# Patent

## Title
Increasing parkin activity by administering a deubiquitinating enzyme inhibitor

## Abstract

Provided herein are methods of promoting parkin activity in a subject, comprising: selecting a subject with a disorder associated with decreased Parkin activity; and administering to the subject an effective amount of a composition that increases parkin activity, wherein the composition is an inhibitor of a deubiquitinating enzyme.

## Background
This application claims the benefit of U.S. Application No. 61/794,338, filed on Mar. 15, 2013, which is hereby incorporated in its entirety by this reference.


STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
This invention was made with government support under grant number AG30378 awarded by the National Institutes of Health. The government has certain rights in the invention.


BACKGROUND
Neurodegenerative diseases include genetic and sporadic disorders associated with progressive nervous system dysfunction. It has been estimated that one of four Americans will develop a neurodegenerative condition in their lifetimes. Generally, however, the underlying mechanisms causing the conditions are not well understood and few effective treatment options are available for preventing or treating neurodegenerative diseases. Similarly, treatment options for myodegenerative disease and prion disease are also limited.
SUMMARY
Provided herein is a method of promoting parkin activity in a subject, comprising: selecting a subject with a disorder associated with decreased parkin activity; and administering to the subject an effective amount of a composition that increases parkin activity, wherein the composition is an inhibitor of a deubiquitinating enzyme.
Further provided is a method of treating or preventing a neurodegenerative disease, a myodegenerative disease or prion disease in a subject, comprising: selecting a subject with a neurodegenerative disease of the central nervous system, a myodegenerative disease, a prion disease or at risk for a neurodegenerative disease of the central nervous system, a myodegenerative disease or a prion disease; and administering to the subject an effective amount of a composition that increases parkin activity, wherein the composition is an inhibitor of a deubiquitinating enzyme.
Also provided is a method of treating or preventing cancer in a subject, comprising selecting a subject with cancer or at risk for cancer; and administering to the subject an effective amount of a composition that increases parkin activity, wherein the composition is an inhibitor of a deubiquitinating enzyme.



