Small molecule drugs that reduce protein aggregation

Abstract

Disclosed herein are small molecule drugs that reduce protein aggregation, and methods of using them. In one aspect of the invention, a method is provided for reducing the abundance of an aggregated protein in a protein aggregate, comprising administering to a subject an effective amount of a compound that stably binds to glial fibrillary acidic protein (GFAP), wherein the aggregated protein comprises BSN, SYN1, MAP2, PLEC, RAB10, MAP1A, DCTN, TUBA4A, SPART, PRKDC, or any combination thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 288,998, filed December 13, 2021, the contents of which are incorporated by reference in their entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under award P01AG01241117A1 from the National Institutes of Health and Merit 2 I01 BX001655 from the Department of Veterans Affairs. The Government has certain rights in the invention.

[0003] Reference to Electronic Sequence Listing The entire contents of the electronic format of the sequence listing (169852.00108.xml; size: 9,341 bytes; creation date: December 12, 2022) are incorporated herein by reference. [Background technology]

[0004] Glial fibrillary acidic protein (GFAP) is a type III intermediate filament structural protein found primarily in astrocytes and has been implicated in several age-related neuropathologies. GFAP has been shown to be associated with progressive neurological disorders such as Alzheimer's disease, Parkinson's disease, and Alexander disease (Helman et al., 2020;Ishiki et al., 2016;Kamphuis et al., 2012;Lee et al., 2017;Middeldorp and Hol, 2011;van Bodegraven et al., 2021). Mutations in GFAP lead to the aggregation of Rosenthal fibers, which is the cause and symptom of Alexander disease (Lee et al., 2017). GFAP expression is regulated at both transcriptional and post-translational levels, affecting important cytoskeletal functions. GFAP is transcriptionally regulated by multiple growth factors and nuclear hormone receptors (Laping et al., 1994). Post-translational modifications (PTMs) of GFAP have been observed, including site-specific phosphorylation by multiple kinases (Battaglia et al., 2019;Clairembault et al., 2014;Herskowitz et al., 2010;Leal et al., 1997;Sullivan et al., 2012), acetylation in amyotrophic lateral sclerosis (ALS) (Liu et al., 2013), and citrullination of five arginine residues in AD (Ishigami et al., 2015). The resulting changes in the structural conformation of GFAP may contribute to traumatic brain injury (Lazarus et al., 2015) or autoimmune diseases (Jin et al., 2013). Several single nucleotide polymorphisms in GFAP are strongly associated with Alexander disease (Lee et al., 2017).

[0005] Thus, there is a need for novel agents that target GFAP for the treatment of neurological disorders such as Alzheimer's disease, Parkinson's disease, and Alexander disease, and their associated dementias. Summary of the Invention

[0006] Small molecule drugs that reduce protein aggregation and methods of their use are disclosed herein. In one aspect of the present invention, a method is provided for reducing the abundance of an aggregated protein in a protein aggregate, comprising administering to a subject an effective amount of a compound that stably binds to glial fibrillary acidic protein (GFAP), wherein the aggregated protein comprises BSN, SYN1, MAP2, PLEC, RAB10, MAP1A, DCTN, TUBA4A, SPART, PRKDC, or any combination thereof. In some embodiments, the subject suffers from a neurodegenerative disease, such as Alzheimer's disease.

[0007] In another aspect of the invention, there is provided a method for increasing the abundance of an aggregating protein in a protein aggregate, the method comprising administering to a subject an effective amount of a compound that stably binds to glial fibrillary acidic protein (GFAP). In some embodiments, the aggregating protein comprises CBX8, TSPYL5, CDK2, KRT33B, or any combination thereof. Non-limiting embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, which are schematic and are not intended to be drawn to scale. Each identical or nearly identical component shown in the drawings is typically represented by the same numeral. Not every component name is shown in every drawing, nor are every component of every embodiment of the present invention shown. This is for the sake of clarity, as description thereof is not necessary for those skilled in the art to understand the invention. [Brief description of the drawings]

