targeting, decreasing or inhibiting phosphatidylinositol metabolism with lipid kinase inhibitors are described herein.

In one aspect, this disclosure provides methods for treating lysosomal storage disorders (e.g. MPS) and/or neurodegenerative diseases (e.g., Alzheimer's disease), which include administering to a subject (e.g., a human patient) in need of such treatment (e.g., identified as being in need of such treatment) an effective amount of one or more compounds targeting, decreasing or inhibiting the activity of PIKfyve. In certain embodiments, the compound is a PIKfyve inhibitor, or a pharmaceutically acceptable salt thereof. In certain embodiments, the PIKfyve inhibitor is administered in an amount effective to modulate TFEB, e.g., in an amount effective to induce TFEB nuclear translocation.

Further provided are methods and compounds that manipulate

phosphatidylinositol metabolism. In some embodiments, the methods and compounds can be used to modulate (e.g., inhibit) the formation of, e.g., PtdIns(3,5)P2 or PtdIns(5)P, e.g., reduce PtdIns(3,5)P2 levels and/or PtdIns(5)P levels. In some embodiments, the methods and compounds can be used to disrupt PtdIns(3,5)P2 signaling. In some embodiments, the methods and compounds can be used to reduce or eliminate phosphorylation of PtdIns3P. In some embodiments, the methods and compounds can be used to target and/or decrease and/or inhibit the activity of PIKfyve.

In certain embodiments, the one or more PIKfyve inhibitors can be administered in the form of a pharmaceutical composition that further includes one or more

pharmaceutically acceptable excipients.

In one aspect, this disclosure provides methods of altering the biogenesis, function, or dynamics of an endosomal or lysosomal system in a subject in need thereof, which include administering to the subject an effective amount of a PIKfyve inhibitor, or a pharmaceutically acceptable salt thereof.

Embodiments can include, e.g., any one or more of the following features.

The PIKfyve inhibitor, or the pharmaceutically acceptable salt thereof can be administered to the subject in an amount effective to treat a lysosomal storage disorder or a neurological disorder; and/or to modulate phosphatidylinositol or the phosphorylated derivatives of phosphatidylinositol; and/or to inhibit biosynthesis of PtdIns5P or

PtdIns(3,5)P2; and/or to reduce amounts of PtdIns5P or PtdIns(3,5)P2; and/or to disrupt PtdIns5P or PtdIns(3,5)P2 signaling; and/or to modulate TFEB; and/or to induce TFEB nuclear translocation; and/or to activate TFEB; and/or to induce TFEB overexpression; and/or to reduce degradation of TFEB.

The PIKfyve inhibitor, or the pharmaceutically acceptable salt thereof can be administered to the subject in an amount effective to treat a lysosomal storage disorder.

The lysosomal storage disorder can be selected from the group consisting of activator deficiency/GM2 gangliosidosis, alpha-mannosidosis, aspartylglucosaminuria, cholesteryl ester storage disease, chronic hemodialy sis-related amyloidosis, chronic hexosaminidase A deficiency, cystinosis, Danon disease, Fabry disease, Farber disease, fucosidosis, 5 galactosialidosis, Gaucher Disease, GM1 gangliosidosis, Infantile Free Sialic Acid Storage Disease/ISSD, juvenile hexosaminidase A deficiency, Krabbe disease, lysosomal acid lipase deficiency, metachromatic leukodystrophy, MPS disorders, Mucolipidosis I/Sialidosis, Mucolipidosis IIIC, Mucolipidosis type IV, multiple sulfatase deficiency, Niemann-Pick Disease, Neuronal Ceroid Lipofuscinoses, CLN6 disease, Batten-Spielmeyer-Vogt/Juvenile NCL/CLN3 disease, Finnish Variant Late Infantile CLN5, Jansky-Bielschowsky disease/Late infantile CLN2/TPP1 Disease, Kufs/Adult- onset NCL/CLN4 disease, Northern Epilepsy/variant late infantile CLN8, Santavuori- Haltia/Infantile CLNl/PPT disease, Beta-mannosidosis, Pompe disease/Glycogen storage disease type II, pycnodysostosis, Sandhoff disease/GM2 Gangliosidosis, Schindler disease, Salla disease/Sialic Acid Storage Disease, Tay-Sachs/GM2 gangliosidosis, and Wolman disease.

The lysosomal storage disorder can be from the group consisting of activator deficiency/GM2 gangliosidosis, Fabry disease, Gaucher Disease, GM1 gangliosidosis, Krabbe disease, metachromatic leukodystrophy, MPS disorders, Mucolipidosis

I/Sialidosis, Mucolipidosis IIIC, Mucolipidosis type IV, Niemann-Pick Disease, Pompe disease, Sandhoff disease, and Tay-Sachs.

The lysosomal storage disorder can be MPS (mucopolysaccharidoses) disorder (e.g., selected from the group consisting of Hurler syndrome (MPS I H), Scheie syndrome (MPS I S), Hurler-Scheie syndrome (MPS I H-S), Hunter syndrome (MPS II), Sanfilippo A (MPS IIIA), Sanfilippo B (MPS IIIB), Sanfilippo C (MPS IIIC), Sanfilippo D (MPS HID)), Morquio A (MPS IVA), Morquio B (MPS IVB)), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), MPS IX (hyaluronidase deficiency), I- cell disease (ML II), and Pseudo-Hurler polydystrophy (ML III).