DESCRIPTION OF DRAWINGS
FIG. 1 is a diagram showing that parkin activity is necessary to clear misfolded proteins via autophagy and the proteasome. Under normal conditions, parkin goes from an inactive to an active state upon ubiquitination. Active parkin facilitates degradation of proteins via autophagy and the proteasome. In certain diseases, for example, a neurodegenerative disease, myodegenerative disease, prion disease or cancer, deubiquitinating enzymes decrease parkin activity, by deubiquitinating parkin, thus decreasing degradation of proteins via autophagy and the proteasome.
FIG. 2 shows that the level of brain deubiquitinases (USP10 and USP13) is increased in post-mortem human Alzheimer's Disease (AD) (FIG. 2A) and Parkinson's Disease (PD) (FIG. 2B) brains. Densitometry analysis of post-mortem cortex in AD (N=12) and age-matched control subjects (N=7) shows increased levels of USP10 and USP13 in AD cortex compared to control (FIG. 2A). Densitometry analysis of post-mortem caudate in PD (N=9) and age-matched control subjects (N=7) shows A) increased levels of USP10 and USP13 in PD compared to control (FIG. 2B). Mann Whitney, p<0.05. mean±SEM.
FIG. 3 shows photomicrographs of control (FIGS. 3A, 3C) and post-mortem human AD (FIGS. 3B, 3D) brains, indicating that parkin interaction with deubiquitinase (USP-10) is increased in AD. A Proximity Ligation Assay (PLA) in situ on 30 mm thick human brain sections shows parkin and ubiquitin specific peptidase 10 (USP10). The interaction between parkin and ubiquitin specific peptidase 10 (USP10) is increased in human hippocampus (Figure A and B) and cortex (Figures C and D) in AD. The increase in USP10 level is paralleled by increased interaction with parkin, indicative of parkin deubiquitination and inactivity, which leads to accumulation and decreased solubility in the human brain.
FIG. 4 shows photomicrographs of control (FIGS. 4A, 4C) and post-mortem human AD (FIGS. 4B, 4D) brains, showing that parkin interaction with deubiquitinase (USP-13) is increased in AD. A Proximity Ligation Assay (PLA) in situ on 30 mm thick human brain sections shows parkin and ubiquitin specific peptidase 10 (USP13). The interaction between parkin and ubiquitin specific peptidase 10 (USP13) is increased in hippocampus (FIGS. 4A, 4C) and cortex (FIGS. 4C and 4D) with AD. The increase in USP13 level is paralleled by increased interaction with parkin, indicative of parkin deubiquitination and inactivity, which leads to accumulation and decreased solubility in the human brain.
FIG. 5 shows photomicrographs of control (FIGS. 5A, 5C) and post-mortem human PD (FIGS. 5B, 5D) brains showing that parkin interaction with deubiquitinase (USP-10/13) is increased in post-mortem human PD brains. A Proximity Ligation Assay (PLA) in situ on 30 mm thick human brain sections shows parkin and ubiquitin specific peptidase 10/13 (USP10/13). Parkin and ubiquitin specific peptidase 10 (USP10) interaction is increased in human PD midbrain (FIG. 5B) as compared to control (FIG. 5A); and parkin and ubiquitin specific peptidase 13 (USP13) interaction is increased in human PD midbrain (FIG. 5D) as compared to control (FIG. 5C). The increase in USP10/13 level is paralleled by increased interaction with parkin, indicative of parkin de-ubiquitination and inactivity, which leads to accumulation and decreased solubility in the human brain.
FIG. 6 shows photomicrographs of wild type mouse brain (FIGS. 6A, 6C) and parkin −/− mouse brain (FIGS. 6B, 6D), showing that parkin interacts with deubiquitinating enzymes in wild type but not parkin −/− mice. A Proximity Ligation Assay (PLA) in situ on 20 mm thick brain sections shows parkin and ubiquitin specific peptidase 13 (USP13) interaction in C57BL/6 mice (FIG. 6A) but not parkin−/− mice (FIG. 6B). PLA in situ on 20 mm thick brain sections shows parkin and ubiquitin specific peptidase 10 (USP10) interaction in C57BL/6 (FIG. 6C) mice but not parkin−/− mice (FIG. 6D). These data indicate that parkin could interact with USP10 and 13 under normal conditions.
FIG. 7 shows photomicrographs of brain sections from Tg-APP mice, indicating that parkin interaction with deubiquitinating enzymes (DUBs) is decreased in Tg-APP mice and tyrosine kinase inhibition increases parkin-DUBs interaction. A Proximity Ligation Assay (PLA) in situ on 20 mm thick brain sections shows parkin and ubiquitin specific peptidase 13 (USP13) in Tg-APP mice treated with DMSO (FIG. 7A), 10 mg/kg Nilotinib (FIG. 7B), and 5 mg/kg Bosutinib (FIG. 7C) daily for 3 weeks. Parkin and ubiquitin specific peptidase 10 (USP10) in Tg-APP mice treated with DMSO (FIG. 7D), 10 mg/kg Nilotinib (FIG. 7E), and 5 mg/kg Bosutinib (FIG. 7F) daily for 3 weeks. Tyrosine kinase inhibition increases the interaction between parkin and USP10 and 13.
FIG. 8 shows that parkin interaction with deubiquitinating enzymes is decreased in Tg-A53T mice and tyrosine kinase inhibition increases parkin-DUBs interaction. A Proximity Ligation Assay (PLA) in situ on 20 μm thick brain sections shows parkin and ubiquitin specific peptidase 13 (USP13) in A53T mice treated with DMSO (FIG. 8A), 10 mg/kg Nilotinib (FIG. 8B), and 5 mg/kg Bosutinib (FIG. 8C) daily for 3 weeks. Parkin and ubiquitin specific peptidase 10 (USP10) in A53T mice treated with DMSO (FIG. 8D), 10 mg/kg Nilotinib (FIG. 8E), and 5 mg/kg Bosutinib (FIG. 8F) daily for 3 weeks. Tyrosine kinase inhibition increases the interaction between parkin and USP10 and 13.
FIG. 9 shows in situ proximity ligation assay (PLA) showing endogenous parkin-beclin-1 complexes in WT C57BL/6 mice (FIG. 9A) (N=5) and parkin−/−mice (FIG. 9B). Also shown is PLA in Tg-APP mice IP injected once daily for 3 weeks with DMSO (FIG. 9C) 5 mg/kg Bosutinib (FIG. 9D) and 10 mg/kg Nilotinib (FIG. 9E) (N=5). Further shown is PLA in human post-mortem brains in the cortex of a normal subject (FIG. 9F) and cortex of an AD patient (FIG. 9G); the hippocampus of a normal subject (FIG. 9H) and of an AD patient (FIG. 9I); the caudate of a normal subject (FIG. 9J) and of an AD/PD patient (FIG. 9K). The increased parkin-USP10/13 interaction is paralleled by decreased parkin-beclin-1 interaction, indicating that DUB deubiquitination of parkin decreases functional parkin-beclin-1 interaction. This data shows that parkin interaction with DUBs, such as ubiquitin specific proteases (USP)-13 and USP-10 regulate parkin activity via deubiquitination.
FIG. 10 is a schematic showing that amyloid accumulation leads to autophagic induction and sequestration in phagophores. In transgenic or amyloid expressing animals parkin interaction with beclin-1 is reduced, leading to decreased maturation of phagophore into autophagosomes and autophagic defects. Kinase inhibition activates parkin and increases its interaction with beclin-1, resulting in maturation of phagophores into phagosomes and clearance. Also in human AD and PD brains parkin-beclin-1 interaction is reduced, indicating that USPs inactivate parkin and prevent functional interaction with beclin-1 to execute autophagic clearance of misfolded amyloid proteins.
FIG. 11 is a bar graph showing that Aβ1-42 accumulates in AV-10 in Tg-APP animals but drug treatment enhances autophagic clearance via deposition of Aβ1-42 in AV-20 and lysosome. Bars show Aβ1-42 in subcellular fractions, including autophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes. Transgenic 3×APP mice were injected IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once a day for 3 consecutive weeks. Brain tissues were fractionated to isolate AVs and human specific ELISA was performed to determine protein contents. N=5 animals per treatment.
FIG. 12 is a bar graph showing that Aβ1-40 accumulates in AV-20 in Tg-APP animals but drug treatment enhances autophagic clearance via deposition of Aβ1-40 in AV-20 and lysosome. Histograms show Aβ1-40 in subcellular fractions, including autophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes. Transgenic 3×APP mice were injected IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once a day for 3 consecutive weeks. Brain tissues were fractionated to isolate AVs and specific ELISA was performed to determine protein contents. N=5 animals per treatment.
FIG. 13 is a bar graph showing that P-Tau accumulates in AV-10 in Tg-APP animals but drug treatment enhances autophagic clearance via deposition of p-Tau in AV-20 and lysosome, which contains degradative enzymes. Histograms show Tau hyper-phosphorylation (p-Tau) at serine 396 in subcellular fractions, including autophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes. Transgenic 3×APP mice were injected IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once a day for 3 consecutive weeks. Brain tissues were fractionated to isolate AVs and mouse-specific ELISA was performed to determine protein contents. N=5 animals per treatment.
FIG. 14 is a bar graph showing that drug treatment increases parkin activity leading to protein clearance including parkin itself. Histograms show parkin in subcellular fractions, including autophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes. Transgenic 3× APP mice were injected IP with 10 mg/kg Nilotinib or 5 mg/kg Bosutinib or DMSO once a day for 3 consecutive weeks. Brain tissues were fractionated to isolate AVs and mouse specific ELISA was performed to determine protein contents. parkin accumulates in AV-10 in Tg-APP animals but drug treatment enhances autophagic clearance via deposition of parkin in AV-20 and lysosome, which contains degradative enzymes. N=5 animals per treatment.
FIG. 15 is a bar graph showing that autophagic clearance is parkin-dependent. Histograms show Aβ1-42 in subcellular fractions, including autophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes. Wild type or parkin−/− mice injected with lentiviral Aβ1-42 for 3 weeks and treated IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once a day for 3 (additional) consecutive weeks. Brain tissues were fractionated to isolate AVs and human specific ELISA was performed to determine protein contents. Aβ1-42 accumulates in AV-10 in lentivirus injected brains but drug treatment enhances autophagic clearance via deposition of Aβ1-42 in AV-20 and lysosome. N=5 animals per treatment.
