Inhibition of β-arrestin oligomerization in tauopathies

Abstract

As disclosed herein, β-arrestin 1 and β-arrestin 2 levels are highly elevated in the brains of FTLD-tau patients, indicating that β-arrestin 1 and β-arrestin 2 are elevated in the brains of patients with AD and FLTD. Current work also shows that tau levels increase when β-arrestin 2 is overexpressed. The data indicate that β-arrestin 2 reduces tau clearance by impairing p62-mediated autophagy, an effect played by the oligomeric form of β-arrestin 2. Therefore, disclosed herein are β-arrestin oligomerization inhibitors that can be used to prevent β-arrestin oligomerization and thereby prevent the accumulation of tau in cells, i.e., tauopathy. Also disclosed is a method for treating tauopathy in a subject, comprising administering to the subject a therapeutically effective amount of a β-arrestin oligomerization inhibitor disclosed herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 971,510, filed on February 7, 2020, which is hereby incorporated by reference in its entirety.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under Grant No. AG059721 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0005] background

[0006] As a cause of early-onset nonvascular dementia, frontotemporal lobar degeneration (FTLD) is an aggressive neurodegenerative disease second only to Alzheimer's disease in incidence, with an unclear pathological basis (Irwin DJ, et al. (2015) Acta Neuropathol 129(4):469-491). Like AD, the most common form of FTLD (FTLD-tau) shows accumulation of hyperphosphorylated tau in neuronal inclusions in affected areas, including the cortex and temporal lobes as well as some subcortical neurons (Cairns NJ, et al. (2007) Acta Neuropathol 114(1):5-22). In contrast to AD, in which amyloid β (Aβ) is a component of tangles, there is no accumulation of Aβ in FTLD neurons, but rather a significant accumulation of tau is observed in most forms. Interestingly, even in AD, tau appears to be essential for Aβ to transduce neurotoxic signals (Vossel KA, et al. (2010) Science 330(6001):198; Jin M, et al. (2011) Proc Natl Acad Sci USA 108(14):5819-5824). In fact, tauopathies are significantly more associated with cognitive deficits in AD than Aβ (Nelson PT, et al. (2010) Brain Pathol 20(1):66-79; Tiraboschi P, et al. (2004) Neurology 62(11):1984-1989; Matthews FE, et al. (2009) PLoS Med 6(11):e1000180). The pathogenic role of tau in AD, together with several observations regarding G protein-coupled receptors (GPCRs) in AD pathology and treatment, suggest the need for a better understanding of how GPCRs and related proteins are integrated into tauopathies in FTLD, for which unique therapeutic strategies have been lacking to date. SUMMARY OF THE INVENTION

[0008] As disclosed herein, β-arrestin 1 and β-arrestin 2 levels are highly elevated in the brains of FTLD-tau patients, suggesting that both β-arrestin 1 and β-arrestin 2 are elevated in the brains of patients with AD and FLTD. The current work also suggests that tau levels are elevated when β-arrestin 2 is overexpressed. The data suggest that β-arrestin 2 reduces tau clearance by impairing p62-mediated autophagy, an effect exerted by oligomeric forms of β-arrestin 2.

[0009] β-Arrestin2 can exist as oligomers in a variety of cell types (Milano SK, et al. (2006) J Biol Chem 281(14):9812-9823; Storez H, et al. (2005) J Biol Chem 280(48):40210-40215). Inositol hexaphosphate (IP6) enhances this self-association of β-arrestin2 by bridging adjacent molecules into a head-to-tail configuration. Positively charged arginine and lysine residues within the N- and C-termini of β-arrestin2 were found to be critical for both IP6 binding and oligomerization. Given the physical overlap of IP6 and GPCR binding sites, binding of β-arrestin2 to activated GPCRs and to IP6 are mutually exclusive, suggesting that in the oligomeric form, β-arrestin2 can serve other purposes besides GPCR binding.

[0010] Therefore, the present invention discloses a β-inhibitor oligomerization inhibitor, which can be used to prevent β-inhibitor oligomerization and thus prevent the accumulation of tau in cells, i.e., tauopathy. Also disclosed is a method for treating tauopathy in a subject, comprising administering to the subject a therapeutically effective amount of a β-inhibitor oligomerization inhibitor disclosed herein.

[0011] β-Arrestin 1 and β-Arrestin 2 form homo- and hetero-oligomers, and inositol hexaphosphate (IP6) promotes these oligomers. Therefore, in some embodiments, the inhibitor of β-arrestin oligomerization is a molecule, such as a peptide, that blocks IP6 binding, thereby reducing the oligomerization of β-arrestin 2. In some embodiments, the inhibitor of oligomerization is a β-arrestin mutant that acts as a dominant negative for homo-hetero-oligomerization of β-arrestin 1 and / or β-arrestin 2.

[0012] The data presented also suggest that pharmacological inhibitors for β-arrestin oligomerization could represent a promising therapeutic strategy to mitigate AD and tauopathies. Therefore, a molecular docking screen using FTMap analysis was performed to screen millions of compounds that block the interface between β-arrestin and β-arrestin 2. Approximately 30 predicted inhibitory compounds were tested using cell-based assays, yielding several small molecule compounds and their analogs that reduce pathogenic tau.

[0013] Regarding the mutants, they were the same size as full-length β-arrestin2. The IP6 binding sites were located at the C-terminus and N-terminus. (Just point mutations). Blocking the binding of IP6 to the C-terminus or N-terminus abolished its oligomerization. So the initial idea was to screen for compounds that block the IP6 binding site, but several compounds were found to bind to the interface between β-arrestin2 and β-arrestin2. These compounds were confirmed to block oligomerization by Co-IP, FRET, and PLA assays.

[0014] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figures 1A to 1I showed that β-arrestin2 was increased in the brains of FTLD-tau patients and tau P301S transgenic mice. Figure 1A Representative immunoblot containing β-arrestin 2 from the frontal cortex of 12 age-matched unaffected controls and 10 FTLD-tau patients. The numbers above the lanes are the sample numbers corresponding to the subjects, and some samples were repeated on a second gel. Figure 1B and 1C Shown is the quantification of β-arrestin2 protein and mRNA levels in the frontal cortex of FTLD-tau patients compared with unaffected controls (N=12 healthy controls, N=10 FTLD-tau, **P<0.005; *P<0.05). Figure 1D The correlation of insoluble tau and β-arrestin 2 expression in FTLD-tau patients (N=10 FTLD-tau) is shown. Each data point represents the average result of repeated measurements from a given subject. The blots are shown in SI Appendix, Figure 1E Contains representative immunoblots of β-arrestin2 in the cortex of mice from 7 month old WT and tau P301S transgenic littermates. Figure 1F and 1G Quantification of β-arrestin2 protein and mRNA levels in the cortex of WT and tau P301S transgenic littermates is shown (N=5 WT, N=4 tau P301S, **P<0.005, *P<0.05). Figure 1H Representative staining for β-arrestin2 (magenta) and total tau (green) using DIV21 hippocampal primary neurons derived from WT and tau P301S littermates is shown (bar = 10 μm). Fig. 1IQuantification of β-arr2 immunoreactivity is shown (N=3 independent experiments, *P<0.05). Box plots show all data points, median and IQR, as well as minimum and maximum values.

[0016] Figures 2A to 2M Overexpression and knockdown of β-arrestin2 were shown to regulate tau levels. Figure 2A Representative immunoblots containing the indicated proteins from Hela-V5-tau cells transiently transfected with GFP or GFP-β-arrestin2. Figures 2B to 2D Quantification of tau, PHF1 and AT8 levels from 6 experiments is shown (*P<0.05, **P<0.005 vs GFP). Figure 2E Shown are representative immunoblots of the indicated proteins in Hela-V5-tau cells transiently transfected with control siRNA or β-arrestin2 siRNA. Figure 2F Quantification of tau levels from 4 experiments is shown (#P<0.0001). Figure 2G Representative staining of tau and AT8 in hippocampal primary neurons from tau P301S-transgenic mice transduced with control (Cont-Ad) or β-arrestin 2 adenovirus (β-arrestin2-Ad) is shown (bar = 10 μm). Figures H and 2I show quantification of AT8 and total tau immunoreactivity from 3 independent experiments, #P<0.0001, ***P<0.0005). Figure 1J shows representative staining of tau in hippocampal primary neurons from tau P301S-transgenic mice transduced with GFP-lenti shRNA or GFP-lenti β-arrestin 2 shRNA. Figure 2K Quantification of total tau immunoreactivity is shown (N=4 independent experiments, #P<0.0001). Figure 2L Shown are representative immunoblots of the indicated proteins from Hela-V5-tau cells transfected with GFP or GFP-β-arrestin2 and treated with cycloheximide (100 μg / ml) for the indicated times. Figure 2M Quantification of tau levels treated with cycloheximide is shown (N=4; **P<0.005, #P<0.0005, *P<0.05, repeated measures ANOVA followed by Bonferroni post hoc test). Box plots show all data points, median and IQR, as well as minimum and maximum values.

[0017] Figure 3A to 3I showed that genetic reduction of β-arrestin2 alleviated neuronal tauopathy. Figure 3AShown are tau P301S, tau P301S / Arrb2 from 7-month-old + / - and tau P301S / Arrb2 - / - Representative immunoblots of sarkosyl-soluble and sarkosyl-insoluble tau from littermate brains. Figure 3B and 3C Quantification of 4 experiments performed as in a) is shown, **P<0.005, #P<0.0001. Figure 3D 7-month-old tau P301S, tau P301S / Arrb2 + / - and tau P301S / Arrb2 - / - Representative staining of phospho-tau, pS199 / 202, in the hippocampus and cortex of mice (bar = 20 μm). Figure 3E and 3F Quantification of pS199 / 202 immunoreactivity in cortex and hippocampus is shown (N=4 / genotype, #P<0.0001). Figure 3G It is shown that the expression of WT, tau P301S, tau P301S / Arrb2 + / - and tau P301S / Arrb2 - / - Input / output (I / O) analysis in acute slices generated with stimulus amplitudes increasing from 1 step to 15 mV. (N=35-46 slices / genotype from 4 mice / genotype). Figure 3H PPF is shown, fEPSP slope is shown as a function of 30-300 ms interstimulus interval (N=38-49 slices / genotype from 4 mice / genotype). FIG3I shows LTP induced by theta burst stimulation, showing tau P301S vs WT, tau P301S / Arrb2 + / - and tau P301S / Arrb2 - / - Significant differences in fEPSP slopes between slice comparisons (N=25-43 slices / genotype from 4 mice / genotype, #P<0.0001). Data shown are box plots or mean±SEM.

[0018] Figures 4A to 4E It was shown that β-arrestin2 oligomerization is required for the inhibition of tau turnover. Figure 4A Shown are representative immunoblots of DIV18 cortical primary neurons from tauP301S transduced with GFP, β-arrestin2ΔIP6C-GFP, or β-arrestin2ΔIP6N-GFP at DIV5. Figure 4B Quantification of tau levels is shown (N=4, #P<0.0001, ***P<0.0005). Figure 4C and 4D Representative immunoblots containing the indicated proteins in Hela-V5-tau cells transfected with control vector, β-arrestin2ΔIP6C, β-arrestin2ΔIP6N and treated with cycloheximide (100 μg / ml) for the indicated times. Figure 4E Quantification of tau levels treated with cycloheximide is shown (N=4; **P<0.005, ***P<0.0005, #P<0.0001, repeated measures ANOVA followed by Bonferroni post-hoc). Data are shown as box plots or mean±SEM.

[0019] Figures 5A to 5N showed that β-arrestin2 oligomerization inhibits p62-mediated tau degradation. Figure 5A Representative images containing LC3-positive autophagosomes from Hela-V5-tau cells transiently transfected with control vector or β-arrestin2-myc with / without 100 nM bafilomycin A1 (bar = 10 μm). Figure 5B Quantification of LC3 puncta is shown (N=4 independent experiments, *P<0.05). Figure 5C Representative images (bar = 10 μm) containing GFP-p62-positive autophagosomes from Hela-V5-tau cells transiently transfected with control vector or β-arrestin2-myc with / without 100 nM bafilomycin A1. Figure 5D Quantification of GFP-p62 puncta is shown (N=4 independent experiments, #P<0.0001, ***P<0.0005). Figure 5E Representative images of DIV21 tau P301S hippocampal primary neurons transduced with mcherry-GFP-p62 AAV9 and control, β-arrestin2, β-arrestin2ΔIP6C, or β-arrestin2ΔIP6N lentiviruses are included at DIV5. Fig. 5F Quantification of mCherry particles only is shown (N=3 independent experiments, **P<0.005, #P<0.0001). Figure 5G Representative immunoblots containing Hela-V5-tau cells transiently transfected with control vector or β-arrestin2-myc with GFP-p62 and / or HA-p62 and lysed followed by co-IP. Figure 5H Quantification of GFP-p62 and HA-p62 interaction by co-IP is shown (N=5 independent experiments, #P<0.0001). Fig.5IRepresentative images of proximity ligation assays (PLA) involving Hela-V5-tau cells transfected with control vector, β-arrestin2, β-arrestin2ΔIP6C, or β-arrestin2ΔIP6N (bar = 10 μm). Figure 5J Quantification of PLA spots is shown (N=4 independent experiments, #P<0.0001, **P<0.005). Figure 5K Shown are 0.4 μg of p62 incubated with / without 0.2 μg of β-arrestin2 for 2 hr at RT before AFM imaging. Figure 5L Quantification of p62 granularity is shown (N=3 independent experiments, *p<0.05). Figure 5M Representative staining of flag in Hela-V5-tau cells transfected with control vector, β-arrestin2-flag, β-arrestin2ΔIP6C-flag, or β-arrestin2ΔIP6N-flag and GFP-p62 with / without 100 nM bafilomycin A treatment is shown (bar = 10 μm). Figure 5N Quantification of GFP-p62 puncta is shown (N=4 independent experiments, #P<0.0001). Data are shown as box plots or mean ± SEM.

[0020] Figures 6A to 6E showed that oligomerization-defective β-arrestin 2 alleviated tauopathy in vivo. Fig. 6A Shown are representative immunoblots of lauroylsarcosine-soluble and lauroylsarcosine-insoluble tau from brains of 7-month-old tau P301S mice that were stereotaxically injected into the hippocampus with rAAV9 encoding GFP, GFP-β-arrestin2ΔIP6C, or GFP-β-arrestin2ΔIP6C at 5 months of age. Figure 6B and 6C Shows from Fig. 6A Quantification of lauroylsarcosine soluble and insoluble tau in stereotaxically injected tauP301S mice as indicated, N=4 / genotype, **P<0.005, ***P<0.0005). Fig.6D Shows Fig. 6A Representative staining of total tau (magenta) in the hippocampus of 7 month old tau P301S injected stereotaxically as shown (bar = 20 μm). Fig. 6A Quantification of total tau immunoreactivity is shown (N=4 / genotype, #P<0.0001). Data are shown as box plots.

[0021] Figure 7is a schematic model of how β-arrestin2 promotes tauopathy in FTLD. In the healthy brain, monomeric β-arrestin2 regulates GPCR trafficking, and there is no excess oligomeric β-arrestin2, so misfolded tau is efficiently ubiquitinated and targeted for autophagic clearance. However, in the FTLD-tau brain, β-arrestin2 oligomers are increased, inhibiting p62-mediated autophagy, resulting in inefficient clearance of misfolded / aggregated tau. In AD, increased β-arrestin2 also leads to an increase in Aβ amyloid (not shown)

[0022] Figure 8 β-Arrestin 2 levels correlated with tau levels in FTLD-tau brains are shown. RIPA-insoluble immunoblots of the indicated proteins from the frontal cortex of 10 FTLD-tau patients.

[0023] Figures 9A to 9F β-Arrestin2 was shown to increase tau levels without altering mRNA. Fig. 9A Representative immunoblots containing the indicated proteins from DIV18 tau P301S cortical primary neurons transduced on DIV5 with control adenovirus (control-Ad) or β-arrestin 2 adenovirus (β-arr2-Ad). Fig. 9B Quantification of total tau levels is shown (N=4, **P<0.005). Fig. 9C Contains representative immunoblots of the indicated proteins from DIV18 tau P301S cortical primary neurons transduced with control shRNA-lentivirus or β-arrestin2 shRNA-lentivirus on DIV5. Fig.9D Quantification of total tau levels is shown (N=4, **P<0.005). Fig.9E Shown are Hela-V5-tau cells transfected with control vector or β-arrestin2 and subjected to qRT-PCR for tau mRNA and normalized to control (N=6 each). Fig.9F Shown are Hela-V5-tau cells transfected with control siRNA or β-arrestin2 siRNA and subjected to qRT-PCR for tau mRNA and normalized to control (N=6 each).

[0024] Figures 10A to 10F showed that genetic reduction of β-arrestin2 reduced lauroylsarcosine-insoluble tau and alleviated synaptic dysfunction in tau P301S primary neurons. Fig. 10A Contains proteins derived from tau P301S and tau P301S;Arrb2 - / -Representative immunoblots of lauroylsarcosine soluble and insoluble tau in DIV18 cortical neurons. Fig. 9A and 9B Shown are derived from tau P301S and tau P301S;Arrb2 - / - Lauroylsarcosine soluble tau ( Fig. 9B )Lauroylsarcosine insoluble tau( Fig. 9C ) (N=3; ***P<0.005). Fig.9D Contains representative confocal images of synaptophysin and drebrin in DIV21 hippocampal primary neurons from WT and tauP301S littermates transduced with GFP-lenti shRNA or GFP-lentiβ-arrestin2 shRNA on DIV5 (scale bar = 10 μm). Fig.9E and 9B Synaptophysin ( Fig.9E ) and brain development regulatory protein ( Fig.9F ) Quantification of immunoreactivity (N=3 independent experiments, #P<0.005).

[0025] Figures 11A to 11E β-Arrestin2 oligomerization mutants were shown to reduce tau aggregates. Fig.11A Representative confocal images containing total tau in Hela-V5-tau cells transfected with YFP, YFP-β-arrestin2ΔIP6C, or YFP-β-arrestin2ΔIP6N (scale bar = 10 μm). Fig. 11B Contains representative confocal images of proximity ligation assay (PLA) in Hela-V5-tau cells transfected with β-arrestin2-flag and β-arrestin2-myc with YFP control, YFP-β-arrestin2ΔIP6C, or YFP-β-arrestin2ΔIP6N. Fig. 11C Quantification of β-arrestin2-flag / β-arrestin2-myc PLA spots / cell is shown (N=3 independent experiments, **P<0.005, ***P<0.0005). Fig.11D Contains representative immunoblots of filter trap assays for tau from Hela-V5-tau cells transfected with control vector, β-arrestin2ΔIP6C-flag, or β-arrestin2ΔIP6N-flag. Fig.11E Quantification of insoluble tau aggregates is shown (N=8 / condition, #P<0.0001).

[0026] Figures 12A to 12Eβ-Arrestin2 was shown to interact with p62 and that β-Arrestin2 oligomerization inhibited p62 self-interaction. Fig. 12A Representative confocal images containing mCherry and EGFP in Hela-V5-tau cells transfected with mCherry-GFP-p62 together with control vector, β-arrestin2-flag or β-arrestin2ΔIP6N-flag (scale bar = 10 μm). Fig. 12B Quantification of mCherry-only particles versus total particles normalized to control is shown (N=3 independent experiments, #P<0.0001).Box and whisker plots show all data points, box plots show all data points, median and IQR, and minimum and maximum values. Fig. 12C Representative immunoblots containing the indicated proteins from Hela-V5-tau cells transfected with GFP or GFP-β-arrestin2 with / without HA-p62. Note that β-arrestin2 was present in HA immune complexes only in the presence of HA-p62, whereas β-arrestin2 was not detected in control IgG beads. Fig.12D Representative immunoblots containing the indicated proteins from Hela-V5-tau cells transfected with control, β-arrestin2-flag, β-arrestin2ΔIP6C-flag, or β-arrestin2ΔIP6N-flag together with HA-p62 and / or GFP-p62. Lysates were processed for IP for HA. Fig.12E Representative confocal images of proximity ligation assays (PLA) involving Hela-V5-tau cells transfected with β-arrestin2-flag, β-arrestin2ΔIP6C-flag, or β-arrestin2ΔIP6N-flag (scale bar = 20 μm). Fig.12F Quantification of flag / p62 PLA spots / cell is shown (N=4 independent experiments, #P<0.0001).

[0027] Figures 13A to 13F showed that mGluR2- and β2AR-mediated increases in pathogenic tau require β-arrestin 1. Fig.13A Hela-V5-tau cells transfected with control siRNA or β-arrestin 1 siRNA are shown. After transfection, cells were treated with vehicle or 10 μM isoproterenol (Iso), lysed and immunoblotted for the indicated proteins. Representative blots are shown. Fig. 13B Quantification of phospho-tau levels is shown. n=3 independent experiments. *p<0.05. 1-way ANOVA with Dunnett's test. Fig. 13CHela-V5-tau cells transfected with control siRNA or β-arrestin 1 siRNA and treated with vehicle, 1 μM or 10 μM LY-379,268, lysed and immunoblotted for the indicated proteins are shown. Representative blots are shown. Fig.13D Quantification of phospho-tau levels is shown. n=4 independent experiments. *p<0.05, **p<0.005. 1-way ANOVA with Dunnett's test. Fig.13E Shown are Hela-V5-tau cells transfected with control siRNA or β-arrestin2 siRNA and treated with vehicle, 10 μM Iso, or 10 μM LY-379,268, lysed, and immunoblotted for the indicated proteins. Fig.13F Quantification of phospho-tau levels is shown. n=4 independent experiments. *p<0.05, **p<0.005. 1-way ANOVA with Dunnett's test.

[0028] Figures 14A to 14G Showing increased β-arrestin 1 in the brains of patients with FTLD-tau. Fig.14A RIPA soluble extracts from the frontal cortex of healthy controls and FTLD-tau patients were immunoblotted for β-arrestin 1 and actin. Representative blots are shown. Fig. 14B Quantification of RIPA soluble β-arrestin 1 levels is shown. Healthy controls (n=12), FTLD-tau patients (n=10). ***p<0.0005. Unpaired t-test. Fig. 14C RIPA-insoluble extracts from the frontal cortex of healthy controls and FTLD-tau patients were immunoblotted for β-arrestin 1 and actin. Representative blots are shown. Fig.14D Quantification of RIPA-insoluble β-arrestin 1 levels is shown. Healthy controls (n=12), FTLD-tau patients (n=10). ***p<0.0005. Unpaired t-test. Fig.14E Shown are RIPA-insoluble extracts from frontal cortex of FTLD-tau patients immunoblotted for β-arrestin 1, tau, and actin. Fig.14F The correlation between RIPA-insoluble tau and β-arrestin 1 in FTLD-tau patients is shown (n = 10 FTLD-tau; R 2 =0.4874, P =0.0248, linear regression). Figure 14GRepresentative Z-stack images of FTLD-tau brains are shown, demonstrating that β-arrestin 1 and AT8+(pS202 / pT205) tau pathology are co-localized (Scale bar = 20 μm). White boxes are magnified below.

[0029] Figures 15A to 15G β-Arrestin 1 was shown to increase tau levels. Fig.15A Shown are DIV5 hippocampal primary neurons derived from P0tauP301S mice that were lentivirally transduced with GFP or β-arrestin 1-shRNA-GFP and immunostained for tau on DIV 21. Representative images are shown (scale bar = 10 μm). Fig. 15B Quantification of tau intensity is shown. n = 4 independent experiments. #p < 0.0001. Unpaired t-test. Fig. 15C DIV5 cortical primary neurons derived from P0tauP301S pups were lentivirally transduced with control or β-arrestin1 shRNA. Cortical neurons were lysed at DIV18 and immunoblotted for β-arrestin1, tau, and actin. Representative blots are shown. Fig.15D Quantification of total tau levels is shown. n = 4 independent experiments. **p < 0.005. Unpaired t-test. Fig.15E Hela-V5-tau cells transfected with control vector or β-arrestin 1 were lysed and immunoblotted for β-arrestin 1, tau, phospho-tau, and actin. Representative blots are shown. Fig.15F and 15G Total tau ( Fig.15F ) and phospho-tau ( Figure 15G ) levels. n = 6 independent experiments. *p < 0.05, #p < 0.0001. Unpaired t-test.

[0030] Figures 16A to 16J showed that genetic reduction of β-arrestin 1 alleviates tauopathy in vivo. Fig.16A Contains 7-month-old tauP301S and tauP301S;Arrb1 + / - Confocal images of littermates stained for pS199 / pS202 tau (scale bar = 10 μm). White boxes are magnified below. Representative images are shown. Fig. 16B Quantification of pS199 / pS202 tau intensity is shown. n=4 / genotype. #p<0.0001. Unpaired t-test. Fig. 16C Shown are tauP301S and tauP301S;Arrb1 from 7-month-old mice + / -Lauroylsarcosine soluble and insoluble extracts of brain were immunoblotted for β-arrestin 1, tau, and actin. Representative blots are shown. Fig.16D and 16E Quantification of lauroylsarcosine soluble and insoluble tau levels is shown. n=5 / genotype. **p<0.005. Unpaired t-test. Fig.16F Shown are samples from 4-month-old WT, tauP301S, and tauP301S;Arrb1 + / - Input-output analysis of acute slices. (WT: 33 slices, 5 mice; tauP301S: 32 slices, 5 mice; tauP301S; Arrb1 + / - (: 30 sections, 4 mice). No significant differences were observed. Figure 16G Shown are samples from 4-month-old WT, tauP301S, and tauP301S;Arrb1 + / - Paired-pulse facilitation (PPF) analysis in acute slices; (WT: 33 slices, 5 mice; tauP301S: 40 slices, 5 mice; tauP301S; Arrb1 + / - : 35 sections, 4 mice). 2-way ANOVA with Dunnett's post hoc test. #p<0.0001: 20-180 ms, WT vs. tauP301S. ***p<0.0005: 20 ms, tauP301S vs. tauP301S; Arrb1 + / - **p<0.005: 200-260ms, WT vs. tauP301S. *p<0.05: 280-300ms, WT vs. tauP301S; 40, 100 and 120ms, tauP301S vs. tauP301S; Arrb1 + / - . Fig.16H Long-term potentiation (LTP) induced by theta burst stimulation is shown. (WT: 31 sections, 5 mice; tauP301S: 29 sections, 5 mice; tauP301S; Arrb1+ / -: 31 sections, 4 mice). 2-way ANOVA with Dunnett's post hoc test. *p<0.0001 at all time points, WT vs. tauP301S and tauP301S Arrb1 + / - All data are expressed as mean ± SEM. Fig.16I Hippocampal primary neurons derived from WT and tauP301S P0 pups were shown to be lentivirally transduced with GFP or β-arrestin 1-shRNA-GFP on DIV 5. Neurons were stained with synaptophysin on DIV 21 (scale bar = 10 μm). Representative images are shown. Fig.16JQuantification of synaptophysin intensity is shown. n=4 independent experiments, #p<0.0001. 1-way ANOVA with Dunnett's test.