The MPS disorder is selected from the group consisting of Hurler syndrome (MPS I H), Scheie syndrome (MPS I S), Hurler-Scheie syndrome (MPS I H-S), Hunter syndrome (MPS II), Sanfilippo A (MPS IIIA), Sanfilippo B (MPS IIIB), Sanfilippo C (MPS IIIC), and Sanfilippo D (MPS HID)), Morquio A (MPS IVA), Morquio B (MPS IVB)), Maroteaux-Lamy syndrome (MPS VI), and Sly syndrome (MPS VII).

The PIKfyve inhibitor, or the pharmaceutically acceptable salt thereof is administered to the subject in an amount effective to treat a neurological disorder.

The neurological disorder can be selected from the group consisting of

Alzheimer's disease, Parkinson's disease, Charcot-Marie-Tooth Type 4J, amyotrophic lateral sclerosis, Huntington's disease, Creutzfeldt-Jakob disease, and Spinocerebellar Ataxia (SCA). The PIKfyve inhibitor is a compound having Formula (I), (II), (III), (IV), or (V), or a pharmaceutically acceptable salt thereof (e.g., a compound described in Tables 1-7, or a pharmaceutically acceptable salt thereof).

The PIKfyve inhibitor, or the pharmaceutically acceptable salt thereof can be administered in the form of a pharmaceutical composition, wherein the pharmaceutical composition includes one or more PIKfyve inhibitors, or the pharmaceutically acceptable salt thereof, and one or more excipients.

The subject can be an animal.

DETAILED DESCRIPTION

Definitions

To facilitate understanding of the disclosure set forth herein, a number of terms are defined below. Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, and pharmacology described herein are those well-known and commonly employed in the art. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

"Halo" refers to a halogen, or fluoro, chloro, bromo, or iodo.

"Alkyl" refers to a straight or branched saturated hydrocarbon radical having 1 to 10 carbon atoms. Illustrative examples include, but are not limited to, methyl, ethyl, n- propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylhexyl, n-heptyl, n-octyl, n-nonyl, and n- decyl. The term "Cx-_y alkyl" refers to alkyl groups that contain from x to y carbons, where x and y are each independently integers from 1 to 10, and y is greater than x.

"Alkoxy" refers to an -OR group where R is alkyl, as defined herein. Illustrative examples include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.

"Amino" refers to an - H2 group.

"Alkylamino" refers to an - HR group, where R is alkyl as defined herein. "Dialkylamino" refers to a - RR' radical where R and R' are each independently alkyl as defined herein.

"Aminoalkyl" refers to an alkyl group substituted with at least one amino.

"(Alkylamino)alkyl" refers to an alkyl group substituted with at least one alkylamino.

"(Dialkylamino)alkyl" refers to an alkyl group substituted with at least one dialkylamino.

The term "acceptable" with respect to a formulation, composition or ingredient, as used herein, means having no persistent detrimental effect on the general health of the subj ect being treated.

The terms "effective amount" or "therapeutically effective amount," refers to a sufficient amount of an agent, a compound, a composition, and/or a formulation being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated.

The term "excipient" or "pharmaceutically acceptable excipient" refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, carrier, solvent, or encapsulating material. In one embodiment, each component is " pharmaceutically acceptable" in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. See, e.g., Remington: The Science and Practice of

Pharmacy, 21st ed.; Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed. ; Rowe et al, Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed. ; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical

Preformulation and Formulation, 2nd ed. ; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009.

The term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable inorganic and organic salts of compounds disclosed herein. In certain embodiments, pharmaceutically acceptable salts are obtained by reacting a compound described herein, with an acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. In some instances, pharmaceutically acceptable salts are obtained by reacting a compound with a base to form a salt such as an ammonium salt, an alkali metal salt, such as a sodium or a potassium salt, an alkaline earth metal salt, such as a calcium or a magnesium salt, a salt of organic bases such as dicyclohexylamine, N- methyl-D-glucamine, tris(hydroxymethyl)methylamine, and salts with amino acids such as arginine, lysine, and the like, or by other methods previously determined.

The term "pharmaceutical composition" refers to a mixture of a compound described herein with other chemical components or excipients, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, or thickening agents. The pharmaceutical composition facilitates administration of the compound to a subject.

The term "subject" refers to a mammal, such as a mouse, guinea pig, rat, dog, or human. In certain embodiments, the subject is a human; or the subject is a human adult; or the subject is a human child.

The terms "treat," "treating," and "treatment," in the context of treating a disease or disorder, are meant to include alleviating or abrogating a disorder, disease, or condition, or one or more of the symptoms associated with the disorder, disease, or condition; or to slowing the progression, spread or worsening of a disease, disorder or condition or of one or more symptoms thereof, or a combination thereof.

PIKfyve Inhibitors

Various classes of compounds targeting, decreasing or inhibiting the activity of PIKfyve have been found to reduce lysosomal accumulation in cellular models of lysosomal storage diseases. These classes of PIKfyve inhibitors include compounds having a structure of Formulae (I) through (V) and the compounds delineated in Tables 1- 7. The compounds in the tables described herein show activity in Assay 1, where activity "A" represents an ICso of less than or equal to 1 μΜ ("A" < 1 μΜ), "B" represents an ICso greater than 1 μΜ to less than or equal to 3 μΜ (1 μΜ < "B" < 3 μΜ), and "C" represents an IC50 greater than 3 μΜ (3 μΜ < "C"). Further, the compounds in the tables described herein show PIKfyve inhibition, where "D" represents a Kd of less than or equal to 1 nM ("D" < 1 nM), "E" represents a Kd of greater than 1 nM to less than or equal to 10 nM (1 nM < "E" < 10 nM), and "F" represents Kd of greater than 10 nM to less than or equal to 60 nM (10 nM < "F" < 60 nM).