FIG. 16 is a bar graph showing that P-Tau at serine 396 accumulates in AV-10 in lentivirus injected brains but drug treatment enhances autophagic clearance via deposition of p-Tau in AV-20 and lysosome, where it is degraded. Histograms show p-Tau in subcellular fractions, including autophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes. Wild type or parkin−/− mice injected with lentiviral Aβ1-42 for 3 weeks and treated IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once a day for 3 (additional) consecutive weeks. Brain tissues were fractionated to isolate AVs and mouse specific. ELISA was performed to determine protein contents. Autophagic clearance is parkin-dependent. N=5 animals per treatment.
FIG. 17 is a bar graph showing that α-synuclein accumulates in AV-10 in lentivirus injected brains but drug treatment enhances autophagic clearance via deposition of α-synuclein in AV-20 and lysosome, which contains degradative enzymes. Histograms show α-synuclein in subcellular fractions, including autophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes. Wild type or parkin−/− mice injected SN with lentiviral α-synuclein for 3 weeks and treated IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once a day for 3 (additional) consecutive weeks. SN tissues were fractionated to isolate AVs and human specific ELISA was performed to determine protein contents. Autophagic cleareance is parkin-dependent. N=5 animals per treatment.
FIG. 18 is a bar graph showing that P-Tau accumulates in AV-10 in lentivirus injected brains but drug treatment enhances autophagic clearance via p-Tau deposition in AV-20 and lysosome, which contains degradative enzymes. Histograms show p-Tau at serine 396 in subcellular fractions, including autophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes. Wild type or parkin−/− mice injected SN with lentiviral α-synuclein for 3 weeks and treated IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once a day for 3 (additional) consecutive weeks. SN tissues were fractionated to isolate AVs and mouse specific ELISA was performed to determine protein contents. Autophagic clearance is parkin-dependent. N=5 animals per treatment.
FIG. 19 is a bar graph showing that α-synuclein accumulates in AV-10 in A53T brains but drug treatment enhances autophagic clearance via α-synuclein deposition in AV-20 and lysosome. Bars show α-synuclein in subcellular fractions, including autophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes, containing digestive enzymes. Transgenic A53T mice were injected IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once a day for 3 consecutive weeks. Brain tissues were fractionated to isolate AVs and human specific ELISA was performed to determine protein contents. N=5 animals per treatment.
FIG. 20 is a bar graph showing that P-Tau accumulates in AV-10 in A53T brains but drug treatment enhances autophagic clearance via p-tau deposition in AV-20 and lysosome. Histograms show p-Tau at Serine 396 in subcellular fractions, including autophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes, containing digestive enzymes. Transgenic A53T mice were injected IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once a day for 3 consecutive weeks. Brain tissues were fractionated to isolate AVs and mouse specific ELISA was performed to determine protein contents. N=5 animals per treatment.
FIG. 21 is a bar graph showing that parkin accumulates in AV-10 in A53T brains but drug treatment enhances autophagic clearance via parkin deposition in AV-20 and lysosome. Bars show parkin in subcellular fractions, including autophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes, containing digestive enzymes. Transgenic A53T mice were injected IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once a day for 3 consecutive weeks. Brain tissues were fractionated to isolate AVs and mouse specific ELISA was performed to determine protein contents. N=5 animals per treatment.
FIG. 22 is a bar graph showing that Tau expression leads to Aβ1-42 accumulation in AV10 and AV20, but not in lysosomes, indicating decreased fusion between autophagosomes and lysosomes. Bars show Aβ1-42 in subcellular fractions, including autophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes. Wild type or parkin−/− mice injected with lentiviral Tau±Aβ1-42 for 3 weeks and treated IP with 10 mg/kg Nilotinib or DMSO once a day for 3 (additional) consecutive weeks. Brain tissues were fractionated to isolate AVs and human specific ELISA was performed to determine Aβ1-42 contents. Aβ1-42 accumulates in AV-10 in lentivirus injected brains but drug treatment enhances autophagic clearance via deposition of Aβ1-42 in AV-20 and lysosome. Autophagic clearance is parkin-dependent. N=5 animals per treatment.
FIG. 23 is a bar graph showing that Aβ1-42 accumulates in AV-10 in lentivirus injected brains but drug treatment enhances autophagic clearance via deposition of Aβ1-42 in AV-20 and lysosome. Autophagic clearance is less efficient in Tau null animals with Aβ1-42 accumulation in AV-10 and AV-20. Bars show Aβ1-42 in subcellular fractions, including autophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes. Wild type or Tau−/− mice injected with lentiviral Aβ1-42 for 3 weeks and treated IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once a day for 3 (additional) consecutive weeks. Brain tissues were fractionated to isolate AVs and human specific ELISA was performed to determine protein contents. N=5 animals per treatment.
FIG. 24 is a bar graph showing that P-Tau at serine 396 accumulates in AV-10 in lentivirus injected brains but drug treatment enhances autophagic clearance via deposition of p-Tau in AV-20 and lysosome, where it is degraded. Bars show p-Tau in subcellular fractions, including autophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes. Wild type or parkin−/− mice injected with lentiviral human Tau±Aβ1-42 for 3 weeks and treated IP with 10 mg/kg Nilotinib or 30 μL DMSO once a day for 3 (additional) consecutive weeks. Brain tissues were fractionated to isolate AVs and mouse specific ELISA was performed to determine protein contents. Autophagic clearance is parkin-dependent. N=5 animals per treatment.
FIG. 25 is a bar graph showing that P-Tau at serine 396 accumulates in AV-10 in lentivirus injected brains but drug treatment enhances autophagic clearance via deposition of p-Tau in AV-20 and lysosome, where it is degraded. Histograms show p-Tau in subcellular fractions, including autophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes. Wild type or parkin−/− mice injected with lentiviral Tau±Aβ1-42 for 3 weeks and treated IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once a day for 3 (additional) consecutive weeks. Brain tissues were fractionated to isolate AVs and human specific ELISA was performed to determine protein contents. Autophagic clearance is parkin-dependent. N=5 animals per treatment.
FIG. 26 is a Western blot analysis of sodium Tris EDTA NP40 (STEN) (soluble) extracts of human M17 cells. Lentiviral USP13 expression (FIG. 26A) (1st blot), endogenous USP10 levels (FIG. 26A) (2nd blot), and lentiviral parkin expression (FIG. 26A) (3rd blot) relative to actin (FIG. 26A) (4th blot) on 10% NuPAGE SDS gel are shown. FIG. 26B shows densitometry results. Western blot analysis of 4 M urea (insoluble) extracts showing parkin levels (FIG. 26C) (1st blot) relative to actin on 10% NuPAGE SDS gel are provided with densitometry results (FIG. 26D). FIG. 26E is a Western blot showing immunoprecipitated (IP) parkin from human M17 cells overexpressing parkin ±USP13 and probed with ubiquitin (1st blot), USP13 (2nd blot) and parkin (3rd blot) relative to actin and input. The graph in FIG. 26F represents parkin activity in human M17 cells as measured by ELISA in the presence of E1-E2 and ATP. N=7 for every treatment; * indicates significantly different, ANOVA, Neuman Keuls multiple comparison, P<0.05.
FIG. 27 is a Western blot analysis of STEN (soluble) extracts of M17 cells. FIG. 27A shows lentiviral USP13 expression (1st blot) and USP13 shRNA named (E08, E07, F04, F03 and F02), endogenous USP10 levels (2nd blot), and parkin expression (3rd blot) relative to actin (4th blot) on a 10% NuPAGE SDS gel. FIG. 27B is a graph representing chymotrypsin-like 26S proteasome activity with lentiviral parkin, USP13 or shRNA. N=4 for every treatment; *indicates significantly different, ANOVA, Neuman Keuls multiple comparison, P<0.05.
FIG. 28 shows that USP13 deletion increases parkin-mediated amyloid clearance. Human M17 cells were transfected with USP13 cDNAs and shRNAs in the presence of αβ42. ELISA was performed showing the level of αβ42 and hyperphosphorylated Tau at Ser 396 in the presence of parkin (FIGS. 28A and B) and in the absence of parkin (FIGS. 28C and D) absence of parkin.
FIG. 29 shows that USP13 deletion can increase parkin-mediated Tau clearance. Human M17 cells were transfected with USP13 cDNAs and shRNAs in the presence of Tau and ELISA was performed showing the level of hyper-phosphorylated Tau at Ser 396 in the presence of parkin (FIG. 29A) and in the absence of parkin (FIG. 29B).
FIG. 30 shows that ubiquitinated parkin is activated in response to amyloid (alpha-Synuclein, beta-amyloid, Tau and TDP-43) accumulation, leading to facilitation of clearance. An increase in USP13 leads to parkin deubiquitination and decreased activity, resulting in misfolded protein accumulation in neurodegenerative diseases (FIG. 30A). It is likely that ubiquitination activates parkin, which is then recycled through the proteasome to prevent its accumulation (FIG. 30B). Activated parkin facilitates autophagic clearance. An increase in USP13 leads to parkin deubiquitination and decreased proteasomal recognition, leading to its instability/insolubility and accumulation. This results in subsequent autophagic failure and death of neurons (FIG. 30B).