[0008] [Figure 1A]Figures 1A-1C show that GFAP is more abundant and more modified in AD aggregates than in AMC. (Figure 1A) The spectral counts of GFAP in sarkosyl-insoluble aggregates isolated by immunopull-down (IP) of Aβ or tau, or in total aggregates without IP, are higher in human AD than in the hippocampus of age-matched controls (AMC). AD and AMC differ by heteroscedastic t-test: *P<0.05; **P<0.005. [Figure 1B] (FIG. 1B) Differences in post-translational modifications (PTMs: phosphorylation of Ser or Thr residues, or oxidation of Met) observed in GFAP isolated from the hippocampus of AMC[ApoE(3,3)] (SEQ ID NO: 7), AD[ApoE(3,3)] (SEQ ID NO: 8), or AD[ApoE(4,4)] (SEQ ID NO: 9). Peptide coverage is highlighted. [Figure 1C] (FIG. 1C) Western blot of phosphorylated GFAP in hippocampal aggregates of AD(3,3) or AD(4,4) compared with AMC(3,3) detected with an antibody against phospho-GFAP (Ser13; ThermoFisher). ***P<0.0001 for differences between each AD group and AMC by two-tailed heteroscedastic t-test. [Figure 2A] 2A-2E show molecular dynamics analysis of the GFAP structure. (FIG. 2A) Initial structural model of GFAP. The internal cavity is a predicted drug-binding pocket. [Figure 2B] (Figure 2B) Prediction of the binding pocket volume of GFAP over a 500 ns span at 50 ns intervals. [Figure 2C] (Figure 2C) Prediction of the cavity for ligand binding at 200 ns. [Figure 2D] (Figure 2D) Change in root mean square deviation (RMSD) of GFAP structure over time when comparing AMC (unmodified) GFAP with GFAP with phosphomimetic substitutions to mimic AD(3,3) and AD(4,4) in a 500 ns in silico simulation. [Figure 2E](FIG. 2E) Distribution of root mean square fluctuations (RMSF) across GFAP (432 residues). Positional fluctuations are shown per residue. (FIGS. 2D and 2E) The keys on the right of FIG. 2D and FIG. 2E show the color codes for RMSD and RMSF values, respectively. [Figure 3A] Figures 3A-3E show the effect of RNAi knockdown targeting GFAP or its putative kinase on aggregation. (Figure 3A) GFAP phosphorylation and its putative kinase observed in human hippocampal aggregates. [Figure 3B] (FIG. 3B) Fluorescence images of Thioflavin T-stained human SH-SY5Y-APPSw cells after liposome-mediated transfection with siRNA constructs targeting GFAP or its candidate kinases. [Figure 3C] (FIG. 3C) Histogram showing the mean ± SEM of Thioflavin T staining per cell for aggregates stained as in panel 3b. [Figure 3D] (FIG. 3D) Thioflavin T stained human T98G cells after transfection with the indicated siRNA constructs. [Figure 3E] (FIG. 3E) Histogram of mean ± SEM for Thioflavin T staining of aggregates similar to those in panel 3d. (FIGS. 3C-3E) Numbers above bars are P values ​​for differences between treated and control groups by two-tailed t-test. Numbers above brackets apply to the group / bar they connect. [Figure 4A] 4A-4B show that ROCK1 protein levels are higher in T98G glioblastoma cells overexpressing the ApoE4 transgene than in T98G cells overexpressing ApoE3 (FIG. 4A) Western blot probed with an antibody against ROCK1 protein. [Figure 4B] (FIG. 4B) Mean ± SEM of band intensities of Western blots of independent T98G cell cultures (N=5 each). E4>E3 (P<0.0001) in t-test. [Figure 5A]Figures 5A-5F illustrate that the drug MSR1 is predicted to specifically bind GFAP, thereby inhibiting the role of GFAP in aggregation. (Figure 5A) Histogram showing GFAP binding stability (Gibbs free energy of binding) predicted by MM-GBSA solvation docking for the top three drugs from the in silico screen. The ChemBridge drug structure library was screened for binding to GFAP. Screening was performed in three stages of increasing stringency, followed by a counterscreen to eliminate drugs with affinity for tubulin. [Figure 5B] (FIG. 5B) SY5Y-APPSw cells were stained with Thioflavin T (green fluorescence) and counterstained with DAPI (not shown). [Figure 5C] (FIG. 5C) Histogram showing a halving of amyloid per cell (Thioflavin T fluorescence divided by the number of DAPI+ nuclei per field as shown in FIG. 5B; ***P≦0.0005). [Figure 5D] (FIG. 5D) Total sarkosyl-insoluble aggregate proteins stained with SYPRO-Ruby after isolation from SY5Y-APPSw cells. [Figure 5E] (Figure 5E) The set of proteins completely removed from aggregates by GFAP siRNA knockdown is nearly identical to the set displaced by MSR1. Venn diagram shows the proteomic overlap of 251 proteins (>7 hits) identified in sarkosyl-insoluble aggregates from untreated SY5Y-APPSw cells, but were not detected 48 hours after transfection with GFAP siRNA or treatment with 1 μM MSR1. Conversely, four proteins that were absent in aggregates from untreated cells were identified in both treated cell groups. [Figure 5F](Figure 5F) Linear regression of log2 (fold change) of aggregate protein abundance after GFAP siRNA treatment (x-axis) and MSR1 treatment (y-axis). Selected proteins are labeled and include those shown to be in close proximity to GFAP by aggregate cross-linking (Balasubramaniam et al., 2019) (red dots including BSN, SYN1, MAP2, PLEC, RAB10, MAP1A, DCTN, TUBA4A, SPART, PRKDC, EEF2, PARP1, NFH, and H14). Dots within the dashed rectangle are those that changed less than 2-fold with either treatment. The regression had an R=0.77 and significance by F-test was P<3E-280. [Figure 6] FIG. 6 shows the molecular structures of MSR-1, MSR-2, and MSR-3. [Figure 7A] Figures 7A-7F illustrate that MSR1 treatment attenuates aggregation and associated phenotypes in a C. elegans model of AD. (Figure 7A) RNAi knockdown of BARK or ROCK1 in the C. elegans AM141 strain (a model of Huntington's disease expressing Q40::YFP in muscle) is compared to the effects of RNAi targeting IFP-1, the closest C. elegans homolog of GFAP. [Figure 7B] (FIG. 7B) RNAi knockdown of ROCK1, BARK, or AKT2 in the C. elegans CL2355 line, an AD model exhibiting pan-neuronal expression of human Aβ1-42. [Figure 7C] (FIG. 7C) RNAi knockdown of ROCK1, AKT2 or BARK in human T98G cells reduces total aggregate burden (measured as total spectral counts of sarkosyl-insoluble material). [Figure 7D] (Figure 7D) MSR1 prevents the aggregation of tau expressed in muscle of the C. elegans VH255 strain. [Figure 7E] (FIG. 7E) Chemotaxis of C. elegans CL2355 strain, an AD model expressing human Aβ1-42 in pan-neuronal tissue. Chemotaxis to n-butanol was measured in adult worms 5 days after hatching. [Figure 7F] (Figure 7F) Calculated mean aggregate fluorescence per worm in C. elegans strain AM141 (q40::yfp), a model for Huntington's disease characterized by muscle expression of a polyglutamine (Q40) fused in frame to a gene encoding yellow fluorescent protein (YFP). YFP-positive aggregates were counted in adult worms 5 days after hatching. (Figures 7A-7F) Differences from controls are significant in two-tailed heteroscedastic t-tests (*P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005; ****P ≤ 0.00005). Experiments were repeated 3-4 times with consistent results. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Disclosed herein are small molecule drugs that target GFAP and reduce protein aggregation. This example shows that GFAP is significantly overexpressed and differentially phosphorylated in the AD hippocampus, particularly in AD subjects with the apolipoprotein E [ε4,ε4] genotype ApoE(4,4), compared to age-matched controls (AMC). Compared to AMC, detergent-insoluble aggregates in AD brains are particularly enriched for hyperphosphorylated GFAP. Four kinases (ROCK1, BARK / GRK, PKA, and AKT2) that may be responsible for the site-specific phosphorylation of GFAP observed in AD brains are upregulated in AD compared to AMC, and are upregulated in SH-SY5Y-APP1, SH-SY5Y-APP2, SH-SY5Y-APP3, SH-SY5Y-APP4, SH-SY5Y-APP5, SH-SY5Y-APP6, SH-SY5Y-APP7, SH-SY5Y-APP8, SH-SY5Y-APP9, SH-SY5Y-APP9, SH-SY5Y-APP10, SH-SY5Y-APP11, SH-SY5Y-APP12, SH-SY5Y-APP13, SH-SY5Y-APP14, SH-SY5Y-APP15, SH-SY5Y-APP16, SH-SY5Y-APP17, SH-SY5Y-APP18, SH-SY5Y-APP19 ... SwKnocking down these kinases in human neuroblastoma cells or T98G human glioblastoma cells significantly reduced amyloid accumulation. Knocking down the orthologous kinases in C. elegans also reduced protein aggregation and associated behavioral characteristics in several models of Alzheimer's-like aggregation and in models of polyglutamine aggregation observed in Huntington's disease. In silico screening identified a drug candidate called MSR1 that stably and specifically binds GFAP. Cellular aggregates are reduced to the same extent by MSR1 exposure or GFAP-specific RNAi knockdown, with a high degree of agreement between the aggregated proteins that are depleted. These examples show that GFAP plays a major role in the increase in neuropathic aggregates. GFAP is both a functional target, a useful biomarker, and a novel therapeutic target to prevent or alleviate neurodegenerative diseases such as AD.