In some embodiments, the PIKfyve inhibitors have a structure of Formula (I):

Formula (I) wherein Q¹ and Q² are each independantly CH or N, where Q¹ and Q² are not both N; each R¹ is independently hydroxy, Ci-4 alkyl, or Ci-4 alkoxy; n is 0, 1, or 2; each R² is independently Ci-4 alkyl or Ci-4 alkoxy; m is 0 or 1; and pharmaceutically acceptable salts thereof.

In certain embodiments, PIKfyve inhibitors having a structure of Formula (I) can be selected from Table 1

Table 1

A-2 B F

In some embodiments, the PIKfyve inhibitors have a structure of Formula (II):

Formula (II) wherein Ar¹ is phenyl or pyridyl, with each optionally independently substituted with 1 or 2 Ci-4 alkoxy; Ar² is phenyl, pyridyl, or pyrimidyl with each optionally independantly substituted with halo, CM alkyl, Ci-4 alkoxy, or C(0) R^2aR² ; R^2a and R² are each independantly H or Ci-4 alkyl; and pharmaceutically acceptable salts thereof.

In certain embodiments, PIKfyve inhibitors having a structure of Formula (II) can be selected from Table 2.

Table 2

In some embodiments, the PIKfyve inhibitors have a structure of Formula (III):

Formula (III) wherein R¹ is hydroxy, Ci-4 alkoxy, or H(CO)R^la; R^la is phenyl or pyridyl, optionally substituted with amino, alkylamino, or dialkylamino; and pharmaceutically acceptable salts thereof.

In certain embodiments, PIKfyve inhibitors having a structure of Formula (III) can be selected from Table 3.

Table 3

In some embodiments, the PIKfyve inhibitors have a structure of Formula (IV):

Formula (IV)

wherein Ar is phenyl or pyridyl, with each optionally independently substituted with 1 or 2 alkyl, aminoalkyl, (alkylamino)alkyl, or (dialkylamino)alkyl; R¹ is hydrogen or alkyl; R² is hydrogen or halo; and pharmaceutically acceptable salts thereof.

In certain embodiments, PIKfyve inhibitors having a structure of Formula (IV) can be selected from Table 4.

Table 4

In some embodiments, the PIKfyve inhibitors have a structure of Formula (V):

Formula (V)

wherein R¹ and R² are each independently hydrogen or Ci-4 alkyl; R³ is hydrogen or Ci-3 alkylenemorpholino; and pharmaceutically acceptable salts thereof.

In certain embodiments, PIKfyve inhibitors having a structure of Formula (V) be selected from Table 5.

Table 5

In some embodiments, the PIKfyve inhibitors can be selected from Table 6.

Table 6

In some embodiments, the PIKfyve inhibitors can be selected from Table 7.

Table 7

In certain embodiments, the PIKfyve inhibitor compounds can be employed in the methods described herein in the free base form. In other embodiments, the PIKfyve inhibitor compounds can be employed in the methods described herein in a salt form, e.g., inorganic acid addition salts such as HBr, HC1, sulfuric, nitric, or phosphoric acid addition salts, or organic acid addition salts such as acetic, proprionic, pyruvic, malanic, succinic, malic, maleic, fumaric, tartaric, citric, benzoic, methanesulfonic, ethanesulforic, stearic or lactic acid addition salt. In still other embodiments, the PIKfyve inhibitor compounds can be employed in the methods described herein as an anhydrate or hydrate of a free form or salt, for example, a hemihydrate, monohydrate, dihydrate, trihydrate, quadrahydrate, pentahydrate; or a solvate of a free form or salt.

Compounds disclosed herein are commercially available or can be readily prepared from commercially available starting materials according to established methodology in the art of organic synthesis. General methods of synthesizing the compound can be found in, e.g., Stuart Warren and Paul Wyatt, Workbook for Organic Synthesis: The Disconnection Approach, second Edition, Wiley, 2010.

Pharmaceutical Compositions

In some embodiments, there is provided a pharmaceutical composition comprising a compound described herein and one or more pharmaceutically acceptable excipients. In some embodiments of the composition, optionally in combination with any or all of the above various embodiments, the composition is formulated for local or systemic delivery. Examples of such formulations are formulations for oral

administration, parenteral administration, topical administration, pulmonary

administration, or an implant. In some or any embodiments, the compound is according to any of the various embodiments described herein.

The pharmaceutical compositions are administered to a subject in need thereof by any route which makes the compound bioavailable. In one embodiment, the composition is a solid formulation adapted for oral administration. In another embodiment, the composition is a tablet, powder, or capsule; or the composition is a tablet. In one embodiment, the composition is a liquid formulation adapted for oral administration. In one embodiment, the composition is a liquid formulation adapted for parenteral administration. In another embodiment, the composition is a solution, suspension, or emulsion; or the composition is a solution. In another embodiment, solid form

compositions can be converted, shortly before use, to liquid form compositions for either oral or parenteral administration. These particular solid form compositions are provided in unit dose form and as such are used to provide a single liquid dosage unit. These and other pharmaceutical compositions and processes for preparing same are well known in the art. (See, for example, Remington: The Science and Practice of Pharmacy (D. B. Troy, Editor, 21st Edition, Lippincott, Williams & Wilkins, 2006).

The dosages may be varied depending on the requirement of the subject, the severity of the condition being treated and the particular compound being employed. Determination of the proper dosage for a particular situation can be determined by one skilled in the medical arts. The total daily dosage may be divided and administered in portions throughout the day or by means providing continuous delivery.