[0031] Figures 17A to 17F β-Arrestin 1 was shown to inhibit tau-induced tubulin polymerization and destabilize microtubules. Fig.17A Shown is a microtubule-bound sedimentation assay using 1 μg of recombinant tau incubated with 2 μg of BSA or 2 μg of recombinant β-arrestin 1. Indicated amounts of β-arrestin 1 or BSA were incubated with or without 0.4 nM prepolymerized microtubules in the presence of tau, and microtubule-bound tau was monitored by co-precipitation and subsequent tau immunoblotting. Representative blots are shown. Fig. 17B Quantification of microtubule-bound tau is shown. n = 5 independent experiments. *p < 0.005. Unpaired t-test. Fig. 17C Shown is a microtubule-bound sedimentation assay using the indicated amounts of recombinant β-arrestin 1, 1 μg of tau, and 0.4 nM prepolymerized microtubules. Tau and β-arrestin 1 in the microtubule-bound pellet and unbound supernatant were monitored by co-precipitation and subsequent immunoblotting. Fig.17D Shown are tubulin polymerization measured by turbidity at 340 nm in the presence of 2 μg of the indicated recombinant proteins. n=4 independent experiments. #p<0.0001. 2-way repeated measures ANOVA. Fig.17E Confocal images of Hela-V5-tau cells transfected with control siRNA or β-arrestin 1 siRNA and treated with 20 μM nocodazole for 30 min and recovered for 1 hour are shown. Cells were fixed and immunostained for tubulin and β-arrestin 1 (scale bar = 10 μm). Enlarged white box on the right. Fig.17F Quantification of transfected cells is shown, with tubulin intensity normalized to control siRNA transfected cells. n=4 independent experiments. #p<0.0001. Unpaired t-test.

[0032] Figures 18A to 18N showed that β-arrestin 1 inhibits autophagy-mediated tau clearance. Fig.18A Shown is the quantification of β-arrestin 1 mRNA levels by qRT-PCR in Hela-V5-tau cells transfected with control vector or β-arrestin 1. n = 6 independent experiments. Fig.18B Shown is the quantification of β-arrestin 1 mRNA levels by qRT-PCR in Hela-V5-tau cells transfected with control siRNA or β-arrestin 1 siRNA. n=6 independent experiments. Fig.18CHela-V5-tau cells transfected with control siRNA or β-arrestin 1 siRNA and treated with cycloheximide (100 μg / ml) for 2 hours and 4 hours are shown. Cells were immunoblotted for β-arrestin 1, tau and actin. Representative blots are shown. Fig.18D Shown is the quantification of remaining tau after cycloheximide treatment. n = 3 independent experiments. *p < 0.05. 2-way repeated measures ANOVA. Fig.18E Confocal images of Hela-V5-tau cells transfected with GFP-p62 and control vector or β-arrestin 1-myc and treated with vehicle or 20 μM nocodazole for 30 minutes. Cells were fixed and immunostained for myc and LC3 (scale bar = 10 μm). Representative images are shown. Fig.18F Quantification of GFP-p62 and LC3 colocalization is shown. n=4 independent experiments. #p<0.0001. 1-way ANOVA with Dunnett's post hoc test. Figure 18G Confocal images of Hela-V5-tau cells transfected with GFP-p62 and vector control or β-arrestin 1-myc and treated with vehicle or 100 nM bafilomycin A1 for 4 hours. Cells were fixed and immunostained for myc and LC3 (scale bar = 10 μm). Representative images are shown. Fig.18H Quantification of GFP-p62 puncta area is shown. n=4 independent experiments. ***p<0.0005, **p<0.005, #p<0.0001. 1-way ANOVA with Dunnett's post hoc test. Fig.18I Quantification of GFP-p62 and LC3 colocalization is shown. n=4 independent experiments. ***p<0.0005, **p<0.005, #p<0.0001. 1-way ANOVA with Dunnett's post hoc test. Fig.18J Confocal images of Hela-V5-tau cells transfected with mCherry-GFP-p62 and control siRNA or β-arrestin 1 siRNA and treated with vehicle or 100 nM bafilomycin A1 for 4 hours. Cells were fixed and immunostained for β-arrestin 1 (scale bar = 10 μm). mCherry was pseudo-colored magenta. Representative images are shown. Figure 18K Quantification of total p62 puncta (mCherry+GFP) normalized to control vehicle treatment is shown. n=4 independent experiments. #p<0.0001, **p<0.005. 1-way ANOVA with Dunnett's test. Figure 18LQuantification of mCherry-only (magenta) spots normalized to control vehicle treatment is shown. n=4 independent experiments. #p<0.0001. 1-way ANOVA with Dunnett's test. Figure 18M Hela-V5-tau cells transiently transfected with control vector or β-arrestin 1-myc together with GFP-p62 and / or HA-p62 and co-immunoprecipitated for HA and immunoblotted for GFP, HA, myc and actin are shown. Representative blots are shown. Fig.18N Quantification of GFP-p62 and HA-p62 interactions n = 3 independent experiments are shown. **p < 0.005. Unpaired t-test.

[0033] Fig.19 Figure 1 is a schematic model of how β-arrestin 1 promotes tauopathy. β-arrestin 1 is upregulated in FTLD-tau and AD brains. Increased β-arrestin 1 can interact with beta-secretase to promote Aβ production. (1) Increased β-arrestin 1 also binds to microtubules and promotes the dissociation of tau from microtubules, thereby destabilizing microtubules and causing tau misfolding. (2) β-arrestin 1 also physically interacts with p62 and inhibits p62 body formation, thereby impairing p62 trafficking and clearance of pathogenic tau. The combined effects of tau displacement, microtubule instability, and tau accumulation promote tauopathy.

[0034] Figures 20A to 20E We show that β-arrestin is required for mGluR2- and β2AR-mediated tau accumulation and phosphorylation. Fig. 20A Shown are DIV18 cortical primary neurons derived from PS19 P0 pups, treated with vehicle or 10 μM isoproterenol (ISO) for 24 hours, lysed and immunoblotted for tau, pS396 / pS404-tau (PHF1), and actin. Representative blots are shown. Fig. 20B Quantification of PHF1 levels is shown. n=4 independent experiments. *p<0.05. Unpaired t-test. All data are expressed as mean ± SEM. FIG21C shows DIV18 cortical primary neurons from PS19P0 pups treated with vehicle, 1 μM or 10 μM LY-379,268, lysed and immunoblotted for tau, PHF1 and actin. Representative blots are shown. Fig.20D Quantification of PHF1 levels is shown. n=4 independent experiments. ***p<0.0005. Unpaired t-test. All data are expressed as mean ± SEM. Fig.20E Arrb2 cells were treated with vehicle, 10 μM ISO or 10 μM LY-379,268.- / - DIV11 cortical primary neurons from P0 pups 24 hours old were lysed and immunoblotted for tau, PHF1, and actin. Representative blots are shown.

[0035] Fig.21A and 21B β-Arrestin 1 shown to co-localize with phospho-tau AT8 in FTLD-tau patients. Fig.21A Confocal images of healthy control brains stained with β-arrestin 1 and AT8 are included (scale bar = 20 μm). Fig. 21B Secondary antibody staining alone showed no immunoreactivity in FTLD-tau brains.

[0036] Fig.22A and 22B Reduced beta-arrestin 1 was shown to reduce tau levels. Fig.22A HeLa-V5-tau cells transfected with control siRNA or β-arrestin 1 siRNA, lysed and immunoblotted for β-arrestin 1, tau and actin are shown. Representative blots are shown. Fig. 22B Quantification of tau is shown. n = 4 independent experiments. #p < 0.0001. Unpaired t-test. All data are expressed as mean ± SEM.

[0037] Figures 23A to 23C showed that increased β-arrestin 1 impairs LC3-mediated autophagy. Fig.23A HeLa-V5-tau cells transfected with control vector or β-arrestin 1-myc and treated with vehicle or 100 nM bafilomycin A1 for 4 hours are shown. Cells were fixed and immunostained for myc and LC3 (scale bar = 10 μm). Representative images are shown. Fig. 23B Quantification of LC3 puncta area normalized to vehicle control vehicle conditions is shown. n=4 independent experiments. #p<0.0001. 1-way ANOVA with Dunnett's post hoc test. All data are expressed as mean ± SEM. Fig.23C Shown are HeLa-V5-tau cells transfected with GFP or GFP-β-arrestin1 and / or HA-p62, co-IPed for HA, and immunoblotted for GFP, HA, and actin.

[0038] Figures 24A to 24Dβ-Arrestin2 oligomerization mutants were shown to impair β-Arrestin1 homo-oligomers and β-Arrestin1 / β-Arrestin2 hetero-oligomers. HeLa-V5-tau cells were co-transfected with vector control (Vec) or β-Arrestin2 oligomerization mutants (ΔIP6C or ΔIP6N) together with β-Arrestin1-GFP+β-Arrestin1-myc or β-Arrestin2-myc. Cells were then subjected to proximity ligation assay (PLA) using antibodies against GFP and myc to detect β-Arrestin1 homo-oligomers ( Fig.24A , 24B) and β-arrestin1 / β-arrestin2 hetero-oligomers and DAPI staining ( Fig.24C , 24D) (spot). The results showed that the oligomerization mutants of β-arrestin 2 (ΔIP6C or ΔIP6N) significantly reduced the homo-oligomerization of β-arrestin 1 ( Fig.24A , 24B ) and β-arrestin1 / β-arrestin2 hetero-oligomers (n=4, 1-way ANOVA, post hoc Dunnett's, #P<0.0001). Cells that received only 1 probe or excluded the primary antibody showed no red PLA spots (negative control).

[0039] Fig.25 The workflow used to screen for compounds that block β-arrestin oligomerization and reduce pathological tau is shown. Based on the crystal structure of β-arrestin 2, we performed an in silico screen of millions of compounds that could dock at the β-arrestin 2 trimer binding interface. The top 30 compounds were then tested for their ability to reduce pathological tau. Those that reduced tau were then tested for their ability to block β-arrestin 2 oligomerization.

[0040] Fig.26 C1 and C2 compounds were shown to reduce tau levels. HeLa-V5-tau cells were treated with 10 μM of a potential inhibitor of β-arrestin 2 oligomerization for 6 hours and subjected to Western blotting. C1 and C2 significantly reduced total tau levels in HeLa-V5-tau cells. (n=4, 1-way ANOVA, post hoc Dunnett's, **P<0.005, ***P<0.0005).

[0041] Fig.27A and 27B The C1 and C2 compounds were shown to reduce pathogenic tau. Fig.27AHeLa-V5-tau cells treated with 20 μM of O1, C1 or C2 compounds for 6 hours and lauroylsarcosine soluble and insoluble fractions were shown. C1 and C2 significantly reduced the level of lauroylsarcosine insoluble tau in HeLa-V5-tau cells. (n=4, 1-way ANOVA, post hoc Dunnett's, **P<0.005, ***P<0.0005). Fig.27B Shown are DIV21 hippocampal primary neurons derived from Tau-P301S mice treated with 20 μM of O1, C1 or C2 compounds for 6 hours and stained for HT7 (total human tau). C1 and C2 significantly reduced HT7 immunoreactivity (n=3, 1-way ANOVA, post hoc Dunnett's, *P<0.05, ***P<0.0005).

[0042] Fig.28A and 28B Compounds C2 and C2 were shown to inhibit β-arrestin 2 oligomerization. Hela-V5-tau cells were transfected with GFP-β-arrestin 2 and Flag-β-arrestin 2 and treated with 20 μM of O1, C1 or C2 for 6 hours. Fig.28A Proximity ligation assay (PLA) showing detection of GFP-β-arrestin2 / Flag-β-arrestin2 complexes using antibodies against GFP and Flag (M2) showed significant reduction in β-arrestin2 oligomerization (n=3, 1-way ANOVA, post hoc Dunnett's, *P<0.05, ***P<0.0005). Inclusion of only 1 PLA probe as a negative control showed no PLA red spots. Fig.28B Cells lysed and co-immunoprecipitated (co-IP) against GFP-β-arrestin2 / Flag-β-arrestin2 complexes are shown, demonstrating that C1 and C2 compounds reduce β-arrestin2 self-interactions to similar background levels as anti-mouse IgG beads alone.

[0043] Fig.29 Compound C2 was shown to inhibit β-arrestin2 oligomerization. Hela-V5-tau cells were transfected with CFP-β-arrestin2 and YFP-β-arrestin2 and treated with vehicle or 20 μM C2 for 30 minutes. β-arrestin2 oligomerization was detected by FRET assay, indicating that C2 compound, but not vehicle, can reduce CFP to YFP FRET signal.

[0044] Detailed description

[0045] Before describing the present disclosure in more detail, it should be understood that the present disclosure is not limited to the particular embodiments described, and as such may of course vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0046] Where a numerical range is provided, it is understood that each intervening value to the tenth of the unit of the lower limit, the upper and lower limits of the range, and any other stated or intervening values ​​in the stated range are included in the disclosure unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included in the disclosure, subject to any specifically excluded limits in the stated range. Where the stated range includes one or both of the limits, ranges excluding one or both of those included limits are also included in the disclosure.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, preferred methods and materials are now described.

[0048] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and is incorporated herein by reference to disclose and describe the methods and / or materials related to the cited publication. Citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. In addition, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0049] As will be apparent to those skilled in the art after reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any enumerated method can be executed in the order of events enumerated or in any other logically possible order.

[0050]

[00136] Embodiments of the present disclosure will employ, unless otherwise indicated, chemical, biological, and other techniques within the skill of the art.

[0051] The following examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use probes disclosed and claimed herein. Efforts have been made to ensure the accuracy of numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, temperatures are in ° C, and pressures are at or near atmospheric pressure. Standard temperature and pressure are defined as 20 ° C and 1 atmosphere.

[0052] Before describing the embodiments of the present disclosure in detail, it should be understood that, unless otherwise stated, the present disclosure is not limited to specific materials, reagents, reaction materials, manufacturing processes, etc., and therefore can vary. It should also be understood that the terms used herein are only used for the purpose of describing specific embodiments and are not restrictive. In the present disclosure, steps can also be performed in different orders where logically feasible.

[0053] definition

[0054] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0055] As used herein, the term "residue" refers to an amino acid incorporated into a polypeptide. The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may include known analogs of natural amino acids that may function in a similar manner to naturally occurring amino acids.

[0056] The term "variant" refers to an amino acid or peptide sequence having conservative amino acid substitutions, non-conservative amino acid substitutions (i.e., degenerate variants), substitutions within the wobble position of each codon (i.e., DNA and RNA) encoding an amino acid, an amino acid added to the C-terminus of a peptide, or a peptide having 60%, 70%, 80%, 90% or 95% homology to a reference sequence.

[0057] "Positively charged" with respect to amino acids refers to those amino acids, amino acid derivatives, amino acid mimetics, and chemical moieties that are positively charged at physiological pH. Positively charged amino acids include, for example, lysine and arginine.

[0058] The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues with similar side chains in proteins. For example, a group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic hydroxyl side chains consists of serine and threonine; a group of amino acids with amide-containing side chains consists of asparagine and glutamine; a group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains consists of lysine, arginine, and histidine; a group of amino acids with acidic side chains consists of glutamic acid and aspartic acid; and a group of amino acids with sulfur-containing side chains consists of cysteine ​​and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0059] The term "percent (%) sequence identity" or "homology" is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical to those in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity. Alignments for determining percent sequence identity can be accomplished in various ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Appropriate parameters for measuring alignments can be determined by known methods, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0060] As used herein, "peptide mimics" refers to mimetics of peptides that include some changes in normal peptide chemistry. Peptide mimics generally enhance some properties of the original peptide, such as increased stability, increased efficacy, enhanced delivery, increased half-life, etc. Methods for preparing peptide mimics based on known polypeptide sequences are described in, for example, U.S. Patent Nos. 5,631,280; 5,612,895; and 5,579,250. The use of peptide mimics may involve the incorporation of non-amino acid residues with non-amide bonds at a given position. One embodiment of the invention is a peptide mimetic, wherein the compound has a bond, a peptide backbone, or an amino acid component substituted by a suitable mimetic. Some non-limiting examples of unnatural amino acids that may be suitable amino acid mimetics include β-alanine, L-α-aminobutyric acid, L-γ-aminobutyric acid, L-α-aminoisobutyric acid, L-ε-aminocaproic acid, 7-aminoheptanoic acid, L-aspartic acid, L-glutamic acid, N-ε-Boc-N-α-CBZ-L-lysine, N-ε-Boc-N-α-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L-norvaline, N-α-Boc-N-δCBZ-L-ornithine, N-δ-Boc-N-α-CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, and Boc-L-thioproline.

[0061] A "vector" or "expression vector" is a replicon, such as a plasmid, phage, virus or cosmid, to which another DNA segment, the "insert", can be attached so as to bring about the replication of the attached segment.

[0062] The term "operably linked to" refers to the functional relationship of a nucleic acid to another nucleic acid sequence. Promoters, enhancers, transcription and translation termination sites, and other signal sequences are examples of nucleic acid sequences that are operably linked to other sequences. For example, operably linked DNA to transcriptional control elements refers to the physical and functional relationship between the DNA and the promoter such that transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA. .

[0063] The term "subject" refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, such as a mammal. Thus, the subject can be a human or veterinary patient. The term "patient" refers to a subject who is being treated by a clinician, such as a physician.

[0064] As used herein, "effective amount" may refer to an amount of a compound or pharmaceutical composition provided herein that is sufficient to achieve a beneficial or desired biological, emotional, medical or clinical response in a cell, tissue, system, animal, or human being. An effective amount may be administered in one or more administrations, applications or dosages. The term may also include within its scope an amount that effectively enhances or restores to substantially normal physiological function. As used herein, "therapeutically effective amount" means an amount sufficient to reduce at least 10%, preferably at least 25%, more preferably at least 50%, and most preferably sufficient to cause an improvement in one or more clinically significant symptoms in a subject.

[0065] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to achieve the desired therapeutic result or to have an effect on undesirable symptoms but generally insufficient to cause adverse side effects. The specific therapeutically effective dosage level for any particular patient will depend on a variety of factors, including the condition being treated and the severity of the condition; the specific composition employed; the patient's age, weight, general health, sex and diet; the time of administration; the route of administration; the excretion rate of the specific compound used; the duration of treatment; the drugs used in combination or concomitantly with the specific compound used, and similar factors within the knowledge and expertise of the health care practitioner, which may be well known in the medical field. In the case of treating a specific disease or condition, in some cases, the desired response may be to inhibit the progression of the disease or condition. This may only involve temporarily slowing the progression of the disease. However, in other cases, it may be necessary to permanently stop the progression of the disease. This can be monitored by conventional diagnostic methods for any specific disease known to those of ordinary skill in the art. The desired response to the treatment of a disease or condition may also be to delay the onset of the disease or condition or even prevent the onset of the disease or condition.

[0066] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0067] The term "pharmaceutically acceptable prodrug" or "prodrug" means those prodrugs of the compounds of the present disclosure which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without excessive exposure to toxicity, irritation, allergic response, etc., commensurate with a reasonable benefit / risk ratio, and effective for their intended use. The prodrugs of the present disclosure can be rapidly converted in vivo to the parent compound having the structure of the disclosed compound, for example, by hydrolysis in the blood. A comprehensive discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, V. 14 of the ACSSymposium Series, and Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press (1987).

[0068] The term "carrier" refers to a compound, composition, substance or structure that, when combined with a compound or composition, aids or promotes the preparation, storage, administration, delivery, effectiveness, selectivity or any other characteristic of the compound or composition for its intended use and purpose. For example, the carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0069] The term "treatment" refers to the medical management of a patient with the intent to cure, alleviate, stabilize or prevent a disease, pathological condition or disorder. The term includes active treatment, i.e., treatment specifically aimed at the improvement of a disease, pathological condition or condition, and also includes causal treatment, i.e., treatment aimed at eliminating the cause of the relevant disease, pathological condition or condition. In addition, the term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure the disease, pathological condition or condition; preventive treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the development of the relevant disease, pathological condition or condition; and supportive treatment, i.e., treatment used to supplement another specific treatment aimed at improving the relevant disease, pathological condition or condition.

[0070] As used herein, "administration" may refer to oral, topical, intravenous, subcutaneous, transdermal, transdermal, intramuscular, intraarticular, parenteral, intraarteriole, intradermal, intraventricular, intraosseous, intraocular, intracranial, intraperitoneal, intralesional, intranasal, intracardiac, intraarticular, intracavernous, intrathecal, intraviral, intracerebral and intraventricular, intratympanic, intracochlear, rectal, vaginal, by inhalation, by catheter, stent, or by implanted reservoir or other device that actively or passively (e.g., by diffusion) administers the composition in the perivascular space and adventitia. For example, a medical device such as a stent may contain a composition or formulation disposed on its surface, which may then be dissolved or otherwise distributed to surrounding tissues and cells. The term "parenteral" may include subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Administration may be continuous or intermittent. In various aspects, the preparation may be administered therapeutically; that is, for the treatment of an existing disease or condition. In further various aspects, the formulations may be administered prophylactically; that is, administered to prevent a disease or condition.

[0071] As used herein, the term "alkyl" is a branched or unbranched saturated hydrocarbon group with 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc. Alkyl can be cyclic or non-cyclic. Alkyl can be branched or unbranched. Alkyl can also be substituted or unsubstituted. For example, alkyl can be substituted by one or more groups, including but not limited to alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxyl, nitro, silyl, sulfo-oxo or mercaptan, as described herein. "low alkyl" is an alkyl containing one to six (e.g., one to four) carbon atoms. The term alkyl may also be C1 alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl and the like up to and including C1-C24 alkyl.

[0072] Throughout the specification, "alkyl" is generally used to refer to both unsubstituted and substituted alkyl; however, substituted alkyl is also specifically mentioned herein by identifying a specific substituent on the alkyl. For example, the term "halogenated alkyl" or "haloalkyl" specifically refers to an alkyl substituted by one or more halides such as fluorine, chlorine, bromine or iodine. Alternatively, the term "monohaloalkyl" specifically refers to an alkyl substituted by a single halide, such as fluorine, chlorine, bromine or iodine. The term "polyhaloalkyl" specifically refers to an alkyl independently substituted by two or more halides, i.e., each halide substituent need not be the same halide as another halide substituent, and multiple instances of halide substituents need not be on the same carbon. The term "alkoxyalkyl" specifically refers to an alkyl substituted by one or more alkoxy groups, as described below. The term "aminoalkyl" specifically refers to an alkyl substituted by one or more amino groups. The term "hydroxyalkyl" specifically refers to an alkyl substituted by one or more hydroxy groups. When "alkyl" is used in one context and a specific term such as "hydroxyalkyl" is used in another context, this does not mean that the term "alkyl" also does not refer to the specific term such as "hydroxyalkyl", etc.

[0073] This approach also applies to other groups described herein. That is, although a term such as "cycloalkyl" refers to unsubstituted and substituted cycloalkyl moieties, the substituted moieties may be otherwise specified herein; for example, a specific substituted cycloalkyl may be referred to as, for example, "alkylcycloalkyl". Similarly, a substituted alkoxy may be specifically referred to as, for example, "haloalkoxy", a specific substituted alkenyl may be, for example, "alkenyl alcohol", etc. Similarly, the use of a general term (e.g., "cycloalkyl") and a specific term (e.g., "alkylcycloalkyl") does not mean that the general term does not also include the specific term.

[0074] As used herein, the term "cycloalkyl" is a non-aromatic carbonyl ring consisting of at least three carbon atoms. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, etc. The term "heterocycloalkyl" is a cycloalkyl as defined above, and is included in the meaning of the term "cycloalkyl", wherein at least one carbon atom of the ring is replaced by a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur or phosphorus. Cycloalkyl and heterocycloalkyl can be substituted or unsubstituted. Cycloalkyl and heterocycloalkyl can be substituted by one or more groups, including but not limited to alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxyl, nitro, silyl, sulfo-oxo or thiol as described herein.

[0075] As used herein, the term "heteroalkyl" refers to an alkyl group containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, and S, wherein nitrogen, phosphorus, and sulfur atoms are optionally oxidized, and nitrogen heteroatoms are optionally quaternized. Heteroalkyl groups may be substituted as defined above for alkyl groups.

[0076] As used herein, the term "heterocycloalkyl" refers to aliphatic, partially unsaturated or fully saturated 3 to 14 ring systems, including monocycles and bicyclic and tricyclic systems of 3 to 8 atoms. The heterocycloalkyl ring system includes one to four heteroatoms independently selected from oxygen, nitrogen and sulfur, wherein nitrogen and sulfur heteroatoms are optionally oxidized and nitrogen heteroatoms are optionally substituted. Representative heterocycloalkyl includes but is not limited to pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl and tetrahydrofuranyl.

[0077] As used herein, the term "aryl" is a group comprising any carbon-based aromatic group, including but not limited to benzene, naphthalene, phenyl, biphenyl, anthracene, etc. Aryl can be substituted or unsubstituted. Aryl can be substituted by one or more groups, including but not limited to alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, -NH2, carboxylic acid, ester, ether, halide, hydroxyl, ketone, azide, nitro, silyl, sulfo-oxo or thiol as described herein. The term "biaryl" is a specific type of aryl, included in the definition of "aryl". In addition, aryl can be a monocyclic structure or contain multiple ring structures, which are either fused ring structures or attached by one or more bridging groups such as carbon-carbon bonds. For example, biaryl is combined with two aryl groups by fused ring structures, such as naphthalene, or connected by one or more carbon-carbon bonds, such as biphenyl. Fused aryl is also considered, including but not limited to indene and naphthyl. Aryl can also be fused with cycloalkyl. For example, a cyclopentyl group can be fused to a phenyl group.

[0078] As used herein, the term "heteroaryl" refers to an aromatic group having at least one heteroatom incorporated into the aromatic group ring. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus, wherein N-oxides, sulfur oxides, and dioxides are permitted heteroatom substitutions. Heteroaryl can be substituted or unsubstituted. Heteroaryl can be substituted by one or more groups, including but not limited to alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxyl, nitro, silyl, sulfo-oxo, or thiol as described herein. Heteroaryl can be a monocyclic or alternatively fused ring system. Heteroaryl groups include, but are not limited to, furanyl, imidazolyl, pyrimidinyl, tetrazolyl, thienyl, pyridinyl, pyrrolyl, N-methylpyrrolyl, quinolyl, isoquinolyl, pyrazolyl, triazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, isothiazolyl, pyridazolyl, pyrazinyl, benzofuranyl, benzodioxazolyl, benzothienyl, indolyl, indazolyl, benzimidazolyl, imidazopyridinyl, pyrazolopyridinyl, and pyrazolopyrimidinyl. Other non-limiting examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, pyrazolyl, imidazolyl, benzo[d]oxazolyl, benzo[d]thiazolyl, quinolinyl, quinazolinyl, indazolyl, imidazo[1,2-b]pyridazinyl, imidazo[1,2-a]pyrazinyl, benzo[c][1,2,5]thiadiazolyl, benzo[c][1,2,5]oxadiazolyl, and pyrido[2,3-b]pyrazinyl.

[0079] As used herein, the terms "halo," "halogen," or "halide" are used interchangeably to refer to F, Cl, Br, or I.

[0080] As described herein, the compounds of the present invention may include "optionally substituted" parts. In general, the term "substituted", whether or not there is the term "optionally" in front, refers to one or more hydrogens of the specified part being replaced by suitable substituents. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted by more than one substituent selected from a specific group, the substituent may be the same or different at each position. The combination of substituents contemplated by the present invention is preferably those that result in the formation of stable or chemically feasible compounds. It is also contemplated that in some aspects, unless explicitly stated to the contrary, a single substituent may be further optionally substituted (i.e., further substituted or unsubstituted).