[0010] Glial fibrillary acidic protein (GFAP) is a type III intermediate filament structural protein found primarily in astrocytes and is involved in several aging-related neuropathologies (Hol and Capetanaki, 2017). GFAP has been functionally linked to animal models of AD (Kamphuis et al., 2012) and Alexander disease (Helman et al., 2020;Lee et al., 2017). Detection of GFAP in human CSF or serum is an important biomarker for neuropathology and contributes to the diagnosis of Alzheimer's disease (AD), Parkinson's disease (PD), dementia with Lewy bodies (DLB), and frontotemporal lobar degeneration (FTLD) (Bartl et al., 2021;Ishiki et al., 2016;Schulz et al., 2021).

[0011] GFAP mutations often lead to aggregation that forms Rosenthal fibers, which are a cause and symptom of chronic gliosis such as that seen in Alexander disease (Lee et al., 2017). Several single nucleotide polymorphisms in GFAP are strongly associated with this demyelinating disorder (Lee et al., 2017). GFAP expression is transcriptionally regulated by multiple growth factors and nuclear hormone receptors (Laping et al., 1994). Previously reported post-translational modifications (PTMs) of GFAP include site-specific phosphorylation associated with Alexander disease (Battaglia et al. 2019), PD (Clairembault et al., 2014), and FTLD (Herskowitz et al., 2010); acetylation of six lysine residues in amyotrophic lateral sclerosis (ALS) (Liu et al., 2013); and citrullination of five arginine residues in AD (Ishigami et al., 2015). Citrullination of GFAP may contribute to traumatic brain injury (Lazarus et al., 2015) and autoimmune diseases (Jin et al., 2013).

[0012] GFAP is encoded by a single gene on chromosome 17 and is expressed as 10 isoforms with distinct splice sites (Brodie et al., 1998;Kamphuis et al., 2012;Moeton et al., 2016;Thomsen et al., 2013). The major isoform, GFAP-α (432 amino acids), is highly expressed in glial cells and neurons in the central nervous system (CNS), whereas the β, γ, ε, κ, and ζ isoforms are expressed in many tissues and cell types in addition to neurons and glia in the CNS (Kamphuis et al., 2012;Moeton et al., 2016;Thomsen et al., 2013). When disease or injury causes neuronal stress, it induces astrocyte activation, resulting in responses such as hypertrophy, proliferation, and increased GFAP expression (de Souza et al., 2020;Fan and He, 2016;Muccigrosso et al., 2016;Nawashiro et al., 1998). The initial glial activation after injury is an acute phase response that allows recovery from brain damage (Dani et al., 2018;Donat et al., 2017). However, long-term neuronal injury or stress leads to chronic neuroinflammation that adversely affects brain function (Calabrese et al., 2018;Streit et al., 2004). AD is diagnosed as dementia accompanied or followed by neuropathology such as tau-specific tangles and amyloid plaques (Dani et al., 2018;Drummond et al., 2018;Hoenig et al., 2020). Increased GFAP expression was observed in the hippocampus of AD patients compared to age-matched controls (Ayyadevara et al., 2016b).

