WO2022261752A1 - Compounds and methods for the treatment of neurodegenerative disorders - Google Patents
Compounds and methods for the treatment of neurodegenerative disorders Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/164—Amides, e.g. hydroxamic acids of a carboxylic acid with an aminoalcohol, e.g. ceramides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/27—Esters, e.g. nitroglycerine, selenocyanates of carbamic or thiocarbamic acids, meprobamate, carbachol, neostigmine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
- C07C271/06—Esters of carbamic acids
- C07C271/40—Esters of carbamic acids having oxygen atoms of carbamate groups bound to carbon atoms of six-membered aromatic rings
- C07C271/42—Esters of carbamic acids having oxygen atoms of carbamate groups bound to carbon atoms of six-membered aromatic rings with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C271/44—Esters of carbamic acids having oxygen atoms of carbamate groups bound to carbon atoms of six-membered aromatic rings with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/78—Ring systems having three or more relevant rings
- C07D311/92—Naphthopyrans; Hydrogenated naphthopyrans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/04—Ortho-condensed systems
Definitions
- the present disclosure generally relates to neurodegenerative disorders, and more specifically to the treatment of neurodegenerative disorders associated with progranulin deficiency.
- Frontotemporal Dementia is a devastating neurodegenerative disorder and the third most common cause of dementia (1).
- FTD is a leading cause of early onset disorder, with patients usually diagnosed between 45 and 65 years of age (1, 2). Unlike other dementias, such as Alzheimer’s disease, FTD is a fast-progressing disease and affected patients usually die 2-5 years after clinical diagnosis.
- Advances in genetic screening techniques have identified many of the causative genes, revealing the complex heterogeneity of the underlying molecular mechanisms of the disease (1-3). Among them are autosomal-dominant heterozygous mutations in the GRN gene (4, 5) causing a severe reduction in the circulating levels of its product, progranulin (PGRN) (6).
- PGRN progranulin
- PGRN-deficient FTD is characterized by neuropathological inclusions of ubiquitinated TAR DNA Binding Protein 43 (TDP-43) in the cytoplasm (4, 5, 7-10).
- TDP-43 ubiquitinated TAR DNA Binding Protein 43
- ALS amyotrophic lateral sclerosis
- the present disclosure provides the following items 1 to 44:
- a method for treating a neurodegenerative disorder associated with progranulin (PGRN) deficiency in a subject comprising administering to the subject an effective amount of (i) a protein kinase C (PKC) inhibitor and/or (ii) an acetylcholinesterase (AChR) inhibitor.
- PKC protein kinase C
- AChR acetylcholinesterase
- a method for treating a neurodegenerative disorder associated with progranulin (PGRN) deficiency in a subject comprising administering to the subject an effective amount of an agent that increases sphingolipid (SL) levels in neural cells from the subject.
- PGRN progranulin
- SL sphingolipid
- PGRN deficiency is caused by a defect or mutation in the GRN gene.
- the neurodegenerative disorder associated with PGRN deficiency is frontotemporal dementia (FTD) or neuronal ceroid lipofuscinosis (NCL).
- An agent for use in treating a neurodegenerative disorder associated with progranulin (PGRN) deficiency in a subject wherein the agent is (i) a protein kinase C (PKC) inhibitor and/or (ii) an acetylcholinesterase (AChR) inhibitor.
- PKC protein kinase C
- AChR acetylcholinesterase
- agent 22 The agent for use of any one of items 17 to 21 , wherein the agent is rottlerin, rivastigmine or a combination thereof.
- SL sphingolipid
- PGRN progranulin
- agent for use of item 24 or 25, wherein the enzyme involved in SL metabolism is acid ceramidase (ASAH-1) or ceramide glucosyltransferase.
- RNA interference agent for use of any one of items 24 to 26, wherein the agent that reduces the expression or activity of an enzyme involved in SL metabolism is an RNA interference agent.
- FTD frontotemporal dementia
- NCL neuronal ceroid lipofuscinosis
- agent for use of any one of items 24 to 29, wherein the agent is for administration into the central nervous system of the subject.
- 31. Use of an agent for the manufacture of a medicament for treating a neurodegenerative disorder associated with progranulin (PGRN) deficiency in a subject, wherein the agent is (i) a protein kinase C (PKC) inhibitor and/or (ii) an acetylcholinesterase (AChR) inhibitor.
- PPC protein kinase C
- AChR acetylcholinesterase
- SL sphingolipid
- PGRN progranulin
- RNA interference agent any one of items 38 to 40, wherein the agent that reduces the expression or activity of an enzyme involved in SL metabolism is an RNA interference agent.
- FIGs. 1A-I show that loss of pgrn-1 results in distinct FTD-like phenotypes including lysosomal dysfunction.
- FIG. 1A pgrn-1 (tm985) mutant animals display age-dependent paralysis which can be rescued by the overexpression of full-length PGRN-1 ::RFP (Mantel-Cox test: N2 vs. pgrn-1 (tm985), 0.0001; N2 vs. pgrn-1 (tm985); pgrn-1::rfp,
- FIG. 1B shows that loss of pgrn-1 results in distinct FTD-like phenotypes including lysosomal dysfunction.
- FIG. 1A pgrn-1 (tm985) mutant animals display age-dependent paralysis which can be rescued by the overexpression of full-length PGRN-1 ::RFP (Mantel-Cox test: N2 vs. pgrn-1 (tm985)
- FIG. 1C Complete loss of pgrn-1 leads to a slight reduction in lifespan, whereas the re-expression of full-length PGRN-1 leads to an extension (Mantel-Cox test: N2 vs. pgrn-1 (tm985), *P ⁇ 0.05, N2 vs. pgrn-1 (tm985); pgrn-1 ::rfp, ***p ⁇ 0.001).
- Mantel-Cox test N2 vs. pgrn-1 (tm985), *P ⁇ 0.05, N2 vs. pgrn-1 (tm985); pgrn-1 ::rfp, ***p ⁇ 0.001).
- FIG. 1D pgrn-1 mutant animals display hypersensitivity to aldicarb, while rescue animals expressing PGRN-1 ::RFP display resistance (Mantel-Cox test: N2 vs. pgrn-1 (tm985), ****p ⁇ 0.0001; N2 vs. pgrn-1 (tm985); pgrn-1 ::rfp, ****P ⁇ 0.0001).
- FIG. 1E Representative images of pgrn-1 (tm985) coelomocyte lysosomes as visualized using an LMP-1::GFP reporter. FIGs.
- FIG. 1F-G Loss of pgrn-1 leads to an increased fluorescence intensity of LMP-1::GFP lysosomes (Student’s t test, ****P ⁇ 0.0001), but smaller ones as evidenced by a reduction in size (Student’s /test, ****P ⁇ 0.0001).
- FIG. 1H Treatment pgrn-1 mutant animals with the proteasome inhibitor, MG-132, leads to a dose-dependent decrease in lifespan (Mantel-Cox tests: Vehicle vs. 20 mM MG-132, *P ⁇ 0.05; Vehicle vs. 40 mM MG-132, ****p ⁇ 0.0001).
- FIG. 1H Treatment pgrn-1 mutant animals with the proteasome inhibitor, MG-132, leads to a dose-dependent decrease in lifespan (Mantel-Cox tests: Vehicle vs. 20 mM MG-132, *P ⁇ 0.05; Vehicle vs. 40 mM MG-132, ****p ⁇ 0.0001).
- Concanamycin A treatment results in a decrease in lifespan in pgrn-1 (tm985) animals but is not dose dependent (Mantel-Cox tests: Vehicle vs. 20 mM MG-132, ****P ⁇ 0.0001; Vehicle vs. 40 mM MG-132, *P ⁇ 0.05).
- FIGs. 2A-J show that mutations in pgrn-1 result in changes in autophagic flux.
- FIG. 2A LGG-1::mCherry reporter shows the formation of distinct puncta in the intestines of WT animals, whereas it remains diffuse in pgrn-1 mutants.
- FIGs. 2B-C Quantification of LGG-1::mCherry puncta at day 5 (FIG. 2B) and day 10 (FIG. 2C) adult animals shows a clear reduction in puncta in both pgrn- 1 mutant strains (Student’s t test. Day 5: WT vs. pgrn-1 (tm985), ****P ⁇ 0.0001, WT vs.
- FIG. 2D Treatment of day 5 pgrn-1 (tm985); lgg-1::mCherry animals with 50 nM Concanamycin A results in an increase in the number of LGG-1::mCherry puncta (Student’s t test, ****p ⁇ 0.0001).
- FIGs. 2G-H Both pgrn-1 mutations also result in an increased number of autophagosomes at day 1 (FIG. 2G), but also a decreased number of autolysosomes (FIG. 2H) (Student’s t test.
- Autophagosome count (FIG. 2G): WT vs. pgrn-1(tm985), 0.0001, WT vs. pgrn-1(gk123284), ****p ⁇ 0.0001.
- Autolysosome count (FIG. 2H): WT vs.
- FIGs. 2I-J At day 1, pgrn- 1 (tm985) animals had no significant change in LGG-1::GFP fluorescence intensity (FIG. 2I) or in the size of their lysosomes (FIG. 2J) (Student’s t test. Fluorescence intensity (FIG. 2I): WT vs. pgrn-1(tm985), n.s.. Lysosome size (FIG. 2J): WT vs. pgrn-1(tm985), n.s.)
- FIGs. 3A-F show that genetic manipulation of the sphingolipid (SL) biosynthetic pathway restores defects in pgrn-1 mutant animals.
- FIG. 3A RNAi-mediated knockdown of genes involved in SL biosynthesis, using ceramide as a reactant, partially restore LGG-1::mCherry puncta formation in the intestine of pgrn- 7-null animals (One-way ANOVA, treated vs. EV control, **p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001).
- FIG. 3A RNAi-mediated knockdown of genes involved in SL biosynthesis, using ceramide as a reactant
- FIGs. 3C-D Knockdown of certain SL biosynthetic genes restore LMP-1::GFP lysosomal intensity and lysosomal width phenotypes in pgrn-1(tm985) mutant animals (One-way ANOVA, treated vs.
- FIG. 3E RNAi knockdown rescues paralysis phenotype of pgrn-1 (tm985) mutant animals (Mantel-Cox test: EV vs. cerk-1 RNAi, n.s.; EV vs. sms- 1 RNAi, ****p ⁇ 0.0001; EV vs. sms-2 RNAi, ****p ⁇ 0.0001; EV vs. cgt-1 RNAi, ****p ⁇ 0.0001; EV vs. cgt-3 RNAi, ****p ⁇ 0.0001; EV vs. asah-1 RNAi, ****p ⁇ 0.0001; EV vs. asah-2 RNAi, ****p ⁇ 0.0001).
- FIG. 3F Summary table of RNAi knockdown effects on different phenotypes tested.
- FIG. 4 shows a schematic representation of SL genes implicated in PGRN-related defects.
- SL genes whose knockdown is able to restore one or more PGRN-related phenotypes are denoted by an asterisk (*), while the ones able to restore all tested phenotypes ( asah-1 and cgt-3) are indicated in grey.
- FIGs. 5A-D show that high-throughput drug screening identifies small molecules able to ameliorate PGRN deficiency in nematodes and mammalian cell lines.
- FIG. 5A Schematic representation of the high-throughput drug screen for PGRN-compensating drugs pgrn-1 (tm) mutant nematodes were treated with -3850 compounds in a phenotypic screen looking to restore motility. Hits were subsequently validated in Grn-deficient NSC34 cell lines to determine their effect in a mammalian model.
- FIG. 5B pgrn-1 (tm) nematodes have a swimming defect in liquid culture when compared to WT animals (Two-way ANOVA, WT vs.
- FIGs. 5C-D Survival of NSC34 cells treated with drug screen hits after 7 (FIG. 5C) and 14 days (FIG. 5D) of low serum growth (7 days, FIG. 5C: one-way ANOVA, drug treatment vs.
- FIG. 5D one-way ANOVA, drug treatment vs. Vehicle; XCT790, n.s.; rottlerin, **p ⁇ 0.01; PAPP, ***p ⁇ 0.001; Daurisoline, **p ⁇ 0.01; Rivastigmine, ****p ⁇ 0.0001; Pirenzepine, *p ⁇ 0.05. 14 days, FIG. 5D: one-way ANOVA, drug treatment vs. Vehicle; XCT790, n.s.; rottlerin, ***p ⁇ 0.001; PAPP, n.s.; Daurisoline, n.s.; Rivastigmine, ****p ⁇ 0.0001; Pirenzepine, n.s.).
- FIGs. 6A-F show that rottlerin and rivastigmine restore behavioral and molecular defects in vivo.
- FIGs. 6A-B When used individually rottlerin or rivastigmine treatment can restore lysosomal LMP-1::GFP fluorescence intensity (FIG. 6A) and lysosomal size (FIG. 6A) in day 5 pgrn-1 (tm985) animals (FIG. 6A, Student’s t test: Vehicle vs. 100 mM rivastigmine, *P ⁇ 0.05; Vehicle vs. 100 pM rottlerin, **P ⁇ 0.01.
- FIG. 6B Student’s t test: Vehicle vs.
- FIG. 6A Student’s t test: 100 pM rottlerin vs. 50 pM riv. + 50 pM rott. ****P ⁇ 0.0001; 100 pM rivastigmine vs. 50 pM riv. + 50 pM rott. ****P ⁇ 0.0001.
- FIG. 6B Student’s t test: 100 pM rottlerin vs.
- FIG. 6C Treatment of pgrn-1 (tm985) animals with 100 pM rottlerin or rivastigmine, or the combination, results in a strong suppression of paralysis (Mantel-Cox test: Vehicle vs. 100 pM rottlerin, ****p ⁇ 0.0001; Vehicle vs. 100 pM rivastigmine, ****P ⁇ 0.0001; Vehicle vs.
- FIG. 6D Both compounds, individually and combined, are able to increase levels of LGG-1::mCherry punctae in pgrn-1 mutant animals’ intestines (Student’s t test: Vehicle vs.
- FIGs. 6E-F Both compounds, individually and combined, are able to influence autophagosome (FIG.
- FIG. 6E shows autolysosome levels of the dual mCherry::LGG-1::GFP reporter in pgrn-1 mutant animals’ neurons
- FIG. 6E Student’s t test: Vehicle vs. 100 pM rivastigmine, ****P ⁇ 0.0001; Vehicle vs. 100 pM rottlerin, ****p ⁇ 0.0001; Vehicle vs. 50 pM riv. + 50 pM rott. ****P ⁇ 0.0001.
- FIG. 6F Student’s / test: Vehicle vs. 100 pM rivastigmine, *P ⁇ 0.05; Vehicle vs. 100 pM rottlerin, ***P ⁇ 0.001; Vehicle vs.
- FIG. 6E Student’s t test: 100 pM rottlerin vs. 50 pM riv. + 50 pM rott., n.s.; 100 pM rivastigmine vs. 50 pM riv. + 50 pM rott ****p ⁇ 0.0001.
- FIG. 6F Student’s t test: 100 pM rottlerin vs. 50 pM riv.
- FIG. 7A Quantitative gene expression reveals that pgrn-1(gk) animals show a marked reduction in pgrn-1 mRNA expression, while it could not be detected in pgrn-1(tm985) mutants (Student’s t test: N2 vs. pgrn-1 (tm985), ****P ⁇ 0.0001; N2 vs. pgrn-1 (gk), ****P ⁇ 0.0001).