[0081] Unless otherwise indicated, chemical formulas in which chemical bonds are shown only as solid lines and not as wedges or dashed lines contemplate every possible isomer, such as each enantiomer and diastereomer, as well as mixtures of isomers, such as racemates or ratio isomer mixtures. The compounds described herein may contain one or more asymmetric centers and may therefore produce diastereomers and optical isomers. Unless otherwise indicated, the present invention includes all such possible diastereomers and their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. Mixtures of stereoisomers as well as isolated specific stereoisomers are also included. During the course of synthetic procedures for preparing such compounds, or in the use of racemization or epimerization procedures known to those skilled in the art, the product of such procedures may be a mixture of stereoisomers. The terms "substantially R" and "substantially S" are defined herein as having at least 70% enantiomeric purity, at least 70% enantiomeric purity, at least 70% enantiomeric purity, at least 70% enantiomeric purity, at least 75% enantiomeric purity, at least 80% enantiomeric purity, at least 85% enantiomeric purity, at least 90% enantiomeric purity, at least 95% enantiomeric purity, at least 99% enantiomeric purity, or 100% enantiomeric purity of the R or S enantiomer.

[0082] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of an active agent prepared with an acid or base, which can be tolerated by a biological system or tolerated by a subject or tolerated by a biological system and tolerated by a biological system when administered in a therapeutically effective amount. When the compounds of the present disclosure contain relatively acidic functionality, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base (pure alkali or in a suitable inert solvent). Examples of pharmaceutically acceptable base addition salts include, but are not limited to, sodium, potassium, calcium, ammonium, organic amines, magnesium salts, lithium salts, strontium salts or similar salts.

[0083] When the compounds of the present disclosure contain relatively basic functionality, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid (pure or in a suitable inert solvent). Examples of pharmaceutically acceptable acid addition salts include, but are not limited to, those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogen carbonic acid, phosphoric acid, monohydrogen phosphoric acid, dihydrogen phosphoric acid, sulfuric acid, monohydrogen sulfuric acid, hydroiodic acid or phosphorous acid, and salts derived from relatively nontoxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, etc. Amino acid salts such as arginine salts, etc., and salts of organic acids such as glucuronic acid or galacturonic acid, etc. are also included.

[0084] The term "pharmaceutically acceptable ester" refers to an ester of a compound of the present disclosure that hydrolyzes in vivo and includes those that are easily decomposed in the human body to leave the parent compound or its salt. Examples of pharmaceutically acceptable non-toxic esters of the present disclosure include C1 to C6 alkyl esters and C5 to C7 cycloalkyl esters, but preferably C1 to C4 alkyl esters. Esters of disclosed compounds can be prepared according to conventional methods. Pharmaceutically acceptable esters can be attached to hydroxyl groups by reacting a compound containing a hydroxyl group with an acid and an alkyl carboxylic acid such as acetic acid or with an acid and an aryl carboxylic acid such as benzoic acid. In the case of a compound containing a carboxylic acid group, a pharmaceutically acceptable ester is prepared by reacting a compound containing a carboxylic acid group with a base such as triethylamine and an alkyl halide, for example methyl iodide, benzyl iodide, cyclopentyl iodide or an alkyl trifluoromethanesulfonate. They can also be prepared by reacting a compound with an acid such as hydrochloric acid and an alcohol such as ethanol or methanol.

[0085] The term "pharmaceutically acceptable amide" refers to the non-toxic amides of the present disclosure derived from ammonia, primary C1 to C6 alkylamines and secondary C1 to C6 dialkylamines. In the case of secondary amines, the amine may also be in the form of a 5- or 6-membered heterocycle containing one nitrogen atom. Preferably, the amides derived from ammonia, C1 to C3 alkyl primary amides and C1 to C2 dialkyl secondary amides. The amides of the disclosed compounds can be prepared according to conventional methods. Pharmaceutically acceptable amides can be prepared from compounds containing primary or secondary amine groups by reacting compounds containing amino groups with alkyl anhydrides, aryl anhydrides, acyl halides or aryl halides. In the case of compounds containing carboxylic acid groups, pharmaceutically acceptable amides are prepared from compounds containing carboxylic acid groups by reacting the compounds with bases such as triethylamine, dehydrating agents such as dicyclohexylcarbodiimide or carbonyldiimidazole, and alkylamines, dialkylamines, such as methylamine, diethylamine and piperidine. They can also be prepared by reacting the compound with an acid such as sulfuric acid and an alkyl carboxylic acid such as acetic acid, or with an acid and an aryl carboxylic acid such as benzoic acid under dehydrating conditions such as the addition of molecular sieves. The composition may comprise a compound of the present disclosure in a pharmaceutically acceptable prodrug form.

[0086] The term "pharmaceutically acceptable prodrug" or "prodrug" means those prodrugs of the compounds of the present disclosure which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and lower animals without toxicity, irritation, allergic response, etc., commensurate with a reasonable benefit / risk ratio, and effective for their intended use. The prodrugs of the present disclosure can be rapidly converted in vivo to the parent compound having the structure of the disclosed compound, for example, by hydrolysis in the blood. A comprehensive discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, V. 14 of the ACSSymposium Series, and Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press (1987).

[0087] β-Arrestin Oligomerization Inhibitors

[0088] Dominant negative peptide

[0089] In some embodiments, the inhibitor of β-arrestin oligomerization is a dominant negative peptide comprising a mutant β-arrestin or a fragment thereof that can block the binding of endogenous β-arrestin 1 and / or β-arrestin 2 to phytate (IP6). Mutant forms of β-arrestin are described in Milano SK, et al. (2006) J Biol Chem 281(14):9812-9823, which is incorporated by reference in its entirety for teaching these mutants. Milano et al. solved the crystal structure of arrestin 2 in complex with IP6 and identified two domains involved in IP6-dependent oligomerization. In some embodiments, the mutant β-arrestin has a mutation in one or more lysine and / or arginine residues in the N-domain and / or C-domain described by Milano SK et al. For example, in some embodiments, the mutant β-arrestin has at least one amino acid substitution corresponding to residue K158, K161, R162, K232, R234, K252, K326, or K328 of wild-type arrestin2.

[0090] Pharmacological inhibitors

[0091] In some embodiments, the β-arrestin oligomerization inhibitor is a compound having the following structure or a pharmaceutically acceptable salt thereof

[0092]

[0093] Where R 1 is hydrogen or alkyl;

[0094] Ar is aryl; and

[0095] When R 1 When it is an alkyl group, a The stereochemistry at is racemic, substantially R, or substantially S.

[0096] In one embodiment, R 1 is hydrogen. In one embodiment, Ar is unsubstituted or substituted phenyl. In another embodiment, Ar is phenyl substituted with one or more halogen atoms. In one embodiment, R 1 C1 to C 10 Alkyl, for example methyl, ethyl, propyl, isopropyl or butyl. a In another embodiment, the stereochemistry at C a In another embodiment, the stereochemistry at C a The stereochemistry at is racemic. In one embodiment, Ar is unsubstituted or substituted phenyl. In one embodiment, Ar is phenyl substituted with one or more alkyl or halogen atoms. In one embodiment, the phenyl is substituted with 1 to 3 halogen atoms. The position of the halogen atoms on the phenyl ring can vary. In another embodiment, Ar is phenyl fused with a cycloalkyl. The cycloalkyl may have 4 to 7 carbon atoms. In one aspect, the phenyl fused with a cycloalkyl has the following structure

[0097]

[0098] In some embodiments, the β-arrestin oligomerization inhibitor is a compound having the following structure or a pharmaceutically acceptable salt thereof

[0099]

[0100] wherein Ar is an aryl group.

[0101] In one embodiment, Ar is unsubstituted or substituted phenyl. In another embodiment, Ar is phenyl substituted with alkyl or cycloalkyl.

[0102] In some embodiments, the β-arrestin oligomerization inhibitor is a compound having the following structure or a pharmaceutically acceptable salt thereof

[0103]

[0104] Where R 2 and R3 are independently hydrogen or alkyl; and

[0105] Ar is aryl.

[0106] In one embodiment, R 2 In another embodiment, R 3 C1 to C 10 Ar is an alkyl group, such as methyl, ethyl, propyl, isopropyl or butyl. In one embodiment, Ar is an unsubstituted or substituted phenyl group. In another embodiment, Ar is a phenyl group substituted by an alkyl group or a halogen atom.

[0107] In some embodiments, the β-arrestin oligomerization inhibitor is a compound having the following structure or a pharmaceutically acceptable salt thereof

[0108]

[0109] Among them, Ar 1 and Ar 2 are independently aryl; and

[0110] X is CH2 or C=O.

[0111] In one embodiment, Ar 1 is unsubstituted or substituted phenyl. In another embodiment, Ar 1 is a phenyl group substituted by an alkyl group or a halogen atom. 1 is unsubstituted or substituted heteroaryl. In another embodiment, Ar 1 is unsubstituted or substituted piperidinyl or pyridazinyl. In one embodiment, Ar 2 is unsubstituted or substituted phenyl. In another embodiment, Ar 2 is a phenyl group substituted by an alkyl group or a halogen atom. 2 is unsubstituted or substituted piperidinyl. In another embodiment, Ar 2 It is a piperidinyl group substituted by an alkyl group or a halogen atom.

[0112] In some embodiments, the β-arrestin oligomerization inhibitor is a compound having the following structure or a pharmaceutically acceptable salt thereof

[0113]

[0114] Among them, Ar 1 and Ar 2 are independently aryl.

[0115] In one embodiment, Ar 1is unsubstituted or substituted phenyl. In another embodiment, Ar 1 is a phenyl group substituted by an alkyl group or a halogen atom. 2 is unsubstituted or substituted phenyl. In another embodiment, Ar 2 It is a phenyl group substituted by an alkyl group or a halogen atom.

[0116] In some embodiments, the β-arrestin oligomerization inhibitor is a compound selected from the group consisting of: (2-[(2,3-dihydro-1H-inden-5-yl)formamido]-N-({imidazo[1,2-a]pyridin-2-yl}methyl)acetamide), N-{2-[(6-methylpyridazin-3-yl)amino]ethyl}-3-phenyl-1,2-oxazole-5-carboxamide, 2-[(2,4-dihydro-1H-inden-5-yl)formamido]-N-({imidazo[1,2-a]pyridin-2-yl}methyl)acetamide, [(3-methylphenyl)formamido]-N-({imidazo[1,2-a]pyridin-2-yl}methyl)acetamide, (2S)-N-({imidazo[1,2-a]pyridin-2-yl}methyl)-3-methyl-2-[(3-methylphenyl)formamido]butanamide, 2-[(3-bromophenyl)formamido]-N-({imidazo[1,2-a]pyridin-2-yl}methyl)acetamide, 2-[(3-chlorophenyl)formamido]-N-({imidazo[1,2-a]pyridin-2-yl}methyl)acetamide, N-{2-[(3-methylpyridin-4-yl)amino]ethyl}-3-phenyl-1,2-oxazole-5-carboxamide, 1-(4-fluorophenyl)-4-[3-(4-

[0063] In some embodiments, the present invention relates to 2-{[3-(4-methylphenyl)-1,2-oxazol-5-yl]formamido}-N-(2,4,6-trimethylphenyl)acetamide, 2-{[3-(4-methylphenyl)-1,2-oxazol-5-yl]formamido}acetamide and 3-phenyl-N-(propan-2-yl)-1,2-oxazole-5-carboxamide.

[0117] In some embodiments, the β-arrestin oligomerization inhibitor is a compound having a structure represented by the following formula:

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124] Pharmaceutical composition

[0125] In various aspects, the present disclosure relates to pharmaceutical compositions comprising a therapeutically effective amount of at least one disclosed compound, at least one product of a disclosed method, or a pharmaceutically acceptable salt thereof. As used herein, "pharmaceutically acceptable carrier" refers to one or more pharmaceutically acceptable diluents, preservatives, antioxidants, solubilizers, emulsifiers, colorants, release agents, coating agents, sweeteners, flavoring agents and flavoring agents and adjuvants. The disclosed pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any method known in the art of pharmacy and pharmaceutical science.

[0126] On the other hand, the disclosed pharmaceutical composition comprises at least one disclosed compound of therapeutically effective amount, the product of at least one disclosed method, or its pharmaceutically acceptable salt as active ingredient, pharmaceutically acceptable carrier, optionally one or more other therapeutic agents, and optional one or more adjuvants. The disclosed pharmaceutical composition includes those suitable for oral, rectal, topical, pulmonary, nasal and parenteral administration, although the most suitable approach in any given case will depend on the nature and severity of the condition to which the specific host and active ingredient are being administered. On the other hand, the disclosed pharmaceutical composition can be formulated to allow oral, nasal, inhaled, parenteral, paracancer, transmucosal, transdermal, intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal, intraventricular, intracranial and intratumoral administration.

[0127] As used herein, "parenteral administration" includes administration by bolus or infusion, as well as administration by intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0128] In various aspects, the present disclosure also relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent, a therapeutically effective amount of a disclosed compound, a product of a disclosed preparation method, a pharmaceutically acceptable salt, a hydrate thereof, a solvate thereof, a polymorph thereof, or a stereochemical isomeric form thereof as an active ingredient. In another aspect, the disclosed compound, the product of the disclosed preparation method, a pharmaceutically acceptable salt, a hydrate thereof, a solvate thereof, a polymorph thereof, or a stereochemical isomeric form thereof, or any subset or combination thereof can be formulated into various pharmaceutical forms for administration.

[0129] Pharmaceutically acceptable salts can be prepared from pharmaceutically acceptable non-toxic bases or acids. For therapeutic use, salts of the disclosed compounds are those in which the counterion is pharmaceutically acceptable. However, salts of non-pharmaceutically acceptable acids and bases may also be used, for example, to prepare or purify pharmaceutically acceptable compounds. The present disclosure encompasses all salts, whether or not pharmaceutically acceptable. Pharmaceutically acceptable acid and base addition salts are intended to include the therapeutically active non-toxic acid and base addition salt forms that the disclosed compounds are able to form.

[0130] In various fields, the disclosed compounds comprising acidic groups or parts, such as carboxylic acid groups, can be used to prepare pharmaceutically acceptable salts. For example, such disclosed compounds can include a separation step, including the treatment with suitable inorganic or organic bases. In some cases, in practice, it may be necessary to first separate the compound from the reaction mixture as a pharmaceutically unacceptable salt, then by treating the latter with an acidic agent, simply converting it back to the free acid compound, and then converting the free acid into a pharmaceutically acceptable base addition salt. These base addition salts can be easily prepared using conventional techniques, for example, by treating the corresponding acidic compound with an aqueous solution containing the required pharmaceutically acceptable cation, and then evaporating the resulting solution to dryness, preferably under reduced pressure. Alternatively, they can also be prepared by mixing the lower alkanol solution of the acidic compound with the required alkali metal alkoxide, and then evaporating the resulting solution to dryness in the same manner as before.

[0131] Bases useful in preparing pharmaceutically acceptable base addition salts of basic compounds are those which form non-toxic base addition salts, i.e., salts containing pharmaceutically acceptable cations such as alkali metal cations (e.g., lithium, potassium and sodium), alkaline earth metal cations (e.g., calcium and magnesium), ammonium or other water-soluble amine addition salts, such as N-methylglucamine-(meglumine), lower alkanolammonium and other such organic amine bases. On the other hand, those derived from pharmaceutically acceptable organic non-toxic bases include primary, secondary and tertiary amines, as well as cyclic amines and substituted amines, such as naturally occurring and synthetic substituted amines. In various aspects, such pharmaceutically acceptable organic non-toxic bases include, but are not limited to, ammonia, methylamine, ethylamine, propylamine, isopropylamine, any of the four butylamine isomers, betaine, caffeine, choline, dimethylamine, diethylamine, diethanolamine, dipropylamine, diisopropylamine, di-n-butylamine, N,N'-dibenzylethylenediamine, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, tromethamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, quinuclidine, pyridine, quinoline and isoquinoline; benzathine, N-methyl-D-glucamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, reduced glucosamine, glucosamine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, hepamine salts and salts with amino acids, for example, histidine, arginine, lysine, etc. Said salt forms can be converted back into the free acid form by treatment with acid.

[0132] In various aspects, the disclosed compounds comprising a protonatable group or part (e.g., amino) can be used to prepare pharmaceutically acceptable salts. For example, such disclosed compounds may include a separation step, including the treatment with a suitable inorganic or organic acid. In some cases, in practice, it may be necessary to first separate the compound from the reaction mixture as a pharmaceutically unacceptable salt, then simply convert the latter back to a free base compound by treating with an alkaline agent, and then convert the free base into a pharmaceutically acceptable acid addition salt. These acid addition salts can be easily prepared using conventional techniques, for example, by treating the corresponding basic compound with an aqueous solution containing a desired pharmaceutically acceptable anion, and then evaporating the resulting solution to dryness, preferably under reduced pressure. Alternatively, they can also be prepared by treating the free base form of the disclosed compound with a suitable pharmaceutically acceptable nontoxic inorganic or organic acid.

[0133] The acid that can be used to prepare the pharmaceutically acceptable acid addition salt of the base compound is an acid that can form a non-toxic acid addition salt, i.e., a salt containing a pharmaceutically acceptable anion formed by its corresponding inorganic acid and organic acid. Exemplary but non-limiting inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Exemplary but non-limiting organic acids include acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid methanesulfonic acid, mucic acid, pamoic acid, pantothenic acid, succinic acid, tartaric acid, p-toluenesulfonic acid, etc. On the other hand, the acid addition salt contains anions formed by hydrobromic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid and tartaric acid.

[0134] In practice, the compounds of the present disclosure or their pharmaceutically acceptable salts can be closely mixed with pharmaceutical carriers as active ingredients according to conventional pharmaceutical compounding techniques. The carrier can take a variety of forms, depending on the form of the preparation required for administration, such as oral or parenteral (including intravenous). Therefore, the pharmaceutical composition of the present disclosure can be presented as discrete units suitable for oral administration, such as capsules, cachets or tablets, each containing a predetermined amount of active ingredients. In addition, the composition can be in the form of powders, granules, solutions, suspensions in aqueous liquids, non-aqueous liquids, oil-in-water emulsions or water-in-oil liquid emulsions. In addition to the common dosage forms listed above, the compounds of the present disclosure and / or their pharmaceutically acceptable salts can also be administered by controlled release means and / or delivery devices. The composition can be prepared by any pharmaceutical method. Generally, such methods include the step of associating the active ingredient with a carrier constituting one or more essential ingredients. Generally, the composition is prepared by uniformly and closely mixing the active ingredient with a liquid carrier or a finely divided solid carrier or both. The product can then be conveniently molded into the desired appearance.

[0135] It is particularly advantageous to formulate the above-mentioned pharmaceutical compositions into unit dosage forms for ease of administration and uniformity of dosage. As used herein, the term "unit dosage form" refers to physically discrete units suitable as unit doses, each unit containing a predetermined amount of active ingredients, calculated to produce the desired therapeutic effect associated with the desired pharmaceutical carrier. That is, "unit dosage form" refers to a single dose in which all active and inactive ingredients are combined in a suitable system so that the patient or the person administering the drug to the patient can open a single container or package containing the entire dose, and it is not necessary to mix any ingredients from two or more containers or packages together. Typical examples of unit dosage forms are tablets (including scored or coated tablets), capsules or pills for oral administration; single-dose vials for injection of solutions or suspensions; suppositories for rectal administration; powder packets; wafers; and separate complexes thereof. This list of unit dosage forms is not intended to be limiting in any way, but only represents typical examples of unit dosage forms.

[0136] Pharmaceutical compositions disclosed herein include compounds of the present disclosure (or their pharmaceutically acceptable salts) as active ingredients, pharmaceutically acceptable carriers and optionally one or more other therapeutic agents. In various aspects, the disclosed pharmaceutical compositions may include pharmaceutically acceptable carriers and disclosed compounds, or pharmaceutically acceptable salts thereof. On the other hand, the disclosed compounds or their pharmaceutically acceptable salts may also be included in pharmaceutical compositions in combination with one or more other therapeutically active compounds. The compositions of the present invention include compositions suitable for oral, rectal, topical and parenteral (including subcutaneous, intramuscular and intravenous) administration, although the most appropriate approach in any given case will depend on a specific host, and the nature and severity of the condition for which the active ingredient is administered. Pharmaceutical compositions may conveniently exist in unit dosage form and be prepared by any method known in the pharmaceutical field.

[0137] Techniques and compositions for preparing dosage forms useful in the materials and methods described herein are described, for example, in the following references: Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; JG Hardy, SSDavis, Clive G. Wilson, Eds.); Modern Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S.Banker, Christopher T. Rhodes, Eds.). .

[0138] The compounds described herein are usually administered in admixture with suitable pharmaceutical diluents, excipients, extenders or carriers (referred to herein as pharmaceutically acceptable carriers or carriers), which are suitably selected and consistent with traditional pharmaceutical practices according to the intended form of administration. The deliverable compound will be in a form suitable for oral, rectal, topical, intravenous injection or parenteral administration. The carrier includes a solid or liquid, and the type of carrier is selected according to the type of administration used. The compound can be administered in a dosage with a known amount of the compound.

[0139] Oral administration can be a preferred dosage form due to ease of administration, and tablets and capsules represent the most advantageous oral dosage unit form, in which case solid drug carriers are obviously used. However, other dosage forms may be suitable, depending on the clinical population (e.g., age and severity of clinical conditions), the solubility of the specific disclosed compound used, etc. Therefore, the disclosed compounds can be used in oral dosage forms, such as pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions. When preparing a composition of an oral dosage form, any convenient pharmaceutical medium can be used. For example, water, glycerol, oil, alcohol, flavoring agents, preservatives, colorants, etc. can be used to form oral liquid preparations such as suspensions, elixirs and solutions; and carriers such as starch, sugar, microcrystalline cellulose, diluents, granulators, lubricants, binders, disintegrants, etc. can be used to form oral solid preparations such as powders, capsules and tablets. Due to ease of administration, tablets and capsules are preferred oral dosage units, in which solid drug carriers are used. Optionally, tablets can be coated by standard aqueous or non-aqueous techniques.

[0140] The disclosed pharmaceutical compositions in oral dosage form may include one or more pharmaceutical excipients and / or additives. Non-limiting examples of suitable excipients and additives include gelatin, natural sugars such as raw sugar or lactose, lecithin, pectin, starch (e.g. corn starch or amylose), dextran, polyvinyl pyrrolidone, polyvinyl acetate, gum arabic, alginic acid, tetraceuticals, talc, lycopodiol, silica gel (e.g. colloids), cellulose, cellulose derivatives (e.g. cellulose ethers in which the cellulose hydroxyl groups are partially etherified with lower saturated fatty alcohols and / or lower saturated fatty hydroxyl alcohols, such as methoxypropylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate), fatty acids and magnesium, calcium or aluminum salts of fatty acids having 12 to 22 carbon atoms, in particular saturated (e.g. stearates), emulsifiers, oils and fats, in particular vegetable (e.g. peanut oil, castor oil, olive oil, sesame oil, cottonseed oil, corn oil, wheat germ oil, sunflower oil, cod liver oil, in each case also optionally hydrated); saturated fatty acids C12 H 24 O2 to C 18 H 36 O2 glycerol esters and polyglycerol esters and mixtures thereof, the glycerol hydroxyl groups can be completely or only partially esterified (for example monoglycerides, diglycerides and triglycerides); pharmaceutically acceptable mono- or polyhydric alcohols and polyethylene glycols, for example polyethylene glycol and its derivatives, esters of aliphatic saturated or unsaturated fatty acids (2-22 carbon atoms, in particular 10-18 carbon atoms) with monovalent fatty alcohols (1-20 carbon atoms) or polyols, for example glycols, glycerol, diethylene glycol, pentaerythritol, sorbitol, mannitol and the like, which can also optionally be etherified, esters of citric acid with primary alcohols, acetic acid, urea, benzyl benzoate, dioxolane, glycerol formal, tetrahydrofurfuryl alcohol, polyethylene glycol ethers with C1-C12-alcohols, dimethylacetamide, lactamide, lactates, ethyl carbonate, siloxanes (in particular polydimethylsiloxanes of medium viscosity), calcium carbonate, sodium carbonate, calcium phosphate, sodium phosphate, magnesium carbonate and the like.

[0141] Other auxiliary substances used in the preparation of oral dosage forms are those which cause disintegration (so-called disintegrants), for example: cross-linked polyvinyl pyrrolidone, sodium carboxymethyl starch, sodium carboxymethyl cellulose or microcrystalline cellulose. Conventional coating substances can also be used for the production of oral dosage forms. For example, polymers and copolymers of acrylic acid and / or methacrylic acid and / or their esters; copolymers of acrylic acid and methacrylic acid esters with a low ammonium content (e.g. Eudragit RRS), copolymers of acrylic acid and methacrylic acid esters with trimethylammonium methacrylate (e.g. Eudragit RRS), RL); polyvinyl acetate; fats, oils, waxes, fatty alcohols; hydroxypropyl methylcellulose phthalate or acetate succinate; cellulose acetate phthalate, acetate phthalate starch and polyvinyl acetate phthalate, carboxymethyl cellulose; methylcellulose phthalate, methylcellulose succinate, methylcellulose phthalate succinate and methylcellulose phthalate half ester; zein; ethyl cellulose and ethylcellulose succinate; shellac, gluten; ethyl carboxyethyl cellulose; ethyl acrylate-maleic anhydride copolymer; maleic anhydride-vinyl methyl ether copolymer; styrene-maleic acid copolymer; 2-ethyl-hexyl acrylate maleic anhydride; crotonic acid-vinyl acetate copolymer; glutamic acid / glutamate copolymer; carboxymethylethylcellulose monocaprylyl glyceride; cellulose acetate succinate; polyarginine.

[0142] Plasticizers that can be considered as coating materials in the disclosed oral dosage forms are: citrate and tartrate esters (acetyl triethyl citrate, acetyl tributyl, tributyl, triethyl citrate); glycerol and glycerides (glycerol diacetate, triacetate, acetylated monoglycerides, castor oil); phthalates (dibutyl phthalate, dipentyl phthalate, diethyl phthalate, dimethyl phthalate, dipropyl phthalate), di(2-methoxy or 2-ethoxyethyl) phthalate, ethyl phthaloyl glycol ... Butyl alcohol and butyl glycolate; alcohols (propylene glycol, polyethylene glycols of various chain lengths), adipates (diethyl adipate, di(2-methoxy or 2-ethoxyethyl) adipate; benzophenone; diethyl and dibutyl sebacate, dibutyl succinate, dibutyl tartrate; diethylene glycol dipropionate; ethylene glycol diacetate, -dibutyrate, -dipropionate; tributyl phosphate, tributyrin; polyethylene glycol sorbitan monooleate (polysorbate, e.g. Polysorbar 50); sorbitan monooleate.

[0143] In addition, suitable binders, lubricants, disintegrants, colorants, flavoring agents, flow inducing agents and melting agents can be included as carriers. The pharmaceutical carrier used can be, for example, solid, liquid or gas. Examples of solid carriers include, but are not limited to, lactose, carclazyte, sucrose, glucose, methylcellulose, dicalcium phosphate, calcium sulfate, mannitol, sorbitol talc, starch, gelatin, agar, pectin, gum arabic, magnesium stearate and stearic acid. Examples of liquid carriers are syrup, peanut oil, olive oil and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0144] In various aspects, binders can include, for example, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, etc. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. On the other hand, disintegrants can include, for example, starch, methylcellulose, agar, bentonite, xanthan gum, etc.