[0013] Examples show the role of GFAP and its PTMs in protein aggregation. Examples show that GFAP is hyperphosphorylated in the AD hippocampus compared to GFAP in age-matched controls (AMC), which lack substantial phosphorylation. AD-specific phosphorylation was reduced by RNAi knockdown of upstream kinases that potentially target the modified GFAP site, and knockdown of each resulted in a marked reduction in amyloid deposition by human neuroblastoma and glioma cells in vitro. We screened approximately 750,000 small molecule structures from the ChemBridge library to identify drug candidates with specific affinity for partially unfolded GFAP. One of these, named MSR1 (Figure 6), was particularly effective in reducing protein aggregation and pathology in various AD models (human cells or C. elegans).

[0014] One aspect of the present invention provides the use of compounds that stably bind to glial fibrillary acidic protein (GFAP). One aspect of the present invention provides a method for reducing the abundance of a protein in an aggregate. The method comprises administering to a subject an effective amount of a compound that stably binds to glial fibrillary acidic protein (GFAP). The term "protein aggregates" refers to aggregates of proteins and other components that are formed in intracellular or extracellular processes. This process causes misfolded or inherently disordered proteins to join together through their conformation, resulting in insoluble aggregates. These aggregates may be globular or may be organized as fibrils. Protein aggregates may be insoluble in medium strength detergents such as Sarkosyl (sodium lauryl sarcosinate). "Aggregated proteins" refers to proteins that are found or may be found in protein aggregates. The term "reducing the abundance of aggregated protein" refers to inhibiting protein aggregation. In some embodiments, protein aggregation is inhibited by 50% or more. In some embodiments, protein aggregation is inhibited by 55%, 60%, 65%, 70%, 75% or more. As shown in the Examples, MSR1 inhibited protein aggregation by 60-75% in various model systems.

[0015] The abundance of multiple different aggregated proteins may be reduced. In some embodiments, 50, 100, 150, 200, or 250 or more aggregated proteins are partially or completely removed from the protein aggregates. Aggregated proteins that may be reduced include, but are not limited to, those shown in FIG. 5F, such as BSN, SYN1, DLG4, ANK2, GJA1, EPB41L3, SLC25A12, PFKP, PC, MAP2, PLEC, GCN1, RAB10, MAP1A, DCTN, TUBA4A, YWHAG, PRKDC, SPART, EEF2, and others. In some embodiments, aggregated proteins may include those in the immediate vicinity of GFAP, such as BSN, SYN1, MAP2, PLEC, RAB10, MAP1A, DCTN, TUBA4A, SPART, PRKDC, EEF2, PARP1, NFH, H14, or any combination thereof. "In close proximity" refers to adjacent within the aggregate. Other proteins may not be in direct contact with GFAP, but may be "linked" to GFAP through other proteins that are closer to GFAP. Aggregated proteins that may be partially or completely removed from protein aggregates include RPS4X, SLC25A5, PSMC1, C11orf98, PRPF40A, TRMT6, CCT5, LARP1, MSH6, UBA1, CSDE1, TARS, EIF2A, UTP20, SNRPE, UQCRC2, CYFIP2, ALDOA, PDS5A, AHCTF1, BANF1, YWHAQ, EP400, SMARCA4, IPO5, DLST, MRPL49, PPP1CB, SLC25A13, GPI, MYH9, GSTM3, RIF1, CHD2, ELAVL4, VARS, SNRPD2, DPYSL3, NSD2, SEPTIN2, PHOX2A, HSPB1, GNAI2, AGO2, FLNB, YWHAB, CHD8, IQGAP1, RBM8A, PDS5B, MTHFD1, MRP L38, EMD, GNAI1, EXOSC7, NPEPPS, CEP170, GAK, PGK1, USP10, XPO5, DPYSL5, LUC7L, FYN, PRMT1, PYGB, NOC3L, ATP2A2, LRPPRC, SF3B1, PDHB, LYN,PDHA1, STRAP, MARS, FLII, FSCN1, TUBB1, TUBB, TUBB3, CDK18, IARS, FLOT2, TUBB6, AGO1, WDR1, RANBP2, CBX1, IPO7, TLN1, NDUFS3, ATAD5, TUBB4B, YWHAG, CRMP1, GLUD1, TUBB2B, NOC2L, GTF3C3, HSPA4, DYNLL2, COX4I1, SEPTIN6, ATP5F1B, SNRPA, RAB39A, TUBB2A, HEATR1, DYNLL1, ZMYM4, CDK12, RAB1B, CHD 7, SEPTIN11, PRDX4, ARHGEF2, CLTC, GNA13, HCFC1, YES1, APEH, SIN3A, COX5A, COX5B, LARS, RPS23, AGO3, WDR33, RAB39B, YWHAH, DYNC1H1, PPP1CC, SEPT IN8、SPTBN1、PPP1CA、SART3、TUBB4A、CDK5、NAP1L1、RAB6A、SLC25A11、TUBA1B、SEPTIN7、EXOSC6、SOGA1、DDX19A、ATP13A1、MYO18A、CBR1、GNB2、TUBAL3、 CAP1, MYO1B, CTNND1, PUF60, CWC22, MARK3, PLP1, USO1, TUBA4A, DPYSL4, ABCF2, CHD4, ALDOC, GCN1, DPYSL2, TUBA8, ZNF462, GDI1, HSPA4L, MBP, DLAT, F LOT1, PPIL1, HRNR, MAST3, YLPM1, MARK2, VDAC3, ACO2, KIF21A, GNAO1, NEFL, RAB10, INA, CAND1, SLC25A22, STXBP1, NEFM, GNB1, ATIC, AP1B1, AP2M1, CAC NA2D1、NIPBL、MAP1B、RAC1、ACTN4、SUCLA2、ATP6AP1、DNAJB2、ALDH2、ATP2B3、CAMK2A、GOT2、DCTN1、AGAP3、MACF1、FARP1、DHX36、CD59、CDC42BPA、SPTA N1, AP2B1, DNM2, GFAP, CAMK2D, PLEC, ACTN1, ATP2B4, THY1, MAP1A, SPTBN2, CKMT1A, TUFM, MAP2, HSPA12A, ATP2B2, CNP, USP5, AP2A2, AARS, ATP1A1, NSF,CAMK2G, LONP1, AP2A1, CKB, ATP2B1, MYCBP2, WDR37, PFKL, EPB41L3, SLC25A12, PFKM, CNTN1, CAMK2B, SPTB, GLS, ATP6V0A1, ATP1A3, PFKP, DNM1, IARS2, ATP1A2, DNM3, DCLK1, PC, GJA1, DLG4, ANK2, CNTNAP1, BSN, SYN1, or any combination thereof.