- FIG. 7A Quantitative gene expression reveals that pgrn-1(gk) animals show a marked reduction in pgrn-1 mRNA expression, while it could not be detected in pgrn-1(tm985) mutants (Student’s t test: N2 vs. pgrn-1 (tm985), ****P ⁇ 0.0001; N2 vs. pgrn-1 (gk), ****P ⁇ 0.0001).
- FIG. 7A Quantitative gene expression reveals that pgr
- pgrn-1 (gk) animals display higher levels of age-dependent paralysis than N2 animals, and heterozygous pgrn-1(gk)/+ animals display the same levels of paralysis as homozygous mutant animals (Mantel-Cox test, N2 vs. pgrn-1 (gk), ****P ⁇ 0.0001; pgrn-1 (gk) vs. pgrn-1(gk)/+, n.s.).
- FIG. 7C Overexpression of pgrn-1 ::rfp in a pgrn-1 (tm985) background display similar levels of paralysis as N2 animals, whereas expressed in a wild-type background results in a slight decrease in paralysis (Mantel-Cox test: N2 vs. pgrn-1 (tm985); pgrn-1::rfp, n.s.; N2 vs. pgrn-1::rfp, *P ⁇ 0.05).
- FIG. 7D Lifespan in unaffected in pgrn-1(gk) animals (Mantel-Cox test, n.s.).
- FIG. 7E pgrn-1(gk) animals display heightened aldicarb hypersensitivity compared to N2 animals (Mantel-Cox test, ****p ⁇ 0.0001).
- FIGs. 7F-G Knockdown of pgrn-1 in N2 animals (FIG. 7F) does not result in an increase in paralysis, while the knockdown in neurons does (FIG. 7G) (Mantel-Cox test: RNAi in N2 (FIG. 7F), n.s.; neuronal RNAi (FIG. 7G), ****P ⁇ 0.0001).
- FIG. 7F-G Knockdown of pgrn-1 in N2 animals (FIG. 7F) does not result in an increase in paralysis, while the knockdown in neurons does (FIG. 7G) (Mantel-Cox test: RNAi in N2 (FIG. 7F), n.s.; neuronal RNAi (FIG. 7G), ****P ⁇ 0.0001).
- FIG. 7F-G Knockdown
- FIG. 7H Nematodes lacking pgrn-1 display an overactive food-seeking behavior and crawl off NGM plates faster than N2 controls; this phenotype is partially rescued by re-expression of pgrn-1::rfp (Mantel-Cox test: N2 vs. pgrn-1 (tm985), ****p ⁇ 0.0001; pgrn-1(tm985) vs. pgrn-1 (tm985); pgrn-1::rfp, **P ⁇ 0.01).
- FIG. 7I Treatment of N2 animals with either 20 or 40 mM MG-132 did not result in a decrease in lifespan (Mantel-Cox test: Vehicle vs.
- FIG. 7J Treatment of pgrn-1::rfp animals with 20 pM MG-132 did not result in a decrease in lifespan but treatment with 40 pM MG-132 did (Mantel- Cox test: Vehicle vs. 20 pM, n.s.; Vehicle vs. 40 pM, ****P ⁇ 0.001.).
- FIG. 7K Treatment of N2 animals with either 50 or 100 nM Concanamycin A did not result in a decrease in lifespan (Mantel-Cox test: Vehicle vs. 50 nM, n.s.; Vehicle vs. 100 nM, n.s.).
- FIG. 7K Treatment of N2 animals with either 50 or 100 nM Concanamycin A did not result in a decrease in lifespan (Mantel-Cox test: Vehicle vs. 50 nM, n.s.; Vehicle vs. 100 nM, n.s.).
- FIG. 8A Both pgrn-1 mutations did not alter LGG-1::mCherry puncta formation at day 1 of adulthood (Student’s t test: WT vs. pgrn-1 (tm985), n.s.; WT vs. pgrn-1 (gk123284), n.s.).
- FIG. 8B Starvation conditions induced autophagy in WT animals, but had no effect on autophagy in both pgrn- 1 mutants (Student’s t test, Fed vs. Starved: WT, ****p ⁇ 0.0001; pgrn-1(tm), n.s.; pgrn-1( gk), n.s.).
- FIG. 9A RNAi knockdown of the corresponding genes did not significantly affect formation of LGG-1::mCherry puncta in pgrn-1 (tm985) mutants.
- FIGs. 9B-C The genetic double mutant, sphk- 1(ok1097); pgrn-1(tm), had restored levels of autophagosomes (FIG. 9B) and autolysosomes (FIG. 9C) in animals’ neurons (One-way ANOVA, autophagosomes: WT vs. double mutant, n.s.; double 0.0001 ; autolysosomes: WT vs. double mutant, n.s.; double mutant vs.
- FIGs. 10A-C Validation of top 17 drugs from the liquid culture screen for their ability to influence lifespan in pgrn-1(tm) mutants (Mantel-Cox test, treatment condition vs. Vehicle control.
- FIGs. 10D-F Validation of top 17 drugs on the paralysis phenotype exhibited by pgrn-1(tm) nematodes (Mantel-Cox test, treatment condition vs. Vehicle control.
- A Resveratrol, ****p ⁇ 0.0001 ; Daurisoline, ****p ⁇ 0.0001; XCT790, ***p ⁇ 0.001; PAPP, ****p ⁇ 0.0001 ; Rivastigmine, ****p ⁇ 0.0001; Rottlerin, **p ⁇ 0.01.
- B 5-Methylhydandoin, n.s.; Bay-11, *p ⁇ 0.05; Tomatidine, n.s.; Eseroline, n.s.; Verruculogen, n.s.; Azatadine, ***p ⁇ 0.001; Pirenzepine, *p ⁇ 0.05.
- C Clonixin, n.s.; Methantheline, ***p ⁇ 0.001; Ethropropazine, ***p ⁇ 0.001; Vigabatrin, ****p ⁇ 0.0001).
- FIGs. 11A-B Dose-dependent testing of rottlerin and rivastigmine against lysosomal phenotypes (Student’s t test, A: DMSO control vs. treatment condition: 100 mM rivastigmine *p ⁇ 0.05, 50 pM rottlerin *p ⁇ 0.05, 100 pM Rottlerin ***p ⁇ 0.001 ; B: DMSO control vs. all treatment conditions, ****p ⁇ 0.0001).
- FIG. 11 C Donepezil and sotrastaurin restore paralysis phenotypes in pgrn-1(tm) animals (Mantel Cox test, ****p ⁇ 0.0001).
- FIGs. 12A-B depict the compounds identified from C. elegans drug screen and their properties.
- the present disclosure provides a method for treating a neurodegenerative disorder associated with progranulin (PGRN) deficiency in a subject, the method comprising administering to the subject an effective amount of an agent that increases sphingolipid (SL) levels in neural cells from the subject.
- PGRN progranulin
- SL sphingolipid
- the present disclosure also provides the use of an agent that increases SL levels in neural cells for the manufacture of a medicament for treating a neurodegenerative disorder associated with PGRN deficiency in a subject.
- the present disclosure also provides agent that increases SL levels in neural cells for use in treating a neurodegenerative disorder associated with PGRN deficiency in a subject.
- neurodegenerative disorder associated with progranulin refers to a neurodegenerative disorder in which a reduced expression and/or activity of PGRN (relative to the normal expression and/or activity of PGRN in a healthy subject) in certain cells from the central nervous system (CNS) leads to a dysfunction and/or death of CNS cells.
- the reduced expression and/or activity of PGRN may be due, e.g., to the absence of one or both copies of the gene encoding PGRN (GRN gene), mutation(s) in the gene encoding PGRN that affect(s) PGRN expression and/or mutation(s) in the PGRN protein that negatively affect(s) PGRN stability, secretion, localization and/or biological activity.
- GNN gene gene encoding PGRN
- mutation(s) in the gene encoding PGRN that affect(s) PGRN expression and/or mutation(s) in the PGRN protein that negatively affect(s) PGRN stability, secretion, localization and/or biological activity.
- GRN neuronal ceroid lipofuscinosis
- the neurodegenerative disorder associated with PGRN deficiency is FTD associated with PGRN deficiency. In another embodiment, the neurodegenerative disorder associated with PGRN deficiency is AD associated with PGRN deficiency. In another embodiment, the neurodegenerative disorder associated with PGRN deficiency is PD associated with PGRN deficiency. In another embodiment, the neurodegenerative disorder associated with PGRN deficiency is CBS associated with PGRN deficiency. In another embodiment, the neurodegenerative disorder associated with PGRN deficiency is LATE associated with PGRN deficiency. In another embodiment, the neurodegenerative disorder associated with PGRN deficiency is HS-aging associated with PGRN deficiency.
- the neurodegenerative disorder associated with PGRN deficiency is ALS associated with PGRN deficiency. In another embodiment, the neurodegenerative disorder associated with PGRN deficiency is NCL. In an embodiment, the methods and uses described herein further comprises identifying a patient with a PGRN deficiency. More than 65 mutations in the GRN gene have been identified in people with neurodegenerative disorder associated with PGRN deficiency such as FTD. One of the most common mutations is Arg493Ter or R493*, which creates a truncated PGRN protein due to a premature stop signal. The mutation A9D is associated with FTD (ubiquitin-positive).
- mutations include IVSODS G-C +5, GLN125TER, MET1THR, MET1ILE, 4-BP INS NT90, 4-BP DEL NT388, IVS8AS G-A +1, 1-BP DEL 998G, 1-BP INS 1145A, IVS7AS A-G -2, 2-BP DEL 675CA, IVS6AS A-G -2, 4-BP DEL 813CACT, 1- BP DEL 102C, 1-BP DEL 154A, 78C-T 3-PRIME UTR, and IVS6AS G-A -1.
- the mutations in the GRN gene are autosomal-dominant heterozygous mutations.
- Agents that may increase SL (e.g., ceramide) levels include SLs perse, precursors thereof, as well as enzymes involved in the synthesis of SLs (e.g., ceramide synthase, ceramide phosphatase, sphingomyelinase or glucosyl ceramidase).
- the levels of enzymes involved in the synthesis of SLs may be increased, for example, by administering the enzymes per se or a nucleic acid encoding the enzymes, or by modification of the promoter(s) and/or enhancer(s) regulating the expression of the genes encoding the enzymes to increase the endogenous expression of the enzymes.
- Such modification may be performed using gene-editing technologies such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases.
- ZFNs zinc-finger nucleases
- TALENs transcription activator-like effector nucleases
- CRISPR clustered regularly interspaced short palindromic repeat
- agents that may increase SL include inhibitors of enzymes involved in the metabolism of SLs (e.g., ceramidase, ceramide kinase, sphingomyelin synthase or ceramide glucosyltransferase/synthase).
- inhibitors include RNA interference (RNAi) agents such as antisense oligonucleotides, shRNAs, siRNAs and miRNAs that targets the mRNA encoding the above-noted enzymes, agents that interferes with the activity of the enzymes, for example agents interfering with binding of the enzyme to its substrate (e.g., small molecules, aptamers, antibodies, etc.).
- the agent is an inhibitor of a ceramidase (ASAH-1) or a ceramide glucosyltransferase (UGCG).
- siRNAs directed against human ASAH-1 are commercially available from various providers such as ThermoFisher Scientific (siRNA ID #119213, 119214, 119215, 14389, 14483 and 14573), Creative Biolabs (Cat. No. SIRGT04294WQ-LN), Santa Cruz Biotechnology Inc. (Cat. No. sc- 105032).
- Suitable RNAi agents directed against transcripts encoding human ASAH-1 may be designed by the skilled person based on the sequences of the transcripts (e.g., RefSeq NM_177924.5 and NM_004315.6).
- ceramidase (ASAH-1) inhibitors include carmofur (1-hexylcarbamoyl-5- fluorouracil), ARN 14988 (5-chloro-3-[(hexylamino)carbonyl]-3,6-dihydro-2,6-dioxo-1(2H)- pyrimidinecarboxylic acid, 2-methylpropyl ester), ARN14974 (6-(4-fluorophenyl)-2-oxo-N-(4- phenylbutyl)-3(2H)-benzoxazolecarboxamide), AC Inhibitor IV, Ceranib-1 , Ceranib-2 (3-[3-(4- methoxyphenyl)-1-oxo-2-propen-1-yl]-4-phenyl-2(1 H)-quinolinone), DP24a, SABRAC, structural analogs of ceramide such as oleoylethanolamide (also called N-oleylethanolamine, o
- ceramidase inhibitors are disclosed in Diamanti et al., Synthesis 2016, 48, 2739-2756.
- siRNAs directed against human UGCG are commercially available from various providers such as ThermoFisher Scientific (siRNA ID #111303, 111304, 111305, 13003, 13096 and 13186), Creative Biolabs (Cat. No. SIRGT20825WQ), Santa Cruz Biotechnology Inc. (Cat. No. sc-45404).
- Suitable RNAi agents directed against transcripts encoding human UGCG may be designed by the skilled person based on the sequences of the transcript (e.g., RefSeq NM_003358.2).
- ceramide glucosyltransferase (or glucosylceramide synthase) inhibitors include PDMP, PPMP, PPPP, Genz-123346, miglustat, eliglustat, the imino sugar N-butyldeoxynojirimycin (NBDNJ).
- NBDNJ imino sugar N-butyldeoxynojirimycin
- the present disclosure provides a method for treating a neurodegenerative disorder associated with PGRN deficiency in a subject, the method comprising administering to the subject an effective amount of (i) a protein kinase C (PKC) inhibitor, such as rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof; and/or (ii) an acetylcholinesterase (AChR) inhibitor, such as rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof.
- PLC protein kinase C
- AChR acetylcholinesterase
- the present disclosure also provides the use of (i) a protein kinase C (PKC) inhibitor, such as rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof; and/or (ii) an acetylcholinesterase (AChR) inhibitor, such as rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a neurodegenerative disorder associated with PGRN deficiency in a subject.
- PLC protein kinase C
- AChR acetylcholinesterase
- the present disclosure also provides an agent for use in treating a neurodegenerative disorder associated with PGRN deficiency in a subject, wherein the agent is (i) a protein kinase C (PKC) inhibitor, such as rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof; and/or (ii) an acetylcholinesterase (AChR) inhibitor, such as rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof.
- PKC protein kinase C
- AChR acetylcholinesterase
- the PKC inhibitor is a PKC- delta (PKC-d) inhibitor.
- AChR inhibitors include Tacrine, 7-methoxytacrine, Donepezil, Galantamine, carbamates ( e.g physostigmine, rivastigmine), huperzine A, protoberbrine alkaloids (e.g., berberine, palmatine, jatrorrhizine, epiberberine), and organophosphorus compounds (e.g., diisopropyl fluorophosphate, echothiophate, trichlorfon) (see, e.g., Colovic et al., Acetylcholinesterase Inhibitors: Pharmacology and Toxicology, Curr Neuropharmacol 2013, 11(3): 315-335).