[0145] In various aspects, oral dosage forms, such as solid dosage forms, can include a disclosed compound as a targetable drug carrier or attached to a polymer as a prodrug. Suitable biodegradable polymers for achieving controlled drug release include, for example, polylactic acid, polyglycolic acid, copolymers of polylactic acid and polyglycolic acid, caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanamides, and hydrogels, preferably covalently crosslinked hydrogels.

[0146] Tablets may contain the active ingredient mixed with non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets. These excipients may be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrants such as corn starch or alginic acid; binders such as starch, gelatin or gum arabic, and lubricants such as magnesium stearate, stearic acid or talc. Tablets may be uncoated, or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a longer lasting action.

[0147] Tablets containing the disclosed compounds can be prepared by compression or molding, optionally with one or more auxiliary ingredients or adjuvants. Compressed tablets can be prepared by compressing the active ingredient in a free-flowing form such as a powder or granules in a suitable machine, optionally mixed with a binder, lubricant, inert diluent, surfactant or dispersant. Molded tablets can be made by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine.

[0148] In various aspects, solid oral dosage forms, such as tablets, can be coated with enteric coatings to prevent easy decomposition in the stomach. In various aspects, enteric coating agents include but are not limited to hydroxypropylmethylcellulose phthalate, methacrylic acid-methacrylate copolymers, polyvinyl acetate-phthalate and cellulose acetate phthalate. AkihikoHasegawa "Application of solid dispersions of Nifedipine with enteric coating agent to prepare a sustained-release dosage form" Chem.Pharm.Bull.33:1615-1619 (1985). Various enteric coating materials can be selected according to the test to achieve the enteric coating dosage form designed from scratch, with a preferred combination of dissolution time, coating thickness and radial crushing strength (for example, see SCPorter et al. "The Properties of Enteric Tablet Coatings Made From Polyvinyl Acetate-phthalateand Cellulose acetate Phthalate", J.Pharm.Pharmacol.22:42p (1970)). In another aspect, the enteric coating can comprise hydroxypropyl-methylcellulose phthalate, methacrylic acid-methacrylate copolymer, polyvinyl acetate-phthalate, and cellulose acetate phthalate.

[0149] In various aspects, oral dosage forms can be solid dispersions with water-soluble or water-insoluble carriers. Examples of water-soluble or water-insoluble carriers include, but are not limited to, polyethylene glycol, polyvinyl pyrrolidone, hydroxypropyl methylcellulose, phosphatidylcholine, polyoxyethylene hydrogenated castor oil, hydroxypropyl methylcellulose phthalate, carboxymethyl ethylcellulose or hydroxypropyl methylcellulose, ethyl cellulose or stearic acid.

[0150] In various aspects, oral dosage forms can be liquid dosage forms, including those that are ingested, or alternatively those that are applied as a mouthwash or gargle. For example, liquid dosage forms can include aqueous suspensions, which contain active substances mixed with excipients suitable for making aqueous suspensions. In addition, oily suspensions can be prepared by suspending the active ingredient in vegetable oils such as peanut oil, olive oil, sesame oil or coconut oil or mineral oils such as liquid paraffin. Oily suspensions may also contain various excipients. Pharmaceutical compositions of the present disclosure can also be in the form of oil-in-water emulsions, which may also contain excipients, such as sweeteners and flavorings.

[0151] For the preparation of solutions or suspensions, for example, water, in particular sterile water, or physiologically acceptable organic solvents, such as alcohols (ethanol, propanol, isopropanol, 1,2-propylene glycol, polyethylene glycol and their derivatives, fatty alcohols, partial glycerides), oils (e.g. peanut oil, olive oil, sesame oil, almond oil, sunflower oil, soybean oil, castor oil, neat's foot oil), paraffin, dimethyl sulfoxide, triglycerides and the like can be used.

[0152] In the case of liquid dosage forms, such as drinkable solutions, the following substances can be used as stabilizers or solubilizers: lower aliphatic mono- and polyhydric alcohols having 2 to 4 carbon atoms, such as ethanol, n-propanol, glycerol, polyethylene glycols with a molecular weight between 200 and 600 (e.g. 1 to 40% aqueous solution), diethylene glycol monoethyl ether, 1,2-propylene glycol, organic amides, such as amides of aliphatic C1-C6-carboxylic acids with ammonia or primary, secondary or tertiary C1-C4-amines or C1-C4-hydroxyamines, such as ureas, carbamates, acetamides, N-methylacetamide, N,N-diethylacetamide, N,N-dimethylacetamide, lower aliphatic amines and diamines having 2 to 6 carbon atoms, such as ethylenediamine, hydroxyethyltheophylline, tromethamine (e.g. as 0.1 to 20% aqueous solution), aliphatic amino acids.

[0153] Solubilizers and emulsifiers may be included in the preparation of the disclosed liquid dosage forms, for example the following non-limiting examples may be used: polyvinylpyrrolidone, sorbitan fatty acid esters such as sorbitan trioleate, phospholipids such as lecithin, gum arabic, gum tragacanth, polyoxyethylated sorbitan monooleate and other ethoxylated fatty acid esters of sorbitan, polyoxyethylated fats, polyoxyethylated triglycerides, linseed oil triglycerides, polyethylene oxide condensation products of fatty alcohols, alkylphenols or fatty acids or also 1-methyl-3-(2-hydroxyethyl)imidazolidinone-(2). In this context, polyoxyethylated means that the substance in question contains polyoxyethylene chains, the degree of polymerization of which is generally between 2 and 40, in particular between 10 and 20. Such polyoxyethylated substances can be obtained, for example, by reaction of hydroxyl-containing compounds (e.g. mono- or diglycerides or unsaturated compounds, such as those containing oleic acid groups) with ethylene oxide (e.g. 40 moles of ethylene oxide per 1 mole of glyceride). Examples of triglycerides are olive oil, peanut oil, castor oil, sesame oil, cottonseed oil, corn oil. See also Dr. HP Fiedler "Lexikon der Hillsstoffe für Pharmazie, Kostnetik und angrenzende Gebiete" 1971, pages 191-195.

[0154] In various aspects, the liquid dosage form may further contain preservatives, stabilizers, buffer substances, flavoring agents, sweeteners, coloring agents, antioxidants and complex formers, etc. Complex formers that may be considered are, for example: chelating agents, such as ethylenediamine kojic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid and salts thereof.

[0155] It may be optionally necessary to stabilize the liquid dosage form with a physiologically acceptable base or buffer to a pH range of about 6 to 9. The pH may preferably be set to neutral or slightly alkaline (up to pH 8).

[0156] In order to enhance the solubility and / or stability of the disclosed compounds in the disclosed liquid dosage forms, parenteral injection dosage forms or intravenous injection dosage forms, it is advantageous to use α-, β- or γ-cyclodextrin or derivatives thereof, particularly hydroxyalkyl-substituted cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin or sulfobutyl-β-cyclodextrin. Cosolvents such as alcohols can also improve the solubility and / or stability of the compounds according to the present disclosure in pharmaceutical compositions.

[0157] In various aspects, the disclosed liquid dosage form, parenteral injection dosage form or intravenous injection dosage form can further comprise a liposome delivery system, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholine.

[0158] Pharmaceutical compositions of the present disclosure are suitable for injection, such as parenteral administration, such as intravenous, intramuscular or subcutaneous administration. Pharmaceutical compositions for injection can be prepared as solutions or suspensions of the active compound in water. Suitable surfactants, such as hydroxypropylcellulose, can be included. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof in oil. In addition, preservatives can be included to prevent harmful growth of microorganisms.

[0159] The pharmaceutical composition of the present disclosure suitable for parenteral administration may include sterile aqueous or oily solutions, suspensions or dispersions. In addition, the composition may be in the form of sterile powders for the temporary preparation of such sterile injectable solutions or dispersions. In some respects, the final injectable form is sterile and must be an effective fluid in order to be used in a syringe. The pharmaceutical composition should be stable under production and storage conditions; therefore, it is preferably preserved to prevent the contamination of microorganisms such as bacteria and fungi. The carrier may be a solvent or a dispersion medium, containing, for example, water, ethanol, a polyol (such as glycerol, propylene glycol and liquid polyethylene glycol), a vegetable oil and a suitable mixture thereof.

[0160] For example, an injectable solution can be prepared in which the carrier comprises a saline solution, a glucose solution, or a mixture of a saline and glucose solution. An injectable suspension can also be prepared, in which case a suitable liquid carrier, a suspending agent, etc. can be used. In some aspects, the disclosed parenteral formulations can include about 0.01-0.1 M, such as about 0.05 M, phosphate buffer. On the other hand, the disclosed parenteral formulations can include about 0.9% saline.

[0161] In various aspects, the disclosed parenteral pharmaceutical composition may include a pharmaceutically acceptable carrier, such as an aqueous or non-aqueous solution, suspension, and emulsion. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include, but are not limited to, water, alcohol / water solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles may include mannitol, normal serum albumin, sodium chloride solution, Ringer's dextrose, glucose and sodium chloride, lactated Ringer's, and fixed oils. Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, finishing agents, inert gases, and the like. On the other hand, the disclosed parenteral pharmaceutical composition may include a small amount of additives, such as substances that enhance isotonicity and chemical stability, such as buffers and preservatives. For injectable pharmaceutical compositions, solid form preparations that are intended to be converted into liquid form preparations shortly before use are also contemplated. Furthermore, other adjuvants may be included to render the formulation isotonic with the blood of the subject or patient.

[0162] In addition to the pharmaceutical compositions described above, the disclosed compounds can also be formulated into long-acting preparations. Such long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. Thus, for example, the compound can be formulated with a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or as a sparingly soluble derivative, e.g., as a sparingly soluble salt.

[0163] Pharmaceutical compositions of the present disclosure can be in a form suitable for topical application. As used herein, the phrase "topical application" refers to application to a biological surface, wherein the biological surface includes, for example, skin areas (e.g., hands, forearms, elbows, legs, faces, nails, anus and genital areas) or mucous membranes. By selecting a suitable carrier and optionally other ingredients that can be included in the composition, as described in detail below, the composition of the present invention can be formulated into any form commonly used for topical application. Topical pharmaceutical compositions can be in the form of creams, ointments, pastes, gels, lotions, emulsions, suspensions, aerosols, sprays, foams, dusting powders, pads and patches. In addition, the composition can be in a form suitable for transdermal devices. These preparations can be prepared using compounds of the present disclosure or their pharmaceutically acceptable salts by conventional processing methods. For example, creams or ointments are prepared by mixing hydrophilic materials and water and compounds of about 5wt% to about 10wt%, to prepare creams or ointments with desired consistency.

[0164] In compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancer and / or a suitable wetting agent, optionally combined in a small proportion with a suitable additive of any nature, which additive does not produce a significant harmful effect on the skin. The additive can promote application to the skin and / or can contribute to the preparation of the required composition. These compositions can be applied in a variety of ways, for example as a transdermal patch, as a spot coating, as an ointment.

[0165] Ointment is a semisolid preparation, usually based on vaseline or petroleum derivatives. The specific ointment base to be used is a kind of active agent selected for a given preparation to provide the best delivery, and preferably, other desired properties (e.g., emollients) are also provided. Like other carriers or vehicles, ointment base should be inert, stable, non-irritating and non-sensitizing. As explained in Remington: The Science and Practice of Pharmacy, 19th Ed., Easton, Pa.: Mack Publishing Co. (1995), pp.1399-1404, ointment base can be divided into four categories: oleaginous base; emulsifiable base; emulsion base; and water-soluble base. Oleaginous ointment base includes, for example, vegetable oil, fat obtained from animals and semisolid hydrocarbons obtained from petroleum. Emulsifiable ointment base, also referred to as absorbent ointment base, is almost free of or free of water, including, for example, hydroxystearin sulfate, anhydrous lanolin and hydrophilic vaseline. Emulsion ointment bases are water-in-oil (W / O) emulsions or oil-in-water (O / W) emulsions, including, for example, cetyl alcohol, glyceryl monostearate, lanolin and stearic acid. Preferred water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weights.

[0166] Lotions are preparations that are applied to the skin surface without friction. Lotions are generally liquid or semi-liquid preparations, in which solid particles including active agents are present in water or alcohol bases. Lotions are generally preferred for treating large areas of the body due to the ease of applying more fluid compositions. Lotions are generally suspensions of solids, and generally contain liquid oily emulsions of the oil-in-water type. It is generally necessary to subdivide the insolubles in the lotion. Lotions generally contain suspending agents to produce better dispersions, as well as compounds for positioning and maintaining active agents in contact with the skin, such as methylcellulose, sodium carboxymethylcellulose, etc.

[0167] Creams are viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are usually water-washable and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase, also referred to as the "internal" phase, is usually composed of petrolatum and / or fatty alcohols such as cetyl alcohol or stearyl alcohol. The aqueous phase usually, but not necessarily, exceeds the oil phase in volume and usually contains a humectant. The emulsifier in cream formulations is usually a nonionic, anionic, cationic, or amphoteric surfactant. Reference may be made to Remington: The Science and Practice of Pharmacy, supra, for more information.

[0168] Pastes are semisolid dosage forms in which the bioactive agent is suspended in a suitable matrix. Depending on the nature of the matrix, pastes are classified as fatty pastes or pastes made from a single-phase hydrogel. The matrix in fatty pastes is generally vaseline, hydrophilic vaseline, etc. Pastes made from a single-phase hydrogel usually contain carboxymethylcellulose, etc. as a matrix. Further reference is made to Remington: The Science and Practice of Pharmacy for more information.

[0169] Gel formulations are semisolid, suspension-type systems. Single-phase gels contain organic macromolecules substantially uniformly distributed throughout a carrier liquid, which is typically aqueous but also preferably contains an alcohol and optionally an oil. Preferred organic macromolecules, i.e., gelling agents, are cross-linked acrylic acid polymers, such as the carbomer polymer family, such as those available under the trademark Carbopol TM Commercially available carboxypolyalkylene. Other types of preferred polymers herein are hydrophilic polymers such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymers and polyvinyl alcohol; modified celluloses such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and methylcellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin. To prepare a uniform gel, a dispersant such as alcohol or glycerol may be added, or the gelling agent may be dispersed by grinding, mechanical mixing or stirring, or a combination thereof.

[0170] Sprays typically provide the active agent in an aqueous and / or alcoholic solution that can be atomized onto the skin for delivery. Such sprays include those formulated to provide a concentration of active agent solution at the site of application after delivery, for example, the spray solution may consist primarily of alcohol or other similar volatile liquids in which the active agent can be dissolved. After delivery to the skin, the carrier evaporates, leaving a concentrated active agent at the site of application.

[0171] Foam compositions are typically formulated as a single-phase or multi-phase liquid form and contained in a suitable container, optionally together with a propellant that facilitates the expulsion of the composition from the container, thereby converting it into a foam upon application. Other foam-forming techniques include, for example, "bag-in-can" formulation techniques. Compositions so formulated typically contain low-boiling hydrocarbons, such as isopropane. Application and agitation of such compositions at body temperature causes the isopropane to evaporate and produce foam in a manner similar to pressurized aerosol foaming systems. Foams can be water-based or aqueous alkanol, but are typically formulated with a high alcohol content, which, once applied to the user's skin, evaporates rapidly, thereby delivering the active ingredient through the upper layers of the skin to the treatment site.

[0172] Skin patches usually include a backing to which a reservoir containing an active agent is attached. For example, the reservoir can be a pad in which an active agent or composition is dispersed or soaked, or a liquid reservoir. The patch also usually includes a water-permeable adhesive on the front side, which adheres and fixes the device to the treatment area. Silicone rubber with self-adhesive properties can be used alternatively. In both cases, a protective permeable layer can be used to protect the adhesive side of the patch before it is used. The skin patch can also include a removable cover for protecting it during storage.

[0173] The example of the patch construction that can be used together with the present invention comprises single layer or multilayer adhesive drug-packed system, it is characterized in that medicine is directly included in the adhesive in contact with the skin.In such transdermal patch design, adhesive is not only used for patch being attached on the skin, but also as formulation basis, medicine and all excipients are included under the single backing film.In the multilayer adhesive drug-packed patch, film is placed between two different adhesive drug-packed layers, or a plurality of adhesive drug-packed layers are incorporated under the single backing film.

[0174] Examples of pharmaceutically acceptable carriers suitable for topical pharmaceutical compositions include well-known carrier materials used in the cosmetic and medical fields as bases for emulsions, creams, aqueous solutions, oils, ointments, pastes, gels, lotions, emulsions, foams, suspensions, aerosols, etc., depending on the final form of the composition. Thus, representative examples of suitable carriers according to the present invention include, but are not limited to, water, liquid alcohols, liquid glycols, liquid polyalkylene glycols, liquid esters, liquid amides, liquid protein hydrolysates, liquid alkylated protein hydrolysates, liquid lanolin and lanolin derivatives, and similar materials commonly used in cosmetic and pharmaceutical compositions. Other suitable carriers according to the present invention include, but are not limited to, alcohols, such as monohydric and polyhydric alcohols, such as ethanol, isopropanol, glycerol, sorbitol, 2-methoxyethanol, diethylene glycol, ethylene glycol, hexylene glycol, mannitol and propylene glycol; ethers, such as ethyl ether or dipropyl ether; polyethylene glycol and methoxypolyoxyethylene (carbon waxes with a molecular weight range of 200 to 20,000); polyoxyethylene glycerol, polyoxyethylene sorbitol, stearyl diacetin, etc.

[0175] If desired, the topical compositions of the present disclosure may be present in a package or dispensing device, such as an FDA-approved kit, which may include one or more unit dosage forms containing the active ingredient. For example, the dispenser device may include a tube. The package or dispensing device may be accompanied by instructions for use. The package or dispensing device may also be accompanied by a notice in a form prescribed by a government agency that manages the manufacture, use, or sale of drugs, which reflects the agency's approval of the form of the composition for human or veterinary use. For example, such a notice may include a label of a prescription drug or an approved product insert approved by the U.S. Food and Drug Administration. Compositions containing the topical compositions of the present invention formulated in a pharmaceutically acceptable carrier may also be prepared, placed in an appropriate container, and labeled for treatment of a specified condition.

[0176] Another kind of patch system configuration that the present invention can use is a reservoir transdermal system design, it is characterized in that comprising a liquid compartment, this liquid compartment comprises a drug solution or a suspension, and this drug solution or a suspension is separated from a release liner by a semipermeable membrane and adhesive. The adhesive component of this patch system can be incorporated between film and the release liner as a continuous layer, or is incorporated around the film with a concentric configuration. Another kind of patch system configuration that the present invention can utilize is a matrix system design, it is characterized in that comprising a semisolid matrix containing a drug solution or a suspension that is in direct contact with the release liner. The component responsible for skin adhesion is incorporated in the cover layer, and forms a concentric structure around the semisolid matrix.

[0177] Pharmaceutical compositions of the present disclosure may be in a form suitable for rectal administration, wherein the carrier is a solid. Preferably, the mixture forms a unit dose suppository. Suitable carriers include cocoa butter and other materials commonly used in the art. Suppositories may be conveniently formed by first mixing the composition with a softened or melted carrier, then cooling and molding in a mold.

[0178] Pharmaceutical compositions containing a compound of the present disclosure and / or a pharmaceutically acceptable salt thereof may also be prepared in powder or liquid concentrate form.

[0179] Pharmaceutical compositions (or formulations) can be packaged in a variety of ways. Typically, articles for distribution include containers containing the pharmaceutical composition in an appropriate form. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), pouches, foil blister packages, etc. The container may also include a tamper-proof component to prevent inadvertent contact with the contents of the package. In addition, the container typically has a label on it that describes the contents of the container and any appropriate warnings or instructions.

[0180] If desired, the disclosed pharmaceutical composition may be present in a package or dispensing device, which may include one or more unit dosage forms containing the active ingredient. The package may, for example, include metal or plastic foil, such as a blister pack. The package or dispensing device may be accompanied by instructions for use. The package or dispensing device may also be accompanied by a notice in a form prescribed by a governmental agency that manages the manufacture, use, or sale of the drug, which reflects the agency's approval of the form of the composition for human or veterinary administration. For example, such a notice may include a label of a prescription drug or an approved product insert approved by the U.S. Food and Drug Administration. Compositions comprising the topical compositions of the present invention formulated in a pharmaceutically acceptable carrier may also be prepared, placed in an appropriate container, and labeled for treatment of a specified condition.

[0181] The exact dosage and frequency of administration depends on the specific disclosed compound, the product of the disclosed preparation process, its pharmaceutically acceptable salt, solvate or polymorph thereof, its hydrate, its solvate, its polymorph, or its stereochemical isomeric form; the specific condition being treated and the severity of the condition being treated; various factors specific to the medical history of the subject to whom the dose is administered, such as age; the weight, sex, degree of disease and general physical condition of the specific subject, and other medications that the individual may be taking; as is well known to those skilled in the art. In addition, it is obvious that the effective daily amount can be lowered or increased, depending on the response of the treated subject and / or on the evaluation of the physician prescribing the disclosed compound.

[0182] Depending on the mode of administration, the pharmaceutical composition will contain 0.05% to 99% by weight, preferably 0.1% to 70% by weight, more preferably 0.1% to 50% by weight of active ingredient, and 1 to 99.95% by weight, preferably 30 to 99.9% by weight, more preferably 50 to 99.9% by weight of a pharmaceutically acceptable carrier, all percentages being based on the total weight of the composition.

[0183] Under the treatment conditions disclosed herein, suitable dosage levels will generally be about 0.01 to 1000 mg per kg of patient body weight per day, and can be administered in single or multiple doses. In various aspects, dosage levels will be about 0.1 to about 500 mg / kg per day, about 0.1 to 250 mg / kg per day, or about 0.5 to 100 mg / kg per day. Suitable dosage levels can be about 0.01 to 1000 mg / kg per day, about 0.01 to 500 mg / kg per day, about 0.01 to 250 mg / kg per day, about 0.05 to 100 mg / kg per day, or about 0.1 to 50 mg / kg per day. Within this range, dosage can be 0.05 to 0.5, 0.5 to 5.0, or 5.0 to 50 mg / kg per day. For oral administration, the composition is preferably provided in the form of tablets containing 1.0 to 1000 mg of active ingredient, particularly 1.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900 and 1000 mg of active ingredient, for symptomatic adjustment of the dosage of the patient to be treated. The compound can be administered 1 to 4 times a day, preferably once or twice a day. The dosage regimen can be adjusted to provide the best therapeutic response.

[0184] Such unit doses as described above and below can be administered more than once a day, for example 2, 3, 4, 5 or 6 times a day. In various aspects, such unit doses can be administered 1 or 2 times a day, so that the total dose for a 70 kg adult is within the range of 0.001 to about 15 mg / kg subject per administration. On the other hand, the dose is 0.01 to about 1.5 mg / kg subject body weight per use, and this therapy can last for weeks or months, and in some cases, years. However, it should be understood that the specific dose level for any particular patient will depend on a variety of factors, including the activity of the specific compound used; the age, weight, general health, sex and diet of the individual being treated; the time and route of administration; the rate of excretion; other drugs previously administered; and the severity of the specific disease being treated, as is well known to those skilled in the art.

[0185] Typical dosages may be 1 mg to about 100 mg tablets or 1 mg to about 300 mg taken once daily, or taken multiple times daily, or a timed release capsule or tablet taken once daily containing a proportionally higher amount of active ingredient. The timed release effect may be obtained by capsule materials that dissolve at different pH values, capsules that release slowly by osmotic pressure, or by any other known controlled release means.

[0186] It may be necessary to use dosages outside of these ranges in certain circumstances, as will be apparent to those skilled in the art. In addition, it is noted that the clinician or treating physician will know how to combine individual patient responses and when to initiate, interrupt, adjust or terminate treatment.

[0187] The present disclosure further relates to a method for preparing a medicine for treating tau disease in a mammal (e.g., a human), comprising combining one or more disclosed compounds, products or compositions with a pharmaceutically acceptable carrier or diluent. Therefore, in one aspect, the present disclosure further relates to a method for preparing a medicine, comprising combining at least one disclosed compound or at least one disclosed product with a pharmaceutically acceptable carrier or diluent.

[0188] The disclosed pharmaceutical compositions may also contain other therapeutically active compounds, which are usually used to treat the above-mentioned pathological or clinical conditions.

[0189] It is to be understood that the disclosed compositions can be prepared from the disclosed compounds. It is also to be understood that the disclosed compositions can be used in the disclosed methods of use.

[0190] As already mentioned, the present disclosure relates to pharmaceutical compositions comprising a therapeutically effective amount of a disclosed compound, a product of a disclosed preparation process, a pharmaceutically acceptable salt, a hydrate thereof, a solvate thereof, a polymorph thereof, and a pharmaceutically acceptable carrier. In addition, the present disclosure relates to a method for preparing such a pharmaceutical composition, characterized in that a pharmaceutically acceptable carrier is intimately mixed with a therapeutically effective amount of a compound according to the present disclosure.

[0191] As already mentioned, the present disclosure also relates to pharmaceutical compositions comprising disclosed compounds, products of disclosed preparation methods, pharmaceutically acceptable salts, hydrates thereof, solvates thereof, polymorphs thereof, and one or more other drugs, treating, preventing, controlling, alleviating or reducing the risk of diseases or conditions for which disclosed compounds or other drugs are available, and the use of such compositions for preparing drugs. The present disclosure also relates to combinations of disclosed compounds, products of disclosed preparation methods, pharmaceutically acceptable salts, hydrates thereof, solvates thereof, or polymorphs thereof. The present disclosure also relates to such combinations used as drugs. The present disclosure also relates to a product comprising (a) disclosed compounds, products of disclosed preparation methods, pharmaceutically acceptable salts, hydrates thereof, solvates thereof, polymorphs thereof, and (b) another therapeutic agent, as a combined preparation for simultaneous, separate or sequential use in the treatment or prevention of a condition in a mammal (including a human), the treatment or prevention of which is affected or promoted by the modulatory effects of the disclosed compounds and another therapeutic agent. The different drugs of such a combination or product may be combined in a single preparation with a pharmaceutically acceptable carrier or diluent, or they may each be present in separate preparations together with a pharmaceutically acceptable carrier or diluent.

[0192] Methods of using the compound

[0193] In another aspect, the present disclosure provides a method of treating tauopathy, comprising administering to a subject in need thereof a therapeutically effective amount of a β-arrestin oligomerization inhibitor disclosed herein.

[0194] Tauopathies are diseases characterized by abnormal levels of tau in individual cells, tissues or body fluids. In some cases, taupathies are characterized by the presence of tau or tau polypeptides and / or pathological forms of tau at elevated (higher than normal) levels in cells, tissues or fluids. For example, in some cases, taupathies are characterized by the presence of elevated levels of tau or tau polypeptides and / or pathological forms of tau in brain tissue and / or cerebrospinal fluid. "Higher than normal" levels of tau in cells, tissues or fluids represent that the tau levels in tissues or fluids are higher than normal control levels, for example, higher than the normal control levels of individuals or individual populations of the same age group. See, for example, Blomberg et al. (2001) "Cerebrospinal fluid tau levels increase with age in healthy individuals" Dement. Geriatr. Cogn. Disord. 12: 127. In some cases, individuals suffering from taupathies show one or more other symptoms (for example, cognitive impairment) of taupathies.

[0195] In other cases, tauopathies are characterized by the presence of below normal levels of tau protein in cells, tissues, or fluids. "Below normal" levels of tau in a tissue or fluid means that the level of tau in the cell, tissue, or fluid is below a normal control level, such as below a normal control level for an individual or group of individuals of the same age group.