[0016] Another aspect of the present invention is to provide a method for increasing the abundance of an aggregated protein in a protein aggregate. The method comprises administering to a subject an effective amount of a compound that stably binds to glial fibrillary acidic protein (GFAP). The term "increasing the abundance of an aggregated protein" refers to inhibiting protein aggregation. In some embodiments, protein aggregation is increased by 50% or more. In some embodiments, protein aggregation is increased by 55%, 60%, 65%, 70%, 75% or more. The abundance of multiple different aggregated proteins may be increased. Aggregated proteins that may be increased include, but are not limited to, CBX8, TSPYL5, CDK2, KRT33B, or any combination thereof. The term "stably bound" refers to the ΔG binding In some embodiments, the ΔG of the compound is less than 0 kcal / mol (i.e., negative energy). binding In some embodiments, the ΔG binding is less than -43 kcal / mol, less than -46 kcal / mol, less than -49 kcal / mol, less than -50 kcal / mol, or less than -52 kcal / mol. "ΔG binding The term "binding affinity" refers to the change in the Gibbs free energy of binding, which estimates the predicted affinity between a ligand and a protein associated with the binding process. The magnitude of binding affinity is a measure of the strength of the interaction between the ligand and the protein, and therefore often directly related to the efficacy of the ligand. The term "negligible affinity" refers to a protein with a ΔG binding This refers to compounds with an average calorie content of -7 kcal / mol or more. Examples of compounds that stably bind to GFAP include, but are not limited to: MSR1, MSR2, or MSR3. The term "MSR1" refers to the compound 3-chloro-N-{[trans-4-(hydroxymethyl)cyclohexyl]methyl}-4-pyrrolidin-1-ylbenzamide, whose molecular formula is C 19 H 27 The structure of MSR1 is shown in Figure 6. The term "MSR2" refers to the compound 3-{[4-(5-chloro-2-pyridinyl)-1-piperazinyl]carbonyl}-5,6,7,8-tetrahydro-2(1H)-quinolinone, whose molecular formula is C 19 H 21 The structure of MSR2 is shown in Figure 6. The term "MSR3" refers to the compound 1-(3-chloro-4-pyrrolidin-1-ylbenzoyl)-4-pyridin-2-ylpiperazine, whose molecular formula is C 20 H 23 The structure of MSR3 is shown in Figure 6.

[0017] In some embodiments, the compounds have negligible affinity for α-tubulin, β-tubulin, or oligomers thereof. The terms "α-tubulin" and "β-tubulin" refer to the α and β subunits of the protein tubulin. The tubulin protein polymerizes into long chains or filaments that form microtubules. Microtubules are hollow fibers that function as the cytoskeletal system within living cells. Microtubules have the ability to adopt a variety of conformations that allow cells to undergo mitosis or perform intracellular transport. α-tubulin is a relatively ubiquitous microtubule component, whereas β-tubulin is specific to neuronal microtubules. Both α- and β-tubulins copolymerize into microtubules. Microtubules are the major components of the eukaryotic cytoskeleton. The terms "oligomer" and "polymer" refer to proteins composed of multiple subunits (polypeptide chains). Thus, oligomeric proteins have a quaternary structure, which is generally considered to be the highest level of organization in the hierarchy of protein structures. Oligomeric proteins may contain only a few copies of identical polypeptide chains, in which case they are called homo-oligomers, or they may contain at least one copy of two or more different types of polypeptide chains (hetero-oligomers).