- carbamates e.g physostigmine, rivastigmine
- huperzine A e.g., protoberbrine alkaloids (e.g., berberine, palmatine, jatrorrhizin
- the AChR inhibitor is a carbamate. In a further embodiment, the AChR inhibitor is rivastigmine, donepezil, or an analog thereof or a pharmaceutically acceptable salt thereof. In a further embodiment, the AChR inhibitor is rivastigmine, or an analog thereof or a pharmaceutically acceptable salt thereof.
- PKC inhibitors examples include rottlerin, calphostin C, 2,6-diamino-/ ⁇ /-([1-oxotridecyl)-2- piperidinyl] methyl) hexanamide, /V-benzyladriamycin- 14-valerate, resveratrol, indolocarbazoles (staurosporine and its analogs such as Go 6976, K252 compounds, 7-hydroxystaurosporine (UCN- 01), and 4’-/V-benzoylstaurosporine (midostaurin)), Maleimide-Based Inhibitors (GF 109203X, Ro SI- 7549, Ro 31-8220, enzastaurin and ruboxistaurin), Sotrastaurin, Balanol and analogs thereof, melittin, pseudosubstrate-derived peptide inhibitors of PKC (e.g., PKC19-36), Chelerythrine, Riluzole,
- the present disclosure provides a method for treating a neurodegenerative disorder associated with PGRN deficiency in a subject, the method comprising administering to the subject an effective amount of (i) rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof; and/or (ii) rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof.
- the present disclosure also provides the use of (i) rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof; and/or (ii) rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating a neurodegenerative disorder associated with PGRN deficiency in a subject.
- the present disclosure also provides agent for use in treating a neurodegenerative disorder associated with PGRN deficiency in a subject, wherein the agent is (i) rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof; and/or (ii) rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof.
- Rottlerin is a polyphenol natural product isolated from the Asian tree Mallotus philippensis that has the following structure:
- rottlerone Another known analog of rottlerin is rottlerone:
- Rivastigmine has the following structure:
- rivastigmine is neostigmine (Prostigmin) neostigmine Another analog of rivastigmine (S-l 26) is described in David et al. , Journal of Enzyme Inhibition and Medicinal Chemistry, Volume 36, 2021 - Issue 1, Pages 491-496:
- pharmaceutically acceptable salt refers to salts of compounds disclosed herein that are pharmacologically acceptable and substantially non-toxic to the subject to which they are administered. More specifically, these salts retain the biological effectiveness and properties of the compounds (e.g., rottlerin, rivastigmine, analogs thereof) and are formed from suitable non-toxic organic or inorganic acids or bases.
- these salts include acid addition salts of the compounds which are sufficiently basic to form such salts.
- Such acid addition salts include acetates, adipates, alginates, lower alkanesulfonates such as a methanesulfonates, trifluoromethanesulfonatse or ethanesulfonates, arylsulfonates such as a benzenesulfonates, 2-naphthalenesulfonates, or toluenesulfonates (also known as tosylates), ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cinnamates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates,
- the salts include base salts formed with an inorganic or organic base.
- Such salts include alkali metal salts such as sodium, lithium, and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; metal salts such as aluminium salts, iron salts, zinc salts, copper salts, nickel salts and a cobalt salts; inorganic amine salts such as ammonium or substituted ammonium salts, such as trimethylammonium salts; and salts with organic bases (for example, organic amines) such as chloroprocaine salts, dibenzylamine salts, dicyclohexylamine salts, dicyclohexylamines, diethanolamine salts, ethylamine salts (including diethylamine salts and triethylamine salts), ethylenediamine salts, glucosamine salts, guanidine salts, methylamine salts (including dimethylamine salts and trimethylamine salts),
- salts can be formed quite readily by those skilled in the art using standard techniques. Indeed, the chemical modification of a pharmaceutical compound (i.e. drug) into a salt is a technique well known to pharmaceutical chemists, (See, e.g., H. Ansel et. al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 196 and 1456-1457). Salts of the compounds disclosed herein may be formed, for example, by reacting the compound with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.
- an amount of acid or base such as an equivalent amount
- the compounds used in the methods and uses described herein are formulated in a pharmaceutical composition.
- Such pharmaceutical compositions typically include one or more pharmaceutically acceptable carriers or excipients, and may be prepared in a manner well known in the pharmaceutical art.
- Supplementary active compounds can also be incorporated into the compositions.
- the carrier/excipient can be suitable, for example, for intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal or pulmonary (e.g., aerosol) administration (see Remington: The Science and Practice of Pharmacy, by Loyd V Allen, Jr, 2012, 22 nd edition, Pharmaceutical Press; Handbook of Pharmaceutical Excipients, by Rowe et al., 2012, 7 th edition, Pharmaceutical Press).
- Therapeutic formulations are prepared using standard methods known in the art by mixing the active ingredient having the desired degree of purity with one or more optional pharmaceutically acceptable carriers, excipients and/or stabilizers.
- the pharmaceutical composition is formulated for administration into the central nervous system, e.g., to the brain.
- excipient has its normal meaning in the art and is any ingredient that is not an active ingredient (drug) itself. Excipients include for example binders, lubricants, diluents, fillers, thickening agents, disintegrants, plasticizers, coatings, barrier layer formulations, lubricants, stabilizing agent, release-delaying agents and other components. "Pharmaceutically acceptable excipient” as used herein refers to any excipient that does not interfere with effectiveness of the biological activity of the active ingredients and that is not toxic to the subject, i.e., is a type of excipient and/or is for use in an amount which is not toxic to the subject.
- the pharmaceutical composition of the present disclosure comprises excipients, including for example and without limitation, one or more binders (binding agents), thickening agents, surfactants, diluents, release-delaying agents, colorants, flavoring agents, fillers, disintegrants/dissolution promoting agents, lubricants, plasticizers, silica flow conditioners, glidants, anti-caking agents, anti-tacking agents, stabilizing agents, anti-static agents, swelling agents and any combinations thereof.
- binders binding agents
- thickening agents surfactants, diluents, release-delaying agents, colorants, flavoring agents, fillers, disintegrants/dissolution promoting agents, lubricants, plasticizers, silica flow conditioners, glidants, anti-caking agents, anti-tacking agents, stabilizing agents, anti-static agents, swelling agents and any combinations thereof.
- any suitable amount of the compound or pharmaceutical composition may be administered to a subject.
- the dosages will depend on many factors including the mode of administration.
- the amount of the compound contained within a single dose will be an amount that effectively prevent, delay or treat the neurodegenerative disorder without inducing significant toxicity.
- the appropriate dosage of the compound/composition will depend on the type of disease or condition to be treated, the severity and course of the disease or condition, whether the compound/composition is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the compound/composition, and the discretion of the attending physician.
- the compound/composition is suitably administered to the patient at one time or over a series of treatments. Preferably, it is desirable to determine the dose-response curve in vitro, and then in useful animal models prior to testing in humans.
- the present disclosure provides dosages for the compounds and compositions comprising same.
- the effective dose may be 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg/ 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, and may increase by 25 mg/kg increments up to 1000 mg/kg, or may range between any two of the foregoing values.
- a typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above.
- the treatment is sustained until a desired suppression of disease symptoms occurs.
- other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
- the optimal daily dose will be determined by methods known in the art and will be influenced by factors such as the age of the patient and other clinically relevant factors.
- patients may be taking medications for other diseases or conditions. The other medications may be continued during the time that the compound disclosed herein is given to the patient, but it is particularly advisable in such cases to begin with low doses to determine if adverse side effects are experienced.
- Genotyping of deletion mutants was done by genomic PCR, whereas genotyping of point mutations was done by high-resolution melting (HRM) using HRM MeltDoctor reagents (Applied Biosystems) and analyzed on HRM software (Applied Biosystems). Verification by Sanger sequencing was performed by Genome Quebec (McGill University). Heterozygous animals were obtained by crossing homozygous mutants with wild-type N2 animals; the progeny from fertilized hermaphrodites were used and immediately frozen in lysis buffer after use for confirmation of their genotype by either PCR or HRM.
- aqueous phase was collected, and extraction was completed using the RNeasy Mini Kit (Qiagen).
- cDNA was synthesized using the Superscript VILO cDNA Synthesis Kit (Invitrogen), and gene expression to quantify pgrn-1 transcript levels was performed using TaqMan probes and standard TaqMan reagents (both probes and reagents were purchased from Applied Biosystems) act-5 was chosen as the housekeeping gene.
- Gene expression assays were run on a QuantStudio 7 Flex (Applied Biosystems) instrument and data analysis was done using QuantStudio Real-Time PCR software.
- Worms were age-matched, washed 3 times in M9 to remove excess bacteria, and placed on NGM plates without bacterial food. Worms were counted daily for the number of worms remaining on the plate, as well as the dead worms found stuck to the side of the plate. Animals that disappeared were censored from statistical analyses. Aldicarb Assays
- Lysosome morphology tests For this assay, only lysosomes from the posterior coelomocytes were considered for this assay as there was minimal obstruction from other background tissues and intestinal fluorescence. Images were taken using the same camera settings across all replicates and were then analyzed in ImageJ. For lysosome intensity analyses, the background signal was subtracted from the lysosomal signal.
- NSC34 cells expressing reduced levels of PGRN were plated in 6 well plates with 20000 cells per well and cultured in DMEM with 10% fetal bovine serum (FBS). After 24 hours cells were replaced with 1% FBS. The experimental drugs were blinded with numbers. Next day the cells were incubated with or without drugs in duplicates. After 7 days and 14 days cells were trypsinized and counted.
- pgrn-1(tm985) mutants will be referred to as pgrn-1(tm)
- pgrn-1 (gk 123284) mutants will be referred to as pgrn-1 (gk)
- pgrn-1 p::pgrn-1::RFP animals will be referred to as “PGRN-1 rescue”.
- Gene expression analysis revealed that pgrn-1 (tm) animals had no residual expression of pgrn-1 while pgrn-1(gk) animals displayed a -50% reduction in pgrn-1 mRNA expression (FIG. 7A).
- This second mutant, pgrn-1(gk) also displayed a paralysis phenotype (FIG. 7A).
- PGRN is a secreted molecule and previous studies have shown the same is true in C. elegans (16).
- C. elegans 16
- Knockdown of pgrn-1 was performed by RNAi in non-neuronal tissues and in neuronal tissues (44- 46) and it was found that only the neuron-specific RNAi resulted in paralysis (FIGs. 7F-G). This suggests that PGRN acts in a cell autonomous manner, though it remains possible that there are feedback mechanisms from other tissues after the neuronal loss of PGRN.
- pgrn-1 mutants displayed an overactive food-seeking behaviour when starved and crawl up the sides of Petri dishes, a phenotype which is also rescued by the pgrn- 1-rescue construct (FIG. 7H), suggesting neuronal deficits in their ability to properly recognize food sources (47).
- mutant worms show changes in lysosome size and fluorescence intensity at day five, but these defects were not seen at day 1 (FIGs. 2I, J).
- FIGs. 2I, J Given the increase in autophagosomes as early as day 1 of adulthood, without the appearance of lysosomal defects, these data point to autophagy defects appearing before lysosomal defects in pgrn-1- deficient nematodes.
- Example 4 Genetically targeting the SL biosynthetic pathway restores defects in pgrn-1 mutant nematodes
- PGRN modulates the maturation and activity of at least two enzymes involved in sphingolipid metabolism, glucocerebrosidase (GBA) (50-53), and hexosaminidase A (54). In addition, it binds to prosaposin, and regulates its trafficking and processing to saponins, a family of non-enzymatic, lysosomal proteins that promote sphingolipid catabolism (35, 53, 55, 56). Disruptions in sphingolipid metabolism may, therefore, contribute to the pathological phenotype of GR/V-related disorders.
- RNAi screen of 17 genes involved in ceramide metabolism available from commercially-available RNAi libraries was performed.
- the genetic double mutant, pgrn-1(tm); sphk-1 (ok1097) was constructed in conjunction with the intestinal LGG-1 ::mCherry reporter, and it was observed that seven of the eight clones tested had an additive effect on the sphk-1 genetic mutant in restoring formation of LGG-1 ::mCherry puncta in a pgrn-1- null background (FIG. 3B).
- RNAi knock-down of several genes rescued the worms’ lysosomal defects by restoring size, LMP-1::GFP fluorescence intensity, or both (FIGs. 3C, D).
- the ability of these RNAi clones to suppress the animals’ paralysis phenotype was also tested (FIG. 3E).
- the summary of the results of these RNAi knock-down assays is depicted in FIG. 3F.
- C. elegans for drug discovery has many advantages since they can be used for high-throughput in vivo drug screening, something that cannot be easily performed with larger animals such as mice. Also, although typical high-throughput drug screens are done using cell-based models, nematodes are able to rapidly assess the efficacy of compounds in the context of a whole organism, complete with multiple cell types and biological complexity. The success of this approach has been validated to identify and translate a compound for ALS into clinical trials using C. elegans (41).
- the primary screen (1 well/compound) resulted in 108 hits, and these were tested in triplicate during a secondary screen (3 wells/compound) to eliminate false positives, which further narrowed down the list to 34 compounds (FIGs. 12A-B).
- the top 17 compounds were selected, regardless of their known function, for further testing in pgrn-1 nematodes for their ability to restore lifespan (FIGs. 10A-C) and suppress paralysis phenotypes (FIGs. 10D-F) in pgrn-1(tm) mutants at a single dose of 20 mM, the same dose used in the high-throughput screen.
- 12 compounds were considered hits from the unbiased, high-throughput screen.
- Rottlerin is a natural product polyphenol isolated from the red kamala tree ( Mallotus philippinensis). Rottlerin has been shown to have various cellular effects including activating autophagy, acting as an antitumor factor, as an anti-proliferative compound, and as an uncoupler of mitochondrial oxidative phosphorylation (60-64). Studies have demonstrated that rottlerin is a protein kinase C delta (PKC5) inhibitor (61 , 62, 64). There is also evidence that rottlerin is a potent large conductance potassium channel (BKCa ++ ) opener (86). The effects of rivastigmine or rottlerin in the context of neurodegenerative diseases associated with PGRN deficiency such as PGRN-deficient FTD has never been reported.
- PKCa ++ protein kinase C delta
- Example 6 Small molecules restore PGRN-deficient phenotypes in C. elegans
- the nematode models were used to further validate the compounds against other phenotypes in the PGRN-1 -deficient nematodes.
- the ability of the compounds to restore lysosomal phenotypes was first assessed, and it was observed that both rottlerin and rivastigmine were able to restore lysosomal fluorescence intensity and size phenotypes seen in the mutant animals (FIGs. 6A, B).
- a dose-dependent testing was performed on both drugs and 100 mM was selected as this was the concentration at which the drugs elicited a statistically significant effect on both phenotypes (FIGs. 11 A, 11B).
- A. W. Kao et al. A neurodegenerative disease mutation that accelerates the clearance of apoptotic cells. PNAS 108, 4441-4446 (2011). 17.
- Z. Ahmed et ai Accelerated lipofuscinosis and ubiquitination in granulin knockout mice suggest a role for progranulin in successful aging. Am J Pathol 177 , 311-324 (2010).
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Abstract
There is currently no effective cure or treatment for neurodegenerative diseases associated with progranulin (PGRN) deficiency, such as frontotemporal dementia (FTD). FTD is a fast-progressing disease, with affected patients usually dying 2-5 years after clinical diagnosis, and is the leading cause of non-Alzheimer dementias. The present application relates to methods and uses for the treatment of neurodegenerative diseases associated with PGRN deficiency such as FTD. Such methods and uses are based on the administration of agents that increase sphingolipid (SL) levels in neural cells from the subject, or of AchR and/or PKC inhibitors such as rottlerin, rivastigmine, or analogs or pharmaceutically acceptable salts thereof.