[0196] Alzheimer's disease and some forms of frontotemporal dementia (Pick's disease, sporadic frontotemporal dementia and frontotemporal dementia with Parkinson's disease linked to chromosome 17) are the most common forms of tauopathy. The present disclosure provides a method of treatment as described above, wherein the tauopathy is Alzheimer's disease, Pick's disease, sporadic frontotemporal dementia and frontotemporal dementia with Parkinson's disease linked to chromosome 17. Other tauopathies include, but are not limited to, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and subacute sclerosing panencephalitis.

[0197] Neurodegenerative tauopathies include Alzheimer's disease, amyotrophic lateral sclerosis / Parkinson's disease-dementia complex, argyrophilic grain dementia, English amyloid angiopathy, cerebral amyloid angiopathy, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangles with calcifications, Down syndrome, frontotemporal dementia (FTD), frontotemporal dementia with Parkinson's disease linked to chromosome 17, frontotemporal lobar degeneration, Gerstmann-Straussler-Scheinke disease, Haller-Schneider disease, and Worden-Spatz disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C, non-Guam motor neuron disease with neurofibrillary tangles, Pick's disease, postencephalitic Parkinson's disease, prion amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangled dementia, multi-infarct dementia, ischemic stroke, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), and stroke.

[0198] Public Aspects

[0199] The present disclosure will be better understood by reading the following numbered aspects, which should not be confused with the claims. In some cases, each numbered aspect described below can be combined with additional aspects described above.

[0200] Aspect 1. A method of treating tauopathy in a subject, the method comprising administering to the human subject a therapeutically effective amount of a β-arrestin oligomerization inhibitor.

[0201] Aspect 2. The method of aspect 1, wherein the tauopathy is selected from the group consisting of Alzheimer's disease, Pick's disease, frontotemporal dementia, chromosome 17-related frontotemporal dementia with Parkinson's syndrome, progressive supranuclear palsy, corticobasal degeneration, subacute sclerosing panencephalitis, amyotrophic lateral sclerosis / Parkinson's-dementia complex, argyrophilic grain dementia, English amyloid angiopathy, cerebral amyloid angiopathy, Creutzfeldt-Jakob disease, pugilistic dementia, diffuse neurofibrillary tangles with calcifications, and Tang et al. syndrome, frontotemporal lobar degeneration, Gerstmann-Straussler-Scheink disease, Hallervorden-Spatz disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C, non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic Parkinson disease, prion amyloid angiopathy, progressive subcortical gliosis, tangled dementia, multi-infarct dementia, ischemic stroke, chronic traumatic encephalopathy, traumatic brain injury, and stroke.

[0202] Aspect 3. The method of Aspect 2, wherein the tauopathy is frontotemporal lobar degeneration.

[0203] Aspect 4. The method of any one of Aspects 1 to 3, wherein the β-arrestin oligomerization inhibitor is a dominant negative mutant of β-arrestin 1 or β-arrestin 2 that blocks endogenous β-arrestin 1 and / or β-arrestin 2 from binding to inositol hexaphosphate (IP6).

[0204] Aspect 5. The method according to any one of aspects 1 to 3, wherein the β-arrestin oligomerization inhibitor is a compound having the following structure or a pharmaceutically acceptable salt thereof:

[0205]

[0206] Where R 1 is hydrogen or alkyl;

[0207] Ar is aryl; and

[0208] When R 1 When it is an alkyl group, a The stereochemistry at is racemic, substantially R, or substantially S.

[0209] Aspect 6. The method of aspect 5, wherein R 1 It's hydrogen.

[0210] Aspect 7. The method of aspect 5 or 6, wherein Ar is unsubstituted or substituted phenyl.

[0211] Aspect 8. The method of aspect 5 or 6, wherein Ar is phenyl substituted with one or more halogen atoms.

[0212] Aspect 9. The method of aspect 5, wherein R 1C1 to C 10 alkyl.

[0213] Aspect 10. The method of aspect 9, wherein in C a The stereochemistry at is essentially R.

[0214] Aspect 11. The method of aspect 9, wherein in C a The stereochemistry at is essentially S.

[0215] Aspect 12. The method of aspect 9, wherein in C a The stereochemistry at is racemic.

[0216] Aspect 13. The method of any one of Aspects 9 to 12, wherein Ar is unsubstituted or substituted phenyl.

[0217] Aspect 14. The method of any one of aspects 9 to 12, wherein Ar is phenyl substituted with one or more halogen atoms.

[0218] Aspect 15. The method according to any one of Aspects 9 to 12, wherein Ar is a phenyl group fused to a cycloalkyl group.

[0219] Aspect 16. The method of any one of Aspects 1 to 3, wherein the compound is C1, C1A, C1B, C1C or C1E.

[0220] Aspect 17. The method of any one of Aspects 1 to 3, wherein the β-arrestin oligomerization inhibitor is a compound having the following structure or a pharmaceutically acceptable salt thereof:

[0221]

[0222] wherein Ar is an aryl group.

[0223] Aspect 18. The method of aspect 17, wherein Ar is unsubstituted or substituted phenyl.

[0224] Aspect 19. The method of aspect 17, wherein Ar is a phenyl group substituted with an alkyl group or a cycloalkyl group.

[0225] Aspect 20. The method of any one of Aspects 1 to 3, wherein the compound is C1D.

[0226] Aspect 21. The method of any one of Aspects 1 to 3, wherein the β-arrestin oligomerization inhibitor is a compound having the following structure or a pharmaceutically acceptable salt thereof:

[0227]

[0228] Where R 2 and R 3 are independently hydrogen or alkyl; and

[0229] Ar is aryl.

[0230] Aspect 22. The method of aspect 121, wherein R 2 It's hydrogen.

[0231] Aspect 23. The method of aspect 21 or 22, wherein R 3 C1 to C 10 alkyl.

[0232] Aspect 24. The method of any one of Aspects 21 to 23, wherein Ar is unsubstituted or substituted phenyl.

[0233] Aspect 25. The method according to any one of Aspects 21 to 23, wherein Ar is a phenyl group substituted with an alkyl group or a halogen atom.

[0234] Aspect 26. The method of any one of Aspects 1 to 3, wherein the compound is C2E.

[0235] Aspect 27. The method of any one of Aspects 1 to 3, wherein the β-arrestin oligomerization inhibitor is a compound having the following structure or a pharmaceutically acceptable salt thereof:

[0236]

[0237] Among them, Ar 1 and Ar 2 are independently aryl; and

[0238] X is CH2 or C=O.

[0239] Aspect 28. The method of aspect 27, wherein Ar 1 is unsubstituted or substituted phenyl.

[0240] Aspect 29. The method of aspect 27, wherein Ar 1 It is a phenyl group substituted by an alkyl group or a halogen atom.

[0241] Aspect 30. The method of aspect 27, wherein Ar 1 is unsubstituted or substituted heteroaryl.

[0242] Aspect 31. The method of aspect 27, wherein Ar 1 is unsubstituted or substituted piperidinyl or pyridazinyl.

[0243] Aspect 32. The method according to any one of Aspects 27 to 31, wherein Ar 2 is unsubstituted or substituted phenyl.

[0244] Aspect 33. The method according to any one of Aspects 27 to 31, wherein Ar 2 It is a phenyl group substituted by an alkyl group or a halogen atom.

[0245] Aspect 34. The method according to any one of Aspects 27 to 31, wherein Ar 2 is unsubstituted or substituted piperidinyl.

[0246] Aspect 35. The method according to any one of Aspects 27 to 31, wherein Ar 2 It is a piperidinyl group substituted by an alkyl group or a halogen atom.

[0247] Aspect 36. The method of any one of Aspects 27 to 35, wherein X is CH2.

[0248] Aspect 37. The method of any one of Aspects 27 to 35, wherein X is C═O.

[0249] Aspect 38. The method of any one of Aspects 1 to 3, wherein the compound is C2, C2A, C2C or C2D.

[0250] Aspect 39. The method of any one of Aspects 1 to 3, wherein the β-arrestin oligomerization inhibitor is a compound having the following structure or a pharmaceutically acceptable salt thereof:

[0251]

[0252] Among them, Ar 1 and Ar 2 are independently aryl.

[0253] Aspect 40. The method of aspect 39, wherein Ar 1 is unsubstituted or substituted phenyl.

[0254] Aspect 41. The method of aspect 39, wherein Ar 1 It is a phenyl group substituted by an alkyl group or a halogen atom.

[0255] Aspect 42. The method according to any one of Aspects 39 to 41, wherein Ar 2 is unsubstituted or substituted phenyl.

[0256] Aspect 43. The method according to any one of Aspects 39 to 41, wherein Ar 2 It is a phenyl group substituted by an alkyl group or a halogen atom.

[0257] Aspect 44. The method of any one of Aspects 1 to 3, wherein the compound is C2B.

[0258] Several embodiments of the present invention have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the present invention. Therefore, other embodiments are within the scope of the following claims. Example

[0259] Example 1: β-Arrestin 2 oligomers impair the clearance of pathological tau and increase tau aggregates.

[0260] Introduction

[0261] GPCRs initiate a wide range of physiological processes, and several have been shown to play a possible role in the pathogenesis of AD (Ni Y, et al. (2006) Nat Med 12(12):1390-1396; AbdAlla S, et al. (2009) J Biol Chem 284(10):6566-6574; Thathiah A, et al. (2009) Science 323(5916):946-951; Bakshi P, et al. (2008) ACS Chem Biol 3(12):777-789; Ray B, et al. (2010) Neurosci Lett 470(1):1-5; Alley GM, et al. (2010) J Neurosci Res 88(1):143-154; Minkeviciene R, et al. (2004) J Pharmacol Exp Ther 311(2):677-682; Scholtzova H, et al. (2008) J Neurosci Res 86(12):2784-2791). Genetic and pharmacological studies have shown that neuronal expression and / or activation of several GPCRs with different structures, endogenous agonists, and cell signaling effects have a positive impact on Aβ and / or tau pathogenesis in animal models. However, it is unclear how this heterogeneous array of GPCRs affects Aβ and tau pathogenesis and neurodegeneration in AD. One potential commonality between these receptors is their interaction with arrestin proteins. After agonist binding (Wilden U, et al. (1986) Proc Natl Acad Sci USA 83(5):1174-1178), most GPCRs are phosphorylated by G protein-coupled receptor kinases, and the phosphorylated receptors are substrates for arrestin binding. These proteins physically block between the receptor and the G protein, partially uncoupling the activated receptor from its functional transducer. This phenomenon is thought to be a mechanism for regulating function within the complex signaling environment of the cell. Agonist activation also induces receptor internalization and promotes additional signaling, which has been shown to be due to arrestins acting as multifunctional adaptor and scaffold proteins.Arrestins constitute a small family of four homologous proteins, called arrestin 1, arrestin 2 (β-arrestin 1), arrestin 3 (β-arrestin 2) and arrestin 4 (Wilden U, et al. (1986) Proc Natl Acad Sci USA 83(5):1174-1178; Moore CA, et al. (2007) Annu Rev Physiol 69:451-482; Gurevich VV & Gurevich EV (2006) Pharmacol Ther 110(3):465-502). While arrestin 1 and arrestin 4 bind to only a few receptors (rhodopsin and color opsins) and are expressed in specific cell types (Shinohara T, et al. (1987) Proc Natl Acad Sci USA 84(20):6975-6979; Yamaki K, et al. (1987) Biochem Biophys Res Commun 142(3):904-910), β-arrestin 1 and β-arrestin 2 are ubiquitously expressed and show the highest levels of expression in the brain and spleen. β-Arrestin exists in three different states in cells: (1) free, (2) GPCR-bound, and (3) microtubule-bound, each with different signaling potentials (Hanson SM, et al. (2006) J Biol Chem 281(14):9765-9772; Hanson SM, et al. (2007) J Mol Biol 368(2):375-387; Nair KS, et al. (2004) J Biol Chem 279(39):41240-41248; Gao H, et al. (2004) Mol Cell 14(3):303-317; Song X, et al. (2007) J Neurochem 103(3):1053-1062; Wu N, et al. (2006) J Mol Biol 364(5):955-96). Previous studies have shown that β-arrestin2 is increased in AD brain (Thathiah A, et al. (2013) Nat Med 19(1):43-49). In addition, genetic studies have shown that endogenous β-arrestin2 promotes Aβ production and deposition by physically interacting with the Aph-1 subunit of the γ-secretase complex (27) linking β-arrestin2 to Aβ pathogenesis (Thathiah A, et al. (2013) Nat Med 19(1):43-49).However, before the current work, it was unclear whether or how β-arrestin2 pathogenically contributes to tauopathy and neurodegeneration in AD or FTLD without Aβ accumulation.

[0262] method

[0263] Mouse

[0264] Arrb2 - / - , tau P301S, and WT mice were bred in a C57BL6 background for at least three generations and then intercrossed. - / - The mouse (Bohn LM, et al. (1999) Science 286(5449):2495-24980) and tau P301S mouse (Yoshiyama Y, et al. (2007) Neuron 53(3):337-351) have been characterized previously. All experiments involving mice were performed at USFHealth according to protocols approved by the Institutional Animal Care and Use Committee (IACUC).

[0265] Patient samples

[0266] Frozen frontal cortex tissue samples were obtained from the Alzheimer's Disease Research Center at Emory University. Samples from patients diagnosed with FTLD-tau (confirmed by pathology) and samples from unaffected (control) patients matched as closely as possible for sex, age, and APOE genotype were used.

[0267] Primary neuronal cultures

[0268] Primary mouse hippocampal and cortical neuronal cultures were previously described (Woo JA, et al. (2019) Commun Biol 2: 112; Woo JA, et al. (2017) Hum Mol Genet 26 (20): 3973-3988; Woo JA, et al. (2015) Cell Death Differ 22 (6): 1069-1070). In brief, primary neurons were prepared from P0 mice. Both the hippocampus and the cortex were dissected separately in HBSS and digested with trypsin. Neurons were plated on glass coverslips coated with poly-D-lysine (Sigma-Aldrich, St Louis, MO, USA) and maintained in neurobasal medium containing glutamax (Invitrogen, Carlsbad, CA, USA) and B27 supplement (Invitrogen, Carlsbad, CA, USA).

[0269] Antibodies and reagents

[0270] Antibodies against total tau (Tau A10, 1:1000 for Western blot, 1:200 for cell / tissue staining, Santa Cruz Biotechnology, Dallas, TX, USA), PHF1 (kind gift from Dr. Peter Davies, 1:20, Albert Einstein College of Medicine), Tau-pS199 / pS202 (1:1000 for Western blot, 1:200 for tissue staining, Invitrogen, Carlsbad, CA, USA), HT7 (1:200 for cell / tissue staining, Invitrogen, Carlsbad, CA, USA), synaptophysin (1:200, Invitrogen, Carlsbad, CA, USA), brain development regulatory protein (1:400, Abcam, Cambridge, MA, USA), actin White (1:5000, Sigma-Aldrich, St. Louis, MO, USA), Flag-M2 (1:1000 for Western blotting, 1:200 for cell staining, Sigma-Aldrich, St. Louis, MO, USA), Myc (1:1000, Cell Signaling, Danvers, MA, USA), HA (1:1000, Cell Signaling, Danvers, MA, USA), GFP (1:1000, Cell Signaling, Danvers, MA, USA).

[0271] DNA constructs, siRNA, shRNA lentivirus

[0272] β-Arrestin2, β-Arrestin2ΔIP6C and β-Arrestin2ΔIP6N constructs were gifts from S. Marullo (Institut Cochin, Paris, France). pCDNA-β-Arrestin2-HA was from X. Xin (Shanghai Institute of Materia Medica, Shanghai, China). The following were obtained from Addgene: β-Arrestin2 GFP WT plasmid #35411 (68), HA-p62 plasmid #28027 (69), pMXs-puro GFP-p62 plasmid #38277 (Itakura E & Mizushima N (2011) J Cell Biol 192(1):17-27), pmRFP-LC3 plasmid #21075 (Kimura S, Noda T, & Yoshimori T (2007) Autophagy 3(5):452-460). β-arrestin 2ON-TARGET plus SMART pool siRNA was purchased from Dharmacon (Lafayette, CO, USA). β-arrestin 2 shRNA plasmid was obtained from Abm (Richmond, BC, Canada). Lentiviral vectors were co-transfected with pVSVG and Pax2 in HEK293T cells overnight using polyethyleneimine (PEI). The medium was removed the next day and replaced with serum-free medium. After 72 hours of incubation, the medium was collected and centrifuged to remove cell debris. The virus was filtered through a syringe filter (0.2-0.45um).

[0273] Proximity ligation assay (PLA)

[0274] The assay was performed using commercially available reagents (Duolink, Sigma-Aldrich, St Louis, MO) and Hela V5-tau cells were transfected with WT β-arrestin2-flag and β-arrestin2-myc, without and with mutant β-arrestin2ΔIP6C or β-arrestin2ΔIP6N constructs. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature and then washed with 0.2% triton in TBS. After washing, cells were blocked with 0.2% Triton in 3% normal goat serum for 1 h at room temperature. After applying primary antibodies overnight at 4°C, cells were washed and incubated with PLA probes at 37°C (1 h), washed with buffer A, incubated with ligation solution at 37°C (30 min), washed with wash buffer A, incubated with amplification solution at 37°C (100 min), and washed with wash buffer B before mounting.

[0275] Immunocytochemistry and immunohistochemistry

[0276] Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, washed with 0.2% triton in TBS, blocked with 3% BSA and 0.1% triton x-100 for 1 hr, incubated with primary antibodies overnight at 4°C and fluorescent secondary antibodies for 45 min at room temperature. For immunohistochemistry, mice were perfused with PBS and fixed with 4% paraformaldehyde. 25 micron sections were blocked for 1 h using normal goat serum and incubated with primary antibodies overnight at 4°C, then incubated with secondary antibodies for 1 h at room temperature before fixation. Images were captured using an Olympus FV10i confocal microscope (Tokyo, Japan), and immunoreactivity was quantified using Image J software (NationalInstitutes of Health, Bethesda, MD). Immunoreactivity was quantified from every 12th serial section through the entire hippocampus. In ICC and IHC experiments, all comparison images were obtained using the same laser intensity, exposure time, and filter. Adjustments to brightness / contrast were also applied to all comparison images. The target regions were randomly selected and the researchers were blinded to the experimental conditions during image acquisition and quantification.

[0277] Cell lysis, immunoblotting, and lauroyl sarcosine extraction

[0278] Brain homogenates and cultured cells were lysed with RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% sodium dodecyl sulfate) plus protease and phosphatase inhibitors. Proteins were extracted and centrifuged at 15000 rpm for 15 min at 4 ° C, and the RIPA insoluble fraction was lysed with 5% SDS and 1% β-mercaptoethanol, 50 mM Tris-HCl pH 6.8, 12.5 mM EDTA, 10% glycerol and 0.02% bromophenol blue. The supernatant was used for western blot analysis. The supernatant was used for western blot analysis. Co-IP experiments were performed from NP40 lysate or indicator buffer, pre-cleared with IgG beads, then IP with IgG beads alone or IgG beads + indicator antibodies, extensively washed (5X) with original buffer, and Western blotted on SDS-PAGE. Lauroylsarcosine extraction was performed as previously described (Hasegawa M, et al. (2007) Brain 130 (Pt 5): 1386-1394). Briefly, brain homogenate was lysed with A68 buffer containing 10 mM Tris-HCl, pH 7.4, 0.8 M NaCl, 10% sucrose, 1 mM EGTA. The samples were centrifuged at 400 g for 20 min at 4 ° C, and 1% lauroylsarcosine was added to the collected supernatant. The samples were incubated for 1.5 h and centrifuged at 80,000 g for 30 min at room temperature. The pellet was resuspended in 100 ul of 50 mM Tris-HCl, pH 7.4.

[0279] Quantitative real-time RT-PCR

[0280] Using Roche Quantitative real-time RT-PCR was performed using the 96 System (Life Science, San Francisco, CA, USA). Total RNA was isolated from human brain or cell lines using Trizol reagent (Invitrogen, CA, USA), reverse transcribed (Superscript III, Invitrogen, CA, USA), and analyzed using Syber green master mix (Invitrogen, CA, USA). Comparative threshold cycle (Ct) values ​​were used to calculate amplification factors, and the relative amount of β-arrestin 2 was normalized to GAPDH.

[0281] Production and stereotaxic injection of rAAV9

[0282] Recombinant rAAV9 viruses were generated by co-transfection of serotype vectors expressing the target gene with pAAV9 and pXX6 in HEK293 cells and purified as previously described (Carty N, et al. (2010) J Neurosci Methods 194(1):144-153). For brain injections, isoflurane-anesthetized mice were injected bilaterally with a 26-gauge needle connected to a 10-□l syringe (Hamilton, Reno, NV, USA) at the following coordinates: 2.7 mm anterior-posterior, 2.7 mm lateral, and 3.0 mm vertical. A total volume of 2 □l purified rAAV9 (1×10 12 The mice were killed 2 months after injection.

[0283] Electrophysiology

[0284] Electrophysiological recordings were performed as described previously (67). Briefly, hippocampal slices were collected from 3-month-old WT, tauP301S, tauP301S / Arrb2 + / - and tauP301S / Arrb2 - / - Mice were prepared and input / output (IO) curves, paired pulse facilitation (PPF), and long-term potentiation (LTP) recordings were performed. The stimulating electrode was placed in the Schaffer collaterals of the hippocampus. The recording glass electrode loaded with ACSF was placed in the CA1 stratum radiatum below the pyramidal cell layer.

[0285] Atomic force microscopy

[0286] Recombinant p62 (4 μg) with / without recombinant β-arrestin 2 (0.2 μg) was prepared in aqueous solution. The samples were incubated in 30 μl of aqueous solution at RT for 2 hr. 10 μl of the solution was dripped on the freshly cut mica surface. After adsorption for 20 min, the excess solution was gently washed from the mica surface with filtered distilled water. The mica was left at room temperature until completely dry before AFM imaging. A commercial PPP-NCHAuD cantilever (Nanosensors, Switzerland) with a nominal spring constant of 42 N / m and a resonance frequency of 330 kHz was used for AFM imaging.

[0287] Recombinant protein

[0288] His-tau BL21 was provided by L.Blair. Tau WT (4RON) was subcloned into pET28a vector and transformed into BL21 competent cells. His-β-arrestin 2 was transformed into Rosetta competent cells. The cells were resuspended in ice-cold lysis buffer (Tris 20mM, pH8.0, NaCl 150mM, imidazole 10mM) with protease inhibitors. The pFast-p62-his construct was transformed into DH10Bac competent cells. After blue-white screening, the DH10Bac strain was selected to express and amplify the recombinant bacmid. Sf9 insect cells transfected with Bacmid (mid-extract from DH10Bac competent cells) were cultured for 3 days with Sf900 II SFM medium, and then the P1 generation virus in the medium was collected and added to new Sf9 cells. After 2 days of culture, Sf9 cells were harvested and lysed with lysis buffer (Tris 20mM, pH7.4, NaCl 150mM, Triton-X100 1%, 10mM imidazole and protease inhibitors). After the cells were sonicated, the cells were centrifuged at 14,000g for 15min, and the supernatant was incubated with Ni Sepharose. The recombinant protein was eluted with ice-cold elution buffer (Tris 20mM, NaCl 150mM, 200mM imidazole) and then dialyzed overnight at 4 degrees in dialysis buffer (Tris 20mM, NaCl 150mM, DTT 1mM).

[0289] Statistical analysis and graphs

[0290] Statistical analysis was performed by Prism 6.0 software (GraphPad Software, San Diego, CA, USA) using paired or unpaired Student's t-test and 1-way or 2-way ANOVA. ANOVA was followed by the indicated post hoc tests. Data are shown as representative experiments, mean ± standard error of the mean (SEM) graphs or box plots showing each data point, median, interquartile range (IQR), and maximum and minimum values.

[0291] result

[0292] Increased levels of β-arrestin 2 in the brains of FTLD-tau patients and tau P301S transgenic mice

[0293] The expression of β-arrestin2 in the frontal cortex of age-matched control subjects (N=12) and patients with FTLD and tauopathy (FTLD-tau, N=10) was compared. Western blotting of RIPA-soluble β-arrestin2 showed an approximately 1.8-fold increase in β-arrestin2 protein in the frontal cortex of FTLD-tau patients compared with healthy controls ( Figure 1A-1B ). Similarly, qRT-PCR showed a similar increase in β-arrestin 2 mRNA in FTLD-tau brain samples compared with non-FTLD-tau controls ( Figure 1C ). It was also confirmed that RIPA-insoluble β-arrestin 2 protein levels were positively correlated with tau levels in FTLD-tau brains ( Figure 1D and 8 ). Next, the expression of β-arrestin 2 in tau P301S transgenic mice was evaluated. These mice overexpress the disease-associated P301S tau in neurons and exhibit pathophysiology and behavior similar to FTLD without Aβ accumulation (Yoshiyama Y, et al. (2007) Neuron 53(3):337-351). β-arrestin 2 protein and mRNA were significantly elevated in the brains of tau P301S transgenic mice compared to non-transgenic littermates ( Figure 1E-1G To confirm these results in a different way, cultured DIV 21 primary hippocampal neurons from tau P301S and wild-type littermates were stained for β-arrestin 2 ( Figure 1H Quantification of β-arrestin 2 confirmed what was observed in homogenous brain tissue in the target cell types ( Fig. 1I ).

[0294] β-Arrestin 2 increases tau stability

[0295] We next determined whether the increase in β-arrestin2 observed in these tauopathy models could regulate tau in a negative (compensatory) or positive (disease enhancing) manner. In Hela cells stably expressing tau (V5-tagged 4R0N tau, referred to as Hela-V5-tau cells), transfected β-arrestin2 significantly increased total tau and phosphorylated tau ( Figures 2A-2D ), and β-arrestin 2 siRNA transfection significantly reduced total tau and phospho-tau ( Figure 2E , 2F). Similarly, immunocytochemical imaging ( Figure 2G-2I ) and immunoblotting ( Figure 8Infection of tau P301S cortical primary neurons with adenovirus packaged with β-arrestin 2 also increased total tau and phospho-tau intensity compared to control adenovirus (control-Ad) conditions, as assessed by imaging (A, 8B). Figure 2J , 2K ) and immunoblotting ( Figure 8 Lentiviral-mediated shRNA knockdown of β-arrestin2 reduced total tau as assessed by β-arrestin2 overexpression or knockdown ( Figure 8 E, 8F) observed no significant differences in tau mRNA levels. The above studies were performed in the absence of any GPCR agonists, focusing on non-receptor mechanisms by which β-arrestin 2 interacts with tau or other partners to induce phenotypes. Tau turnover was then examined as a potential mechanism by which increased β-arrestin 2 increases the effective level of tau at steady state. Treatment of Hela-V5-tau cells with cycloheximide to block protein translation showed that transfected β-arrestin 2 significantly delayed tau turnover ( Figure 2L , 2M), which is consistent with β-arrestin 2 stabilizing tau:protein interactions via a scaffolding protein.