Methods of Treatment

In certain embodiments, provided herein is a method of treating or ameliorating a disease comprising administering to a subject in need of treatment a therapeutically effective amount of a compound targeting, decreasing or inhibiting the activity of PIKfyve. In some embodiments, the compound is a PIKfyve inhibitor. In some embodiments, the compound is a compound having any of one of Formulas (I), (II), (III), (IV), or (V) or a compound delineated in any one of Tables 1-7 optionally as a single stereoisomer or mixture of stereoisomers thereof and additionally optionally as a pharmaceutically acceptable salt thereof. In certain embodiments, the subject is a mammal, or the subject is a human.

In certain embodiments, the disease is selected from amyloid diseases (such as

Alzheimer's disease, Parkinson's disease, Huntington's disease, type 2 diabetes, diabetic amyloidosis and chronic hemodialysis-related amyloid), multiple sclerosis, and an MPS disorder (such as MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS HID, MPS IVA, MPS IVB, MPS VI, MPS VII, or MPS IX). In some embodiments, the diseases are autoimmune disorders (such as multiple sclerosis, rheumatoid arthritis, juvenile chronic arthritis, Ankylosing spondylitis, psoriasis, psoriatic arthritis, adult still disease, Becet syndrome, familial Mediterranean fever, Crohn's disease, leprosy, osteomyelitis, tuberculosis, chronic bronchiectasis, Castleman disease), or CNS disorders (such as spongiform encephalopathies (Creutzfeld- Jakob, Kuru, Mad Cow)).

In certain embodiments, there are provided methods for reducing the storage of material that is normally degraded in the lysosome and can abnormally accumulate in individuals with lysosomal storage diseases comprising administering to a subject in need of treatment a therapeutically effective amount of a compound targeting, decreasing or inhibiting the activity of PIKfyve. In some embodiments, the compound is a PIKfyve inhibitor. In some embodiments, the compound targeting, decreasing or inhibiting the activity of PIKfyve alters the biogenesis, function or dynamics of the endosomal or lysosomal systems in a way that reduces the abundance of the material abnormally stored in the lysosome in lysosomal storage diseases. In some embodiments, the compound targeting, decreasing or inhibiting the activity of PIKfyve alters the biogenesis, functions, or dynamics of the endoplasmic reticulum or Golgi apparatus in a way that reduces the abundance of the material abnormally stored in the lysosome in lysosomal storage diseases. In some embodiments, the compound targeting, decreasing or inhibiting the activity of PIKfyve alters the expression or function of cellular metabolic and catabolic pathways ways that reduce the abundance of the material abnormally stored in the lysosome in lysosomal storage diseases. In certain embodiments, provided herein are methods of treating or ameliorating a disease comprising administering to a subject in need of treatment a therapeutically effective amount of a compound that inhibits the biosynthesis of PtdIns(3,5)P2 in said subject. In some embodiments, the disease is a lysosomal storage disorder. In other embodiments, the disease is a neurological disorder.

In certain embodiments, provided herein are methods of treating or ameliorating a disease comprising administering to a subject in need of treatment a therapeutically effective amount of a compound that reduces the amount of PtdIns(3,5)P2 in said subject. In some embodiments, the disease is a lysosomal storage disorder. In other embodiments, the disease is a neurological disorder.

In certain embodiments, provided herein are methods of treating or ameliorating a disease comprising administering to a subject in need of treatment a therapeutically effective amount of a compound that disrupts PtdIns(3,5)P2 signaling in said subject. In some embodiments, the disease is a lysosomal storage disorder. In other embodiments, the disease is a neurological disorder.

In certain embodiments, provided herein are methods of treating or ameliorating a disease comprising administering to a subject in need of treatment a therapeutically effective amount of a compound that inhibits the biosynthesis of PtdIns(5)P in the subject. In some embodiments, the disease is a lysosomal storage disorder. In other embodiments, the disease is a neurological disorder.

In certain embodiments, provided herein are methods of treating or ameliorating a disease comprising administering to a subject in need of treatment a therapeutically effective amount of a compound that reduces the amount of PtdIns(5)P in said subject. In some embodiments, the disease is a lysosomal storage disorder. In other embodiments, the disease is a neurological disorder.

In certain embodiments, provided herein are methods of treating or ameliorating a disease comprising administering to a subject in need of treatment a therapeutically effective amount of a compound that disrupts PtdIns(5)P signaling in said subject. In some embodiments, the disease is a lysosomal storage disorder. In other embodiments, the disease is a neurological disorder. In certain embodiments, provided herein are methods of treating or ameliorating a lysosomal storage disorder or a neurological disorder comprising administering to a subject in need of treatment a PIKfyve inhibitor in an amount effective to modulate TFEB. In some embodiments, the disease is a lysosomal storage disorder. In other embodiments, the disease is a neurological disorder.

In certain embodiments, provided herein are methods of treating or ameliorating a lysosomal storage disorder or a neurological disorder comprising administering to a subject in need of treatment a PIKfyve inhibitor in an amount effective to induce TFEB nuclear translocation. In some embodiments, the disease is a lysosomal storage disorder. In other embodiments, the disease is a neurological disorder.

In certain embodiments, provided herein are methods of treating or ameliorating a lysosomal storage disorder or a neurological disorder comprising administering to a subject in need of treatment a PIKfyve inhibitor in an amount effective to activate TFEB. In some embodiments, the disease is a lysosomal storage disorder. In other embodiments, the disease is a neurological disorder.

In certain embodiments, provided herein are methods of treating or ameliorating a lysosomal storage disorder or a neurological disorder comprising administering to a subject in need of treatment a PIKfyve inhibitor in an amount effective to induce TFEB overexpression. In some embodiments, the disease is a lysosomal storage disorder. In other embodiments, the disease is a neurological disorder.