Description
TITLE OF INVENTION
COMPOUNDS AND METHODS FOR THE TREATMENT OF NEURODEGENERATIVE DISORDERS
CROSS REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of U.S. provisional patent application No. 63/202,491 filed on June 14, 2021, which is incorporated herein by reference in its entirety.
TECHNICAL FIELD
The present disclosure generally relates to neurodegenerative disorders, and more specifically to the treatment of neurodegenerative disorders associated with progranulin deficiency.
BACKGROUND ART
Frontotemporal Dementia (FTD) is a devastating neurodegenerative disorder and the third most common cause of dementia (1). FTD is a leading cause of early onset disorder, with patients usually diagnosed between 45 and 65 years of age (1, 2). Unlike other dementias, such as Alzheimer’s disease, FTD is a fast-progressing disease and affected patients usually die 2-5 years after clinical diagnosis. Advances in genetic screening techniques have identified many of the causative genes, revealing the complex heterogeneity of the underlying molecular mechanisms of the disease (1-3). Among them are autosomal-dominant heterozygous mutations in the GRN gene (4, 5) causing a severe reduction in the circulating levels of its product, progranulin (PGRN) (6). PGRN-deficient FTD is characterized by neuropathological inclusions of ubiquitinated TAR DNA Binding Protein 43 (TDP-43) in the cytoplasm (4, 5, 7-10). The presence of these inclusions links FTD and amyotrophic lateral sclerosis (ALS) at the molecular level, as mutations in TDP-43 also cause and inclusions of the protein are also found in 97% of ALS cases (11). Although more than a decade has passed since the identification of GRN’s involvement in FTD, there is no consensus on the molecular mechanisms linking low PGRN levels and disease pathology. Inversely, the overexpression of PGRN has been found to be protective in multiple models of neurodegeneration, such as ALS (7), Alzheimer’s (12), Parkinson’s (13), and Huntington’s diseases (14).
There is a need for the development of novel therapeutic approaches for the treatment of neurodegenerative disorders such as FTD.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
SUMMARY
The present disclosure provides the following items 1 to 44:
1. A method for treating a neurodegenerative disorder associated with progranulin (PGRN) deficiency in a subject, the method comprising administering to the subject an effective amount of (i) a protein kinase C (PKC) inhibitor and/or (ii) an acetylcholinesterase (AChR) inhibitor.
2. The method of item 1 , wherein the PKC inhibitor is rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof.
3. The method of item 1 or 2, wherein the AChR inhibitor is rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof
4. The method of any one of items 1 to 3, comprising administering to the subject an effective amount of rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof.
5. The method of item 4, comprising administering to the subject an effective amount of rottlerin.
6. The method of any one of items 1 to 5, comprising administering to the subject an effective amount of rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof.
7. The method of item 6, comprising administering to the subject an effective amount of rivastigmine.
8. The method of any one of items 1 to 7, wherein an effective amount of rottlerin and rivastigmine is administered.
9. A method for treating a neurodegenerative disorder associated with progranulin (PGRN) deficiency in a subject, the method comprising administering to the subject an effective amount of an agent that increases sphingolipid (SL) levels in neural cells from the subject.
10. The method of item 9, wherein the agent reduces the expression or activity of an enzyme involved in SL metabolism in the neural cells from the subject.
11. The method of item 9 or 10, wherein the SL is ceramide.
12. The method of item 10 or 11 , wherein the enzyme involved in SL metabolism is acid ceramidase (ASAH-1) or a ceramide glucosyltransferase.
13. The method of any one of items 10 to 12, wherein the agent that reduces the expression or activity of an enzyme involved in SL metabolism is an RNA interference agent.
14. The method of any one of items 1 to 13, wherein the PGRN deficiency is caused by a defect or mutation in the GRN gene.
15. The method of any one of items 1 to 14, wherein the neurodegenerative disorder associated with PGRN deficiency is frontotemporal dementia (FTD) or neuronal ceroid lipofuscinosis (NCL).
16. The method of any one of items 1 to 13, wherein the PKC inhibitor, AChR inhibitor and/or agent that increases SL levels is administered into the central nervous system of the subject.
17. An agent for use in treating a neurodegenerative disorder associated with progranulin (PGRN) deficiency in a subject, wherein the agent is (i) a protein kinase C (PKC) inhibitor and/or (ii) an acetylcholinesterase (AChR) inhibitor.
18. The agent for use of item 17, wherein the PKC inhibitor is rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof.
19. The agent for use of item 18, wherein the PKC inhibitor is rottlerin.
20. The agent for use of any one of items 17 to 19, wherein the AChR inhibitor is rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof.
21. The agent for use of item 20, wherein the AChR inhibitor is rivastigmine.
22. The agent for use of any one of items 17 to 21 , wherein the agent is rottlerin, rivastigmine or a combination thereof.
23. An agent that increases sphingolipid (SL) levels in neural cells for use in treating a neurodegenerative disorder associated with progranulin (PGRN) deficiency in a subject.
24. The agent for use of item 23, wherein the agent reduces the expression or activity of an enzyme involved in SL metabolism in the neural cells from the subject.
25. The agent for use of item 23 or 24, wherein the SL is ceramide.
26. The agent for use of item 24 or 25, wherein the enzyme involved in SL metabolism is acid ceramidase (ASAH-1) or ceramide glucosyltransferase.
27. The agent for use of any one of items 24 to 26, wherein the agent that reduces the expression or activity of an enzyme involved in SL metabolism is an RNA interference agent.
28. The agent for use of any one of items 24 to 27, wherein the PGRN deficiency is caused by a defect or mutation in the GRN gene.
29. The agent for use of any one of items 24 to 28, wherein the neurodegenerative disorder associated with PGRN deficiency is frontotemporal dementia (FTD) or neuronal ceroid lipofuscinosis (NCL).
30. The agent for use of any one of items 24 to 29, wherein the agent is for administration into the central nervous system of the subject.
31. Use of an agent for the manufacture of a medicament for treating a neurodegenerative disorder associated with progranulin (PGRN) deficiency in a subject, wherein the agent is (i) a protein kinase C (PKC) inhibitor and/or (ii) an acetylcholinesterase (AChR) inhibitor.
32. The use of item 31, wherein the PKC inhibitor is rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof.
33. The use of item 32, wherein the PKC inhibitor is rottlerin.
34. The use of any one of items 31 to 33, wherein the AChR inhibitor is rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof.
35. The use of item 34, wherein the AChR inhibitor is rivastigmine.
36. The use of any one of items 31 to 35, wherein the agent is rottlerin, rivastigmine or a combination thereof.
37. Use f an agent that increases sphingolipid (SL) levels in neural cells for use in treating a neurodegenerative disorder associated with progranulin (PGRN) deficiency in a subject.
38. The use of item 37, wherein the agent reduces the expression or activity of an enzyme involved in SL metabolism in the neural cells from the subject.
39. The use of item 37 or 38, wherein the SL is ceramide.
40. The use of item 38 or 39, wherein the enzyme involved in SL metabolism is acid ceramidase (ASAH-1) or ceramide glucosyltransferase.
41. The use of any one of items 38 to 40, wherein the agent that reduces the expression or activity of an enzyme involved in SL metabolism is an RNA interference agent.
42. The use of any one of items 38 to 41 , wherein the PGRN deficiency is caused by a defect or mutation in the GRN gene.
43. The use of any one of items 38 to 42, wherein the neurodegenerative disorder associated with PGRN deficiency is frontotemporal dementia (FTD) or neuronal ceroid lipofuscinosis (NCL).
44. The use of any one of items 38 to 43, wherein the agent is for administration into the central nervous system of the subject.
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
BRIEF DESCRIPTION OF DRAWINGS
In the appended drawings:
FIGs. 1A-I show that loss of pgrn-1 results in distinct FTD-like phenotypes including lysosomal dysfunction. FIG. 1A: pgrn-1 (tm985) mutant animals display age-dependent paralysis which can be rescued by the overexpression of full-length PGRN-1 ::RFP (Mantel-Cox test: N2 vs. pgrn-1 (tm985),
0.0001; N2 vs. pgrn-1 (tm985); pgrn-1::rfp,
FIG. 1B:
Heterozygous pgrn-1 (tm985)/+ animals display the same levels of paralysis as homozygous mutant animals (Mantel-Cox test, n.s.). FIG. 1C: Complete loss of pgrn-1 leads to a slight reduction in lifespan, whereas the re-expression of full-length PGRN-1 leads to an extension (Mantel-Cox test: N2 vs. pgrn-1 (tm985), *P< 0.05, N2 vs. pgrn-1 (tm985); pgrn-1 ::rfp, ***p< 0.001). FIG. 1D: pgrn-1 mutant animals display hypersensitivity to aldicarb, while rescue animals expressing PGRN-1 ::RFP display resistance (Mantel-Cox test: N2 vs. pgrn-1 (tm985), ****p< 0.0001; N2 vs. pgrn-1 (tm985); pgrn-1 ::rfp, ****P<0.0001). FIG. 1E: Representative images of pgrn-1 (tm985) coelomocyte lysosomes as visualized using an LMP-1::GFP reporter. FIGs. 1F-G: Loss of pgrn-1 leads to an increased fluorescence intensity of LMP-1::GFP lysosomes (Student’s t test, ****P<0.0001), but smaller ones as evidenced by a reduction in size (Student’s /test, ****P<0.0001). FIG. 1H: Treatment pgrn-1 mutant animals with the proteasome inhibitor, MG-132, leads to a dose-dependent decrease in lifespan (Mantel-Cox tests: Vehicle vs. 20 mM MG-132, *P< 0.05; Vehicle vs. 40 mM MG-132, ****p<0.0001). FIG. 11: Concanamycin A treatment results in a decrease in lifespan in pgrn-1 (tm985) animals but is not dose dependent (Mantel-Cox tests: Vehicle vs. 20 mM MG-132, ****P<0.0001; Vehicle vs. 40 mM MG-132, *P< 0.05).
FIGs. 2A-J show that mutations in pgrn-1 result in changes in autophagic flux. FIG. 2A: LGG-1::mCherry reporter shows the formation of distinct puncta in the intestines of WT animals, whereas it remains diffuse in pgrn-1 mutants. FIGs. 2B-C: Quantification of LGG-1::mCherry puncta at day 5 (FIG. 2B) and day 10 (FIG. 2C) adult animals shows a clear reduction in puncta in both pgrn- 1 mutant strains (Student’s t test. Day 5: WT vs. pgrn-1 (tm985), ****P< 0.0001, WT vs. pgrn- 1(gk123284), ****P<0.0001. Day 10: WT vs. pgrn-1 (tm985), ****P<0.0001, WT vs. pgrn-1 (gk 123284), ****p< 0.0001). FIG. 2D: Treatment of day 5 pgrn-1 (tm985); lgg-1::mCherry animals with 50 nM Concanamycin A results in an increase in the number of LGG-1::mCherry puncta (Student’s t test, ****p<0.0001). FIGs. 2E-F: Visualization of neuronal autophagy using a dually tagged LGG-1 reporter, mCherry::lgg-1::gfp, shows that both pgrn-1 mutants display an increased number of autophagosomes (FIG. 2E), but no change in the number of autolysosomes (FIG. 2F) at day 5 of adulthood (Student’s t test. Autophagosomes count (FIG. 2E): WT vs. pgrn-1 (tm985), ****P< 0.0001, WT vs. pgrn-1 (gk123284), ****p< 0.0001. Autolysosome count (FIG. 2F): WT vs. pgrn-1 (tm985), n.s., WT vs. pgrn-1 (gk123284), n.s.). FIGs. 2G-H: Both pgrn-1 mutations also result in an increased number of autophagosomes at day 1 (FIG. 2G), but also a decreased number of autolysosomes (FIG.
2H) (Student’s t test. Autophagosome count (FIG. 2G): WT vs. pgrn-1(tm985),
0.0001, WT vs. pgrn-1(gk123284), ****p< 0.0001. Autolysosome count (FIG. 2H): WT vs. pgrn-1(tm985), ****P<0.0001, WT vs. pgrn-1(gk123284), ****P<0.0001). FIGs. 2I-J: At day 1, pgrn- 1 (tm985) animals had no significant change in LGG-1::GFP fluorescence intensity (FIG. 2I) or in the size of their lysosomes (FIG. 2J) (Student’s t test. Fluorescence intensity (FIG. 2I): WT vs. pgrn-1(tm985), n.s.. Lysosome size (FIG. 2J): WT vs. pgrn-1(tm985), n.s.)
FIGs. 3A-F show that genetic manipulation of the sphingolipid (SL) biosynthetic pathway restores defects in pgrn-1 mutant animals. FIG. 3A: RNAi-mediated knockdown of genes involved in SL biosynthesis, using ceramide as a reactant, partially restore LGG-1::mCherry puncta formation in the intestine of pgrn- 7-null animals (One-way ANOVA, treated vs. EV control, **p<0.01, *** p<0.001, **** p<0.0001). FIG. 3B: RNAi-mediated knockdown of genes involved in SL biosynthesis have an additive effect on LGG-1::mCherry puncta formation in a pgrn-1 (tm985); sphk-1(ok1097) genetic background (One-way ANOVA, sphk-1(ok1097); pgrn-1 (tm985) treated vs. EV control, *** p<0.001, **** p<0.0001). FIGs. 3C-D: Knockdown of certain SL biosynthetic genes restore LMP-1::GFP lysosomal intensity and lysosomal width phenotypes in pgrn-1(tm985) mutant animals (One-way ANOVA, treated vs. EV control, *p< 0.05, *** p<0.001). FIG. 3E: RNAi knockdown rescues paralysis phenotype of pgrn-1 (tm985) mutant animals (Mantel-Cox test: EV vs. cerk-1 RNAi, n.s.; EV vs. sms- 1 RNAi, ****p<0.0001; EV vs. sms-2 RNAi, ****p<0.0001; EV vs. cgt-1 RNAi, ****p<0.0001; EV vs. cgt-3 RNAi, ****p<0.0001; EV vs. asah-1 RNAi, ****p<0.0001; EV vs. asah-2 RNAi, ****p<0.0001). FIG. 3F: Summary table of RNAi knockdown effects on different phenotypes tested.
FIG. 4 shows a schematic representation of SL genes implicated in PGRN-related defects. SL genes whose knockdown is able to restore one or more PGRN-related phenotypes are denoted by an asterisk (*), while the ones able to restore all tested phenotypes ( asah-1 and cgt-3) are indicated in grey.