[0296] Genetic reduction of β-arrestin2 alleviates tauopathy and synaptic dysfunction in tau P301S mice

[0297] The above data suggest that increased tau increases β-arrestin2, which in turn further enhances tau-mediated events by stabilizing the protein, thus indicating a vicious positive pathogenic feedback loop. This suggests a therapeutic attack point, whether mice will exhibit the expected pathological phenotype when β-arrestin2 is genetically reduced. Therefore, to assess the physiological relevance of endogenous β-arrestin2 in tau regulation in vivo, tau P301S transgenic mice were crossed with β-arrestin2- / - (Arrb2 - / - ) mice. First, 7-month-old tau P301S, tau P301S / Arrb2 + / - and tau P301S / Arrb2 - / - Brains of littermates were fractionated for lauroylsarcosine soluble and insoluble tau. Although lauroylsarcosine soluble tau did not change significantly, tau P301S / Arrb2 + / - and tau P301S / Arrb2 - / - Compared with control tau P301S transgenic mice, the amount of lauroylsarcosine-insoluble tau was significantly reduced in mice ( Figures 3A-3C ). 7-month-old WT, tau P301S, tau P301S / Arrb2+ / - and tau P301S / Arrb2 - / - Cortex of littermates ( Figure 3D , 3E ) and hippocampus ( Figure 3D , 3F ) for immunohistochemistry of phospho-tau (pS199 / pS202). + / - and tau P301S / Arrb2 - / - Brain sections from tau P301S mice showed approximately 50% less phospho-tau immunoreactivity compared to those from tau P301S mice ( Figure 3D-3F ). It has been confirmed that it originates from tau P301S / Arrb2 - / - Knockout of β-arrestin2 reduces lauroylsarcosine-insoluble tau in cultured primary neurons from mouse brain compared to those from tau P301S mice ( Figures 10A-10C ).

[0298] To determine functional changes in synaptic plasticity, electrophysiological recordings were performed on the CA3-CA1 Schaffer collateral pathway in acute brain slices. Input-output (IO) analysis showed that WT, tau P301S, tau P301S / Arrb2 + / - and tau P301S / Arrb2 - / - There were no significant differences between sections from the same litter ( Figure 3G ), as expected from previous electrophysiological studies of tau P301S mice (Yoshiyama Y, et al. (2007) Neuron 53(3):337-351; Woo JA, et al. (2019) Commun Biol 2:112; Woo JA, et al. (2017) Hum Mol Genet 26(20):3973-3988). In paired pulse facilitation (PPF) experiments, a slight but significant decrease in the slope of fEPSPs was detected in tau P301S slices compared with WT slices at all stimulus intervals (except 260-280 ms) ( Figure 3H ), of which tau P301S / Arrb2 + / - and tau P301S / Arrb2 - / - The slices were not significantly different from those of tau P301S ( Figure 3H ). In long-term potentiation (LTP) recordings using theta pulse stimulation, tau P301S slices were significantly impaired in the induction and maintenance of LTP compared to WT slices. + / -and tau P301S / Arrb2 - / - LTP in the slices was significantly restored to levels very similar to those in WT slices (Figure 3I). These results suggest that loops can be broken at the β-arrestin2 interface. As further evidence for the beneficial effects of reducing β-arrestin2, and consistent with the LTP data, silencing of β-arrestin2 by shRNA lentivirus significantly rescued synaptophysin (presynaptic; Fig. 10D , 10E ) and brain development regulatory protein (postsynaptic; Fig. 10D , 10F ) consumption.

[0299] β-Arrestin 2 oligomerization is required for tau stability

[0300] It has been shown that β-arrestin 2 exists as an oligomer in a variety of cell types (Milano SK, et al. (2006) J Biol Chem 281(14):9812-9823; Storez H, et al. (2005) J Biol Chem 280(48):40210-40215). Inositol hexaphosphate (IP6) enhances this self-association of β-arrestin 2 by bridging adjacent molecules into a head-to-tail configuration. Positively charged arginine and lysine residues within the N- and C-termini of β-arrestin 2 were found to be critical for both IP6 binding and oligomerization. Given the physical overlap of IP6 and GPCR binding sites, the binding of β-arrestin2 to activated GPCRs and IP6 is mutually exclusive, suggesting that in the oligomeric form, β-arrestin2 may serve other purposes besides GPCR binding (Dinculescu A, et al. (2002) J Biol Chem 277(14):11703-11708; Breitman M, et al. (2012) J Biol Chem 287(23):19653-19664; Gurevich VV & Benovic JL (1993) J Biol Chem 268(16):11628-11638; Gurevich VV, et al. (1995) J Biol Chem 270(2):720-731; Hanson SM, et al. (2007) Proc Natl Acad Sci USA 104(9):3125-3128; Kim M, et al. (2012) Proc Natl Acad Sci USA 109 (45): 18407-18412; Palczewski K, et al. (1994) Protein Sci 3 (2): 314-324; Pulvermuller A, et al. (2000) J Biol Chem 275(48):37679-37685; Vishnivetskiy SA, et al. (2011) J Biol Chem 286 (27): 24288-24299; Zhuang T, et al. (2013) Proc Natl Acad Sci USA 110 (3): 942-947; BoularanC, et al. al.(2007)Proc Natl Acad Sci USA 104(46):18061-1806).

[0301] The hypothesis that β-arrestin2 self-oligomerization affects tau stability and tauopathy was tested using β-arrestin2 N-terminal domain mutants (K158A, K161A and R162A, termed β-arrestin2ΔIP6N) and C-terminal domain mutants (K232A, R234A, K252A, K326A and K328A, termed β-arrestin2ΔIP6C); these mutants were unable to form oligomers as determined by BRET and co-immunoprecipitation assays (Milano SK, et al. (2006) J Biol Chem 281(14):9812-9823; Storez H, et al. (2005) J Biol Chem 280(48):40210-40215), but still bound activated GPCP with WT affinity (Gaidarov I, et al. (1999) EMBO J 18(4):871-881).

[0302] When DIV 18 tau P301S cortical primary neurons were transduced with β-arrestin2ΔIP6C or ΔIP6N lentivirus, a 50% reduction in tau was evident compared to controls ( Figure 4A , 4B ), which represents a key finding relevant to the mechanism of action and potential therapeutic strategies. Consistent with these findings, YFP-β-arrestin2ΔIP6C and YFP-β-arrestin2ΔIP6N expression in Hela-V5-tau cells also resulted in a significant reduction in tau as assessed by immunocytochemistry ( Fig.11A ). To show that these mutants act as dominant negatives for oligomerization, proximity ligation assays (PLA) were performed to examine the interaction between β-arrestin2-flag and β-arrestin2-myc in the presence of control vector or β-arrestin2ΔIP6C or β-arrestin2ΔIP6N. Indeed, β-arrestin2ΔIP6C and β-arrestin2ΔIP6N significantly reduced the interaction between β-arrestin2-flag and β-arrestin2-myc ( Fig. 11B , 11C). These results confirm the oligomerization of WT β-arrestin2 in a control setting and show that WT β-arrestin2 is displaced from oligomers by expression of ΔIP6C or ΔIP6N mutants ( Fig. 11B , 11C). We also investigated whether β-arrestin2 oligomerization is required to slow tau turnover by disrupting oligomerization using these mutants in several models. Studies with the β-arrestin2ΔIP6C and β-arrestin2ΔIP6N mutants showed enhanced tau turnover in cycloheximide chase experiments ( Figure 4C-4ETo determine whether the β-arrestin2 oligomerization state alters tau aggregation itself, Hela-V5-tau cells transfected with β-arrestin2 variants were subjected to a filter capture assay in which tau aggregates from detergent-soluble and -insoluble lysates were captured on cellulose acetate membranes and assessed by immunoblotting. While detergent-soluble lysates showed little or no aggregates, detergent-insoluble lysates derived from β-arrestin2ΔIP6C or ΔIP6N transfected cells showed significantly reduced tau aggregates compared to control vector-transfected cells ( Fig.11D , 11E ).

[0303] β-Arrestin 2 oligomers interact with p62 and inhibit p62 self-association

[0304] Hyperphosphorylated (and therefore more "ordered") tau is thought to be cleared via the autophagy-lysosomal pathway (Dolan PJ & Johnson GV (2010) J Biol Chem 285(29):21978-21987; Wong ES, et al. (2008) Hum Mol Genet 17(16):2570-2582; Kim S, et al. (2016) Sci Rep 6:24933; Kruger U, et al. (2012) Neurobiol Aging 33(10):2291-2305; Wang Y, et al. (2010) Neurodegener Dis 7(1-3):103-107). To understand the mechanistic basis of β-arrestin2 in tau stabilization and accumulation, we tested whether β-arrestin2 affects autophagy using bafilomycin A, a lysosomal inhibitor known to activate autophagy and promote the accumulation of LC3-positive autophagosomes. Interestingly, overexpression of β-arrestin2 in Hela-V5-tau cells significantly suppressed the increase of LC3-positive puncta induced by bafilomycin A (BafA). Figure 5A , 5B ), indicating that β-arrestin2 inhibits autophagy at or upstream of LC3.

[0305] P62 / SQSTM1 is a key autophagic cargo receptor that regulates autophagosome formation by tethering its cargo (i.e., misfolded tau or Aβ) to LC3-positive autophagosomes. In fact, p62 is associated with neurofibrillary tangles (Caccamo A, et al. (2017) Mol Psychiatry 22(6):865-873; King A, et al. (2013) Acta Neuropathol 125(2):303-310; Kuusisto E, et al. (2002) Neuropathol Appl Neurobiol 28(3):228-237; Terni B, et al. (2007) Acta Neuropathol 113(4):403-416), and soluble cytoplasmic p62 levels are significantly reduced in AD brains (Caccamo A, et al. (2017) Mol Psychiatry 22(6):865-873; Zheng X, et al. (2012) Neural Regen Res 7(17):1304-1311). Increased p62 expression improves cognitive impairment in AD animal models by enhancing autophagy induction, and genetic deletion of p62 leads to significant accumulation of tau and neurodegeneration (Caccamo A, et al. (2017) Mol Psychiatry 22(6):865-873; Zheng X, et al. (2012) Neural Regen Res 7(17):1304-1311; Ramesh Babu J, et al. (2008) J Neurochem 106(1):107-120). In addition, a recent study showed that p62 expression is associated with the clearance of insoluble tau (Xu Y, et al. (2019) Autophagy 15(4):583-598).

[0306] P62 forms particles through self-interaction via its N-terminal PB1 domain, which is essential for its activity and can be observed as spots of different sizes in cells (Wurzer B, et al. (2015) Elife 4:e08941; Itakura E & Mizushima N (2011) J Cell Biol 192(1):17-27). In Hela V5-tau cells transfected with GFP-p62, it is expected that GFP-p62 spots will increase after Bafilomycin A (BafA) treatment ( Figure 5C , 5D), however, overexpression of β-arrestin2 significantly reduced GFP-p62 puncta in both steady-state and after bafilomycin, such that bafilomycin A no longer increased GFP-p62 puncta ( Figure 5C , 5D ). This suggests that β-arrestin 2 inhibits the flow of p62 particles from their target substrates to lysosomes. To directly test this hypothesis, an mCherry-EGFP-p62 autophagy flow reporter was used. This reporter exploits the sensitivity of GFP to low pH (it quenches the signal) and the insensitivity of mCherry to low pH. Therefore, co-localized red and green spots indicate non-lysosomal p62 in a stable state. However, after fusion with lysosomes (autolysosomes), red spots persist, while green spots dim and disappear (Larsen KB, et al. (2010) Autophagy 6(6):784-793; Pankiv S, et al. (2007) J Biol Chem 282(33):24131-24145). In primary neurons transduced with control or β-arrestin variant lentiviruses and high-titer rAAV9 mCherry-EGFP-p62, β-arrestin2 expression significantly reduced the ratio of mCherry-only (mCherry-only) to total puncta ( Figure 5E , 5F Importantly, the β-arrestin2ΔIP6C and ΔIP6N mutants, which act as dominant negatives of WT β-arrestin2 oligomerization, significantly increased this measure ( Figure 5E , 5F ). Thus, these β-arrestin2 mutants that are unable to self-oligomerize (and inhibit WT-β-arrestin2 oligomerization) failed to reduce flux, supporting the notion that oligomerized, non-GPCR-bound β-arrestin2 is acting to promote tauopathy. Similar results were observed for p62 autophagic flux with β-arrestin2 and β-arrestin2ΔIP6N in Hela-V5-Tau cells ( Fig. 12A , 12B ).

[0307] Given the effect on p62 autophagic flux, we tested whether β-arrestin2 physically interacts with p62. Co-immunoprecipitation (co-IP) experiments indeed showed that β-arrestin2 forms a complex with p62 ( Fig. 12C). The self-association of p62 through its N-terminal PB1 domain is essential for its cargo receptor activity by allowing its ubiquitinated cargo to have more interaction sites (multiple binding) and to bind to multiple LC3 proteins simultaneously (Wurzer B, et al. (2015) Elife 4:e08941; Itakura E & Mizushima N (2011) J Cell Biol 192(1):17-27). Therefore, whether β-arrestin 2 affects p62 self-interaction was tested by co-IP experiments using HA-p62 and GFP-p62 constructs. β-arrestin 2 reduced p62 self-association as detected by co-IP of GFP-p62 and HA-p62 ( Figure 5G , 5H ). Given that the β-arrestin2 oligomerization mutants ΔIP6C and ΔIP6N reduce tau aggregates by slowing tau turnover, we next tested whether the β-arrestin2ΔIP6C and ΔIP6N mutants affect p62 self-association by proximity ligation assay (PLA) as well as co-IP. Although β-arrestin2 significantly reduced the HA-p62 / GFP-p62 composite PLA signal (p62 self-interaction), neither β-arrestin2ΔIP6C nor β-arrestin2ΔIP6N reduced HA-p62 / GFP-p62 PLA spots ( Fig.5I , 5J ). It was also confirmed that although β-arrestin2 significantly reduced the HA-p62 and GFP-p62 interaction, neither β-arrestin2ΔIP6C nor β-arrestin2ΔIP6N alone could reduce the HA-p62 and GFP-p62 interaction by co-IP ( Fig.12D In fact, both β-arrestin2ΔIP6C and β-arrestin2ΔIP6N interact much less with p62 than β-arrestin2 ( Fig.12E , 12F ). Recent studies have shown that purified recombinant p62 spontaneously forms globular oligomers (Zaffagnini G, et al. (2018) EMBO J 37(5)). To test whether β-arrestin2 can directly affect p62 oligomerization in vitro, purified recombinant p62, β-arrestin2, and p62 mixed with β-arrestin2 were observed by atomic force microscopy. Recombinant p62 spontaneously formed visible spherical particles with diameters between 5 and 100 nm ( Figure 5K ), whereas only very few small β-arrestin 2 particles were visible under the same conditions ( Figure 5K). When p62 was mixed with β-arrestin2, the size of the particles was significantly reduced (Figure 5k, l), indicating that inhibition of p62 self-association by β-arrestin2 reduced the size of p62 particles. We next tested whether bafilomycin A-induced p62 puncta formation requires β-arrestin2 oligomerization. As expected, in cells transfected with the control vector, GFP-p62 puncta increased after Bafilomycin A (BafA) treatment ( Figure 5M , 5N ). However, β-arrestin2 significantly reduced GFP-p62 puncta both at steady state and after bafilomycin A ( Figure 5M , 5N Interestingly, both β-arrestin2ΔIP6C- and β-arrestin2ΔIP6N-transfected cells showed increased GFP-p62 puncta at steady state, suggesting that β-arrestin2ΔIP6C and β-arrestin2ΔIP6N increased the flux of p62 particles and thus increased autophagic flux ( Figure 5M , 5N ).

[0308] Mutant β-arrestin 2 viral therapy suppresses tauopathy in vivo

[0309] Given the above results, indicating that the oligomeric form of β-arrestin2 is required in tauopathies, and the dominant negative effect of mutant β-arrestin2 that is unable to oligomerize, we tested whether the β-arrestin2ΔIP6C and β-arrestin2ΔIP6N mutants reduce tauopathy in an in vivo setting as a potential therapeutic strategy. High titers (>1×10 12 vg -1 )rAAV9. AAV was then stereotaxically injected bilaterally into the hippocampus of 5-month-old tau P301S mice. Two months after injection, the mouse brains were probed for GFP and tau. These studies showed that injection of GFP-β-arrestin2ΔIP6N or GFP-β-arrestin2ΔIP6C significantly reduced lauroylsarcosine-insoluble tau ( Fig. 6A , 6C However, lauroylsarcosine-soluble tau was not significantly altered by the β-arrestin 2 mutants ( Fig. 6A , 6B Furthermore, neurons expressing GFP-β-arrestin2ΔIP6N and GFP-β-arrestin2ΔIP6C showed a significant reduction in HT7-immunoreactive tau compared to GFP-expressing controls or adjacent uninfected neurons ( Fig.6D ,6E ). Given the dominant negative effects of these mutants ( Figure 4A , 4B , 11B, 11C), these results are consistent with its therapeutic mechanism, namely, effective reduction of increased endogenous neuronal β-arrestin 2 oligomerization in tau P301S mice. Furthermore, since injections were performed at 5 months of age, when tau accumulation was well underway, it is clear that this approach can reduce pre-existing tau tangle pathology.

[0310] discuss

[0311] FTLD represents a distinct clinical and pathological dementia, but is often misdiagnosed as AD, or treated in a similar manner to AD. The most obvious difference between the pathology of AD and FTLD is the absence of Aβ accumulation in FTLD. In its most common form, FTLD-tau has accumulation of tau as a sharp feature. Given that tau levels (in AD) appear to be a better predictor of cognitive deficits, tau accumulation in FTLD is thought to be a key factor in neurodegeneration in this disease as well. Several GPCR agonists and antagonists have been proposed as potential therapies for AD (Ni Y, et al. (2006) Nat Med 12(12):1390-1396; Melancon BJ, et al. (2013) Drug Discov Today 18(23-24):1185-1199; Angulo E, et al. (2003) Brain Pathol 13(4):440-451; Mogi M & Horiuchi M (2009) Hypertens Res 32(9):738-740), and given the involvement of tau in AD and FTLD, approaches to modulate GPCR signaling through two β-arrestin proteins associated with most GPCRs have been considered. Increased β-arrestin 2 in human FTLD brain and tau-overexpressing cells suggests that this may be a useful approach to understand pathogenesis and locate therapeutic blockade points. Further studies revealed some unexpected findings. First, it is clear that β-arrestin2 can upregulate tau and tau can upregulate β-arrestin2. This suggests that β-arrestin2 upregulation in human FTLD brain is maladaptive rather than a compensatory or beneficial event that might eliminate tau accumulation. Therefore, this effect is a positive feedback loop that, once initiated, promotes further deleterious effects. Furthermore, it is clear that oligomerized β-arrestin2 (not interacting with GPCRs) is responsible for tau accumulation, which led us to examine non-receptor functions of β-arrestin2 in the context of FTLD. Notably, β-arrestin2 has previously been shown to positively regulate Aβ production by regulating γ-secretase processing of APP through interaction with Aph-1 (Thathiah A, et al. (2013) Nat Med 19(1):43-49). The same study showed that β-arrestin2 was significantly increased in AD brain and APP transgenic mice (Thathiah A, et al. (2013) Nat Med 19(1):43-49). The current work shows that beta-arrestin2 is elevated in cells and mice with elevated tau. Furthermore, tau levels are elevated when beta-arrestin2 is overexpressed. Based on unchanged tau mRNA levels, tau clearance is proposed as a mechanism by which beta-arrestin2 regulates its levels.Data suggest that β-arrestin2 reduces tau clearance by impairing p62-mediated autophagy, an effect exerted by oligomeric forms of β-arrestin2. Specifically, the results suggest that β-arrestin2 oligomers increase tau levels by blocking p62 self-interactions, an essential initial step in p62-mediated autophagic flux. Genetic reduction or elimination of β-arrestin2 significantly reduced lauroylsarcosine-insoluble tau and alleviated tauopathy in vivo. Furthermore, β-arrestin2 mutants that are unable to form oligomers actually reduced insoluble tau. These effects were demonstrated to be due to the dominant-negative “anti-oligomer” properties of these mutants, which reduced tauopathy in cultured model cells and neurons, as well as in the FTLD-tau mouse model in vivo. These data highlight a novel mechanism by which β-arrestin2 regulates tau and provide a proof-of-concept strategy to alleviate tauopathy by targeting β-arrestin2 oligomerization. Figure 7 ).

[0312] Boularan et al. found that β-arrestin 2 □IP6N and □IP6C mutants do not form oligomers, but instead are normal in localization and binding to (and mediating internalization of) GPCRs and other partners such as AP2, Filamin A, and MAPK. To date, no previous studies have shown that oligomeric β-arrestin 2 is involved in p62-mediated autophagy. These findings suggest that β-arrestin 2 reduces p62 self-interactions, the number of p62 particles, and alters tau clearance. Furthermore, expression of β-arrestin 2 ΔIP6N and ΔIP6C mutants increases p62 self-interactions and the number of p62 particles by impairing oligomerization of WT (endogenous) β-arrestin 2. These changes are directly associated with tau turnover and accumulation of insoluble tau, consistent with the role of p62 in preferentially reducing insoluble tau (Xu Y, et al. (2019) Autophagy 15(4): 583-598). In addition, insoluble p62 itself is associated with neurofibrillary tangles (Caccamo A, et al. (2017) Mol Psychiatry 22(6):865-873; King A, et al. (2013) Acta Neuropathol 125(2):303-310; Kuusisto E, et al. (2002) Neuropathol Appl Neurobiol 28(3):228-237; Terni B, et al. (2007) Acta Neuropathol 113(4):403-416), while soluble cytoplasmic p62 levels are significantly reduced in AD brains (Caccamo A, et al. (2017) Mol Psychiatry 22(6):865-873; Zheng X, et al. (2012) Neural Regen Res 7(17):1304-1311). Increased p62 expression improves cognitive impairment in AD animal models by enhancing autophagy induction, and genetic deletion of SQSTM1 / p62 leads to tau accumulation and neurodegenerative brain (Caccamo A, et al. (2017) Mol Psychiatry 22(6):865-873; Zheng X, et al. (2012) Neural Regen Res 7(17):1304-1311; Ramesh Babu J, et al. (2008) J Neurochem 106(1):107-120).p62 self-interaction represents the first step in p62 particle formation, which is essential for p62-mediated autophagy. Regulation of p62 by β-arrestin2 at this step provides an opportunity for therapeutic intervention. The observation that both IP6-binding mutants enhance p62 self-interaction and reduce insoluble tau suggests that, while β-arrestin2 oligomers block p62-mediated autophagy, β-arrestin2 monomers promote p62-mediated autophagy. If so, the transition from β-arrestin2 oligomers to monomers, and vice versa, could serve as a tunable molecular switch to switch p62-mediated autophagy.

[0313] In the current experiment, the mutant β-arrestin 2 protein was very effective in reducing tau in neurons. In single brain cells where the expression of ΔIP6N or ΔIP6C was confirmed (by their GFP tags), tau levels were essentially undetectable, in contrast to the remaining cells with high levels of tau. For gene therapy of human FTLD-tau, mutants with this inhibitory ability reduced to some extent may be needed so that there are certain levels of oligomers to perform other functions. In fact, oligomeric β-arrestin 2 has been shown to promote nuclear-cytoplasmic shuttling of proteins (Boularan C, et al. (2007) Proc Natl Acad Sci USA 104 (46): 18061-18066). Similarly, small molecule inhibitors of β-arrestin 2 oligomerization, used to treat or prevent FTLD-tau, can be designed to avoid the complete loss of oligomers in cells. Importantly, these strategies are not expected to change neuronal GPCR signaling pathways because the monomeric form of β-arrestin 2 will be retained. Based on these findings, it is anticipated that inhibiting the oligomerization of β-arrestin2 will not only inhibit the development of new tau tangles, but also clear existing tau accumulation due to this mechanism of enhanced tau clearance. Therefore, this therapeutic strategy could be used to prevent those at risk for or with mild cognitive impairment by reducing existing tau tangles, as well as treat those with overt FTLD-tau. In addition to tauopathies, it is conceivable that this strategy could also prove beneficial in other neurodegenerative diseases with proteinopathies that are cleared by p62.

[0314] Example 2: β-Arrestin 1 drives tauopathy by destabilizing microtubules and impairing p62 / SQSTM1-mediated tau clearance.

[0315] Introduction

[0316] The defining pathological features of Alzheimer's disease (AD) are the accumulation of amyloid β (Aβ) and hyperphosphorylated tau in the brain. Various G protein-coupled receptors (GPCRs) have been shown to play an integral role in the pathogenesis of AD (Ni, Y. et al. Nat Med (2006) 12: 1390-1396; AbdAlla, S. et al. J Biol Chem (2009) 284: 6566-6574; AbdAlla, S. et al. J Biol Chem (2009) 284: 6554-6565; Thathiah, A. et al. Science (2009) 323: 946-951; Bakshi, P., et al. ACS Chem Biol (2008) 3: 777-789; Alley, G. M. et al. J Neurosci Res (2010) 88: 143-154; Minkeviciene, R., et al. J Pharmacol Exp Ther (2004) 311: 677-682; Dobarro, M., et al. Int J Neuropsychopharmacol (2013) 16: 2245-2257; Wisely, EV, et al. Hum Mol Genet (2014) 23: 4024-4034; Luong, K. & Nguyen, LTAm J Alzheimers Dis Other Demen (2013) 28: 427-439; Lee, HG et al. Acta Neuropathol (2004) 107: 365-371; Lee, HG et al. Brain Res (2009) 1249: 244-250; Sun, L. et al. FEBSLett (2005) 579: 251-258). However, it is unclear how different GPCRs similarly affect the pathogenesis of Aβ and tau. GPCRs share a common mechanism of action through inhibitory protein scaffold signaling complexes that desensitize GPCRs through internalization and degradation via the endolysosomal pathway (Lohse, MJ, et al. Science (1990) 248: 1547-1550).In addition, arrestins (arrestin 1, arrestin 2, arrestin 3 and arrestin 4) (Wilden, U., et al. Proc Natl Acad Sci USA (1986) 83: 1174-1178; Moore, CA, et al. Annu Rev Physiol (2007) 69: 451-482; Gurevich, VV & Gurevich, EV Pharmacol Ther (2006) 110: 465-502) act as multifunctional adaptor proteins that regulate a variety of different signaling pathways (Lefkowitz, RJ, et al. Mol Cell (2006) 24: 643-652; Lefkowitz, R. J Prog Mol Biol Transl Sci (2013) 118: 3-18). While arrestin 1 and arrestin 4 bind to only a few receptors (rhodopsin and color opsins) and are expressed in specific cell types (Shinohara, T. et al. Proc Natl Acad Sci USA (1987) 84:6975-6979; Yamaki, K., et al. Biochem Biophys Res Commun (1987) 142:904-910), arrestin 2 (β-arrestin 1) and arrestin 3 (β-arrestin 2) show the highest expression in the brain and spleen, but are also ubiquitously expressed (Lohse, MJ, et al. Science (1990) 248:1547-1550; Attramadal, H. et al. J Biol Chem (1992) 267:17882-17890).

[0317] Microtubule-associated protein tau (MAPT) plays an important role in many neurodegenerative diseases (Goedert, M., et al. Proc Natl Acad Sci USA (1988) 85: 4051-4055; von Bergen, M. et al. J Biol Chem (2001) 276: 48165-48174; Lashley, T., et al. Neuropathol Appl Neurobiol (2015) 41: 858-881), and pathogenic substances of tau form neurotoxic aggregates, which are associated with cognitive deficits and neurodegeneration in humans and animal models of tauopathy (Wang, Y. & Mandelkow, E. Nat Rev Neurosci (2016) 17: 5-21; Ward, SM, et al. Biochem Soc Trans (2012) 40: 667-671; Patterson, K. et al. J Biol Chem (2013) 41 ... Biol Chem (2011) 286:23063-23076). Therefore, reducing pathogenic tau represents an attractive therapeutic strategy.