[0018] The compounds that stably bind to GFAP used in the methods disclosed herein may be formulated as pharmaceutical compositions comprising: (a) a therapeutically effective amount of one or more protein degraders described herein; and (b) one or more pharma- ceutically acceptable carriers, excipients, or diluents. The pharmaceutical compositions may comprise the compounds in the range of about 0.1-5000 mg (preferably about 0.5-500 mg, more preferably about 1-100 mg). The pharmaceutical compositions may be administered to provide a daily dose of the compounds of about 0.1-500 mg / kg body weight (preferably about 0.5-20 mg / kg body weight, more preferably about 0.1-10 mg / kg body weight). In some embodiments, the pharmaceutical compositions may further comprise a bioactive agent. The compounds utilized in the methods disclosed herein may be formulated as pharmaceutical compositions in solid dosage form, but any pharma- ceutically acceptable dosage form may be utilized. Examples of solid dosage forms include, but are not limited to, tablets, capsules, sachets, lozenges, powders, pills, or granules. Examples of solid dosage forms include fast-dissolving dosage forms, controlled release dosage forms, lyophilized dosage forms, delayed release dosage forms, sustained release dosage forms, pulsed release dosage forms, and mixed dosage forms of fast-release dosage forms and controlled release dosage forms, or combinations thereof. The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition containing a carrier, which may be selected from the group consisting of, for example, proteins, carbohydrates, sugars, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste. The compounds utilized in the methods disclosed herein may be formulated as pharmaceutical compositions comprising one or more of binders, fillers, lubricants, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Suitable diluents can be, for example, pharma- ceutically acceptable inert fillers such as microcrystalline cellulose, lactose, calcium hydrogen phosphate, sugars, and mixtures of any of the foregoing. Suitable disintegrants include lightly cross-linked polyvinylpyrrolidone, corn starch, potato starch, maize starch, modified starches, croscarmellose sodium, crospovidone, sodium starch glycolate, and mixtures thereof. Examples of effervescent couples include an organic acid and a carbonate or bicarbonate, or alternatively, only the sodium bicarbonate component of the effervescent couple may be present. The compounds utilized in the methods disclosed herein may be formulated as pharmaceutical compositions for delivery by any suitable route.For example, pharmaceutical compositions may be administered orally, intravenously, intramuscularly, subcutaneously, topically, and pulmonary.Examples of pharmaceutical compositions for oral administration include capsules, syrups, concentrates, powders, and granules. The compounds utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredients with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art, which may involve mixing, granulating, compressing or dissolving the ingredients as appropriate to the desired preparation. Pharmaceutical compositions containing the compounds may be adapted for administration by any suitable route, including oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, or transdermal), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal) routes, intraperitoneal injection, and topical administration such as by eye drops. Such formulations may be prepared by any method known in the art of pharmacy, for example by combining the active ingredient with the carrier or excipient. The formulations may be presented in unit-dose or multi-dose containers.

[0019] The compounds used in the compositions and methods disclosed herein may be administered as pharmaceutical compositions, and thus pharmaceutical compositions incorporating the compounds are considered to be embodiments of the compositions disclosed herein. Such compositions may be in any pharma-ceutically acceptable physical form, for example, pharmaceutical compositions for oral administration. Such pharmaceutical compositions contain an effective amount of the disclosed compounds, which is related to the daily dose of the compounds administered. Each dosage unit may contain a daily dose of a particular compound. Alternatively, each dosage unit may contain a portion of a daily dose, such as one-half or one-third. The amount of each compound contained in each dosage unit may depend in part on the particular compound selected for treatment, and other factors, such as for what indication the compound is being administered. The pharmaceutical compositions disclosed herein may be formulated using well-known procedures to provide immediate, sustained, or delayed release of the active ingredient after administration to a patient. The compounds for use according to the methods disclosed herein may be administered as a single compound or as a combination of compounds. For example, the compounds may be administered as a single compound or in combination with other compounds that have the same or different pharmacological activity.

[0020] As indicated above, pharma- ceutically acceptable salts of the compounds are contemplated and may be utilized in the disclosed methods. As used herein, the term "pharma- ceutically acceptable salts" refers to salts of compounds that are substantially non-toxic to living organisms. Exemplary pharma- ceutical acceptable salts include salts prepared by reacting the compounds disclosed herein with pharma- ceutical acceptable mineral or organic acids, or organic or inorganic bases. Such salts are known as acid addition salts and base addition salts. Those skilled in the art will understand that most or all of the compounds disclosed herein can form salts, and that in many cases, salt forms of pharmaceuticals are more commonly used because they are easier to crystallize and purify than the free acids or bases. The particular counterion forming a part of any salt of a compound disclosed herein may not be important to the activity of the compound, so long as the salt, as a whole, is pharmacologically acceptable and the counterion does not impart undesirable properties to the salt, as a whole, which may include undesirable solubility or toxicity. Pharmaceutically acceptable esters and amides of compounds can also be used in the compositions and methods disclosed herein.Suitable examples of esters include alkyl, aryl, and aralkyl esters, such as methyl esters, ethyl esters, propyl esters, dodecyl esters, and benzyl esters.Suitable examples of amides include unsubstituted amides, monosubstituted amides, and disubstituted amides, such as methyl amides, dimethyl amides, and methylethyl amides. Additionally, the methods disclosed herein may be practiced with solvated forms of the compounds, or their salts, esters, and / or amides, including ethanol solvates and hydrates.

[0021] One aspect of the present technology provides a method for the treatment of a subject in need of a compound that stably binds to GFAP. Suitably, the method may include administering to the subject an effective amount of a compound that stably binds to GFAP. As used herein, the terms "treating" or "to treat" refer to alleviating symptoms, temporarily or permanently eliminating the cause of symptoms, and / or preventing or delaying the onset or reversing the progression or severity of symptoms caused by a particular disease or disorder, respectively. Thus, the methods disclosed herein encompass both therapeutic and prophylactic administration. As used herein, "subject" may be interchangeable with "patient" or "individual" and refers to an animal in need of treatment, which may be a human or non-human animal. A "subject in need of treatment" includes a subject having a disease, disorder, or condition that responds to treatment with a compound disclosed herein alone or in combination with another bioactive agent. Examples of diseases, disorders, or conditions include, but are not limited to, neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease, traumatic brain injury, other neurological conditions, or cardiovascular conditions. The term "bioactive agent" is used to refer to an agent other than a conjugate that is used in combination with the compound as a biologically active agent to help achieve the intended treatment, inhibition, and / or prevention / prophylaxis of the compound.