FIGs. 5A-D show that high-throughput drug screening identifies small molecules able to ameliorate PGRN deficiency in nematodes and mammalian cell lines. FIG. 5A: Schematic representation of the high-throughput drug screen for PGRN-compensating drugs pgrn-1 (tm) mutant nematodes were treated with -3850 compounds in a phenotypic screen looking to restore motility. Hits were subsequently validated in Grn-deficient NSC34 cell lines to determine their effect in a mammalian model. FIG. 5B: pgrn-1 (tm) nematodes have a swimming defect in liquid culture when compared to WT animals (Two-way ANOVA, WT vs. pgrn-1 (tm), ****p<0.0001) but PGRN-1 rescue animals did not (Two-way ANOVA, WT vs. pgrn-1 p::pgrn-1 ::rfp, n.s.). FIGs. 5C-D: Survival of NSC34 cells treated with drug screen hits after 7 (FIG. 5C) and 14 days (FIG. 5D) of low serum growth (7 days, FIG. 5C: one-way ANOVA, drug treatment vs. Vehicle; XCT790, n.s.; rottlerin, **p<0.01; PAPP,
***p<0.001; Daurisoline, **p<0.01; Rivastigmine, ****p<0.0001; Pirenzepine, *p<0.05. 14 days, FIG. 5D: one-way ANOVA, drug treatment vs. Vehicle; XCT790, n.s.; rottlerin, ***p<0.001; PAPP, n.s.; Daurisoline, n.s.; Rivastigmine, ****p<0.0001; Pirenzepine, n.s.).
FIGs. 6A-F show that rottlerin and rivastigmine restore behavioral and molecular defects in vivo. FIGs. 6A-B: When used individually rottlerin or rivastigmine treatment can restore lysosomal LMP-1::GFP fluorescence intensity (FIG. 6A) and lysosomal size (FIG. 6A) in day 5 pgrn-1 (tm985) animals (FIG. 6A, Student’s t test: Vehicle vs. 100 mM rivastigmine, *P<0.05; Vehicle vs. 100 pM rottlerin, **P<0.01. FIG. 6B: Student’s t test: Vehicle vs. 100 pM rivastigmine, ****P<0.0001; Vehicle vs. 100 uM rottlerin, ****P<0.0001). When used simultaneously, the combination has a greater effect than each drug individually on fluorescence intensity (FIG. 6A), but not on lysosomal width (FIG. 6B) (FIG. 6A: Student’s t test: 100 pM rottlerin vs. 50 pM riv. + 50 pM rott. ****P<0.0001; 100 pM rivastigmine vs. 50 pM riv. + 50 pM rott. ****P<0.0001. FIG. 6B: Student’s t test: 100 pM rottlerin vs. 50 pM riv. + 50 pM rott. ***P<0.001; 100 pM rivastigmine vs. 50 pM riv. + 50 pM rott. *P<0.05). FIG. 6C: Treatment of pgrn-1 (tm985) animals with 100 pM rottlerin or rivastigmine, or the combination, results in a strong suppression of paralysis (Mantel-Cox test: Vehicle vs. 100 pM rottlerin, ****p<0.0001; Vehicle vs. 100 pM rivastigmine, ****P<0.0001; Vehicle vs. 50 pM rivastigmine + 50 pM rottlerin ****P<0.0001), and the simultaneous treatment had a stronger effect than the individual drugs (Mantel-Cox test: 100 pM rottlerin vs. 50 pM riv. + 50 pM rott. **P<0.01; 100 pM rivastigmine vs. 50 pM riv. + 50 pM rott. ***P<0.001). FIG. 6D: Both compounds, individually and combined, are able to increase levels of LGG-1::mCherry punctae in pgrn-1 mutant animals’ intestines (Student’s t test: Vehicle vs. 100 pM rivastigmine, ****P<0.0001; Vehicle vs. 100 pM rottlerin, ****P<0.0001; Vehicle vs. 50 pM riv. + 50 pM rott. ****P<0.0001), and the combination had a stronger effect than the individual treatments (Student’s t test: 100 pM rottlerin vs. 50 pM riv. + 50 pM rott. ****P<0.0001; 100 pM rivastigmine vs. 50 pM riv. + 50 pM rott. ****P<0.0001). FIGs. 6E-F: Both compounds, individually and combined, are able to influence autophagosome (FIG. 6E) and autolysosome (FIG. 6F) levels of the dual mCherry::LGG-1::GFP reporter in pgrn-1 mutant animals’ neurons (FIG. 6E: Student’s t test: Vehicle vs. 100 pM rivastigmine, ****P<0.0001; Vehicle vs. 100 pM rottlerin, ****p<0.0001; Vehicle vs. 50 pM riv. + 50 pM rott. ****P<0.0001. FIG. 6F: Student’s / test: Vehicle vs. 100 pM rivastigmine, *P< 0.05; Vehicle vs. 100 pM rottlerin, ***P<0.001; Vehicle vs. 50 pM riv. + 50 pM rott. **P<0.01). The combination of both drugs had a stronger effect than rivastigmine alone on autophagosomes (FIG. 6E), but did not have an effect on autolysosome levels (FIG. 6E: Student’s t test: 100 pM rottlerin vs. 50 pM riv. + 50 pM rott., n.s.; 100 pM rivastigmine vs. 50 pM riv. + 50 pM rott ****p<0.0001. FIG. 6F: Student’s t test: 100 pM rottlerin vs. 50 pM riv. + 50 pM rott., n.s.; 100 pM rivastigmine vs. 50 pM riv. + 50 pM rott., n.s.)
FIG. 7A: Quantitative gene expression reveals that pgrn-1(gk) animals show a marked reduction in pgrn-1 mRNA expression, while it could not be detected in pgrn-1(tm985) mutants (Student’s t test: N2 vs. pgrn-1 (tm985), ****P<0.0001; N2 vs. pgrn-1 (gk), ****P<0.0001). FIG. 7B: pgrn-1 (gk) animals display higher levels of age-dependent paralysis than N2 animals, and heterozygous pgrn-1(gk)/+ animals display the same levels of paralysis as homozygous mutant animals (Mantel-Cox test, N2 vs. pgrn-1 (gk), ****P<0.0001; pgrn-1 (gk) vs. pgrn-1(gk)/+, n.s.). FIG. 7C: Overexpression of pgrn-1 ::rfp in a pgrn-1 (tm985) background display similar levels of paralysis as N2 animals, whereas expressed in a wild-type background results in a slight decrease in paralysis (Mantel-Cox test: N2 vs. pgrn-1 (tm985); pgrn-1::rfp, n.s.; N2 vs. pgrn-1::rfp, *P<0.05). FIG. 7D: Lifespan in unaffected in pgrn-1(gk) animals (Mantel-Cox test, n.s.). FIG. 7E: pgrn-1(gk) animals display heightened aldicarb hypersensitivity compared to N2 animals (Mantel-Cox test, ****p<0.0001). FIGs. 7F-G: Knockdown of pgrn-1 in N2 animals (FIG. 7F) does not result in an increase in paralysis, while the knockdown in neurons does (FIG. 7G) (Mantel-Cox test: RNAi in N2 (FIG. 7F), n.s.; neuronal RNAi (FIG. 7G), ****P<0.0001). FIG. 7H: Nematodes lacking pgrn-1 display an overactive food-seeking behavior and crawl off NGM plates faster than N2 controls; this phenotype is partially rescued by re-expression of pgrn-1::rfp (Mantel-Cox test: N2 vs. pgrn-1 (tm985), ****p< 0.0001; pgrn-1(tm985) vs. pgrn-1 (tm985); pgrn-1::rfp, **P< 0.01). FIG. 7I: Treatment of N2 animals with either 20 or 40 mM MG-132 did not result in a decrease in lifespan (Mantel-Cox test: Vehicle vs. 20 pM, n.s.; Vehicle vs. 40 pM, n.s.). FIG. 7J: Treatment of pgrn-1::rfp animals with 20 pM MG-132 did not result in a decrease in lifespan but treatment with 40 pM MG-132 did (Mantel- Cox test: Vehicle vs. 20 pM, n.s.; Vehicle vs. 40 pM, ****P<0.001.). FIG. 7K: Treatment of N2 animals with either 50 or 100 nM Concanamycin A did not result in a decrease in lifespan (Mantel-Cox test: Vehicle vs. 50 nM, n.s.; Vehicle vs. 100 nM, n.s.). FIG. 7F: Treatment of pgrn-1::rfp animals with either 50 or 100 nM Concanamycin A did not result in a decrease in lifespan (Mantel-Cox test: Vehicle vs. 50 nM, n.s.; Vehicle vs. 100 nM, n.s.).
FIG. 8A: Both pgrn-1 mutations did not alter LGG-1::mCherry puncta formation at day 1 of adulthood (Student’s t test: WT vs. pgrn-1 (tm985), n.s.; WT vs. pgrn-1 (gk123284), n.s.). FIG. 8B: Starvation conditions induced autophagy in WT animals, but had no effect on autophagy in both pgrn- 1 mutants (Student’s t test, Fed vs. Starved: WT, ****p<0.0001; pgrn-1(tm), n.s.; pgrn-1( gk), n.s.).
FIG. 9A: RNAi knockdown of the corresponding genes did not significantly affect formation of LGG-1::mCherry puncta in pgrn-1 (tm985) mutants. FIGs. 9B-C: The genetic double mutant, sphk- 1(ok1097); pgrn-1(tm), had restored levels of autophagosomes (FIG. 9B) and autolysosomes (FIG. 9C) in animals’ neurons (One-way ANOVA, autophagosomes: WT vs. double mutant, n.s.; double
0.0001 ; autolysosomes: WT vs. double mutant, n.s.; double mutant vs.
FIGs. 10A-C: Validation of top 17 drugs from the liquid culture screen for their ability to influence lifespan in pgrn-1(tm) mutants (Mantel-Cox test, treatment condition vs. Vehicle control. A: Resveratrol, n.s.; Daurisoline, **p<0.01; XCT790, n.s.; PAPP, n.s.; Rivastigmine, n.s.; Rottlerin, n.s. B: 5-Methylhydandoin, n.s.; Bay-11, n.s.; Tomatidine, n.s.; Eseroline, n.s.; Verruculogen, n.s.; Azatadine, n.s.; Pirenzepine, *p< 0.05. C: Clonixin, n.s.; Methantheline, **p<0.01; Ethropropazine, ****p<0.0001; Vigabatrin, n.s.). FIGs. 10D-F: Validation of top 17 drugs on the paralysis phenotype exhibited by pgrn-1(tm) nematodes (Mantel-Cox test, treatment condition vs. Vehicle control. A: Resveratrol, ****p<0.0001 ; Daurisoline, ****p<0.0001; XCT790, ***p<0.001; PAPP, ****p<0.0001 ; Rivastigmine, ****p<0.0001; Rottlerin, **p<0.01. B: 5-Methylhydandoin, n.s.; Bay-11, *p< 0.05; Tomatidine, n.s.; Eseroline, n.s.; Verruculogen, n.s.; Azatadine, ***p<0.001; Pirenzepine, *p< 0.05. C: Clonixin, n.s.; Methantheline, ***p<0.001; Ethropropazine, ***p<0.001; Vigabatrin, ****p<0.0001).
FIGs. 11A-B: Dose-dependent testing of rottlerin and rivastigmine against lysosomal phenotypes (Student’s t test, A: DMSO control vs. treatment condition: 100 mM rivastigmine *p<0.05, 50 pM rottlerin *p<0.05, 100 pM Rottlerin ***p<0.001 ; B: DMSO control vs. all treatment conditions, ****p<0.0001). FIG. 11 C: Donepezil and sotrastaurin restore paralysis phenotypes in pgrn-1(tm) animals (Mantel Cox test, ****p<0.0001).
FIGs. 12A-B depict the compounds identified from C. elegans drug screen and their properties.
DETAILED DISCLOSURE
The use of the terms "a" and "an" and "the" and similar referents in the context of describing the technology (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
The terms "comprising", "having", "including", and "containing" are to be construed as open- ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
The use of any and all examples, or exemplary language (“e.g", "such as") provided herein, is intended merely to better illustrate embodiments of the claimed technology and does not pose a limitation of the scope unless otherwise claimed.
No language in the specification should be construed as indicating any non-claimed element as essential to the practice of embodiments of the claimed technology.
Herein, the term "about" has its ordinary meaning. The term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% of the recited values (or range of values).
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.
Where features or aspects of the disclosure are described in terms of Markush groups or list of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member, or subgroup of members, of the Markush group or list of alternatives.
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).
Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present). In the studies described herein, the present inventors have identified a link between pgrn-1/GRN mutations, sphingolipid metabolism, and autophagy, and have shown that inhibiting the expression of certain genes involved in SL metabolism attenuated several pgrn-1/GRN phenotypes in vivo. The present inventors have performed high-throughput drug screening in a nematode model of GRN-deficient FTD and have demonstrated that rottlerin and rivastigmine are able to reverse the biochemical, cellular and functional phenotypes of GRN deficiency.
Accordingly, in a first aspect, the present disclosure provides a method for treating a neurodegenerative disorder associated with progranulin (PGRN) deficiency in a subject, the method comprising administering to the subject an effective amount of an agent that increases sphingolipid (SL) levels in neural cells from the subject. The present disclosure also provides the use of an agent that increases SL levels in neural cells for the manufacture of a medicament for treating a neurodegenerative disorder associated with PGRN deficiency in a subject. The present disclosure also provides agent that increases SL levels in neural cells for use in treating a neurodegenerative disorder associated with PGRN deficiency in a subject.
The expression “neurodegenerative disorder associated with progranulin (PGRN, UniProtKB accession No. P28799) deficiency” as used herein refers to a neurodegenerative disorder in which a reduced expression and/or activity of PGRN (relative to the normal expression and/or activity of PGRN in a healthy subject) in certain cells from the central nervous system (CNS) leads to a dysfunction and/or death of CNS cells. The reduced expression and/or activity of PGRN may be due, e.g., to the absence of one or both copies of the gene encoding PGRN (GRN gene), mutation(s) in the gene encoding PGRN that affect(s) PGRN expression and/or mutation(s) in the PGRN protein that negatively affect(s) PGRN stability, secretion, localization and/or biological activity. Mutations in the GRN gene have been found in certain forms/subtypes of neurodegenerative diseases including FTD, Alzheimer’s disease (AD), Parkinson’s disease (PD), corticobasal syndrome (CBS), limbic- predominant age-related TDP-43 encephalopathy (LATE), Hippocampal sclerosis of aging (HS- aging), neuronal ceroid lipofuscinosis 11 (CLN11), Frontotemporal Lobar Degeneration (FLD), aphasia (progressive non-fluent aphasia), Pick disease, Lewy Body dementia and amyotrophic lateral sclerosis (ALS). Loss of both GRN alleles leads to the development of neuronal ceroid lipofuscinosis (NCL). In an embodiment, the neurodegenerative disorder associated with PGRN deficiency is FTD associated with PGRN deficiency. In another embodiment, the neurodegenerative disorder associated with PGRN deficiency is AD associated with PGRN deficiency. In another embodiment, the neurodegenerative disorder associated with PGRN deficiency is PD associated with PGRN deficiency. In another embodiment, the neurodegenerative disorder associated with PGRN deficiency is CBS associated with PGRN deficiency. In another embodiment, the neurodegenerative disorder associated with PGRN deficiency is LATE associated with PGRN deficiency. In another embodiment, the neurodegenerative disorder associated with PGRN deficiency is HS-aging associated with PGRN deficiency. In another embodiment, the neurodegenerative disorder associated with PGRN deficiency is ALS associated with PGRN deficiency. In another embodiment, the neurodegenerative disorder associated with PGRN deficiency is NCL. In an embodiment, the methods and uses described herein further comprises identifying a patient with a PGRN deficiency.