DETAILED DESCRIPTION
Provided herein is a method of promoting parkin activity in a subject, comprising selecting a subject with a disorder associated with decreased Parkin activity and administering to the subject an effective amount of a composition that increases parkin activity, wherein the composition is an inhibitor of a deubiquitinating enzyme. A disorder associated with decreased parkin activity can be, but is not limited to, a neurodegenerative disease, a myodegenerative disease, prion disease or cancer. In the methods provided herein, neurodegenerative diseases of the central nervous system include, but are not limited to, Amyotrophic Lateral Sclerosis, Alzheimer's Disease, Parkinson's Disease, frontotemporal dementia, Huntington's Disease, Mild Cognitive Impairment, an α-Synucleinopathy, a Tauopathy or a pathology associated with intracellular accumulation of TDP-43.
In the methods provided herein, myodegenerative diseases include, but are not limited to, inclusion body myositis (IBM), spinal-bulbar muscular atrophy (SBMA), and motor neuron disease (MND). In the methods provided herein, prion diseases or transmissible spongiform encephalopathies (TSEs) include, but are not limited to, Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease (vCJD), Gerstmann-Straussler-Scheinker Syndrome, Fatal Familial Insomnia and Kuru in humans. Animal prion diseases include, but are not limited to, Scrapie, Bovine Spongiform Encephalopathy (BSE), Chronic Wasting Disease (CWD), Transmissible mink encephalopathy, Feline spongiform encephalopathy and Ungulate spongiform encephalopathy.
As used throughout, cancer refers to any cellular disorder in which the cells proliferate more rapidly than normal tissue growth. A proliferative disorder includes, but is not limited to, neoplasms, which are also referred to as tumors. A neoplasm can include, but is not limited to, pancreatic cancer, breast cancer, brain cancer (e.g., glioblastoma), lung cancer, prostate cancer, colorectal cancer, thyroid cancer, renal cancer, adrenal cancer, liver cancer, neurofibromatosis 1, and leukemia. A neoplasm can be a solid neoplasm (e.g., sarcoma or carcinoma) or a cancerous growth affecting the hematopoietic system (e.g., lymphoma or leukemia). Other proliferative disorders include, but are not limited to neurofibromatosis.
One of skill in the art would know how to select a subject with a disorder associated with decreased parkin activity. For example, and not to be limiting, one of skill in the art knows how to diagnose a subject with or at risk of developing a neurodegenerative disease. For example, one or more of the follow tests can be used genetic test (e.g., identification of a mutation in TDP-43 gene) or familial analysis (e.g., family history), central nervous system imaging (e.g., magnetic resonance imaging and positron emission tomography), clinical or behavioral tests (e.g., assessments of muscle weakness, tremor, or memory), laboratory tests.
One of skill in the art would also know how to identify a subject with decreased parkin activity. Methods for measuring parkin activity are known in the art. See, for example, Schlossmacher and Shimura (“Parkinson's disease: assays for the ubiquitin ligase activity of neural Parkin,” Methods Mol. Biol. 301: 351-69 (2005)); Morrison et al. (“A simple cell based assay to measure Parkin activity,” J. Neurochem. 116(3): 342-9 (2011)) and Burns et al. (Hum. Mol. Genet. 18 3206-3216 (2009)).
One or more inhibitors of deubiquitinating enzymes (DUBs) can be used to increase parkin activity. Deubiquitinizing enzymes include, but are not limited to, ubiquitin carboxyl-terminal hydrolase 10 (USP10) and ubiquitin carboxyl-terminal hydrolase 13 (USP13). USP10 and USP13 are enzymes that specifically cleave ubiquitin from ubiquitin-conjugated protein substrates. Protein sequences for USP10 are provided herein as SEQ ID NO: 6 and SEQ ID NO: 7. SEQ ID NO: 6 corresponds to the protein sequence for isoform 1 of human USP10, which can be found under GenBank Accession No. NP_001259004.1. SEQ ID NO: 7 corresponds to the protein sequence for isoform 2 of human USP10, which can be found under GenBank Accession No. NP_005144.2. Nucleic acid sequences encoding USP10 isoform 1 and USP10 isoform 2 are provided herein as SEQ ID NO: 8 (GenBank Accession No. NM_001272075.1) and SEQ ID NO: 9 (GenBank Accession No. NM_005153.2), respectively. SEQ ID NO: 10 corresponds to the protein sequence for human USP13 found under GenBank Accession No. NP_003931.2. Nucleic acid sequences encoding USP13 are provided herein as SEQ ID NO: 11 (GenBank Accession No. NM_003940.2) and SEQ ID NO: 12. The coding sequence for USP13 can comprise nucleic acids 82-2673 of SEQ ID NO: 11.
As set forth above, DUBs, such as USP10 and USP13, deubiquitinate parkin, thus reducing parkin activity. Upon reduction of parkin activity, the interaction between parkin and beclin-1 is also reduced. Therefore, inhibition of DUBs prevents deubiquitination of parkin, which leads to increased parkin activity and an increase in the interaction between parkin and beclin-1. Optionally, these inhibitors can cross the blood brain barrier.
For example, one or more inhibitors of USP10 and/or USP13 can be used in the methods set forth herein. An inhibitor can be a chemical, a small or large molecule (organic or inorganic), a drug, a protein, a peptide, a cDNA, an antibody, a morpholino, a triple helix molecule, an siRNA, a shRNA, an miRNA, an antisense RNA, a ribozyme or any other compound now known or identified in the future that inhibits at least one activity of USP10 and/or USP13, for example, deubiquitination of parkin.
For example, the inhibitor can be a compound represented by formula I:




or a pharmaceutically acceptable salt, biologically active metabolite, solvate, hydrate, prodrug, enantiomer or stereoisomer thereof, wherein

n is 0, 1, 2, 3 or 4;
Y is —C(R1)═ or —N═;
R is —H, lower alkyl, —CH3, lower fluoroalkyl, —CH2F, —CHF2, —CF3, —NO2, —OH, —NH2, NH(lower alkyl), —N(lower alkyl)2, or lower alkynyl;
R1 is independently selected for each occurrence from the group consisting of —H, —F, —Cl, —Br, —I, —NO2, —OH, —NH2, —NH(lower alkyl), —N(lower alkyl)2, —CH3, —CF3, —C(═O)(lower alkyl), —CN, —O(lower alkyl), —O(lower fluoroalkyl), —S(═O)(lower alkyl), —S(═O)2lower alkyl) and C(═O)O(lower alkyl);
R2 and R3 are independently selected from the group consisting of —H, lower alkyl, lower fluoroalkyl, lower alkynyl and hydroxyalkyl;
X is —O—, —S—, —N(H)—, —N(lower alkyl)-, —CH2—, —CH2CH2—, —CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2— or —CH2CH2CH2CH2CH2CH2—; and
Z is phenyl, pyridyl, vinyl, morphinyl, phenanthrolinyl, naphthyl, furyl or benzo[d]thiazolyl; and optionally substituted with one or more substitutents selected from the group consisting of —CH3, lower alkyl, fluoroalkyl, —OCH3, —OCF3, lower fluoroalkoxy, —F, —Cl, —Br, —I, —NO2, lower alkyoxy, —NH(lower alkyl), —N(lower alkyl)2, —CF3, and 3,4-methylene dioxy. Optionally, the compound of formula I is not




wherein J is Cl, OCHF2, OCH2CH3, OCH2CF3, O(CH2)2CH3, OCH(CH3)2, O(CH2)3CH3, or O(cyclopentyl).

A non-limiting example of Formula 1 that can be used in the method set forth herein is spautin-1, or a derivative thereof. Pharmaceutical salts of spautin-1 and its derivatives are also provided herein.