In certain embodiments, provided herein are methods of treating or ameliorating a lysosomal storage disorder or a neurological disorder comprising administering to a subject in need of treatment a PIKfyve inhibitor in an amount effective to reduce degradation of TFEB. In some embodiments, the disease is a lysosomal storage disorder. In other embodiments, the disease is a neurological disorder.

In certain embodiments, provided herein are methods of treating or ameliorating a disease comprising administering to a subject in need of treatment a therapeutically effective amount of a compound that reduces or eliminates phosphorylation of PtdIns3P in said subject. In some embodiments, the disease is a lysosomal storage disorder. In other embodiments, the disease is a neurological disorder. In certain embodiments, provided herein is a method of treating or ameliorating a lysosomal storage disorder or a neurological disorder comprising administering to a subject in need of treatment a therapeutically effective amount of a compound targeting, decreasing or inhibiting the activity of PIKfyve. In certain embodiments, the compound is a PIKfyve inhibitor. In certain embodiments, the disease is a lysosomal storage disorder. In other embodiments, the disease is a neurological disorder. In certain embodiments, the subject is a mammal, or the subject is a human.

Lysosomal storage disorders include, but are not limited to, activator

deficiency/GM2 gangliosidosis (also known as GM2 gangliosidosis, AB variant), alpha- mannosidosis, aspartylglucosaminuria, cholesteryl ester storage disease, chronic hemodialysis-related amyloidosis, chronic hexosaminidase A deficiency, cystinosis, Danon disease, Fabry disease, Farber disease, fucosidosis, galactosialidosis, Gaucher Disease (such as Type I, Type II, or Type III), GM1 gangliosidosis (such as infantile, late infantile, juvenile, adult, or chronic), Infantile Free Sialic Acid Storage Disease/ISSD, juvenile hexosaminidase A deficiency, Krabbe disease (such as infantile onset or late onset), lysosomal acid lipase deficiency (such as early onset or late onset), metachromatic leukodystrophy, MPS disorders, Mucolipidosis I/Sialidosis, Mucolipidosis IIIC,

Mucolipidosis type IV, multiple sulfatase deficiency, Niemann-Pick Disease (such as Type A, Type B, or Type C), Neuronal Ceroid Lipofuscinoses, CLN6 disease (such as atypical late infantile, late onset variant, early juvenile forms), Batten-Spielmeyer- Vogt/Juvenile NCL/CLN3 disease, Finnish Variant Late Infantile CLN5, Jansky- Bielschowsky disease/Late infantile CLN2/TPP1 Disease, Kufs/ Adult-onset NCL/CLN4 disease, Northern Epilepsy/variant late infantile CLN8, Santavuori-Haltia/Infantile CLN1/PPT disease, Beta-mannosidosis, Pompe disease/Glycogen storage disease type II, pycnodysostosis, Sandhoff disease/GM2 Gangliosidosis (such as adult onset, infantile, or juvenile forms), Schindler disease, Salla disease/Sialic Acid Storage Disease, Tay- Sachs/GM2 gangliosidosis, and Wolman disease.

In certain embodiments, the lysosomal storage disorder is an MPS disorder. MPS disorders (mucopolysaccharidoses) are caused by the inability to produce specific enzymes, which in turn leads to an abnormal storage of mucopolysaccharides. MPS disorders include conditions such as Hurler syndrome (MPS I H), Scheie syndrome (MPS I S), Hurler-Scheie syndrome (MPS I H-S), Hunter syndrome (MPS II), Sanfilippo syndrome (e.g. Sanfilippo A (MPS IIIA), Sanfilippo B (MPS IIIB), Sanfilippo C (MPS IIIC), and Sanfilippo D (MPS HID)), Morquio syndrome (e.g. Morquio A (MPS IV A) and Morquio B (MPS IVB)), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), MPS IX (hyaluronidase deficiency), I-cell disease (ML II), and Pseudo-Hurler polydystrophy (ML III).

Neurodegenerative diseases include, but are not limited to, the following:

Alzheimer's disease, Parkinson's disease, Huntington's disease, Creutzfeldt-Jakob disease, and Spinocerebellar Ataxia (SCA).

BIOLOGICAL EXAMPLES

The following describes ways in which the compounds described herein were tested to measure in vitro activity in cell-based assays. A person of ordinary skill in the art would know that variations in the assay conditions could be used to determine the activity of the compounds. See Nat. Chem Biol. 2012, 8, 197.

Assay 1: MPS Cellular Assay

Dermal fibroblasts from humans with an MPS disorder and from healthy donors are grown. Cells are expanded and kept in culture for analysis. Human foreskin fibroblasts can be obtained from the American Type Culture Collection (CRL-1634). Human dermal fibroblasts derived from biopsies of individuals are purchased from Coriell Institute: clinically healthy individuals, GM00408, GM00409, GM00200

(clinically unaffected sibling of an individual with metachromatic leukodystrophy), GM05659, GM08398, and GM15871 (clinically unaffected sibling of an individual with unclassified Ehlers-Danlos syndrome); heterozygous MPS I carriers, GM01392,

GM01393 and GM0003; homozygous individuals affected with MPS I, GM06214, GM11495, GM00034, GM01391 and GM01256; individuals with MPS II, GM01896, GM03181, GM00615 and GM00298; individuals with MPS IIIA, GM00879, GM00643, GM00934, GM06110 and GM00629; MPS IIIB, GM01426; MPS IIIC, GM05157; MPS HID, GM05093; and MPS VII, GM00121. All cells are grown in DMEM containing 50 U ml-1 penicillin [please check units], 50 μg ml-1 streptomycin [please check units], 2 mM glutamine and 10% FBS. Cells are seeded on 15 -cm-diameter tissue culture dishes, grown to confluence and maintained in culture for analysis. Sulfamidase activity in cell extracts is measured with 4-methylumbelliferyl-a-D-N-sulfoglucosaminide according the vendors instructions substituting Tris-acetate buffer (pH 6.5; Moscerdam).