More than 65 mutations in the GRN gene have been identified in people with neurodegenerative disorder associated with PGRN deficiency such as FTD. One of the most common mutations is Arg493Ter or R493*, which creates a truncated PGRN protein due to a premature stop signal. The mutation A9D is associated with FTD (ubiquitin-positive). Other mutations include IVSODS G-C +5, GLN125TER, MET1THR, MET1ILE, 4-BP INS NT90, 4-BP DEL NT388, IVS8AS G-A +1, 1-BP DEL 998G, 1-BP INS 1145A, IVS7AS A-G -2, 2-BP DEL 675CA, IVS6AS A-G -2, 4-BP DEL 813CACT, 1- BP DEL 102C, 1-BP DEL 154A, 78C-T 3-PRIME UTR, and IVS6AS G-A -1. In an embodiment, the mutations in the GRN gene are autosomal-dominant heterozygous mutations.
Agents that may increase SL (e.g., ceramide) levels include SLs perse, precursors thereof, as well as enzymes involved in the synthesis of SLs (e.g., ceramide synthase, ceramide phosphatase, sphingomyelinase or glucosyl ceramidase). The levels of enzymes involved in the synthesis of SLs may be increased, for example, by administering the enzymes per se or a nucleic acid encoding the enzymes, or by modification of the promoter(s) and/or enhancer(s) regulating the expression of the genes encoding the enzymes to increase the endogenous expression of the enzymes. Such modification may be performed using gene-editing technologies such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases.
Alternatively, agents that may increase SL (e.g., ceramide) levels include inhibitors of enzymes involved in the metabolism of SLs (e.g., ceramidase, ceramide kinase, sphingomyelin synthase or ceramide glucosyltransferase/synthase). Such inhibitors include RNA interference (RNAi) agents such as antisense oligonucleotides, shRNAs, siRNAs and miRNAs that targets the mRNA encoding the above-noted enzymes, agents that interferes with the activity of the enzymes, for example agents interfering with binding of the enzyme to its substrate (e.g., small molecules, aptamers, antibodies, etc.). In an embodiment, the agent is an inhibitor of a ceramidase (ASAH-1) or a ceramide glucosyltransferase (UGCG). siRNAs directed against human ASAH-1 are commercially available from various providers such as ThermoFisher Scientific (siRNA ID #119213, 119214, 119215, 14389, 14483 and 14573), Creative Biolabs (Cat. No. SIRGT04294WQ-LN), Santa Cruz Biotechnology Inc. (Cat. No. sc- 105032). Suitable RNAi agents directed against transcripts encoding human ASAH-1 may be designed by the skilled person based on the sequences of the transcripts (e.g., RefSeq NM_177924.5 and NM_004315.6).
Examples of ceramidase (ASAH-1) inhibitors include carmofur (1-hexylcarbamoyl-5- fluorouracil), ARN 14988 (5-chloro-3-[(hexylamino)carbonyl]-3,6-dihydro-2,6-dioxo-1(2H)- pyrimidinecarboxylic acid, 2-methylpropyl ester), ARN14974 (6-(4-fluorophenyl)-2-oxo-N-(4-
phenylbutyl)-3(2H)-benzoxazolecarboxamide), AC Inhibitor IV, Ceranib-1 , Ceranib-2 (3-[3-(4- methoxyphenyl)-1-oxo-2-propen-1-yl]-4-phenyl-2(1 H)-quinolinone), DP24a, SABRAC, structural analogs of ceramide such as oleoylethanolamide (also called N-oleylethanolamine, OE), (1S,2R)-D- erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-e-MAAP) and its derivative (1R, 2R)-2-(N- tetradecanoylamino)-1-(4-nitrophenyl)-1, 3-propanediol (B-13 or D-NMAPPD), as well as B-13 analogs such as LCL204, LCL385, LCL464 and LCL521. Several ceramidase inhibitors are disclosed in Diamanti et al., Synthesis 2016, 48, 2739-2756. siRNAs directed against human UGCG are commercially available from various providers such as ThermoFisher Scientific (siRNA ID #111303, 111304, 111305, 13003, 13096 and 13186), Creative Biolabs (Cat. No. SIRGT20825WQ), Santa Cruz Biotechnology Inc. (Cat. No. sc-45404). Suitable RNAi agents directed against transcripts encoding human UGCG may be designed by the skilled person based on the sequences of the transcript (e.g., RefSeq NM_003358.2).
Examples of ceramide glucosyltransferase (or glucosylceramide synthase) inhibitors include PDMP, PPMP, PPPP, Genz-123346, miglustat, eliglustat, the imino sugar N-butyldeoxynojirimycin (NBDNJ). Several ceramide glucosyltransferase inhibitors are also disclosed in U.S. Patent Nos. 5,302,609; 5,472,969; 5.525,616; 5,916,911; 5,945,442; 5,952,370; 6,030,995; 6,051,598; 6,255,336; 6,569,889; 6,610,703; 6,660,794; 6,855,830; 6,916,802; 7,253,185; 7,196,205; and 7,615,573; PCT publications Nos. WO 2008/150486; WO 2009/117150; WO 2010/014554 and WO 2013/059119.
The present disclosure provides a method for treating a neurodegenerative disorder associated with PGRN deficiency in a subject, the method comprising administering to the subject an effective amount of (i) a protein kinase C (PKC) inhibitor, such as rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof; and/or (ii) an acetylcholinesterase (AChR) inhibitor, such as rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof. The present disclosure also provides the use of (i) a protein kinase C (PKC) inhibitor, such as rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof; and/or (ii) an acetylcholinesterase (AChR) inhibitor, such as rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a neurodegenerative disorder associated with PGRN deficiency in a subject. The present disclosure also provides an agent for use in treating a neurodegenerative disorder associated with PGRN deficiency in a subject, wherein the agent is (i) a protein kinase C (PKC) inhibitor, such as rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof; and/or (ii) an acetylcholinesterase (AChR) inhibitor, such as rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof. In an embodiment, the PKC inhibitor is a PKC- delta (PKC-d) inhibitor.
Examples of AChR inhibitors include Tacrine, 7-methoxytacrine, Donepezil, Galantamine, carbamates ( e.g physostigmine, rivastigmine), huperzine A, protoberbrine alkaloids (e.g., berberine, palmatine, jatrorrhizine, epiberberine), and organophosphorus compounds (e.g., diisopropyl fluorophosphate, echothiophate, trichlorfon) (see, e.g., Colovic et al., Acetylcholinesterase Inhibitors: Pharmacology and Toxicology, Curr Neuropharmacol 2013, 11(3): 315-335). In an embodiment, the AChR inhibitor is a carbamate. In a further embodiment, the AChR inhibitor is rivastigmine, donepezil, or an analog thereof or a pharmaceutically acceptable salt thereof. In a further embodiment, the AChR inhibitor is rivastigmine, or an analog thereof or a pharmaceutically acceptable salt thereof.
Examples of PKC inhibitors include rottlerin, calphostin C, 2,6-diamino-/\/-([1-oxotridecyl)-2- piperidinyl] methyl) hexanamide, /V-benzyladriamycin- 14-valerate, resveratrol, indolocarbazoles (staurosporine and its analogs such as Go 6976, K252 compounds, 7-hydroxystaurosporine (UCN- 01), and 4’-/V-benzoylstaurosporine (midostaurin)), Maleimide-Based Inhibitors (GF 109203X, Ro SI- 7549, Ro 31-8220, enzastaurin and ruboxistaurin), Sotrastaurin, Balanol and analogs thereof, melittin, pseudosubstrate-derived peptide inhibitors of PKC (e.g., PKC19-36), Chelerythrine, Riluzole, auranofin and sodium aurothiomalate, [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3- dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1), 8-hydroxy-1,3,6-naphthalenetrisulfonic acid, 2-acetyl-1,3-cyclopentanedione) and DNDA (3,4-diaminonaphthalene-2,7-disulfonic acid (ACPD) (see, e.g., Kawano et al., Activators and Inhibitors of Protein Kinase C (PKC): Their Applications in Clinical Trials, Pharmaceutics 2021, 13(11): 1748). In an embodiment, the PKC inhibitor is sotrastaurin, rottlerin, or an analog thereof or a pharmaceutically acceptable salt thereof. In an embodiment, the PKC inhibitor is rottlerin, or an analog thereof or a pharmaceutically acceptable salt thereof.
The present disclosure provides a method for treating a neurodegenerative disorder associated with PGRN deficiency in a subject, the method comprising administering to the subject an effective amount of (i) rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof; and/or (ii) rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof. The present disclosure also provides the use of (i) rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof; and/or (ii) rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating a neurodegenerative disorder associated with PGRN deficiency in a subject. The present disclosure also provides agent for use in treating a neurodegenerative disorder associated with PGRN deficiency in a subject, wherein the agent is (i) rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof; and/or (ii) rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof.
Rottlerin (mallotoxin) is a polyphenol natural product isolated from the Asian tree Mallotus philippensis that has the following structure:
Synthetic analogs of rottlerin are known in the art. For example, US Patent No. 8,586,768 discloses the following rottlerin analogs:
An analog of rivastigmine is neostigmine (Prostigmin)
neostigmine
Another analog of rivastigmine (S-l 26) is described in David et al. , Journal of Enzyme Inhibition and Medicinal Chemistry, Volume 36, 2021 - Issue 1, Pages 491-496:
The term "pharmaceutically acceptable salt" refers to salts of compounds disclosed herein that are pharmacologically acceptable and substantially non-toxic to the subject to which they are administered. More specifically, these salts retain the biological effectiveness and properties of the compounds (e.g., rottlerin, rivastigmine, analogs thereof) and are formed from suitable non-toxic organic or inorganic acids or bases.
For example, these salts include acid addition salts of the compounds which are sufficiently basic to form such salts. Such acid addition salts include acetates, adipates, alginates, lower alkanesulfonates such as a methanesulfonates, trifluoromethanesulfonatse or ethanesulfonates, arylsulfonates such as a benzenesulfonates, 2-naphthalenesulfonates, or toluenesulfonates (also known as tosylates), ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cinnamates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiodides, hydrogen sulphates, 2-hydroxyethanesulfonates, itaconates, lactates, maleates, mandelates, methanesulfonates, nicotinates, nitrates, oxalates, pamoates, pectinates, perchlorates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates, sulfonates, tartrates, thiocyanates, undecanoates and the like.
Additionally, acids which are generally considered suitable for the formation of pharmaceutically useful salts from basic pharmaceutical compounds are discussed, for example, by P. Stahl et al, Camille G. (eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use. (2002) Zurich: Wiley-VCH; S. Berge et al, Journal of Pharmaceutical Sciences (1977) 66(1) 1-19; P. Gould, International J. of Pharmaceutics (1986) 33 201-217; Anderson et al, The Practice of Medicinal Chemistry (1996), Academic Press, New York; and in The Orange Book (Food & Drug Administration, Washington, D.C. on their website).
Also, where the compounds are sufficiently acidic, the salts include base salts formed with an inorganic or organic base. Such salts include alkali metal salts such as sodium, lithium, and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; metal salts such
as aluminium salts, iron salts, zinc salts, copper salts, nickel salts and a cobalt salts; inorganic amine salts such as ammonium or substituted ammonium salts, such as trimethylammonium salts; and salts with organic bases (for example, organic amines) such as chloroprocaine salts, dibenzylamine salts, dicyclohexylamine salts, dicyclohexylamines, diethanolamine salts, ethylamine salts (including diethylamine salts and triethylamine salts), ethylenediamine salts, glucosamine salts, guanidine salts, methylamine salts (including dimethylamine salts and trimethylamine salts), morpholine salts, morpholine salts, N,N'-dibenzylethylenediamine salts, N-benzyl-phenethylamine salts, N- methylglucamine salts, phenylglycine alkyl ester salts, piperazine salts, piperidine salts, procaine salts, t-butyl amines salts, tetramethylammonium salts, t-octylamine salts, tris-(2-hydroxyethyl)amine salts, and tris(hydroxymethyl)aminomethane salts.
Such salts can be formed quite readily by those skilled in the art using standard techniques. Indeed, the chemical modification of a pharmaceutical compound (i.e. drug) into a salt is a technique well known to pharmaceutical chemists, (See, e.g., H. Ansel et. al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 196 and 1456-1457). Salts of the compounds disclosed herein may be formed, for example, by reacting the compound with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.
In an embodiment, the compounds used in the methods and uses described herein are formulated in a pharmaceutical composition. Such pharmaceutical compositions typically include one or more pharmaceutically acceptable carriers or excipients, and may be prepared in a manner well known in the pharmaceutical art. Supplementary active compounds can also be incorporated into the compositions. The carrier/excipient can be suitable, for example, for intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal or pulmonary (e.g., aerosol) administration (see Remington: The Science and Practice of Pharmacy, by Loyd V Allen, Jr, 2012, 22nd edition, Pharmaceutical Press; Handbook of Pharmaceutical Excipients, by Rowe et al., 2012, 7th edition, Pharmaceutical Press). Therapeutic formulations are prepared using standard methods known in the art by mixing the active ingredient having the desired degree of purity with one or more optional pharmaceutically acceptable carriers, excipients and/or stabilizers. In an embodiment, the pharmaceutical composition is formulated for administration into the central nervous system, e.g., to the brain.
An "excipient," as used herein, has its normal meaning in the art and is any ingredient that is not an active ingredient (drug) itself. Excipients include for example binders, lubricants, diluents, fillers, thickening agents, disintegrants, plasticizers, coatings, barrier layer formulations, lubricants,
stabilizing agent, release-delaying agents and other components. "Pharmaceutically acceptable excipient" as used herein refers to any excipient that does not interfere with effectiveness of the biological activity of the active ingredients and that is not toxic to the subject, i.e., is a type of excipient and/or is for use in an amount which is not toxic to the subject. Excipients are well known in the art, and the pharmaceutical composition of the present disclosure is not limited in these respects. In certain embodiments, the pharmaceutical composition of the present disclosure comprises excipients, including for example and without limitation, one or more binders (binding agents), thickening agents, surfactants, diluents, release-delaying agents, colorants, flavoring agents, fillers, disintegrants/dissolution promoting agents, lubricants, plasticizers, silica flow conditioners, glidants, anti-caking agents, anti-tacking agents, stabilizing agents, anti-static agents, swelling agents and any combinations thereof. As those of skill would recognize, a single excipient can fulfill more than two functions at once, e.g., can act as both a binding agent and a thickening agent. As those of skill will also recognize, these terms are not necessarily mutually exclusive.
Any suitable amount of the compound or pharmaceutical composition may be administered to a subject. The dosages will depend on many factors including the mode of administration. Typically, the amount of the compound contained within a single dose will be an amount that effectively prevent, delay or treat the neurodegenerative disorder without inducing significant toxicity.
For the prevention, treatment or reduction in the severity of a given disease or condition (neurodegenerative disorder), the appropriate dosage of the compound/composition will depend on the type of disease or condition to be treated, the severity and course of the disease or condition, whether the compound/composition is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the compound/composition, and the discretion of the attending physician. The compound/composition is suitably administered to the patient at one time or over a series of treatments. Preferably, it is desirable to determine the dose-response curve in vitro, and then in useful animal models prior to testing in humans. The present disclosure provides dosages for the compounds and compositions comprising same. For example, depending on the type and severity of the disease, about 1 pg/kg to to 1000 mg per kg (mg/kg) of body weight per day. Further, the effective dose may be 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg/ 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, and may increase by 25 mg/kg increments up to 1000 mg/kg, or may range between any two of the foregoing values. A typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs.