[0022] The term "effective amount" as used herein refers to an amount or dose of a compound that produces a desired effect, such as when administered once or multiple times to a subject. For example, in the context of treating cancer, an effective amount refers to an amount of a therapeutic agent that reduces tumor growth rate, reduces tumor burden, reduces the number of metastases, increases the time to tumor progression, or increases survival time by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. In the context of sensitization, an effective amount refers to an amount of a therapeutic agent that produces sensitization of the subject or cell as described above. The effective amount can be determined by the attending diagnostician, who is skilled in the art, by observing the results obtained under similar circumstances using known techniques. When determining the effective amount or dosage of the compound to be administered, the attending diagnostician can consider many factors, including, for example, the species of the subject; the size, age, and general health of the subject; the relevance or severity of the disease or disorder involved; the response of the individual subject; the particular compound to be administered; the mode of administration; the bioavailability characteristics of the formulation to be administered; the selected dosage; the use of concomitant drugs; and other relevant circumstances.

[0023] In some embodiments, the subject has a neurodegenerative disease. The term "neurodegenerative disease" refers to a type of disease in which nerve cells in the brain or peripheral nervous system lose function over time and eventually die. Neurodegenerative diseases are characterized by a breakdown in protein homeostasis, as indicated by the accumulation of insoluble protein aggregates in the brain. Common neurodegenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, prion disease, motor neuron disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, amyotrophic lateral sclerosis, Friedreich's ataxia, Lewy body disease, multiple system atrophy, and progressive supranuclear palsy. In some embodiments, the subject has Alzheimer's disease (AD). The term "Alzheimer's disease" refers to a progressive neurological disorder in which the brain shrinks (atrophies) and brain cells die. AD is the most common cause of dementia and the continuing decline in thinking, behavior, and socialization. In AD patients, brain cell connections and the cells themselves degenerate and die, ultimately destroying memory and other important mental functions.

[0024] Unless otherwise specified or indicated by context, the terms "a," "an," and "the" mean "one or more." For example, "a molecule" should be interpreted as meaning "one or more molecules." As used herein, the terms "about," "approximately," "substantially," and "significantly" will be understood by those of ordinary skill in the art and may vary to some extent depending on the context in which the words are used. If the context in which a term is used would make its use unclear to a person of ordinary skill in the art, "about" and "approximately" will mean within ±10% of the particular term, and "substantially" and "significantly" will mean more than ±10% of the particular term. In this specification, the words "include" and "including" have the same meaning as "comprise" and "comprising". The terms "comprise" and "comprising" should be interpreted as "open" transitional terms that allow for the inclusion of additional elements beyond the elements recited in the claims. The terms "consist" and "consisting of" should be interpreted as "closed" transitional terms that do not allow for the inclusion of additional elements beyond the elements recited in the claims. The term "consisting essentially of" should be interpreted as being partially closed, allowing for the inclusion of only additional elements that do not fundamentally change the nature of the subject matter recited in the claims. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any examples provided herein, or the use of exemplary language (such as, for example, "such as"), are intended only to better illustrate the invention and do not limit the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. All references cited herein, including publications, patent applications, and patents, are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference in its entirety. Preferred embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect that such variations will be adopted by those skilled in the art as appropriate, and the inventors intend the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, this invention encompasses any combination of the above-described elements in all possible variations thereof unless otherwise indicated herein or clearly contradicted by context. EXAMPLES

[0025] method C. elegans strains: All nematode strains used in this study were obtained from the Caenorhabditis Genetics Center (CGC; Minneapolis, MN). The nematode model of Alzheimer's-like amyloidosis is based on human Aβ 1-42 CL4176[smg-1 ts ;myo-3p::Aβ 1-42 ::let-851 3'-UTR;rol-6(su1006)]; and human Aβ 1-42 CL2355[smg-1 ts ;snb-1::Aβ 1-42The AM141 strain expressed a polyglutamine reporter (Q40::YFP) in muscle, making it a model for glutamine tract aggregation similar to that observed in Huntington's disease and several other neuropathologies (Morley et al., 2002). Unless otherwise stated, all strains were grown at 20°C on 2% (w / v) agar plates containing nematode growth medium (NGM) overlaid with Escherichia coli OP50.

[0026] Chemotaxis and paralysis assays in Aβ transgenic C. elegans strains CL2355 and CL4176. 1-42 Transgenic C. elegans strains expressing human Aβ were grown at 20 °C in sufficient E. coli (OP50) and lysed on adult day 1 to release eggs to generate synchronized cohorts. Eggs were then placed on 100 mm NGM agar dishes seeded with bacteria expressing RNAi targeting GFAP orthologues (E. coli HT115) or empty vector control bacteria. Worms at the L3-L4 transition were upshifted to 25.5 °C to express human Aβ. 1-42 Transgene expression was induced and assayed 48 hours later. Chemotaxis (Dosanjh et al., 2010) and paralysis (Dostal and Link, 2010) assays were performed as previously described (Ayyadevara et al., 2017;Ayyadevara et al., 2016b;Ayyadevara et al., 2016d;Kakraba et al., 2019).

[0027] Paralysis assay using human tau-expressing C. elegans VH255 strain. C. elegans VH255 strain expressing human tau in a pan-neuronal manner (Brandt et al., 2009) was maintained on agar plates overlaid with a lawn of E. coli (OP50) at 25°C. Unladen eggs were obtained by lysing the worms with an alkaline hypochlorite solution and used to generate synchronized cultures. The eggs were then transferred to 100 mm NGM agar plates and MSR1 or MSR2 was added to the plates at a final concentration of 1 μM. For RNAi knockdown of ifp-1, a lawn consisting of the HT115 strain expressing an exon segment of double-stranded ifp-1 RNA was used (see next section). Worms were washed every other day and added to new drug-equilibrated plates. Assays were performed on 3-day-old adults to assess the percentage of paralyzed worms.