Identification of additional inhibitors can be determined using methods known in the art. See, for example, Lee et al. “A High-Throughput Screening Method for Identification of Inhibitors of the Deubiquitinating Enzyme USP14,” Curr. Protoc. Chem. Biol. 4: 311-330 (2012); Tian et al. “Characterization of selective ubiquitin and ubiquitin-like protease inhibitors using a fluorescence-based multiplex assay format,” Assay Drug Dev. Technol. 9(2): 165-173 (2011), both of which are incorporated herein in their entireties by this reference.
Other examples of inhibitors include, but are not limited to, shRNA sequences that inhibit USP13, such as, SEQ ID NOs: 1-4, set forth in the Examples. Additional examples of shRNA sequence that can be used to inhibit USP13 include, but are not limited to, SEQ ID NO: 13 (5′AAATCGTCATCAGTATCTA3′) and SEQ ID NO: 14 (5′CCATGTGAACAATGATCCA3′). Other antisense, siRNA or shRNA sequences can be readily obtained by one of skill in the art based on the known coding sequences of USP10 and USP13 provided herein.
The methods set forth herein optionally include administering a second therapeutic agent to the subject. For example, in order to treat a neurodegenerative disorder, the second therapeutic agent can be selected from the group consisting of levadopa, a dopamine agonist, an anticholinergic agent, a monoamine oxidase inhibitor, a COMT inhibitor, amantadine, rivastigmine, an NMDA antagonist, a cholinesterase inhibitor, riluzole, an anti-psychotic agent, an antidepressant, and tetrabenazine. In another example, in order to treat cancer, the second therapeutic agent, can be, for example, a chemotherapeutic agent or radiation.
As used throughout, an increase in parkin activity, and/or an increase in the interaction between parkin and beclin-1 in a subject can be an increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90%, 100% or greater as compared to a control. For example, the increase in parkin activity and/or the increase in the interaction between parkin and beclin-1 can be an increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90%, 100% or greater as compared to a subject that was not administered a deubiquitinating enzyme inhibitor or a control value.
The amount of inhibition or reduction of activity of the deubiquitinating enzyme does not have to be complete as this can range from a decrease to complete ablation of enzymatic activity. For example, the reduction can be about a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between Inhibition or reduction of activity of the deubiquitinating enzyme can be due to a decrease in mRNA expression, a decrease in protein expression, and/or a decrease in the enzymatic activity of the deubiquitinating enzyme.
Further provided is a method of treating or preventing a neurodegenerative disease, a myodegenerative disease or prion disease in a subject, comprising: selecting a subject with a neurodegenerative disease of the central nervous system, a myodegenerative disease, a prion disease or at risk for a neurodegenerative disease of the central nervous system, a myodegenerative disease or a prion disease; and administering to the subject an effective amount of a composition that increases parkin activity, wherein the composition is an inhibitor of a deubiquitinating enzyme.
Also provided is a method of treating or preventing cancer in a subject, comprising: selecting a subject cancer or at risk for cancer; and administering to the subject an effective amount of a composition that increases parkin activity, wherein the composition is an inhibitor of a deubiquitinating enzyme.
The deubiquitinating enzyme inhibitors can be in a pharmaceutically acceptable carrier. The term carrier means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject. Such pharmaceutically acceptable carriers include sterile biocompatible pharmaceutical carriers, including, but not limited to, saline, buffered saline, artificial cerebral spinal fluid, dextrose, and water.
Modes of administration of the compositions used in the invention are exemplified below. Any of the inhibitors described herein can be delivered by any of a variety of routes including: by injection (e.g., subcutaneous, intramuscular, intravenous, intra-arterial, intraperitoneal), by continuous intravenous infusion, cutaneously, dermally, transdermally, orally (e.g., tablet, pill, liquid medicine, edible film strip), by implanted osmotic pumps, by suppository, or by aerosol spray. Routes of administration include, but are not limited to, topical, intradermal, intrathecal, intralesional, intratumoral, intrabladder, intravaginal, intra-ocular, intrarectal, intrapulmonary, intracranial, intraventricular, intraspinal, dermal, subdermal, intra-articular, placement within cavities of the body, nasal inhalation, pulmonary inhalation, impression into skin, and electroporation.
In an example in which a nucleic acid is employed, such as an antisense, shRNA, or siRNA molecule, the nucleic acid can be delivered intracellularly (for example by expression from a nucleic acid vector or by receptor-mediated mechanisms), or by an appropriate nucleic acid expression vector which is administered so that it becomes intracellular, for example by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (such as a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (for example Joliot et al., Proc. Natl. Acad. Sci. USA 1991, 88:1864-8). Antisense or siRNA carriers also include, polyethylene glycol (PEG), PEG-liposomes, branched carriers composed of histidine and lysine (HK polymers), chitosan-thiamine pyrophosphate carriers, surfactants, nanochitosan carriers, and D5W solution. The present disclosure includes all forms of nucleic acid delivery, including synthetic oligos, naked DNA, plasmid and viral delivery, integrated into the genome or not.
As mentioned above, vector delivery can be via a viral system, such as a retroviral vector system which can package a recombinant retroviral genome (see e.g., Pastan et al., Proc. Natl. Acad. Sci. U.S.A. 85:4486, 1988; Miller et al., Mol. Cell. Biol. 6:2895, 1986). The exact method of introducing the altered nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are widely available for this procedure including the use of adenoviral vectors (Mitani et al., Hum. Gene Ther. 5:941-948, 1994), adeno-associated viral (AAV) vectors (Goodman et al., Blood 84:1492-1500, 1994), lentiviral vectors (Naidini et al., Science 272:263-267, 1996), and pseudotyped retroviral vectors (Agrawal et al., Exper. Hematol. 24:738-747, 1996). It is also possible to deliver short hairpin RNAs (shRNAs) via vector delivery systems in order to inhibit gene expression (See Pichler et al. “In vivo RNA interference-mediated ablation of MDR1 P-glycoprotein.” Clin Cancer Res. Jun. 15, 2005; 11(12):4487-94; Lee et al. “Specific inhibition of HIV-1 replication by short hairpin RNAs targeting human cyclin T1 without inducing apoptosis.” FEBS Lett. Jun. 6, 2005; 579(14):3100-6.).
Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanisms (see, for example, Schwartzenberger et al., Blood 87:472-478, 1996) to name a few examples. This invention can be used in conjunction with any of these or other commonly used gene transfer methods.
The effective amount of an inhibitor can depend on the nature of the disease and can be determined by standard clinical techniques. Therefore, these amounts will vary. For example, the dosage can be anywhere from 0.01 mg/kg to 100 mg/kg. Multiple dosages can also be administered depending on the disease, and the subject's condition. In addition, in vitro assays can be employed to identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each subject's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
The disclosure also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. Instructions for use of the composition can also be included.
Depending on the intended mode of administration, the pharmaceutical composition can be in the form of solid, semi-solid, or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, aerosols, or suspensions, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include a therapeutically effective amount of the compound described herein or derivatives thereof in combination with a pharmaceutically acceptable carrier and, in addition, can include other medicinal agents, pharmaceutical agents, carriers, or diluents. By pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, which can be administered to an individual along with the selected compound without causing unacceptable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained.
As used herein, the term carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005. Examples of physiologically acceptable carriers include buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN® (ICI, Inc.; Bridgewater, N.J.), polyethylene glycol (PEG), and PLURONICSTM (BASF; Florham Park, N.J.).
Compositions containing one or more of the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof suitable for parenteral injection can comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
These compositions can also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be promoted by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, for example, sugars, sodium chloride, and the like can also be included. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
Solid dosage forms for oral administration of the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds described herein or derivatives thereof is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example, paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms can also comprise buffering agents.
Solid compositions of a similar type can also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like.
Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art. They can contain opacifying agents and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration of the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures of these substances, and the like.
Besides such inert diluents, the composition can also include additional agents, such as wetting, emulsifying, suspending, sweetening, flavoring, or perfuming agents.
Suspensions, in addition to the active compounds, can contain additional agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
Compositions of the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof for rectal administrations are optionally suppositories, which can be prepared by mixing the compounds with suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component.
As used throughout, treat, treating, and treatment refer to a method of reducing or delaying one or more effects or symptoms of a disease or disorder, for example, a neurodegenerative disease or cancer. The subject can be diagnosed with a disease or disorder. Treatment can also refer to a method of reducing the underlying pathology rather than just the symptoms. The effect of the administration to the subject can have the effect of, but is not limited to, reducing one or more symptoms of the disease or disorder, a reduction in the severity of the disease or disorder, the complete ablation of the 1 disease or disorder, or a delay in the onset or worsening of one or more symptoms. For example, a disclosed method is considered to be a treatment if there is about a 10% reduction in one or more symptoms of the disease in a subject when compared to the subject prior to treatment or when compared to a control subject or control value. Thus, the reduction can be about a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between.
As utilized herein, by prevent, preventing, or prevention is meant a method of precluding, delaying, averting, obviating, forestalling, stopping, or hindering the onset, incidence, severity, or recurrence of the neurodegenerative disease or disorder. For example, the disclosed method is considered to be a prevention if there is a reduction or delay in onset, incidence, severity, or recurrence of neurodegeneration or one or more symptoms of neurodegeneration (e.g., tremor, weakness, memory loss, rigidity, spasticity, atrophy) in a subject susceptible to neurodegeneration as compared to control subjects susceptible to neurodegenration that did not receive an a deubiquitinating enzyme inhibitor that increases parkin activity. The disclosed method is also considered to be a prevention if there is a reduction or delay in onset, incidence, severity, or recurrence of neurodegeneration or one or more symptoms of neurodegeneration in a subject susceptible to neurodegeneration after receiving an agent that promotes parkin activity as compared to the subject's progression prior to receiving treatment. Thus, the reduction or delay in onset, incidence, severity, or recurrence of neurodegeneration can be about a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between.
As used throughout, by subject is meant an individual. Preferably, the subject is a mammal such as a primate, and, more preferably, a human. Non-human primates are subjects as well. The term subject includes domesticated animals, such as cats, dogs, etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.). Thus, veterinary uses and medical formulations are contemplated herein.
Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed and a number of modifications that can be made to a number of molecules including in the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.
Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties. A number of embodiments have been described. Nevertheless, it will be understood that various modifications may be made. Accordingly, other embodiments are within the scope of the following claims.
Example I