Cells are treated with a compound of the invention at a concentration at or below 30 μΜ for 4 to 21 days. Cell monolayers are washed with phosphate buffered saline and detached by treatment with GIBCO trypsin-EDTA solution (Invitrogen). After centrifugation, the supernatant is removed and the cells are lysed in 0.5 mL of 0.1 N sodium hydroxide. The amount of protein is determined by bicinchoninic acid (BCA assay, Thermo Scientific). Glucosaminoglycans ("GAGs") are isolated by anion exchange chromatography and digested with glycosaminoglycan lyases (heparinases I, II, and III).

Enzymatically depolymerized GAG preparations are differentially mass labeled by reductive amination with [¹²C6]aniline. Briefly, heparan sulfate disaccharides (1-10 pmol) are dried down in a centrifugal evaporator and reacted with [¹²C₆]aniline or

[¹³C₆]aniline (15 pL, 165 pmol) and 15 pL of 1 M NaCNBft (Sigma-Aldrich) freshly prepared in dimethylsulfoxide/acetic acid (7:3, v/v) is added to each sample. Reactions are carried out at 37 °C for 16 h and then dried in a centrifugal evaporator. Unsubstituted amines are reacted with propionic anhydride (Sigma-Aldrich). Dried samples are reconstituted in 20 pL of 50% methanol, and 3 pL of propionic anhydride (Sigma- Aldrich, 23.3 pmol) is added. Reactions are carried out at ~23 °C for 2 h. Acylated disaccharides are subsequently aniline tagged as described above. Each sample is mixed with commercially available standard unsaturated disaccharides (Seikagaku), standard N- sulfoglucosamine, glucosamine-6-sulfate, N-acetylgalactosamine-4-sulfate and N- acetylgalactosamine-6-sulfate (Sigma-Aldrich), and/or synthesized a-L- idopyranosyluronate-(l,4)-2-N-acetyl-2-deoxy-a/p-D-glucopyranoside (I0S0). All standards are tagged with [¹³C6]aniline (Sigma-Aldrich). Samples are then analyzed by LC-MS using an LTQ Orbitrap Discovery electrospray ionization mass spectrometer (Thermo Scientific) equipped with quaternary HPLC pump (Finnigan Surveyor MS pump) and a reverse-phase capillary column as described in J. Biol. Chem. 2008,

283(48), 33674.

MPS II: Assay 1, as described above, was used to determine activity for MPS II using GM01896 cells. The cells were treated with a compound of the invention and the heparan sulfate disaccharide detected was [¹²C₆] aniline-tagged I2a4, 12a6, 12S0 and/or I2S6. Extracted ion chromatograms were obtained. Cell viability was monitored using alamar blue or ATP quantification. IC50's were calculated using curve fitting within the Chemlnnovation CBIS database system. Data is given in Table 2.

Assay 1 can be adapted for use in testing cellular activity of compounds of the invention for treating MPS I where the biomarker detected is I0a6, 10a4, IOalO, I0S0 and/or I0S6; MPS IIIA where the biomarker detected is SO, S0U0S6, S0U2A6, S0U2S0, S0U2S6, S6U0A6, S6U0S0, S6U0S6, S6U2A6, S6U2S0, and/or S6U2S6; MPSIIIB where the biomarker detected is A0U0A6, A0U0S6, A0U2A0, A0U2S0, and/or A0U2S6; MPS IIIC where the biomarker detected is H0U0S6, H0U2A6, H0U2S0, H0U2S6, H6U0A6, H6U0S0, H6U0S6, H6U2A6, H6U2S0, and/or H6U2S6; MPS HID where the biomarker detected is H6; and MPS VII where the biomarker detected is GOaO, G0a4, GOalO, G0a6, GOAO, G0A6, G0S0, and/or G0S6. Saccharide structure naming code is as described in Lawrence et al. Nature Methods 2008, 5(4), 291. Certain embodiments have been evaluated in Assay I in MPSIIIA cells and demonstrated activity in the range of about 0.2 μΜ to about 2 μΜ.

The compounds described herein have the ability to reduce heparan sulfate accumulation as shown by Assay 1 in which the compounds reduced biomarker(s) indicative of abnormal HS accumulation in MPS II cells. Certain embodiments have also been evaluated in MPS IIIA cells and demonstrated a reduction in biomarker(s) indicative of abnormal HS accumulation. A person of ordinary skill in the art would recognize that the compounds have the ability to modulate HS biosynthesis in general which enables the degradation of the heparan sulfate polymer and therefore are useful for treating diseases associated with abnormal HS accumulation. MPS IIIA Disease Mouse Model

MPS IIIA mice (Sgsh-/-) are obtained from Jackson Laboratory (B6.Cg-Sgsh) and were housed in Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-approved vivaria. MPS IIIA mice (see Bhaumik et al. Glycobiol 1999, 9, 1389) are injected with an amount (e.g., about 100 mg/kg/day) of a PIKfyve inhibitor.