However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
These are simply guidelines since the actual dose must be carefully selected and titrated by the attending physician based upon clinical factors unique to each patient or by a nutritionist. The optimal daily dose will be determined by methods known in the art and will be influenced by factors such as the age of the patient and other clinically relevant factors. In addition, patients may be taking medications for other diseases or conditions. The other medications may be continued during the time that the compound disclosed herein is given to the patient, but it is particularly advisable in such cases to begin with low doses to determine if adverse side effects are experienced.
EXAMPLES
The present invention is illustrated in further details by the following non-limiting examples.
Example 1: Materials and Methods
C. elegans strains and maintenance
All nematode strains were cultured and handled as per standard methods. All experiments were carried out at 20°C, and were repeated a minimum of three times. The following strains were used: Bristol N2 wild-type, XQ561 pgrn-1(tm985), XQ592 pgrn-1(gk123284), CF3778 pgrn-1(tm985); pgrn-1p::pgm-1::rfp, RT258 pwls50 [Imp-1 ::GFP + Cbr-unc-119(+)], TU3311 uls60 [unc-119p::YFP + unc-119p::sid-1], VK1093 vkEx1093 [nhx-2p::mCherry::lgg-1], MAH508 sqEx67 [rgef- 1p::mCherry::GFP::lgg-1 + rol-6]. Genetic mutant worms were outcrossed 5 times to wild-type N2 worms before use, and pgrn-1(gk) animals were outcrossed 7 times. Genotyping of deletion mutants was done by genomic PCR, whereas genotyping of point mutations was done by high-resolution melting (HRM) using HRM MeltDoctor reagents (Applied Biosystems) and analyzed on HRM software (Applied Biosystems). Verification by Sanger sequencing was performed by Genome Quebec (McGill University). Heterozygous animals were obtained by crossing homozygous mutants with wild-type N2 animals; the progeny from fertilized hermaphrodites were used and immediately frozen in lysis buffer after use for confirmation of their genotype by either PCR or HRM.
RNAi Experiments
All C. elegans RNAi experiments were administered through feeding using standard protocols. For all assays, worms were grown on standard NGM plates, synchronized, and eggs were placed on NGM plates with either empty vector (EV) bacteria or bacterial clones expressing dsRNA against the target gene. Worms were grown on RNAi plates and the second generation was used for subsequent assays.
Gene expression assays
Synchronized, age-matched animals were collected in M9 buffer at day 1 of adulthood. Animals were washed with buffer 4 times to remove excess bacteria and the supernatant was removed after the last wash step. Worms were then flash-frozen at -80°C in 500 mI_ TRIzol Reagent (Thermo Fisher Scientific). After thawing, worms were homogenized using a 27 ½ G needle with a syringe and another 500 mI_ of TRIzol was added. Samples were let incubate at room temperature for 5 minutes before adding 200 mI_ of chloroform and letting them sit for an additional 2 minutes. Samples were then centrifuged at 12,000 g for 15 minutes allowing the phases to separate. The aqueous phase was collected, and extraction was completed using the RNeasy Mini Kit (Qiagen). cDNA was synthesized using the Superscript VILO cDNA Synthesis Kit (Invitrogen), and gene expression to quantify pgrn-1 transcript levels was performed using TaqMan probes and standard TaqMan reagents (both probes and reagents were purchased from Applied Biosystems) act-5 was chosen as the housekeeping gene. Gene expression assays were run on a QuantStudio 7 Flex (Applied Biosystems) instrument and data analysis was done using QuantStudio Real-Time PCR software.
Paralysis Assays
For paralysis assays, 25-30 L4 larval animals were placed onto NGM plates and scored daily starting the following day, at day 1 of adulthood. Worms were counted as paralyzed if they failed to move their body when prodded with a platinum worm pick. Worms were considered dead if they failed to respond to heat stimuli or if exhibited no pharyngeal pumping; dead worms were censored from statistical analyses. For each paralysis assay, a minimum of 200 animals were scored per genotype and per condition. For paralysis assays with compound treatments, the compounds were mixed directly into the NGM to the appropriate concentration.
Lifespan Assays
As with paralysis assays, 25-30 L4 animals were picked onto NGM plates and then scored from the 1st day of adulthood until death. Worms were considered dead if they failed to respond to mechanical or heat stimuli, and if they showed no pharyngeal pumping. A minimum of 200 animals were scored per genotype or per condition. For assays done with compound treatments, the compounds were mixed directly into the NGM to the appropriate concentration.
Overactive Food-Seeking Behaviour
Worms were age-matched, washed 3 times in M9 to remove excess bacteria, and placed on NGM plates without bacterial food. Worms were counted daily for the number of worms remaining on the plate, as well as the dead worms found stuck to the side of the plate. Animals that disappeared were censored from statistical analyses.
Aldicarb Assays
Animals were picked onto NGM plates supplemented with 1mM aldicarb at day 1 of adulthood. Paralysis was scored every 30 minutes for 2 hours. Animals were considered paralyzed if they failed to respond to gentle prodding by a platinum pick. A minimum of 200 animals were counted per genotype.
Microscopy Experiments
All microscopy experiments were carried out by a Zeiss Axio Observer inverted microscope. For all microscopy experiments, animals were mounted onto 2% agarose pads and immobilized in 5 mM levamisole.
Intestinal autophagy counts. Worms were synchronized on standard NGM plates until the L4 stage and were then maintained until the appropriate day. Worms were placed on empty NGM plates for about 30 minutes before experiments were carried out in order to get rid of excess intestinal bacteria. For RNAi experiments, animals were synchronized on standard NGM plates until the L4 stage and were then transferred onto RNAi plates until the appropriate day. For starvation assays, animals were maintained on standard NGM plates and were transferred to empty plates for 24 hours before experiments were carried out. During data acquisition, fluorescent LGG-1 ::mCherry puncta were counted manually, and 50 animals were counted per genotype or condition.
Neuronal autophagy counts (Dual GFP/mCherry LGG-1 reporter). Synchronized worms were raised on standard NGM plates and maintained until the appropriate day and visualized by fluorescent microscopy. Total numbers of red and green puncta were then counted manually. Since green puncta were indicative of APs only and red puncta were indicative both of APs and ALs, the number of ALs was calculated by AL= (Total # of red punctae)-(Total # of green punctae).
Lysosome morphology tests. For this assay, only lysosomes from the posterior coelomocytes were considered for this assay as there was minimal obstruction from other background tissues and intestinal fluorescence. Images were taken using the same camera settings across all replicates and were then analyzed in ImageJ. For lysosome intensity analyses, the background signal was subtracted from the lysosomal signal.
Liquid culture motility testing
Animals were synchronized to age-match the populations and grown on standard NGM plates until day 1 of adulthood. Worms were collected and washed with M9 buffer to remove excess bacteria. They were then placed in standard 96-well plates in M9 buffer, to a number of -50-70 worms/well. Motility was recorded unbiasedly using a WMicrotracker ONE instrument.
High-throughput, unbiased drug screening in C. elegans
Small molecule libraries from the Prestwick Chemical Library, Sigma Aldrich LOPAC Library, Microsource Drug Library, and the BML Natural Products Library from Enzo Life Sciences (together, totalling 3942 molecules) were selected for screening pgrn-1 mutant animals were grown on standard NGM plates and the efficacy of the drugs were monitored using WMicrotracker ONE instruments. Drug treatment was acute, and all compounds were tested at 20 uM. Animals were only put in contact with the drugs at the start of the assay for an acute treatment. Each compound was tested once (1 drug/well). Molecules were considered as hits if they were able to increase motility of pgrn-1 (tm985) animals based on a yes/no criteria. Hits were then re-tested in 3 wells/drug in a secondary screen to remove any false positives.
Drug efficacy testing in NSC34 cells
NSC34 cells expressing reduced levels of PGRN (shPGRN-NSC34) were plated in 6 well plates with 20000 cells per well and cultured in DMEM with 10% fetal bovine serum (FBS). After 24 hours cells were replaced with 1% FBS. The experimental drugs were blinded with numbers. Next day the cells were incubated with or without drugs in duplicates. After 7 days and 14 days cells were trypsinized and counted.
Example 2: Loss of pgrn-1 results in distinct phenotypes and recapitulates key features of
FTD
Characterization of pgrn-1 (tm985) animals revealed that they exhibited an age-dependent paralysis phenotype, which could be rescued by the re-expression of full-length PGRN-1 ::RFP under the control of the endogenous pgrn-1 promoter (FIG. 1A). As the tm985 allele of pgrn-1 is a full deletion resulting in a null allele, the effect of a substitution mutation in the gene, which resulted in a missense change at the protein level, was also tested. The gk123284 allele was selected, generated by the Million Mutation Project (42), which resulted in a G119E change in the 2nd exon of pgrn-1. For simplicity, pgrn-1(tm985) mutants will be referred to as pgrn-1(tm), pgrn-1 (gk 123284) mutants will be referred to as pgrn-1 (gk), and pgrn-1 p::pgrn-1::RFP animals will be referred to as “PGRN-1 rescue”. Gene expression analysis revealed that pgrn-1 (tm) animals had no residual expression of pgrn-1 while pgrn-1(gk) animals displayed a -50% reduction in pgrn-1 mRNA expression (FIG. 7A). This second mutant, pgrn-1(gk), also displayed a paralysis phenotype (FIG. 7A). Interestingly, in both cases the mutant pgrn-1 alleles were dominant, as heterozygous animals displayed equal rates of paralysis as the homozygous mutants (FIG. 1B, FIG. 7B). Overexpression of PGRN-1::RFP in pgrn- 1(tm) animals displayed no paralysis phenotype compared to wild-type N2 animals, while its overexpression in a wild-type background resulted in a slight decrease in spontaneous age-related
paralysis (FIG. 7C). Analysis of the lifespan of these animals revealed that pgrn-1(tm) mutants showed a slight, but statistically significant decrease in lifespan, whereas the rescue construct, showed a slight increase in lifespan compared to WT animals (FIG. 1C), whereas pgrn-1(gk) mutants showed no lifespan effects (FIG. 7D). Pgrn-1(tm) mutants were then subjected to aldicarb, a potent acetylcholinesterase inhibitor commonly used to study neurotransmission in C. elegans (43). Hypersensitivity to aldicarb can be indicative of increased synaptic acetylcholine release, whereas the resistance to it can suggest the opposite. Here, it was observed that both pgrn-1 mutant alleles displayed a heightened sensitivity to aldicarb, with the gk123284 point mutation being slightly less sensitive than the pgrn-1 (tm) mutants (FIGs. 1D, 7E). However, this phenotype was not as severe as that of unc-47(e307) control animals, which have a mutation in their GABA transporter.
In humans, PGRN is a secreted molecule and previous studies have shown the same is true in C. elegans (16). As a result, it was investigated whether the paralysis phenotypes that were observed were due to PGRN-1 acting in a cell autonomous or a non-cell autonomous manner. Knockdown of pgrn-1 was performed by RNAi in non-neuronal tissues and in neuronal tissues (44- 46) and it was found that only the neuron-specific RNAi resulted in paralysis (FIGs. 7F-G). This suggests that PGRN acts in a cell autonomous manner, though it remains possible that there are feedback mechanisms from other tissues after the neuronal loss of PGRN. Furthermore, pgrn-1 mutants displayed an overactive food-seeking behaviour when starved and crawl up the sides of Petri dishes, a phenotype which is also rescued by the pgrn- 1-rescue construct (FIG. 7H), suggesting neuronal deficits in their ability to properly recognize food sources (47).
One of the key features observed in FTD cases due to GRN mutations is lysosomal dysfunction suggesting a link between PGRN and lysosomal function. The presence of lysosomal dysfunction was therefore investigated in the nematode model. Using a fluorescent reporter for Imp- 7/I-AMP1, LMP-1::GFP which localizes to lysosomal membranes, higher levels of GFP fluorescence were observed in the coelomocytes of pgrn-1 (tm) mutant animals relative to WT animals (FIGs. 1E, F) at 5 days of adulthood, and that they had correspondingly smaller-sized lysosomes (FIG. 1G). Other studies in human cells have shown that loss of PGRN results in increased lysosomal biogenesis (33), and therefore, it is possible the increase in LMP-1::GFP intensity could be analogous. To confirm the presence of lysosomal dysfunction, it was hypothesised that these worms would be sensitive to impairment of proteasomal degradation (by MG 132, a proteasome inhibitor) or through inhibition of lysosomal function (by Concanamycin A, a potent inhibitor of lysosomal vATPases and prevents acidification of the lysosomal lumen). Treatment of worms with MG132 reduced the lifespan of pgrn-1 (tm) mutants in a dose-dependent manner (FIG. 1H) but had little effect on the survival of WT and pgrn- 7-rescue animals (FIG. 7I-J). It was observed that pgrn-1 (tm) mutant
animals displayed reduced lifespan when exposed to ConcA (FIG. 11), but WT and pgrn-1- rescue animals were unaffected (FIGs. 7K-L). Altogether, these results suggest that pgrn-1 mutant C. elegans recapitulate key features of FTD and are therefore a biologically relevant model to model this human disease.
Example 3: Mutations in pgrn-1 alter autophagy in C. elegans
Given the presence of lysosomal defects in pgrn-1 mutants, it was sought to better understand its consequences on the autophagic process. A fluorescent reporter of lgg-1 /LC3 (LGG- 1 ::mCherry), which is expressed in the intestinal cells of C. elegans, was utilized. Under normal autophagy conditions this transgenic reporter will form distinct red puncta in intestinal cells. However, this was not the case in pgrn-1 mutant animals: at days 5 and 10 of adulthood, LGG-1 ::mCherry remains diffuse in pgrn-1 mutants (FIG. 2A) and forms significantly fewer puncta than in WT animals (FIGs. 2B, C). There was no change in puncta formation at day 1 (FIG. 8A). Normally, autophagy levels increase upon conditions of nutrient deprivation, such as starvation (48). However, in both pgrn-1 mutants, starvation did not increase levels of LGG-1 ::mCherry puncta (FIG. 8B). However, treatment of pgrn-1 mutant worms with ConcA for 24 hours led to increased numbers of LGG- 1 ::mCherry puncta (FIG. 2D). Together, these results suggest that pgrn-1 mutations result in changes in autophagic degradation and may be independent of classical autophagy-inducing mechanisms.
T o see if these changes were due to changes in autophagic flux, a tandem-tagged Igg- 1/ LC3 reporter, GFP::mCherry::LGG-1, was used. This reporter can provide important information on the degradation process by measuring conversion of autophagosomes to autolysosomes (49). Since GFP emission is limited in low pH environments, red and green puncta indicate an autophagosome, whereas red-only puncta indicate an autolysosome. Furthermore, this transgene is expressed only in C. elegans neurons, providing important information on the autophagic process specifically in these cells. Autophagosomes were quantified by counting green puncta in the worms’ nervous system and it was observed that at day 5, there was an increase in autophagosomes in both pgrn-1 mutant animals compared to WT (FIG. 2E). There was, however, no significant change in the number of autolysosomes (FIG. 2F). At day 1, an increase in autophagosomes (FIG. 2G) as well as a decrease in autolysosome numbers were observed (FIG. 2H). This suggests that by day 5, pgrn-1 mutant worms exhibit defects in autophagosome-lysosome fusion which is not apparent at day 1. As seen in FIGs. 1E and 1F, mutant worms show changes in lysosome size and fluorescence intensity at day five, but these defects were not seen at day 1 (FIGs. 2I, J). Given the increase in autophagosomes as early as day 1 of adulthood, without the appearance of lysosomal defects, these data point to autophagy defects appearing before lysosomal defects in pgrn-1- deficient nematodes.