Parkin activity is necessary to clear misfolded proteins via autophagy and the proteasome. As set forth herein, parkin must be ubiquitinated in order to be in an active form that effects degradation of its substrates. In neurodegenerative diseases, including Alzheimer's and Parkinson's disease brains, parkin is insoluble likely due to lack of activity or lack of degradation. Parkin must be ubiquitinated to be degraded by the proteasome and must be ubiquitinated to be active. Therefore, the balance between parkin activity and re-cycling via degradation is a critical mechanism to keep parkin functioning. As shown is FIG. 1, parkin activity is necessary to clear misfolded proteins via autophagy and the proteasome. Under normal conditions, parkin goes from an inactive to an active state upon ubiquitination. Active parkin facilitates degradation of proteins via autophagy and the proteasome. In certain diseases, for example, a neurodegenerative disease, myodegenerative disease, prion disease or cancer, deubiquitinating enzymes decrease parkin activity, by deubiquitinating parkin, thus decreasing degradation of proteins via autophagy and the proteasome.
Using proximity ligation assay (PLA), which allows direct observation of individual endogenous protein complexes in situ, it was found that parkin interacts with at least two ubiquitin specific proteases (USP)-13 and 10 (FIGS. 3-5), which are deubiquitination enzymes involved in detaching ubiquitin from parkin, leading to decreased activity or lack of proteasomal degradation. The levels of USP-13 and USP-10 are increased in post-mortem AD and PD brains compared to age-matched controls (FIG. 2). In parallel with the alteration in deubiquitinase levels, parkin soubility is decreased and interaction with beclin-1 is greatly reduced (FIG. 9). Functional parkin-beclin-1 interaction is also lost in Tg-APP and A53T mice (FIGS. 7 and 8). Further, parkin interacts with deubiquitinating enzymes in wild type but not parkin −/− mice (FIG. 6).
FIG. 10 is a schematic diagram showing that amyloid accumulation leads to autophagic induction and sequestration in phagophores. In transgenic or amyloid expressing animals parkin interaction with beclin-1 is reduced, leading to decreased maturation of phagophore into autophagosomes and autophagic defects. Kinase inhibition activates parkin and increases its interaction with beclin-1, resulting in maturation of phagophores into phagosomes and clearance. Also, in human AD and PD brains, parkin-beclin-1 interaction is reduced, indicating that USPs inactivate parkin and prevent functional interaction with beclin-1 to execute autophagic clearance of misfolded amyloid proteins.
Thus, increased levels of USP-13 and 10 in neurodegeneration reduces parkin function and prevents its recycling via proteasomal degradation, leading to decreased solubility and accumulation in disease Inhibition of USP-13 and/or USP-10 and perhaps other deubiquitination enzymes (DUBs) in neurodenegerative diseases, myodegenerative diseases, prion disease and cancer leads to increased parkin activity as a therapeutic strategy.
Also provided herein are the results of experiments showing that treatment with Nilotinib or Bosutinib enhances autophagic clearance (FIGS. 11-25).
Example II
To determine if USP13 deubiquitinates parkin and decreases its stability and E3 ubiquitin ligase activity, several studies were performed.
Parkin levels, solubility and ubiquitination were examined via Western blot (WB), ELISA, Proximity Ligation Assay (PLA) and immunoprecipitation. To evaluate USP13 effects on endogenous parkin in A53T, parkin solubility was determined via fractionation of soluble (supernatant) and insoluble (pellet re-suspended in 4M urea) parkin. Quantitative ELISA (My BioSource, San Diego, Calif. (MBS723678)) and WB was performed with anti-parkin antibodies. To determine whether parkin level correlates with its enzymatic activity, parkin (1:100) was immunoprecipitated with anti-parkin antibody (PRK8) from mouse brain lysates and its E3 ubiquitin ligase activity (with E2:UbcH7) was measured using the E3LITE customizable ubiquitin ligase kit (LifeSensors, Malvern, Pa. (Cat #UC101)).
Immunohistochemistry of Brain Sections
Animals were deeply anesthetized with a mixture of Xylazine and Ketamine (1:8), washed with 1× saline for 1 min and then perfused with 4% paraformaldehyde (PFA) for 15-20 min. Brains were quickly dissected out and immediately stored in 4% PFA for 24 h at 4° C., and then transferred to 30% sucrose at 4° C. for 48 h. Tissues were cut using a cryostat at 4° C. into 20 μm thick sections and stored at −20° C.
Subcellular Fractionation for Isolation of Autophagic Compartments
Animal brains (0.5 grams) were homogenized at low speed (Cole-Palmer, Vernon Hills, Ill. (LabGen 7 Series Homogenizer, 115 Vac)) in 1×STEN buffer and centrifuged at 1,000 g for 10 minutes to isolate the supernatant from the pellet. The pellet was resuspended in 1×STEN buffer and centrifuged once to increase the recovery of lysosomes. The pooled supernatant was centrifuged at 100,000 rpm for 1 hour at 4° C. to extract the pellet containing autophagic vacuoles (AVs) and lysosomes. The pellet was resuspended in 10 ml (0.33 g/ml) 50% Metrizamide and 10 ml deionized water in cellulose nitrate tubes. A discontinuous Metrizamide gradient was constructed in layers from bottom to top as follows: 6 ml of pellet suspension, 10 ml of 26%; 5 ml of 24%; 5 ml of 20%; and 5 ml of 10% Metrizamide. After centrifugation at 10,000 rpm for 1 hour at 4° C., the fraction floating on the 10% layer (Lysosome) and the fractions banding at the 24%/20% (AV 20) and the 20%/10% (AV10) Metrizamide interphases were collected by syringe and examined.
Proximity Ligation Assay
Primary 1:100 mouse anti-parkin and rabbit 1:100 anti-ubiquitin antibodies were applied to 20 μm thick sections of mouse brain overnight at 4° C. A Duolink® In Situ Red Starter Kit (Sigma Aldrich, St. Louis, Mo. (Cat #92101-KI01)) containing species-specific secondary antibodies or PLA probes, each with a unique short DNA strand attached to it (Axxora, LLC, Farmingdale, N.Y.) was used as described in the manufacturer's protocol. When the PLA probes are in close proximity, the DNA strands interact through subsequent addition of two other circle-forming DNA oligonucleotides. After joining of the two added oligonucleotides by enzymatic ligation, they are amplified via rolling circle amplification using a polymerase to highlight the interaction. Fluorescence in each single-molecule amplification product is easily visible as a distinct bright spot when viewed with a fluorescence microscope.
USP 13 and Parkin
Ubiquitination involves the attachment of a 76-amino acid ubiquitin moiety to a protein via an isopeptide linkage that is formed between the C-terminal glycine of ubiquitin and the ε-amino acid side chain of a Lys residue within the target protein. Conjugation of ubiquitin to a protein is a multistep process requiring three different enzymes, including the E1 ubiquitin-activating and E2 conjugating enzymes and interaction with the E3 ubiquitin ligase. Parkin is an E3 RING ubiquitin ligase, which provides a scaffold through which the E3 interacts with the E2-ubiquitin to facilitate the transfer of ubiquitin from the E2 onto the substrate protein.
Human M17 neuroblastoma cells, which express parkin mRNA but quickly degrade the protein, were transfected with lentiviral plasmids containing LacZ, parkin and USP13 cDNAs (3 μg) in 12 well dishes for 48 hr. Western blot (WB) analysis of soluble (STEN) extracts showed that USP13 expression was significantly (84%) increased relative to actin in transfected cells (FIGS. 26A&B, 1st blot, p<0.05) but the level of USP10 was unaffected (FIG. 26A, 2nd blot) compared to LacZ transfected cells. The expression level of parkin was undetected in LacZ transfected cells (FIG. 26A, 3rd blot), but co-expression of parkin and USP13 resulted in a significant increase of soluble parkin relative to actin (210%) compared to parkin transfected cells (FIGS. 26A and D), suggesting that USP13 increases parkin level (N=7, P<0.05). USP13 could alter parkin levels via de-ubiquitination and decreased protein solubility, therefore the insoluble fraction was extracted from the pellets in 4M urea. Co-expression of parkin and USP13 also led to a significant increase (160%) in insoluble parkin relative to actin (FIGS. 26C and D) compared to cells transfected with parkin alone (N=7, p<0.05). To determine whether USP13 can deubiquitinate parkin, rat B35 neuroblastoma cells were transfected with lentiviral wild type or the non-catalytic mutant T240R parkin with and without USP13. This cell line was used instead of the human M17 cells in order to immunoprecipitate parkin without overexpression or proteasomal inhibition. Parkin immunoprecipitation and Western blot with anti-ubiquitin antibodies showed decreased ubiquitinated parkin level when parkin was co-expressed with USP13 (FIG. 26E, 1st blot) compared to parkin expression alone or the input (N=7). However, the level of ubiquitinated proteins when mutant T240R parkin was expressed was unaltered in the presence or absence of USP13 compared to wild type parkin (FIG. 26E), showing that wild type but not mutant T240R parkin can be deubiquitinated by USP13.
To verify that USP13 is a parkin deubiquitinase, parkin and USP13 were separately immunoprecipitated from transfected cells and incubated with recombinant E1, E2 and ATP using a Customizable Ubiquitin Ligase E3LITE Kit (Life Sensors, UC#101), which measures the mechanisms of E1-E2-E3 activity. Parkin and USP13 were added to UbcH7 as an E2 that provides maximum activity with parkin E3 ligase in the presence of E1 and recombinant ubiquitin, including WT containing all seven possible surface Lys or no Lys mutant (K0, negative control). The mix was added to an ELISA microplate that captures polyubiquitin chains formed in the E3-dependent reaction. Poly-ubiquitin chains were used as positive controls (FIG. 26F). No parkin activity was detected with K0 ubiquitin, but parkin was ubiquitinated using WT ubiquitin suggesting parkin activity. However, USP13 significantly decreased (36%) parkin ubiquitination (FIG. 26F, p<0.05, N=7), showing decreased parkin activity in the presence of USP13.
To further determine whether USP13 targets parkin, M17 cells were transfected with 3 μg lentiviral plasmids containing parkin, USP13 or different shRNA USP13 clones (FIG. 27A) for 48 hr. The expression level of the shRNAs was determined by routine stereology of the GFP and VS-tagged lentivirus to ensure equal expression of lentiviral plasmids. USP13 gene expression level is verified by PCR. Co-expression of USP13 and parkin led to an increase (134%) in parkin level compared to parkin alone (FIG. 27A, 1st blot, N=4), but shRNA expression reduced USP13 and parkin levels relative to actin (FIG. 27A). E08 shRNA (5′ TATTCGTTGCTCGTAGTGC 3′(SEQ ID NO: 5)) did not produce any significant effects on USP13 (FIG. 27A, 1st blot) or USP10 (2nd blot) or parkin (3rd blot), but 4 other shRNAs including, E07 (5′ TTTCCAGTGAAGTACACAG 3′ (SEQ ID NO: 1)); F04 (5′AACTTAGTCATCTGTGTGT 3′ (SEQ ID NO: 2)); F03 (5′ TTTCTCGCAGGTCGCATCT 3′ (SEQ ID NO: 3)) and F02 (5′ TCTTGTAGACCCTGTCGCC 3′ (SEQ ID NO: 4)) significantly reduced USP13 (60-70%), without any effects on USP10, but also significantly reduced (55-70%) parkin levels relative to actin compared to USP13 (FIG. 3A, p<0.05, N=4). These data show that USP13 overexpression increases parkin levels, while USP13 blockage with shRNA decreases parkin, perhaps due to increased protein degradation. To ascertain that manipulation of USP13 expression affects protein clearance, the activity of the 20S proteasome activity was measured using chymotrypsin-like assays. Proteasome activity was inhibited after 2 hr treatment with 20 μM proteasome inhibitor MG132 compared to control (LacZ) or parkin alone (FIG. 27B, N=4). Co-expression of USP13 with parkin significantly reduced proteasome activity (34%) compared to parkin (N=4, p<0.05) and MG132 further inhibited the proteasome. However, blocking USP13 with shRNA (F03) significantly increased proteasome activity compared to control (14%) and both F03 and F02 completely reversed the effects of USP13 overexpression on proteasome activity, while MG132 blocked these effects. Taken together, these data show that USP13 expression can deubiquitinate parkin, leading to decreased protein solubility and accumulation, whereas blocking USP13 expression reduces parkin level via decreased deubiquitination and enhanced proteasomal clearance.
Human M17 cells were transfected with USP13 cDNAs and shRNAs in the presence of Tau and ELISA was performed showing the level of hyperphosphorylated Tau at Ser 396 in the presence of parkin (FIG. 28A) the presence and B) absence of parkin. These data show that USP13 deletion can increase parkin-mediated Tau clearance.
Human M17 cells were transfected with USP13 cDNAs and shRNAs in the presence of αβ42. ELISA was performed showing the level of αβ42 and hyperphosphorylated Tau at Ser 396 in the presence of parkin (FIGS. 29A and B) and in the absence of parkin (FIGS. 29C and D). These data show that USP13 deletion can increase parkin-mediated amyloid clearance.
In summary, ubiquitinated parkin is activated in response to amyloid (alpha-Synuclein, beta-amyloid, Tau and TDP-43) accumulation, leading to facilitation of clearance. An increase in USP13 leads to parkin deubiquitination and decreased activity, resulting in misfolded protein accumulation in neurodegenerative diseases (FIG. 30A). It is likely that ubiquitination activates parkin, which is then recycled through the proteasome to prevent its accumulation (FIG. 30B). Activated parkin facilitates autophagic clearance. The studies provided herein show that an increase in USP13 leads to parkin deubiquitination and decreased proteasomal recognition, leading to its instability/insolubility and accumulation. This results in subsequent autophagic failure and death of neurons. Therefore, inhibitors of USP13 are useful for the treatment of diseases such as neurodegenerative diseases, a myodegenerative diseases, prion diseases or cancer.