GAGs are isolated by anion exchange chromatography and digested with glycosaminoglycan lyases (heparinases I, II, and III). Enzymatically depolymerized GAG preparations are differentially mass labeled by reductive amination with [¹²C6]aniline. Briefly, heparan sulfate disaccharides (1-10 pmol) are dried down in a centrifugal evaporator and reacted with [¹²C6]aniline or [¹³C6]aniline (15 [iL, 165 μιηοΐ) and 15 [iL of 1 M NaC BFL (Sigma-Aldrich) freshly prepared in dimethylsulfoxide/acetic acid (7:3, v/v) is added to each sample. Reactions are carried out at 37 °C for 16 h and then dried in a centrifugal evaporator. Unsubstituted amines are reacted with propionic anhydride (Sigma-Aldrich). Dried samples are reconstituted in 20 [iL of 50% methanol, and 3 μΐ_^ of propionic anhydride (Sigma-Aldrich, 23.3 μιηοΐ) is added. Reactions are carried out at ~23 °C for 2 h. Acylated disaccharides are subsequently aniline tagged as described above. Each sample is mixed with commercially available standard unsaturated disaccharides (Seikagaku), standard N-sulfoglucosamine, glucosamine-6-sulfate, N- acetylgalactosamine-4-sulfate and N-acetylgalactosamine-6-sulfate (Sigma-Aldrich), and/or synthesized a-L-idopyranosyluronate-(l,4)-2-N-acetyl-2-deoxy-a/p-D- glucopyranoside (I0S0). All standards are tagged with [¹³C6]aniline (Sigma-Aldrich). Samples are then analyzed by LC-MS using an LTQ Orbitrap Discovery electrospray ionization mass spectrometer (Thermo Scientific) equipped with quaternary HPLC pump (Finnigan Surveyor MS pump) and a reverse-phase capillary column as described in J. Biol. Chem. 2008, 283(48), 33674.

Substrate Reduction Therapy

The non-specific inhibitor of sulfation, sodium chlorate, is used for validation or substrate reduction therapy (SRT). 30-60 mM sodium chlorate inhibits the synthesis of PAPs, the sulfate donor used in all cellular sulfation reactions including heparan sulfate biosynthesis. Cells grown in the presence of sodium chlorate produce heparan sulfate with reduced sulfation. In certain instances, in vitro MPS models are based on measuring the accumulation of GAG fragments in cultured primary human fibroblast from MPS patients. In some instances, the GAGs that accumulate in MPS patients are much smaller than normal tissue GAGs and they lack a core protein on their reducing termini. Based on these features the in vitro MPS model is based on a method of tagging reducing ends of the GAGs with a detectable label and analyzing (i.e., detecting and/or measuring) the detectable labels using a device suitable for detecting the label (e.g., an HPLC with a fluorimeter).

PIKfyve Inhibition Assay

The compounds described herein were tested for PIKfyve inhibitory activity using the KdELECT Kinase Assay testing specifically for PIKfyve.

A known active binder of PIKfyve, PI-103, is immobilized in a well. A test compound is then added to the well followed by DNA-tagged PIKfyve. The test compound competes with the immobilized PI-103 to bind to PIKfyve. The amount of PIKfyve bound to the immobilized PI-103 is then measured via qPCR of the DNA tag.

In particular, Streptavidin coated magnetic beads treated with biotinylated PIKfyve ligands for 30 minutes at room temperature. The bead-bound ligands were blocked with excess biotin and washed with blocking buffer (SeaBlock from Pierce; 1% BSA, 0.0% Tween 20, 1 mM DTT) to remove unbound ligand and reduce non-specific binding. The assay is started by combining the bead-bound ligands, DNA-tagged

PIKfyve and test compound in binding buffer (20% SeaBlock, 0.17x PBS, 0.05% Tween 20, 6 mM DTT) in polystyrene 96-well plate, with a final volume of 135 μΐ. The plate was incubated for 1 hour at room temperature with shaking. The beads were washed with wash buffer (lx PBS, 0.05% Tween 20). The beads were then resuspended in elution buffer (lx PBS, 0.05% Tween 20, 0.5 uM non-biotinylated affinity ligand). The plate was incubated for 30 minutes at room temperature with shaking. The concentration of DNA- tagged PIKfyve bound to PI-103 was measured via qPCR. A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of altering the biogenesis, function, or dynamics of an endosomal or lysosomal system in a subject in need thereof, the method comprising administering to the subject an effective amount of a PIKfyve inhibitor, or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the PIKfyve inhibitor, or the pharmaceutically acceptable salt thereof is administered to the subject in an amount effective to treat a lysosomal storage disorder or a neurological disorder.

3. The method of claim 1, wherein the PIKfyve inhibitor, or the pharmaceutically acceptable salt thereof is administered to the subject in an amount effective to modulate phosphatidylinositol or the phosphorylated derivatives of phosphatidylinositol.

4. The method of claim 1, wherein the PIKfyve inhibitor, or the pharmaceutically acceptable salt thereof is administered to the subject in an amount effective to inhibit biosynthesis of PtdIns5P or PtdIns(3,5)P2.

5. The method of claim 1, wherein the PIKfyve inhibitor, or the pharmaceutically acceptable salt thereof is administered to the subject in an amount effective to reduce amounts of PtdIns5P or PtdIns(3,5)P2.

6. The method of claim 1, wherein the PIKfyve inhibitor, or the pharmaceutically acceptable salt thereof is administered to the subject in an amount effective to disrupt PtdIns5P or PtdIns(3,5)P2 signaling.

7. The method of claim 1, wherein the PIKfyve inhibitor, or the pharmaceutically acceptable salt thereof is administered to the subject in an amount effective to modulate TFEB. The method of claim 1 , wherein the PIKfy ve inhibitor, or the pharmaceutically acceptable salt thereof is administered to the subject in an amount effective to induce TFEB nuclear translocation.