[0318] Interestingly, β-arrestin 1 (Liu, X. et al. Cell Res (2013) 23: 351-3650) and β-arrestin 2 (Thathiah, A. et al. Nat Med (2013) 19: 43-49) are significantly increased in AD brains and play an important role in promoting Aβ production by interacting with the β-secretase subunit Aph-1 (Liu, X. et al. Cell Res (2013) 23: 351-365; Thathiah, A. et al. Nat Med (2013) 19: 43-49), thus linking β-arrestin 1 and β-arrestin 2 to Aβ pathogenesis. A recent study showed that β-arrestin 2 is also increased in patients with frontotemporal lobar degeneration (FTLD-tau), and genetic reduction of β-arrestin 2 significantly alleviated tau pathology in vivo.

[0319] However, it is unknown whether and how β-arrestin1 contributes to the pathological progression of tauopathies. Furthermore, it is unclear whether the effects of GPCR stimulation on tauopathies require β-arrestin1 and / or β-arrestin2. In this study, β-arrestin1 was found to be significantly increased in patients with frontotemporal lobar degeneration-tau (FTLD-tau), a degenerative disease defined as tauopathy in the absence of Aβ deposits, and elevated β-arrestin1 promoted tau accumulation and tauopathy in vitro and in vivo through two different mechanisms. Furthermore, both β-arrestin1 and β-arrestin2 have been shown to mediate the effects of GPCR stimulation on tauopathies. Thus, reducing either β-arrestin1 or β-arrestin2 is sufficient to block the effects of GPCR stimulation on tauopathies. β-arrestin2 increases pathogenic tau by impairing autophagic flux. Here, the molecular mechanistic basis of β-arrestin1 in tauopathies was further defined by demonstrating that β-arrestin1 not only induces tau dissociation from microtubules but also inhibits tau-induced microtubule assembly. Furthermore, we found that β-arrestin1 and β-arrestin2 share a common mechanism to promote the aggregation of pathogenic tau by blocking the autophagic cargo receptor p62. Indeed, genetic reduction of β-arrestin1 significantly restored synaptic dysfunction and significantly alleviated tauopathy in tauP301S transgenic mice in vivo.

[0320] result

[0321] β-Arrestin 1 and β-Arrestin 2 are required for mGluR2- and β2AR-mediated increases in pathogenic tau

[0322] To date, two GPCRs have been implicated in tau phosphorylation and accumulation: the β2-adrenergic receptor (β2AR) (Dobarro, M., et al. Int J Neuropsychopharmacol (2013) 16:2245-2257; Wisely, EV, et al. Hum Mol Genet (2014) 23:4024-4034; Luong, K. & Nguyen, LTAm J Alzheimers Dis Other Demen (2013) 28:427-439) and the metabotropic glutamate receptor 2 (mGluR2) (Lee, HG et al. Acta Neuropathol (2004) 107:365-371; Lee, HG et al. Brain Res (2009) 1249:244-250). Compared with controls, β2AR in the frontal cortex and hippocampus of AD brains increased significantly (Kalaria, RN et al. J Neurochem (1989) 53: 1772-1781; Kalaria, RN & Harik, SI Neurosci Lett (1989) 106: 233-238). Genetic studies have shown that polymorphisms in β2AR are associated with a higher risk of developing sporadic AD (Rosenberg, PB et al. Am J Geriatr Psychiatry (2008) 16: 883-892; Yu, JT et al. Brain Res (2008) 1210: 216-222), and genetic reduction of β2AR significantly alleviates tauopathy in vivo (Wisely, EV, et al. Hum Mol Genet (2014) 23: 4024-4034). The classic β2AR agonist isoproterenol significantly increases tau phosphorylation, thereby inducing memory deficits in rats (Sun, L. et al. FEBS Lett (2005) 579: 251-258). mGluR2 is also significantly increased in AD, and mGluR2 expression is closely related to hyperphosphorylated tau deposition (Lee, HG et al. Acta Neuropathol (2004) 107: 365-371; Lee, HG et al. Brain Res (2009) 1249: 244-250). It is reported that the mGluR2 agonist LY-379,628 increases tau phosphorylation through ERK activation (Lee, HG et al. Brain Res (2009) 1249: 244-250).Based on these previous observations, the goal was to confirm these findings in tauP301S transgenic primary neurons and Hela cells stably expressing tau (V5-tagged 4R0N tau, Hela-V5-tau cells). As expected, 10 μM isoproterenol (Iso) treatment significantly increased phospho-tau in tauP301S cortical primary neurons. Fig.21A , 21B 10 μM LY-379,628 treatment also increased phospho-tau in tauP301S cortical neurons ( FIGS. 21C , 21D ).

[0323] At present, it is not clear how different GPCRs, such as β2AR and mGluR2, and their agonists change tau phosphorylation. A common point of downstream signaling between these GPCRs is β-arrestin 1 and β-arrestin 2, which were originally identified and characterized according to their functions of internalizing and desensitizing GPCRs (including β2AR) (Lohse, MJet al.JBiol Chem (1992) 267: 8558-8564) and mGluR2 (lacovelli, Let al.Mol Pharmacol (2009) 75: 991-1003). In order to test whether β2AR-mediated tau regulation requires β-arrestin 1 and / or β-arrestin 2, Hela-V5-tau cells were transfected with control siRNA or β-arrestin 1 siRNA and treated with vehicle or 10 μM Iso. While Iso was expected to increase tau phosphorylation in control siRNA-transfected cells, cells transfected with β-arrestin1 siRNA failed to respond to Iso, indicating that β-arrestin1 is required for the Iso-induced increase in phospho-tau ( Fig.13A , 13B Interestingly, β-arrestin 1 siRNA was also found to significantly reduce total tau levels ( Fig.13A , 13B ). Next, we assessed whether β-arrestin1 is required for mGluR2-mediated tau phosphorylation. LY-379,268 treatment increased phospho-tau; however, siRNA-mediated knockdown of β-arrestin1 again resulted in a failure of the response to LY-379,268, such that total and phospho-tau levels were similarly reduced with or without LY-379,268 treatment ( Fig. 13C , 13D). Next, Hela-V5-tau cells were transfected with control siRNA or β-arrestin 2 siRNA and treated with vehicle, 10 μM Iso, or LY-379,268. As expected, both Iso and LY-379,268 treatment increased tau phosphorylation; however, siRNA-mediated knockdown of β-arrestin 1 failed to increase tau phosphorylation ( Fig.13E , 13F These data suggest that both β-arrestin1 and β-arrestin2 are required for mGluR2- and β2AR-mediated increases in pathogenic tau.

[0324] Elevated β-arrestin 1 and colocalization with pathogenic tau (AT8) in FTLD-tau

[0325] The finding that β-arrestin1 and β-arrestin2 mediate the increase in pathogenic tau in response to GPCR stimulation prompted us to evaluate β-arrestin1 levels in FTLD-tau patients. β-arrestin2 levels were significantly increased in the frontal cortex of FTLD-tau patients compared with control subjects. However, β-arrestin1 levels had not been examined. Therefore, β-arrestin1 levels were assessed in the frontal cortex of FTLD-tau patients (n=10) compared with control subjects (n=12). Interestingly, FTLD-tau brains (n=10) had increased levels of β-arrestin2 in RIPA-soluble extracts ( Fig.14A , 14B ) and RIPA-insoluble extracts ( Fig. 14C , 14D ) showed >50% increase in β-arrestin 1. Interestingly, the levels of insoluble β-arrestin 1 closely reflected the levels of insoluble tau in FTLD-tau, such that by linear regression analysis, insoluble β-arrestin 1 and tau levels were correlated with R 2 =0.4874 is significantly correlated ( Fig.14E , 14F), suggesting a functional link between β-arrestin 1 and tau in the brain. To assess the spatial relationship between β-arrestin 1 and tau, the frontal gyrus of FTLD-tau was next stained for phospho-tau (AT8 antibody: pS202 / pT205-tau) and β-arrestin 1. Surprisingly, confocal images of AT8+tau aggregates and β-arrestin 1 showed almost perfect colocalization ( Figure 14G ), as confirmed by Z-stack images at 1 μm increments ( Figure 14G ). Although β-arrestin 1 staining was prominent in the same sections ( Fig.22A ), while secondary antibody staining alone did not show any immunoreactivity ( Fig. 22B) but confirmed the absence of AT8+tau pathology in control brains.

[0326] β-Arrestin 1 promotes accumulation of pathogenic tau in primary neurons

[0327] Given that β-arrestin1 is significantly increased in FTLD-tau patients, we next assessed whether endogenous β-arrestin1 increases tau levels. Hela-V5-tau cells were transfected with control siRNA or β-arrestin1 siRNA. As shown by immunoblotting, β-arrestin1 depletion significantly reduced tau ( Fig.13A , 13C , 23A, 23B). To confirm the relevance of these results in neurons, β-arrestin 1 was knocked down in tauP301S hippocampal primary neurons using lentivirus-mediated shRNA. Compared with control shRNA-transduced neurons, tauP301S neurons transduced with β-arrestin 1 shRNA showed a significant decrease of tau immunoreactivity in neuronal cell bodies and processes by approximately 50% ( Fig.15A , 15B ). It was also confirmed by Western blotting that tauP301S cortical primary neurons transduced with β-arrestin 1 shRNA showed significantly reduced tau levels ( Fig. 15C , 15D ). As β-arrestin 1 levels are increased in FTLD-tau, the next question to be determined is whether increased β-arrestin 1 would in turn increase tau levels. Transient transfection of β-arrestin 1 (β-arr1) in Hela-V5-tau cells significantly increased total tau by approximately 50% ( Fig.15E , 15F ) and phospho-tau by nearly 2 times ( Fig.15E , 15G Together, these results suggest that β-arrestin1 not only increases and colocalizes with pathogenic tau in FTLD-tau brains, but also that β-arrestin1-mediated tau regulation underlies both steady-state and GPCR (β2AR and mGluR2) agonist-induced tau increases.

[0328] Genetic reduction of β-arrestin 1 alleviates tauopathy in vivo

[0329] The above data show that increased β-arrestin1 increases tau and decreased β-arrestin1 decreases tau. Therefore, the next step was to assess the physiological effects of reducing endogenous β-arrestin1 on tauopathy in vivo. + / (Arrb1 + / -) mice were crossed to generate tauP301S and tauP301S;Arrb1 + / - Mice. TauP301S mice begin to show tauopathy at 4 months of age, which gradually worsens (Yoshiyama, Y. et al. Neuron (2007) 53: 337-351). Immunohistochemistry was first performed to detect tauP301S and tauP301S from 7-month-old mice; Arrb1 + / - Phospho-tau (pS199 / 202) in the hippocampus of littermates. In fact, tauP301S / Arrb1 + / - Mice showed approximately 60% reduction in phospho-tau immunoreactivity ( Fig.16A , 16B This finding was further confirmed using lauroylsarcosine extracts. Consistent with the immunohistochemical results, tauP301S / Arrb1 + / - The lauroyl sarcosine-insoluble tau in the Fig. 16C , 16E ). Compared with tauP301S littermates, tauP301S / Arrb1 + / - The lauroyl sarcosine-soluble tau in the Fig. 16C , 16D ).

[0330] Genetic reduction of β-arrestin1 rescues functional synaptic defects in tauP301S mice

[0331] Initial characterization of tauP301S mice demonstrated impaired paired pulse facilitation (PPF) and long-term potentiation (LTP) at 6 months of age (Yoshiyama, Y. et al. Neuron (2007) 53: 337-351). Later studies showed that tauP301S mice exhibited significant LTP defects even at 3 months of age (Woo, JA et al. Hum Mol Genet (2017) 26: 3973-3988; Woo, JA et al. Commun Biol (2019) 2: 112). To assess functional changes in synaptic plasticity, 4-month-old wild-type, tauP301S, and tauP301; Arrb1 + / - Electrophysiological recordings were performed on the CA3-CA1 Schaffer collateral pathway in acute brain slices of mice. Initial input-output (IO) analysis showed that WT, tauP301S, tauP301S / Arrb1 + / - There were no significant differences between sections from the same litter ( Fig.16F In the PPF experiment, a significant decrease in the fEPSP slope in tauP301S slices was observed at all stimulus intervals compared to wild-type slices, which was more prominent in the early stimulus intervals ( Figure 16G ), which is similar to previous reports (Woo, JA et al. Hum Mol Genet (2017) 26: 3973-3988; Woo, JA et al. Commun Biol (2019) 2: 112). + / - In slices, PFFs were significantly stronger in the 20–120 ms interstimulus interval range in tauP301S compared to wild-type slices. Long-term potentiation (LTP) recordings using theta burst stimulation revealed that tauP301S slices were severely impaired in both the induction and maintenance of LTP compared to wild-type slices ( Fig.16H ). However, compared with tauP301S littermates, tauP301S / Arrb1 + / - Slices showed a significant restoration of LTP, almost reaching the level of wild-type slices ( Fig.16H ). These functional synaptic plasticity results were confirmed in mature DIV21 primary hippocampal neurons stained for synaptophysin. Specifically, tauP301S hippocampal primary neurons (control GFP transduced) exhibited significantly reduced synaptophysin immunoreactivity in primary neurites compared to wild-type neurites (control GFP transduced). In contrast, tauP301S neurons transduced with β-arrestin 1-shRNA-GFP significantly restored synaptophysin immunoreactivity, approaching wild-type control levels ( Fig.16I , 16J)

[0332] β-Arrestin 1 promotes tau dissociation from microtubules and inhibits tau-induced microtubule assembly

[0333] Tau is a microtubule-associated protein that stabilizes microtubules (Cleveland, DW, et al. J Mol Biol (1977) 116: 207-225). However, in tauopathies including AD, tau first dissociates from microtubules, mislocalizes from somatoxonal to somatodendritic compartments (Ballatore, C., et al. Nat Rev Neurosci (2007) 8: 663-672; Biernat, J. & Mandelkow, EM Mol Biol Cell (1999) 10: 727-740) and gradually becomes insoluble to eventually form filamentous aggregates (Alonso, AD, et al. Proc Natl Acad Sci USA (1997) 94: 298-303). Arrestin exists in three different states in cells: (1) free, (2) GPCR-bound, and (3) microtubule-bound, each with different signaling capabilities (Hanson, SM et al. J Mol Biol (2007) 368: 375-387; Gao, H. et al. Mol Cell (2004) 14: 303-317; Song, X., et al. J Neurochem (2007) 103: 1053-1062). Specifically, β-arrestin 1 directly binds to microtubules and recruits Mdm2 and ERK2 (Hanson, SM et al. J Mol Biol (2007) 368: 375-387; Gurevich, VV & Gurevich, EV Curr Protoc Pharmacol (2014) 67, Unit 210 11-19). Therefore, it was first assessed whether the binding of β-arrestin1 to microtubules would alter the binding of tau to microtubules. Therefore, microtubule binding experiments were performed using recombinant proteins. 1 μg of recombinant His-tau (4R) was incubated with purified microtubules plus BSA control or recombinant β-arrestin1 for 30 minutes. After incubation, the samples were centrifuged at 100,000g. The supernatant contained microtubule-unbound proteins and the pellet contained microtubule-bound proteins. Notably, β-arrestin1 significantly reduced the amount of tau bound to the microtubule pellet by approximately 45%, while increasing the amount of free tau in the supernatant ( Fig.17A , 17B The inhibitory effect of β-arrestin 1 on tau binding to microtubules was dose-dependent, as increasing the amount of β-arrestin 1 gradually reduced the amount of tau bound to microtubules ( Fig. 17C). Next, we assessed whether β-arrestin1 alters tau-induced microtubule assembly in vitro. As expected, tubulin alone exhibited a time-dependent polymerization into microtubules, which was greatly accelerated by the addition of recombinant tau ( Fig.17D However, the addition of β-arrestin1 together with tau completely suppressed the tau-induced acceleration of microtubule assembly ( Fig.17D Addition of β-arrestin1 alone to tubulin also weakly reduced tubulin polymerization compared with tubulin alone, suggesting that β-arrestin1 / tubulin binding may have a minor inhibitory effect in microtubule assembly.

[0334] To determine whether this inhibitory effect of β-arrestin 1 on tau-dependent microtubule assembly in vitro could be observed in cells, Hela-V5-tau cells were transfected with control siRNA or β-arrestin 1 siRNA. After transfection, cells were treated with nocodazole for 30 min, which induces rapid disassembly of microtubules (Woo, JA et al. Commun Biol (2019) 2:112). After 30 minutes, the medium containing nocodazole was washed away and the cells were allowed to recover for 1 hour. After nocodazole treatment, staining for tubulin appeared highly scrambled in both control and β-arrestin 1 siRNA transfection conditions ( Fig.17E 1 hour after nocodazole washout, microtubule reassembly was readily visible as evidenced by prominent filamentous microtubule staining in the perinuclear region, which was significantly increased by nearly 3-fold in β-arrestin 1 siRNA-transfected cells compared with control siRNA-transfected cells ( Fig.17E , 17F ). Thus, these results in transfected cells and in vitro suggest that β-arrestin 1 promotes the dissociation of tau from microtubules, which both inhibits microtubule assembly and misfolds tau.

[0335] β-Arrestin 1 does not alter tau mRNA but increases tau stability by disrupting p62 self-interactions and impeding p62 flux

[0336] Although tau dissociation from microtubules deregulates microtubule dynamics and is essential for tau mismatch and aggregation (Wang, Y. & Mandelkow, E. Nat Rev Neurosci (2016) 17:5-21), this mechanism cannot easily explain the increase in total tau secondary to increased β-arrestin 1. Therefore, we tested whether β-arrestin 1 alters tau mRNA levels by qRT-PCR of tau mRNA. However, no changes in tau mRNA were observed after β-arrestin 1 overexpression or knockdown ( Fig.18A , 18B). We next assessed whether endogenous β-arrestin1 would alter tau turnover. Indeed, cycloheximide (CHX) chase experiments showed that β-arrestin1 siRNA significantly promoted tau turnover ( Fig. 18C , 18D ), suggesting that endogenous β-arrestin 1 enhances tau levels by increasing its stability.

[0337] Several studies have shown that microtubule instability impairs autophagosome maturation and autophagy-mediated protein degradation (Fass, E., et al. J Biol Chem (2006) 281: 36303-36316; Aplin, A., et al. J Cell Physiol (1992) 152: 458-466), because microtubule-based transport is required for the delivery of autophagosomes to lysosomes (Farfel-Becker, T. et al. Cell Rep (2019) 28: 51-64e54; Boecker, CA & Holzbaur, EL Curr Opin Neurobiol (2019) 57: 94-101). In order to clear misfolded proteins by autophagy, autophagic cargo receptors such as p62 / SQSTM1 must first sequester cargo and connect polyubiquitinated cargo to LC3+ autophagosomes, and then deliver them together to lysosomes for fusion and degradation (Katsuragi, Y., et al. FEBS J (2015) 282: 4672-4678; Pankiv, S. et al. J Biol Chem (2007) 282: 24131-24145). Since β-arrestin 1 displaces tau from microtubules, destabilizes microtubules and increases tau stability, it was initially hypothesized that β-arrestin 1-mediated microtubule destabilization may disrupt the delivery of p62 to LC3+ autophagosomes, thereby increasing tau stability. Hela-V5-tau cells were transfected with GFP-p62 together with control vector or β-arrestin 1, and the cells were treated with vehicle or nocodazole for 30 minutes. In cells transfected with the control vector, GFP-p62 puncta of varying sizes were present (green), whereas endogenous LC3 (magenta) was observed as small punctate staining ( Fig.18E GFP-p62 is often co-localized with LC3 (white spots) ( Fig.18E As expected, nocodazole treatment significantly reduced the colocalization of GFP-p62 with endogenous LC3-positive puncta in cells transfected with the vector control ( Fig.18E , 18F). However, β-arrestin1-overexpressing cells showed a surprisingly strong disruption of GFP-p62 colocalization with LC3 at steady state, to an extent comparable to that of nocodazole treatment ( Fig.18E , 18F). Thus, nocodazole treatment of β-arrestin1-expressing cells did not further disrupt GFP-p62 / LC3 colocalization ( Fig.18E , 18F ). This suggests that β-arrestin 1-mediated microtubule destabilization is as severe as nocodazole treatment (unlikely), or that other factors may play a role in this strong disruption of p62-LC3 colocalization. To examine LC3 and p62 in a different way, the effect of β-arrestin 1 on LC3 and GFP-p62 puncta was assessed with or without treatment with bafilomycin A1, a potent lysosomal inhibitor known to promote the accumulation of both LC3 and p62 puncta (Yamamoto, A. et al. Cell Struct Funct (1998) 23: 33-42; Mauvezin, C. & Neufeld, TP Autophagy (2015) 11: 1437-1438; Yoshimori, T., et al. J Biol Chem (1991) 266: 17707-17712). Overexpression of β-arrestin 1 in Hela V5-tau cells not only reduced steady-state LC3 puncta but also significantly attenuated the increase in LC3 puncta induced by bafilomycin A1 ( Fig.24A , 24B ), indicating that β-arrestin1 blocks autophagy at the level of LC3 or upstream. Similarly, overexpression of β-arrestin1 also reduced steady-state GFP-p62 puncta and significantly attenuated the increase in GFP-p62 puncta induced by bafilomycin A1 ( Figure 18G , 18H). In addition, although bafilomycin A treatment significantly increased the colocalization of GFP-p62 and LC3 in cells transfected with the vector control, β-arrestin 1 transfection significantly attenuated the increase in GFP-p62 / LC3 colocalization ( Figure 18G , 18I ), together suggesting that β-arrestin 1 blocks autophagy at the level of p62 or upstream, and probably not directly on LC3.

[0338] P62 is associated with neurofibrillary tangles (King, A. et al. Acta Neuropathol (2013) 125: 303-310; Kuusisto, E., et al. Neuropathol Appl Neurobiol (2002) 28: 228-237), and soluble cytoplasmic p62 levels are significantly reduced in AD brains (Caccamo, A., et al. Mol Psychiatry (2017) 22: 865-873; Zheng, X. et al. Neural Regen Res (2012) 7: 1304-1311). Increased p62 expression improves cognitive impairment in AD animal models by enhancing autophagy induction (Caccamo, A., et al. Mol Psychiatry (2017) 22: 865-873; Zheng, X. et al. Neural Regen Res (2012) 7: 1304-1311; Ramesh Babu, J. et al. J Neurochem (2008) 106: 107-120). To further investigate the p62 changes induced by β-arrestin 1, the p62 flow was assessed using the mCherry-GFP-p62 reporter. This reporter uses the sensitivity of GFP (green) and the insensitivity of mCherry (pseudo-coloring of magenta) to low pH, so that the circulation of p62 to lysosomes can be tracked (Pankiv, S. et al. J Biol Chem (2007) 282: 24131-24145; Larsen, KB et al. Autophagy (2010) 6: 784-793). Therefore, perfectly co-localized mCherry and GFP (white or light green) represent non-lysosomal LC3. However, after fusion with acidified lysosomes (autolysosomes), mCherry spots persist, while GFP spots disappear (thus only magenta). Hela-V5 cells were co-transfected with mCherry-GFP-p62 and control siRNA or β-arrestin 1 siRNA, and total mCherry+GFP spots (white / light green) and only mCherry (magenta) spots were quantified. As expected, Bafilomycin A (BafA) treatment increased the total mCherry+GFP puncta in cells transfected with the vector control (Veh) ( Fig.18J , 18K ). However, cells transfected with β-arrestin1 siRNA showed a significant increase in total mCherry+GFP puncta at steady state, and bafilomycin A treatment did not significantly increase this measure further ( Fig.18J , 18K). The percentage of acidified mCherry-only (magenta) puncta in total p62 puncta increased approximately 2-fold in β-arrestin1 siRNA compared with control siRNA-transfected cells, and almost all of them were abolished by 4 h bafilomycin A treatment, indicating that the loss of β-arrestin1 promotes p62 turnover ( Fig.18J , 18L In co-immunoprecipitation (co-IP) experiments from Hela-V5-tau cells, β-arrestin 1 formed a specific complex with HA-p62 ( Fig.24C ), suggesting that β-arrestin 1 may directly alter p62 activity through physical interactions. To question this possibility, the known ability of p62 to form particles by self-interaction through its N-terminal PB1 domain was utilized, which allows the formation of p62 helical filaments arranged in a head-to-tail configuration (Ciuffa, R. et al. Cell Rep (2015) 11: 748-758), a step that is critical for its cargo receptor activity (Wurzer, B. et al. Elife (2015) 4: e08941; Itakura, E. & Mizushima, NJ Cell Biol (2011) 192: 17-27). Therefore, it was tested whether the β-arrestin 1 / p62 interaction would alter the ability of p62 to form self-interacting complexes using HA-p62 and GFP-p62 constructs. The specific presence of GFP-p62 in HA-p62 immune complexes was observed, which was significantly reduced by >60% due to β-arrestin1 overexpression under conditions where similar amounts of HA-p62 were pulled down in HA immune complexes ( Figure 18M , 18N ). Thus, these results suggest that increased β-arrestin 1, as seen in brains of FTLD-tau and AD, strongly blocks p62 self-interaction, an initial step required for p62-mediated clearance of cargo, including misfolded tau (Caccamo, A., et al. Mol Psychiatry (2017) 22:865-873; Zheng, X. et al. Neural Regen Res (2012) 7:1304-1311; Ramesh Babu, J. et al. J Neurochem (2008) 106:107-120; Wurzer, B. et al. Elife (2015) 4:e08941; Itakura, E. & Mizushima, NJ Cell Biol (2011) 192:17-27).

[0339] method

[0340] Patient samples

[0341] Frontal cortex tissue samples of pathologically confirmed FTLD-tau and age-matched non-demented controls were obtained from Emory ADRC (P50 AG025688).

[0342] Animal models

[0343] The following mouse strains were used in this study: WT C57BL / 6, tauP301S, and Arrb1 + / - Mouse. C57BL / 6J (Jackson Laboratory line 000664), tauP301S (Jackson Laboratory line 008169) and Arrb1 - / - (Jackson Laboratory line 011131) were obtained from Jackson Laboratory. - / - The mouse (AbdAlla, S. et al. J Biol Chem (2009) 284: 6554-6565) and tauP301S (Yoshiyama, Y. et al. Neuron (2007) 53: 337-351) mice have been characterized. The mice were housed under pathogen-free conditions, and all experiments involving mice were performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of South Florida.

[0344] Primary neuronal cultures

[0345] Primary neurons were obtained from postnatal day 0 mice. The cortex and hippocampus were dissected in cold HBSS and digested with 0.25% trypsin. Neurons were plated on poly-D-lysine-coated plates or coverslips and maintained in neurobasal medium with Glutamax and B27 supplements as previously described (Woo, JA et al. Nat Commun (2017) 8: 15558).

[0346] DNA transfection, constructs and siRNA

[0347] pcDNA3β-arrestin 1-HA (addgene, 14693) (Luttrell, LM et al. Science (1999) 283: 655-661), pcDNA3β-arrestin 1-Flag (addgene, 14687) (Luttrell, LM et al. Science (1999) 283: 655-661), pMXs-puro GFP-p62 (Addgene, 38277) (Itakura, E. & Mizushima, N. J Cell Biol (2011) 192: 17-27) were obtained from addgene. β-arrestin 1-GFP and β-arrestin 1-myc constructs were obtained. β-arrestin 1 ON-TARGET plus SMART pool siRNA was purchased from Dharmacon. DNA constructs and siRNA were transiently transfected with Lipofectamine 2000 and Opti-MEM.