## Claims

1. A method of promoting parkin activity in a subject that has a neurodegenerative disease of the central nervous system, a myodegenerative disease or a prion disease, comprising:
a) selecting a subject with a disorder associated with decreased parkin activity, wherein the disorder is a neurodegenerative disease of the central nervous system, a myodegenerative disease or a prion disease, wherein the neurodegenerative disease is selected from the group consisting of Amyotrophic Lateral Sclerosis, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, and Mild Cognitive Impairment, an α-Synucleinopathy and a Tauopathy; and
b) administering to the subject an effective amount of a composition that increases parkin activity, wherein the composition is an inhibitor of a deubiquitinating enzyme.

2. The method of claim 1, wherein the deubiquitinating enzyme is USP-10 or USP-13.

3. The method of claim 1, wherein the composition increases parkin activity by promoting the interaction between parkin and beclin-1.

4. The method of claim 1, wherein the composition inhibits deubiquitination of parkin.

5. A method of treating a neurodegenerative disease, a myodegenerative disease or prion disease in a subject, comprising:
a) selecting a subject with a neurodegenerative disease of the central nervous system, a myodegenerative disease, or a prion disease, wherein the neurodegenerative disease is selected from the group consisting of Amyotrophic Lateral Sclerosis, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, and Mild Cognitive Impairment, an α-Synucleinopathy and a Tauopathy; and
b) administering to the subject an effective amount of a composition that increases parkin activity, wherein the composition is an inhibitor of a deubiquitinating enzyme.

6. The method of claim 5, wherein the deubiquitinating enzyme is USP-10 or USP-13.

7. The method of claim 5, wherein the composition promotes the interaction between parkin and beclin-1.

8. The method of claim 5, wherein the composition inhibits deubiquitination of parkin.