The method of claim 1 , wherein the PIKfy ve inhibitor, or the pharmaceutically acceptable salt thereof is administered to the subject in an amount effective to activate TFEB.

The method of claim 1 , wherein the PIKfy ve inhibitor, or the pharmaceutically acceptable salt thereof is administered to the subject in an amount effective to induce TFEB overexpression.

The method of claim 1 , wherein the PIKfy ve inhibitor, or the pharmaceutically acceptable salt thereof is administered to the subject in an amount effective to reduce degradation of TFEB.

The method of claim any one of claims 1-11, wherein the PIKfy ve inhibitor, or the pharmaceutically acceptable salt thereof is administered to the subject in an amount effective to treat a lysosomal storage disorder.

The method of claim 12, wherein the lysosomal storage disorder is selected from the group consisting of activator deficiency/GM2 gangliosidosis, alpha- mannosidosis, aspartylglucosaminuria, cholesteryl ester storage disease, chronic hemodialysis-related amyloidosis, chronic hexosaminidase A deficiency, cystinosis, Danon disease, Fabry disease, Farber disease, fucosidosis, 5 galactosialidosis, Gaucher Disease, GM1 gangliosidosis, Infantile Free Sialic Acid Storage Disease/ISSD, juvenile hexosaminidase A deficiency, Krabbe disease, lysosomal acid lipase deficiency, metachromatic leukodystrophy, MPS disorders, Mucolipidosis I/Sialidosis, Mucolipidosis IIIC, Mucolipidosis type IV, multiple sulfatase deficiency, Niemann-Pick Disease, Neuronal Ceroid Lipofuscinoses, CLN6 disease, Batten-Spielmeyer- Vogt/Juvenile NCL/CLN3 disease, Finnish Variant Late Infantile CLN5, Jansky-Bielschowsky disease/Late infantile CLN2/TPP1 Disease, Kufs/Adult- onset NCL/CLN4 disease, Northern Epilepsy/variant late infantile CLN8, Santavuori-Haltia/Infantile CLN1/PPT disease, Beta-mannosidosis, Pompe disease/Glycogen storage disease type II, pycnodysostosis, Sandhoff disease/GM2 Gangliosidosis, Schindler disease, Salla disease/Sialic Acid Storage Disease, Tay-Sachs/GM2 gangliosidosis, and Wolman disease.

The method of claim 13, wherein the lysosomal storage disorder is selected from the group consisting of activator deficiency/GM2 gangliosidosis, Fabry disease, Gaucher Disease, GM1 gangliosidosis, Krabbe disease,

metachromatic leukodystrophy, MPS disorders, Mucolipidosis I/Sialidosis, Mucolipidosis IIIC, Mucolipidosis type IV, Niemann-Pick Disease, Pompe disease, Sandhoff disease, and Tay-Sachs.

The method of claim 12, wherein the lysosomal storage disorder is an MPS (mucopolysaccharidoses) disorder.

The method of claim 15, wherein the MPS disorder is selected from the group consisting of Hurler syndrome (MPS I H), Scheie syndrome (MPS I S), Hurler-Scheie syndrome (MPS I H-S), Hunter syndrome (MPS II), Sanfilippo A (MPS IIIA), Sanfilippo B (MPS IIIB), Sanfilippo C (MPS IIIC), Sanfilippo D (MPS HID)), Morquio A (MPS IV A), Morquio B (MPS IVB)), Maroteaux- Lamy syndrome (MPS VI), Sly syndrome (MPS VII), MPS IX (hyaluronidase deficiency), I-cell disease (ML II), and Pseudo-Hurler poly dystrophy (ML III).

The method of claim 16, wherein the MPS disorder is selected from the group consisting of Hurler syndrome (MPS I H), Scheie syndrome (MPS I S), Hurler-Scheie syndrome (MPS I H-S), Hunter syndrome (MPS II), Sanfilippo A (MPS IIIA), Sanfilippo B (MPS IIIB), Sanfilippo C (MPS IIIC), and Sanfilippo D (MPS HID)), Morquio A (MPS IV A), Morquio B (MPS IVB)), Maroteaux-Lamy syndrome (MPS VI), and Sly syndrome (MPS VII).

18. The method any one of claims 1-1 1, wherein the PIKfyve inhibitor, or the pharmaceutically acceptable salt thereof is administered to the subject in an amount effective to treat a neurological disorder.

19. The method of any one of claims 1 -1 1, wherein the neurological disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Charcot-Marie-Tooth Type 4J, amyotrophic lateral sclerosis, Huntington's disease, Creutzfeldt- Jakob disease, and Spinocerebellar Ataxia (SCA).

20. The method of any one of claims 1 -19, wherein the PIKfyve inhibitor is a compound having Formula (I), (II), (III), (IV), or (V), or a pharmaceutically acceptable salt thereof.

21. The method of any one of claims 1 -19, wherein the PIKfyve inhibitor is a compound described in Tables 1 -7, or a pharmaceutically acceptable salt thereof.

22. The method of any one of claims 1 -21 , wherein the PIKfyve inhibitor, or the pharmaceutically acceptable salt thereof is administered in the form of a pharmaceutical composition, wherein the pharmaceutical composition comprises one or more PIKfyve inhibitors, or the pharmaceutically acceptable salt thereof, and one or more excipients.

23. The method of any one of claims 1 -22, wherein the subj ect is an animal.

24. The method of any one of claims 1 -22, wherein the subj ect is a human.