[0348] Recombinant protein

[0349] The pFast-tau-his and pFast-β-inhibitory protein 1-his constructs were transformed into DH10Bac competent cells. After blue-white screening, the DH10Bac strain was selected to express the recombinant bacmid. The Sf9 insect cells transfected with Bacmid were cultured with Sf900 IISFM medium for 3 days, and then the P1 generation virus in the medium was collected and added to new Sf9 cells. After 2 days of culture, the Sf9 cells were harvested and lysed with lysis buffer (Tris 20mM, pH7.4, NaCl 150mM, Triton-X100 1%, 10mM imidazole and protease inhibitors). After centrifugation at 12,000g for 15 minutes, the supernatant was collected and shaken with GE Healthcare NiSepharose at 4°C for 1 hour. The bound proteins on the agarose were washed three times with ice-cold lysis buffer, the recombinant proteins were eluted with ice-cold elution buffer (Tris 20 mM, NaCl 150 mM, 200 mM imidazole), and then the proteins were dialyzed in dialysis buffer (Tris 20 mM, NaCl 150 mM, DTT 1 mM) at 4°C overnight.

[0350] SDS-PAGE and Western blotting

[0351] Mouse brain extracts or cells were lysed in RIPA buffer (1% NP-40, 0.1% sodium dodecyl sulfate, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA) with protease and phosphatase inhibitors. After equilibration of protein concentration using the BCA assay, the lysate was mixed with 4X LDS sample buffer and loaded onto an SDS-PAGE gel. The membrane was blocked with TBS-T containing 5% milk for 1 hour at room temperature. After blocking, the membrane was probed with the designated primary antibody at 4°C overnight and incubated with horseradish peroxidase-linked secondary antibodies for 2-4 hours at room temperature.

[0352] Immunoprecipitation

[0353] Cells were lysed with CHAPS buffer (30 mM Tris-Cl pH 7.5, 150 mM NaCl, 1% CHAPS) with protease and phosphatase inhibitors. After equilibration of protein concentration, the lysate was pre-incubated with IgG beads for 1 hour and washed with CHAPS buffer. The lysate was incubated with the designated primary antibody and IgG beads at 4°C overnight, and then the protein was eluted with 4X LDS sample buffer and boiled for subsequent SDS-PAGE and Western blotting.

[0354] Insoluble and soluble extracts of lauroyl sarcosine

[0355] Lauroylsarcosine extraction was performed as previously described (Woo, JA et al. Commun Biol (2019) 2: 112). Briefly, brain homogenate was lysed with A68 buffer containing 10 mM Tris-HCl, pH 7.4, 0.8 M NaCl, 10% sucrose, 1 mM EGTA. Samples were centrifuged at 400 g for 20 min at 4 °C. After centrifugation, 1% lauroylsarcosine was added to the supernatant. The samples were incubated for 1 hour and 30 minutes and centrifuged at 80,000 g for 30 minutes at room temperature. The pellet was resuspended in 100 ul of 50 mM Tris-HCl, pH 7.4 and subjected to SDS-PAGE.

[0356] Tubulin polymerization assay

[0357] Tubulin polymerization was measured by absorbance reading at 340 nm using a Tubulin Polymerization Assay Kit (Cytoskeleton Inc., Denver, CO, USA). Tubulin was at a concentration of 3 mg / ml in 0.5 mM EGTA, 2 mM MgCl2, 1 mM GTP, 80 mM PIPES pH 6.9 in a total polymerization volume of 100 μl.

[0358] Microtubule binding assay

[0359] The microtubule binding assay was performed by the microtubule binding protein spin-down assay kit (Cytoskeleton Inc., Denver, CO, USA). Stable microtubules between 5-10 μm in length were used for the assay. After incubating the stable microtubules with the recombinant proteins, the microtubule-associated proteins were pulled down at 100,000×g.

[0360] Immunofluorescence

[0361] Cells were washed with ice-cold PBS and fixed with 4% paraformaldehyde at room temperature. After fixation, cells were washed with 0.2% triton in TBS. Mice were perfused with PBS and fixed with 4% paraformaldehyde. 25 μm sections were washed with 0.2% triton in TBS. After washing, cells and tissue sections were blocked with 3% normal goat serum and 0.1% triton X-100 for 1 hour at room temperature and incubated with the indicated primary antibodies overnight at 4°C. After washing three times with PBS, cells and tissue sections were incubated with secondary antibodies for 45 min at room temperature. Images were acquired using an Olympus FV10i confocal microscope (Tokyo, Japan) or a Zeiss LSM880 confocal microscope (Germany). Immunoreactivity was quantified using Image J software (National Institutes of Health, Bethesda, MD). All images for comparison were acquired with the same laser intensity and exposure time. The researchers were blinded to the experimental conditions during image acquisition and quantification.

[0362] Generation of β-arrestin 1 shRNA lentivirus

[0363] β-Arrestin 1 shRNA plasmid was obtained from Abm (Richmond, BC, Canada). Lentiviral vectors were co-transfected with pVSVG and Pax2 in HEK293 cells using polyethyleneimine (PEI) overnight. The medium was removed the next day and replaced with serum-free medium. After 72 hours of incubation, the medium was collected and centrifuged to remove cell debris. The virus was filtered through a syringe filter (0.2-0.45 μm).

[0364] Electrophysiology

[0365] Electrophysiological recordings were performed as described (Woo, JA et al. Hum Mol Genet (2017) 26: 3973-3988; Woo, JA et al. Commun Biol (2019) 2: 112; Woo, JA, et al. Cell Death & Differentiation (2015) 2015 Feb 20). Briefly, 4-month-old WT, tauP301S and tauP301S / Arrb1 + / - Brain slices were prepared from mice and input / output (IO) curves, paired pulse facilitation (PPF) and long-term potentiation (LTP) were measured.

[0366] Quantitative real-time RT-PCR

[0367] Using Roche Quantitative real-time RT-PCR was performed using the 96 System (Life Science, San Francisco, CA, USA). Total RNA was isolated using Trizol reagent (Invitrogen, CA, USA), reverse transcribed, and quantitative PCR analysis was performed using Syber green master mix (Invitrogen, CA, USA). Comparison of threshold cycle (Ct) values ​​was used to calculate the amplification factor, and the relative amount of tau was normalized to GAPDH.

[0368] Statistical analysis and data presentation

[0369] Statistical analysis was performed by GraphPad Prism 7.0 software (GraphPad Software, San Diego, CA, USA) using paired or unpaired Student's t-test and 1-way or 2-way ANOVA with specified post hoc tests. Data are shown as a representative experiment. Box plots and whisker plots represent all data points, and the mean is ± SEM. P < 0.05 was considered statistically significant.

[0370] discuss

[0371] The GPCRs in the AD and the GPCRs (Ni, Y. et al. Nat Med(2006) 12:1390-1396;AbdAlla, S. J. Biol. Chem(2009)284:6566-6574;AbdAlla,S.et al.JBiol Chem(2009)284:6554-6565;Thathiah,A.et al.Science(2009)323:946-951;Bakshi,P,ACS et al. Biol(2008)3:777-789:Alley,GMet al.J Neurosci Res(2010)88:143-154;Minkeviciene,R.,et al.J Pharmacol Exp Ther(2004)311:677-682;Dobarro,Met al.Int. Neuropsychopharmacol(2013)16:2245-2257;Wisely,EV,etal.Hum Mol Genet(2014)23:4024-4034;Luong,K.&Nguyen,LTAm J Alzheimers DisOther Demen(2013)28:427-224-HGet;Lee al.Acta Neuropathol(2004)107:365-371:Lee,HGet al.Brain Res(2009)1249:244-250;Sun,L.et al.FEBS Lett(2005)579:251-258),including βARM,Jet,Int. Neuropsychopharmacol(2013)16:2245-2257;Wisely,EV,et al.Hum Mol Genet(2014)23:4024-4034;Luong,K.&Nguyen,LTAmJ Alzheimers Dis Other Demen(2013)28:427-28.Kalaria, JRN et al. Neurochem(1989)53:1772-1781;Kalaria,RN&Harik,SINeurosci Lett(1989)106:233-238)andmGluR2(Lee,HGet al.Acta Neuropathol (2004) 107: 365-371; Lee, H. G. et al. Brain Res (2009) 1249: 244-250). However, it is still unclear how different classes of GPCRs act similarly to influence the pathogenesis of AD.

[0372] Since β-arrestin1 and β-arrestin2 were originally identified and named based on their ability to terminate GPCR signaling through agonist-induced receptor internalization and desensitization (Lohse, MJ, et al. Science (1990) 248:1547-1550; Wilden, U., et al. Proc Natl Acad Sci USA (1986) 83:1174-1178; Moore, CA, et al. Annu Rev Physiol (2007) 69:451-482; Gurevich, VV & Gurevich, EV Pharmacol Ther (2006) 110:465-502), the first goal was to determine whether β-arrestin1 and / or β-arrestin2 represent a common convergence point for β2AR and mGluR2 agonists to alter tau phosphorylation. Our results demonstrate that loss of either β-arrestin1 or β-arrestin2 abolishes the ability of β2AR or mGluR2 agonists to increase phospho-tau, suggesting that β-arrestin1 / 2 are an important node of convergence for GPCR-mediated tau regulation. Although it remains to be determined whether other AD-implicated GPCRs (i.e., ADRB, GPR3, AT2R, CXCR2, etc.) (AbdAlla, S. et al. J Biol Chem (2009) 284:6566-6574; AbdAlla, S. et al. J Biol Chem (2009) 284:6554-6565; Thathiah, A. et al. Science (2009) 323:946-951; Bakshi, P., et al. ACS Chem Biol (2008) 3:777-789) require the pathogenic activity of β-arrestin 1 and / or β-arrestin 2, these preliminary observations led us to examine changes in β-arrestin 1 levels in the brains of FTLD-tau patients. Interestingly, previous studies reported that β-arrestin 1 (Liu, X. et al. Cell Res (2013) 23: 351-365) and β-arrestin 2 (Thathiah, A. et al. Nat Med (2013) 19: 43-49) were significantly increased in the brains of AD patients, and both β-arrestin 1 and β-arrestin 2 interacted with the Aph-1 subunit of the □-secretase complex to increase Aβ production, thereby linking β-arrestin 1 / 2 to Aβ pathogenesis. β-arrestin 2 was significantly elevated in the brains of FTLD-tau patients, and increased β-arrestin 2 promoted tau aggregation in the absence of GPCR stimulation.As disclosed herein, β-arrestin 1 levels are highly elevated in the brains of FTLD-tau patients, suggesting that both β-arrestin 1 and β-arrestin 2 are elevated in the brains of AD and FTD patients. Whether β-arrestin 1 and / or β-arrestin 2 levels increase during aging has not been studied. In addition, whether β-arrestin 1 and β-arrestin 2 contribute to the progression of other neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson's disease, needs to be exploited.

[0373] β-Arrestin 1 levels are also highly elevated in the brains of patients with FTLD-tau, a disease pathologically defined by tauopathy lacking Aβ deposits (Irwin, DJ et al. Acta Neuropathol (2015) 129: 469-491), suggesting that tau, rather than the Aβ component of AD pathology, is required for elevated β-Arrestin 1 levels in the brain. Furthermore, the observation that insoluble tau levels correlated closely with insoluble β-Arrestin 1 levels and that AT8-positive phospho-tau aggregates colocalized almost perfectly with β-Arrestin 1 suggest a functional pathogenic relationship between β-Arrestin 1 and tau pathogenesis in FTLD-tau.

[0374] The above findings led us to hypothesize that increased β-arrestin levels promote tau accumulation and tauopathy, whereas reduced β-arrestin levels counteract this phenotype in primary neurons and in vivo. Indeed, this hypothesis was confirmed in primary neurons by β-arrestin1 overexpression and RNAi-mediated silencing experiments. In vivo, genetic reduction of β-arrestin1 not only alleviated tauopathy in tauP301S transgenic mice, but also functionally rescued the significant defects in synaptic plasticity (i.e., PPF and LTP) and synaptic integrity in tauP301S acute slices and neurons. Thus, these findings suggest that pathogenic tau accumulation upregulates β-arrestin1 through an as yet unclear mechanism, which in turn further drives tauopathy by increasing β-arrestin1 in a maladaptive positive feedback loop of progressive pathogenesis. Thus, the observation that a 50% reduction in β-arrestin1 alleviates tauopathy and associated synaptic dysfunction demonstrates that β-arrestin1 represents a viable therapeutic blockade point to break this pathogenic feed-forward loop. However, as a homozygous β-arrestin1 null mutant was not used in this study, it remains to be determined whether the complete absence of β-arrestin1 provides further benefit in the setting of tauopathy.

[0375] The main biological function of tau is attributed to its ability to bind and stabilize microtubules and promote their assembly (Cleveland, DW, et al. J Mol Biol (1977) 116: 207-225). However, in AD and other tauopathies, tau dissociates from microtubules, causing it to mismatch from the body axon to the body dendrite region (Ballatore, C., et al. Nat Rev Neurosci (2007) 8: 663-672; Biernat, J. & Mandelkow, EMM Mol Biol Cell (1999) 10: 727-740; Hoover, BR et al. Neuron (2010) 68: 1067-1081). This event occurs early in the disease process and is considered to be necessary for its hyperphosphorylation and self-assembly into aggregates (Wang, Y. & Mandelkow, E. Nat Rev Neurosci (2016) 17: 5-21). Since a large amount of β-arrestin 1 directly binds to microtubules (Hanson, SM et al. J Mol Biol (2007) 368: 375-387; Gurevich, VV & Gurevich, EV Curr Protoc Pharmacol (2014) 67, Unit 2 10 11-19), β-arrestin 1 bound to microtubules was shown to promote the dissociation of tau from microtubules in a dose-dependent manner, thereby effectively inhibiting tau-mediated microtubule assembly in vitro and in transfected cells. This effect of β-arrestin 1 is reminiscent of the way that the actin-binding protein cofilin displaces tau from microtubules, inhibits tau-induced microtubule assembly, and promotes tau pathology (Woo, JA et al. Commun Biol (2019) 2: 112). Interestingly, β-arrestin 1 and β-arrestin 2 bind to cofilin and scaffold the interaction with the cofilin-activating phosphatase chronophin to enhance cofilin activation (Zoudilova, M. et al. J Biol Chem (2007) 282: 20634-20646; Zoudilova, M. et al. J Biol Chem (2010) 285: 14318-14329). The interaction of β-arrestin 2 with cofilin also plays an important role in the translocation of activated cofilin to dendritic spines to regulate spine morphology (Pontrello, CG et al. Proc Natl Acad Sci USA (2012) 109: E442-451). However, β-arrestin 1 inhibits tau microtubule binding and tau-induced microtubule assembly in the context of purified recombinant protein lacking cofilin.Thus, this inhibitory effect of β-arrestin1 does not require cofilin per se and supports the view that the ability of β-arrestin1 to displace tau from microtubules (with or without cofilin) ​​contributes to the propensity of tau to mislocalize and subsequently self-assemble into aggregates.

[0376] Microtubule dynamics and autophagy are intricately linked in cells, especially in neurons, because autophagosomes formed in distal neurites or axons must be combined with mature lysosomes that are relatively enriched in somatic cells (Lee, S., et al. J Neurosci (2011) 31: 7817-7830; Maday, S., et al. J Cell Biol (2012) 196: 407-417; Cheng, XT, et al. Autophagy (2015) 11: 1434-1436; Wang, T. et al. J Neurosci (2015) 35: 6179-6194). Therefore, this spatial difference requires microtubule-based autophagosomes and lysosomes to be transported over relatively long distances. Indeed, impaired microtubule dynamics disrupt autophagic clearance (Fass, E., et al. J Biol Chem (2006) 281: 36303-36316; Aplin, A., et al. J Cell Physiol (1992) 152: 458-466; Farfel-Becker, T. et al. Cell Rep (2019) 28: 51-64e54; Boecker, CA & Holzbaur, EL Curr Opin Neurobiol (2019) 57: 94-101), and defects in autophagy contribute to AD pathogenesis (Nixon, RA et al. J Neuropathol Exp Neurol (2005) 64: 113-122; Yang, D Set al. Am J Pathol (2008) 173: 665-681; Sanchez-Varo, R. et al. Acta Neurol (2008) 174: 665-681; Sanchez-Varo, R. et al. Acta Neurol (2019) 153: 665-681). Neuropathol (2012) 123: 53-70) by promoting the accumulation of Aβ and tau (Caccamo, A., et al. Mol Psychiatry (2017) 22: 865-873; Zheng, X. et al. Neural Regen Res (2012) 7: 1304-1311; Ramesh Babu, J. et al. J Neurochem (2008) 106: 107-120; Xu, Y., et al. Autophagy (2019) 15: 583-598).Observing that β-arrestin 1 does not alter tau mRNA but increases tau protein stability, the initial focus was on the p62-LC3 autophagy mechanism, because microtubule-based transport promotes the proximity of p62-bound cargo to LC3+ autophagosomes (Lee, S., et al. J Neurosci (2011) 31: 7817-7830; Maday, S., et al. J Cell Biol (2012) 196: 407-417; Cheng, XT, et al. Autophagy (2015) 11: 1434-1436; Wang, T. et al. J Neurosci (2015) 35: 6179-6194). In addition, β-arrestin 2 disrupts p62-mediated tau clearance. Therefore, it is assumed that β-arrestin 1 can inhibit p62-mediated tau clearance, because they share multiple biological functions with 78% sequence identity.

[0377] p62 / SQSTM1 knockout mice exhibit severe neurodegeneration as well as hyperphosphorylated tau and neurofibrillary tangles (Ramesh Babu, J. et al. J Neurochem (2008) 106: 107-120), and p62 overexpression strongly reduces pathogenic tau in transfected cells and in vivo (Xu, Y., et al. Autophagy (2019) 15: 583-598). Overexpression of β-arrestin 1 alone is as effective as nocodazole treatment (an effective microtubule destabilizer) in disrupting the colocalization of p62-LC3 at steady state, suggesting that β-arrestin 1 is as effective as nocodazole in destabilizing microtubules (which is unlikely) or other mechanisms that also cause this powerful destruction. In fact, the finding that β-arrestin 1 reduces both LC3 and p62 spots and significantly attenuates the accumulation of both LC3 and p62 spots induced by bafilomycin suggests that β-arrestin 1 inhibits autophagy at the level of p62 itself or upstream. P62 flow and co-IP experiments confirmed that β-arrestin 1 inhibits autophagy at the p62 level, because β-arrestin 1 not only blocks p62 flow, but also binds to p62 and interferes with p62 self-association, which is a necessary step for the formation of p62 bodies (Ciuffa, R. et al. Cell Rep (2015) 11: 748-758). This self-association of p62 through its N-terminal PB1 domain is essential for its cargo receptor activity, by enabling stronger connections (multiple binding) to its ubiquitinated cargo and simultaneous binding to multiple LC3 proteins (Wurzer, B. et al. Elife (2015) 4: e08941; Itakura, E. & Mizushima, NJ Cell Biol (2011) 192: 17-27), which helps explain the loss of p62 spots caused by β-arrestin 1 overexpression. In addition, cargo-bound p62 promotes autophagosome formation by enhancing the conversion of LC3 to its active lipidated form LC3-II (Cha-Molstad, H. et al. Nat Commun (2017) 8: 102), which may explain the observation that β-arrestin 1 overexpression also reduces LC3 spots and reduces p62-LC3 colocalization. This mechanism of β-arrestin 1 binding to p62 and interfering with p62 self-oligomerization and destabilization of microtubules is consistent with the observed role of β-arrestin 1 in impeding p62 trafficking and impairing the clearance of misfolded tau.

[0378] To date, no previous studies have implicated β-arrestin 1 in tauopathy, microtubule dynamics, or p62-mediated autophagy. Together, these findings suggest that β-arrestin 1 is involved in microtubule destabilization, promoting tau mislocalization, and inhibiting p62-mediated tau clearance. In addition to these activities found in this study, β-arrestin 1 also promotes Aβ production and deposition in vivo (Liu, X. et al. Cell Res (2013) 23: 351-365). Therefore, targeting β-arrestin 1 represents a promising therapeutic intervention point that could simultaneously mitigate Aβ and tau pathogenesis. Although β-arrestin 1 - / - The mice showed impaired desensitization to β-adrenergic receptor stimulation in the heart, but they were generally normal, fertile, and did not show any physical or behavioral abnormalities (Conner, DA et al. Circ Res (1997) 81: 1021-1026). Therefore, reducing β-arrestin 1 levels or activity may be a beneficial strategy to alleviate tau pathogenic diseases including AD.

[0379] Example 3: Pharmacological Inhibitors

[0380] β-Arrestin 1 and β-Arrestin 2 form homo- and hetero-oligomers, and inositol hexaphosphate (IP6) promotes these oligomers. Therefore, in some embodiments, the inhibitor of β-arrestin oligomerization is a molecule, such as a peptide, that blocks IP6 binding, thereby reducing the oligomerization of β-arrestin 2. In some embodiments, the inhibitor of oligomerization is a β-arrestin mutant that acts as a dominant negative for homo-hetero-oligomerization of β-arrestin 1 and / or β-arrestin 2.

[0381] The data presented also suggest that pharmacological inhibitors for β-arrestin oligomerization could represent a promising therapeutic strategy to mitigate AD and tauopathies. Therefore, a molecular docking screen using FTMap analysis was performed to screen millions of compounds that block the interface between β-arrestin2 and β-arrestin2. Approximately 30 predicted inhibitory compounds were tested using cell-based assays, yielding several small molecule compounds and their analogs that reduced pathogenic tau. Next, we tested whether these inhibitors that reduced pathogenic tau also abolished β-arrestin2 oligomerization. Co-immunoprecipitation, fluorescence resonance energy transfer (FRET), and proximity ligation assay (PLA) data demonstrated that these compounds indeed blocked β-arrestin2 oligomerization.

[0382] Figures 24A to 24Dβ-Arrestin2 oligomerization mutants were shown to impair β-Arrestin1 homo-oligomers and β-Arrestin1 / β-Arrestin2 hetero-oligomers. HeLa-V5-tau cells were co-transfected with vector control or β-Arrestin2 oligomerization mutants (ΔIP6C or ΔIP6N) together with β-Arrestin1-GFP+β-Arrestin1-myc or β-Arrestin2-myc. Cells were then subjected to proximity ligation assay (PLA) using antibodies against GFP and myc to detect β-Arrestin1 homo-oligomers ( Fig.24A , 24B ) and β-arrestin 1 / β-arrestin 2 hetero-oligomers and DAPI staining ( Fig.24C , 24D )(spots). The results showed that the oligomerization mutants of β-arrestin 2 (ΔIP6C or ΔIP6N) significantly reduced the homo-oligomerization of β-arrestin 1 ( Fig.24A , 24B ) and β-arrestin1 / β-arrestin2 hetero-oligomers (n=4, 1-way ANOVA, post hoc Dunnett's, #P<0.0001). Cells that received only 1 probe or excluded the primary antibody showed no red PLA spots (negative control).

[0383] Fig.25 The workflow for screening compounds that block β-arrestin oligomerization and reduce pathological tau is shown. Based on the crystal structure of β-arrestin 2, we performed an in silico screening of millions of compounds that could dock on the β-arrestin 2 trimer binding interface. The top 30 compounds were then tested for their ability to reduce pathological tau. The tau-reducing compounds (synthesized by MolPort) provided in the table below were then tested for their ability to block β-arrestin 2 oligomerization:

[0384]

[0385]

[0386] Fig.26 C1 and C2 compounds were shown to reduce tau levels. HeLa-V5-tau cells were treated with 10 μM potential inhibitor of β-arrestin 2 oligomerization for 6 hours and subjected to western blotting. C1 and C2 significantly reduced total tau levels in HeLa-V5-tau cells (n=4, 1-way ANOVA, post hoc Dunnett's, **P<0.005, ***P<0.0005).

[0387] Fig.27A and 27B The C1 and C2 compounds were shown to reduce pathogenic tau. Fig.27AHeLa-V5-tau cells treated with 20 μM of O1, Cl or C2 compounds for 6 hours and fractionated for lauroylsarcosine soluble and insoluble tau. C1 and C2 significantly reduced lauroylsarcosine insoluble tau levels in HeLa-V5-tau cells. (n=4, 1-way ANOVA, post hoc Dunnett's, **P<0.005, ***P<0.0005). Fig.27B Shown are DIV21 hippocampal primary neurons derived from Tau-P301S mice treated with 20 μM of O1, C1 or C2 compounds for 6 hours and stained for HT7 (total human tau). C1 and C2 significantly reduced HT7 immunoreactivity (n=3, 1-way ANOVA, post hoc Dunnett's, *P<0.05, ***P<0.0005).

[0388] Fig.28A and 28B Compounds C2 and C2 were shown to inhibit β-arrestin 2 oligomerization. Hela-V5-tau cells were transfected with GFP-β-arrestin 2 and Flag-β-arrestin 2 and treated with 20 μM of O1, C1 or C2 for 6 hours. Fig.28A Proximity ligation assay (PLA) using antibodies against GFP and Flag (M2) to detect GFP-β-arrestin2 / Flag-β-arrestin2 complexes showed a significant reduction in β-arrestin2 oligomerization (n=3, 1-way ANOVA, post hoc Dunnett's, *P<0.05, ***P<0.0005). Inclusion of only 1 PLA probe as a negative control showed no PLA red spots. Fig.28B Cells lysed and co-immunoprecipitated (co-IP) the GFP-β-arrestin2 / Flag-β-arrestin2 complex are shown, demonstrating that the C1 and C2 compounds reduced β-arrestin2 self-interaction to similar background levels as anti-mouse IgG beads alone.

[0389] Fig.29 Compound C2 was shown to inhibit β-arrestin2 oligomerization. Hela-V5-tau cells were transfected with CFP-β-arrestin2 and YFP-β-arrestin2 and treated with vehicle or 20 μM C2 for 30 min. β-arrestin2 oligomerization was detected by FRET assay, showing that C2 compound, but not vehicle, reduced the CFP to YFP FRET signal.

[0390] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs.Publications cited herein and the materials in which they are cited are specifically incorporated by reference.

[0391] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. Use of a β-arrestin oligomerization inhibitor in the preparation of a medicament for treating tauopathy in a subject, wherein the inhibitor is a compound having a structure selected from the following or a pharmaceutically acceptable salt thereof:

2. The method of claim 1, wherein the tauopathy is selected from the group consisting of: Alzheimer's disease, Pick's disease, frontotemporal dementia, chromosome 17-related frontotemporal dementia with Parkinson's syndrome, progressive supranuclear palsy, corticobasal degeneration, subacute sclerosing panencephalitis, amyotrophic lateral sclerosis / Parkinson-dementia complex, argyrophilic grain dementia, English amyloid angiopathy, cerebral amyloid angiopathy, Creutzfeldt-Jakob disease, pugilistic dementia dementia, diffuse neurofibrillary tangles with calcifications, Down syndrome, Gerstmann-Straussler-Scheink disease, Hallervorden-Spatz disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C, non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic Parkinson's disease, prion amyloid angiopathy, progressive subcortical gliosis, tangled dementia, multi-infarct dementia, traumatic brain injury, and stroke.

3. The use of claim 1, wherein the tau disease is frontotemporal lobar degeneration.

4. The use according to claim 2, wherein the stroke is ischemic stroke.

5. The use according to claim 2, wherein the traumatic brain injury is chronic traumatic encephalopathy.

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