Example 4: Genetically targeting the SL biosynthetic pathway restores defects in pgrn-1 mutant nematodes
PGRN modulates the maturation and activity of at least two enzymes involved in sphingolipid metabolism, glucocerebrosidase (GBA) (50-53), and hexosaminidase A (54). In addition, it binds to prosaposin, and regulates its trafficking and processing to saponins, a family of non-enzymatic, lysosomal proteins that promote sphingolipid catabolism (35, 53, 55, 56). Disruptions in sphingolipid metabolism may, therefore, contribute to the pathological phenotype of GR/V-related disorders. To further explore this and to better understand the nature of the interaction between pgrn-1 and the SL pathway, a small-scale RNAi screen of 17 genes involved in ceramide metabolism available from commercially-available RNAi libraries was performed. The formation of intestinal LGG-1::mCherry puncta in pgrn-1 (tm) animals in the presence of the RNAi after 5 days of treatment was scored. Since nematode neurons are mostly resistant to RNAi treatment, this intestinal autophagy marker was used as a proxy for neuronal knockdown of the genes. It was observed that a small subset of eight RNAis resulted in a partial increase of LGG-1 puncta (FIG. 3A). Interestingly, there was common thread linking all the genes whose RNAi knock-down increased puncta formation, as they all coded for enzymes that use ceramide as a substrate to convert it to a downstream product. Likewise, the RNAi knock-down of genes that catalyzed the reverse reactions and regenerate ceramide had no effect on puncta formation (FIG. 9A). This genetically was also verified in neurons in the double GFP::mCherry::LGG-1 reporter by introducing a SL metabolic mutation, sphk-1(ok1097), in a pgrn- 1(tm) background. It was observed that the loss of sphk-1 restores autophagosome (FIG. 9B) and autolysosome numbers (FIG. 9C) in pgrn-1 mutants. It was next assessed targeting a combination of genes in the SL pathways could have an additive effect on restoring these phenotypes. Treating worms with two RNAis simultaneously is not a common methodological approach since it is difficult to control the efficacy of the knockdown of each individual RNAi, or to control for the amount of each bacterium eaten by the animals. Therefore, a double mutant between pgrn-1(tm) and sphk-1 (one of the genes that was identified in initial screen) was generated, and then these double mutants were treated with individual RNAi. Thus, the genetic double mutant, pgrn-1(tm); sphk-1 (ok1097), was constructed in conjunction with the intestinal LGG-1 ::mCherry reporter, and it was observed that seven of the eight clones tested had an additive effect on the sphk-1 genetic mutant in restoring formation of LGG-1 ::mCherry puncta in a pgrn-1- null background (FIG. 3B).
The eight genes of interest were further tested against other behavioural and molecular phenotypes characteristic of the pgrn-1 (tm985) animals. Among these, the ability of the RNAi clones to restore lysosomal defects seen in these animals was tested. RNAi knock-down of several genes
rescued the worms’ lysosomal defects by restoring size, LMP-1::GFP fluorescence intensity, or both (FIGs. 3C, D). The ability of these RNAi clones to suppress the animals’ paralysis phenotype was also tested (FIG. 3E). The summary of the results of these RNAi knock-down assays is depicted in FIG. 3F. It was observed that the knockdown of only 2 of the 8 genes tested, cgt-3 (corresponding to ceramide glucosyltransferase (UGCG) in humans) and asah-1, leads to the restoration all the tested phenotypes in pgrn-1(tm) animals (FIG. 4).
Example 5: High-throughput, unbiased drug screening in PGRN-deficient models
With the unmet need for new therapies to treat individuals with GR/V-deficit mutations, the nematode model was applied to try to close this gap and identify novel small molecules capable of compensating for the loss of PGRN. Using C. elegans for drug discovery has many advantages since they can be used for high-throughput in vivo drug screening, something that cannot be easily performed with larger animals such as mice. Also, although typical high-throughput drug screens are done using cell-based models, nematodes are able to rapidly assess the efficacy of compounds in the context of a whole organism, complete with multiple cell types and biological complexity. The success of this approach has been validated to identify and translate a compound for ALS into clinical trials using C. elegans (41).
Since pgrn-1(tm) nematodes are fully deficient in PGRN-1, the unbiased drug screening approach sought to chemically replace PGRN-Ts action. To do so, -3850 small molecule compounds from the Prestwick, Sigma Aldrich LOPAC, Microsource, and the BML Natural Products drug libraries were screened in C. elegans, followed by validation in previously characterized GR/V-deficient NSC34 cells (57) (FIG. 5A). The screening in C. elegans was based on the ability to restore the swimming defect in pgrn-1(tm) animals (FIG. 5B). The primary screen (1 well/compound) resulted in 108 hits, and these were tested in triplicate during a secondary screen (3 wells/compound) to eliminate false positives, which further narrowed down the list to 34 compounds (FIGs. 12A-B). In keeping with the unbiased nature of the screen, the top 17 compounds were selected, regardless of their known function, for further testing in pgrn-1 nematodes for their ability to restore lifespan (FIGs. 10A-C) and suppress paralysis phenotypes (FIGs. 10D-F) in pgrn-1(tm) mutants at a single dose of 20 mM, the same dose used in the high-throughput screen. In total, 12 compounds were considered hits from the unbiased, high-throughput screen.
To validate the translational ability of the compounds and to see if they could also function in a mammalian system, the 12 compounds were tested in NSC34 cell line models of PGRN deficiency. These cells have been validated for relevant phenotypes after silencing of GRN expression by shRNA (57). The 12 drugs were tested for their ability to restore cell survival, and it
was determined that 6 reduced cell survival and were excluded. Of the other 6, 5 had the ability to promote cell growth after 7 days (FIG. 5C), but only two, rottlerin and rivastigmine, had a prolonged effect and significantly increased cell growth after 14 days (FIG. 5D). Rivastigmine is an acetylcholinesterase inhibitor (FIG. 5D). Rottlerin, on the other hand, is a natural product polyphenol isolated from the red kamala tree ( Mallotus philippinensis). Rottlerin has been shown to have various cellular effects including activating autophagy, acting as an antitumor factor, as an anti-proliferative compound, and as an uncoupler of mitochondrial oxidative phosphorylation (60-64). Studies have demonstrated that rottlerin is a protein kinase C delta (PKC5) inhibitor (61 , 62, 64). There is also evidence that rottlerin is a potent large conductance potassium channel (BKCa++) opener (86). The effects of rivastigmine or rottlerin in the context of neurodegenerative diseases associated with PGRN deficiency such as PGRN-deficient FTD has never been reported.
Example 6: Small molecules restore PGRN-deficient phenotypes in C. elegans
With the final two hits identified from the drug screen, the nematode models were used to further validate the compounds against other phenotypes in the PGRN-1 -deficient nematodes. The ability of the compounds to restore lysosomal phenotypes was first assessed, and it was observed that both rottlerin and rivastigmine were able to restore lysosomal fluorescence intensity and size phenotypes seen in the mutant animals (FIGs. 6A, B). A dose-dependent testing was performed on both drugs and 100 mM was selected as this was the concentration at which the drugs elicited a statistically significant effect on both phenotypes (FIGs. 11 A, 11B). Interestingly, the combination of both drugs at a half-dose, 50 pM, had a stronger effect than either drug individually on lysosomal size (FIG. 6A), but for lysosomal width (FIG. 6B), the combination did not have this additive effect at both 50 and 100 pM. The compounds were then tested against paralysis (FIG. 6C) and a significant effect was observed for the individual drugs, and a stronger effect from the combination of both (at 50 pM) relative to controls, though the combination at 100 pM was not. The compounds’ influence on autophagy in pgrn-1(tm985) animals was assessed, beginning with the intestinal LGG-1 ::mCherry reporter, and both drugs were able to increase LGG-1 ::mCherry puncta formation and the combination at 50 pM had a stronger effect, but at 100 pM did not (FIGs. 6D). Finally, using the tandem-tagged autophagy reporter, GFP::mCherry::LGG-1, a restoration of autophagosomes (FIG. 6E) and autolysosomes (FIG. 6F) was also observed. Here, however, the combination of both drug treatments at 50 pM had no additive effect on autolysosomes, and only had a greater effect than rivastigmine alone on autophagosomes, likely because the rottlerin-only treatment had already reached a plateau. However, at 100 pM, the combination of both compounds had no effect on autolysosome formation. While the biological activity of rivastigmine is well-known (AChR inhibitor),
rottlerin’s still remains complex as noted above. Another known AChR inhibitor, donepezil, and a PKC inhibitor, sotrastaurin, were tested in pgrn-1(tm) worms and both compounds were able to reduce paralysis phenotypes (FIG. 11C). Together, these data suggest that rottlerin and rivastigmine are promising compounds with therapeutic applications in PGRN-deficient FTD. Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word "comprising" is used as an open- ended term, substantially equivalent to the phrase "including, but not limited to". The singular forms "a", "an" and "the" include corresponding plural references unless the context clearly dictates otherwise.
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Claims
WHAT IS CLAIMED IS:
I. A method for treating a neurodegenerative disorder associated with progranulin (PGRN) deficiency in a subject, the method comprising administering to the subject an effective amount of (i) a protein kinase C (PKC) inhibitor and/or (ii) an acetylcholinesterase (AChR) inhibitor.
2. The method of claim 1, wherein the PKC inhibitor is rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof.
3. The method of claim 1 or 2, wherein the AChR inhibitor is rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof
4. The method of any one of claims 1 to 3, comprising administering to the subject an effective amount of rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof.
5. The method of claim 4, comprising administering to the subject an effective amount of rottlerin.
6. The method of any one of claims 1 to 5, comprising administering to the subject an effective amount of rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof.
7. The method of claim 6, comprising administering to the subject an effective amount of rivastigmine.
8. The method of any one of claims 1 to 7, wherein an effective amount of rottlerin and rivastigmine is administered.
9. A method for treating a neurodegenerative disorder associated with progranulin (PGRN) deficiency in a subject, the method comprising administering to the subject an effective amount of an agent that increases sphingolipid (SL) levels in neural cells from the subject.
10. The method of claim 9, wherein the agent reduces the expression or activity of an enzyme involved in SL metabolism in the neural cells from the subject.
I I . The method of claim 9 or 10, wherein the SL is ceramide.
12. The method of claim 10 or 11, wherein the enzyme involved in SL metabolism is acid ceramidase (ASAH-1) or a ceramide glucosyltransferase.
13. The method of any one of claims 10 to 12, wherein the agent that reduces the expression or activity of an enzyme involved in SL metabolism is an RNA interference agent.
14. The method of any one of claims 1 to 13, wherein the PGRN deficiency is caused by a defect or mutation in the GRN gene.
15. The method of any one of claims 1 to 14, wherein the neurodegenerative disorder associated with PGRN deficiency is frontotemporal dementia (FTD) or neuronal ceroid lipofuscinosis (NCL).
16. The method of any one of claims 1 to 13, wherein the PKC inhibitor, AChR inhibitor and/or agent that increases SL levels is administered into the central nervous system of the subject.
17. An agent for use in treating a neurodegenerative disorder associated with progranulin (PGRN) deficiency in a subject, wherein the agent is (i) a protein kinase C (PKC) inhibitor and/or (ii) an acetylcholinesterase (AChR) inhibitor.
18. The agent for use of claim 17, wherein the PKC inhibitor is rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof.
19. The agent for use of claim 18, wherein the PKC inhibitor is rottlerin.
20. The agent for use of any one of claims 17 to 19, wherein the AChR inhibitor is rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof.
21. The agent for use of claim 20, wherein the AChR inhibitor is rivastigmine.
22. The agent for use of any one of claims 17 to 21, wherein the agent is rottlerin, rivastigmine or a combination thereof.
23. An agent that increases sphingolipid (SL) levels in neural cells for use in treating a neurodegenerative disorder associated with progranulin (PGRN) deficiency in a subject.
24. The agent for use of claim 23, wherein the agent reduces the expression or activity of an enzyme involved in SL metabolism in the neural cells from the subject.
25. The agent for use of claim 23 or 24, wherein the SL is ceramide.
26. The agent for use of claim 24 or 25, wherein the enzyme involved in SL metabolism is acid ceramidase (ASAH-1) or ceramide glucosyltransferase.
27. The agent for use of any one of claims 24 to 26, wherein the agent that reduces the expression or activity of an enzyme involved in SL metabolism is an RNA interference agent.
28. The agent for use of any one of claims 24 to 27, wherein the PGRN deficiency is caused by a defect or mutation in the GRN gene.
29. The agent for use of any one of claims 24 to 28, wherein the neurodegenerative disorder associated with PGRN deficiency is frontotemporal dementia (FTD) or neuronal ceroid lipofuscinosis (NCL).
30. The agent for use of any one of claims 24 to 29, wherein the agent is for administration into the central nervous system of the subject.
31. Use of an agent for the manufacture of a medicament for treating a neurodegenerative disorder associated with progranulin (PGRN) deficiency in a subject, wherein the agent is (i) a protein kinase C (PKC) inhibitor and/or (ii) an acetylcholinesterase (AChR) inhibitor.
32. The use of claim 31, wherein the PKC inhibitor is rottlerin, an analog thereof or a pharmaceutically acceptable salt thereof.
33. The use of claim 32, wherein the PKC inhibitor is rottlerin.
34. The use of any one of claims 31 to 33, wherein the AChR inhibitor is rivastigmine, an analog thereof or a pharmaceutically acceptable salt thereof.
35. The use of claim 34, wherein the AChR inhibitor is rivastigmine.
36. The use of any one of claims 31 to 35, wherein the agent is rottlerin, rivastigmine or a combination thereof.
37. Use f an agent that increases sphingolipid (SL) levels in neural cells for use in treating a neurodegenerative disorder associated with progranulin (PGRN) deficiency in a subject.
38. The use of claim 37, wherein the agent reduces the expression or activity of an enzyme involved in SL metabolism in the neural cells from the subject.
39. The use of claim 37 or 38, wherein the SL is ceramide.
40. The use of claim 38 or 39, wherein the enzyme involved in SL metabolism is acid ceramidase (ASAH-1) or ceramide glucosyltransferase.
41. The use of any one of claims 38 to 40, wherein the agent that reduces the expression or activity of an enzyme involved in SL metabolism is an RNA interference agent.
42. The use of any one of claims 38 to 41 , wherein the PGRN deficiency is caused by a defect or mutation in the GRN gene.
43. The use of any one of claims 38 to 42, wherein the neurodegenerative disorder associated with PGRN deficiency is frontotemporal dementia (FTD) or neuronal ceroid lipofuscinosis (NCL).
44. The use of any one of claims 38 to 43, wherein the agent is for administration into the central nervous system of the subject.
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