[0028] RNAi in C. elegans. RNA-mediated interference (RNAi) is achieved by feeding bacteria expressing double-stranded RNA corresponding to exonic fragments of mRNA targets (Ayyadevara et al., 2016c;Fire et al., 1991;Fire et al., 1998;Kakraba et al., 2019). Briefly, worm cultures were synchronized by alkaline hypochlorite lysis to release unlaid eggs. Prehatched eggs or late L4 stage larvae were placed on IPTG-containing NGM plates seeded with bacteria (E. coli HT115[DE3]) harboring the empty vector L4440 (pPD129.36) or bacterial clones expressing ifp-1 (homologous to human GFAP), let-502 (orthologous to human ROCK1), akt-2 (AKT2), ​​kin-1 (PKA), or grk-1 (BARK). Day 3 adult worms (5.5 days after hatching) were imaged to assess total aggregate fluorescence (strain AM141) or to assess paralysis (VH255) or chemotaxis to n-butanol (CL2355).

[0029] siRNA knockdown and Thioflavin T staining of human cells. SH-SY5Y-APP from logarithmic cultures. SwCells were trypsinized, rinsed, and plated at 10,000 cells per well in 96-well plates and grown for 16 h at 37°C in DMEM+F12 (Life Technologies) supplemented with 10% fetal bovine serum (FBS). Cells at approximately 40% confluence were transfected with short interfering RNA (siRNA) constructs targeting GFAP (SAS1_Hs01 00227618), AKT2 (SAS1_Hs01 00035058), ROCK1 (SAS1 Hs 00065571), BARK (SAS1 Hs 00039321), or PKA (SAS1 Hs 00217223), all from Millipore-Sigma (St. Louis, USA). Transfection of the siRNAs was performed using RNAiMax reagent (Life Technologies) according to the manufacturer's instructions. 48 hours after transfection, cells were fixed with 4% (v / v) formaldehyde and stained with 0.1% (w / v) Thioflavin T in a dark container. Cells were washed four times in PBS, then covered with Antifade+DAPI (EMD-Millipore), and fluorescence from nine fields of each well was captured in the green and blue channels using a Keyence fluorescence microscope with a motorized stage for automated well-by-well imaging. Thioflavin T fluorescence intensity was measured using the DAPI staining of each well. + Amyloid ratios per cell were determined by dividing by the number of nuclei and summarized as mean ± SD.

[0030] SH-SY5Y-APP Sw MSR1 treatment of human neuroblastoma (SH-SY5Y-APP Sw ) cells were grown as previously described (Kakraba et al., 2019; Liu et al., 2005). SH-SY5Y-APP expressing the aggregation-prone "Swedish" double mutant amyloid precursor protein (APPSw) was SwCells were cultured in DMEM + 10% (v / v) FBS at 37°C. Cells were suspended in trypsin / EDTA and rinsed in buffer before replating or harvesting. Immediately prior to the assay, cells were grown for 48 hours in the presence of 10 μM MSR1 dissolved in DMSO (final concentration 0.02%) or in 0.02% DMSO (solvent only) for control cells. Cells were harvested, total protein was isolated and aggregated proteins were purified as follows.

[0031] Western blot analysis of glial (T98G) cells for pGFAP and ROCK1. Human glioblastoma cells (T98G) were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen / Life Technologies, Grand Island, NY) supplemented with 10% FBS (v / v). Cells were harvested and cellular proteins were extracted in lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% (w / v) Nonidet P40, 0.1% SDS, 0.5% sodium deoxycholate) and quantified using Bradford reagent (Bio-Rad). Protein aliquots (50 μg) were electrophoresed on a 4-20% gradient Bis-Tris acrylamide gel (BioRad Life Science, Hercules, CA) at 100 V for 2 h and transferred to nitrocellulose membranes. Blots were preincubated with BSA blocker (Pierce) and then probed with rabbit antibodies against pGFAP or ROCK-1 (Cell Signaling, 1:100 dilution) at 4°C overnight. After washing, the membranes were incubated with secondary antibodies (HRP-conjugated goat anti-rabbit IgG (AbCam, 1:10,000 dilution) or rabbit anti-goat IgG (Rockland Immuno-chemicals, Gilbertsville, PA)) for 1 h at room temperature and developed with an ECL chemiluminescence detection kit (Pierce). Data were digitized and analyzed using ImageJ software (NIH).

[0032] Isolation of aggregated proteins. Cultured human cells were harvested, flash frozen in liquid nitrogen, and homogenized at 0°C in a buffer containing non-ionic detergent (1% (v / v) NP40, 20 mM Hepes (pH 7.4), 300 mM NaCl, 2 mM MgCl2, and protease / phosphatase inhibitors [CalBiochem]) [11, 12, 37]. The lysate was centrifuged (5 min, 3000 rpm, 4°C) to remove debris. After removing cytoplasmic proteins (soluble in 1% NP40 non-ionic detergent) by centrifugation (18 min, 13,000 × g, 4°C), the protein pellet was brought to pH 7.4 in 0.1 M HEPES buffer containing 1% (v / v) sarkosyl (sodium lauryl sarcosinate) and 5 mM EDTA and centrifuged at 100,000 × g for 30 min. Pelleted proteins (sarkosyl-insoluble fraction) were resuspended in Laemmli loading buffer (containing 50 mM dithiothreitol and 2% (v / v) SDS (sodium dodecyl sulfate)) and proteins soluble in this buffer were dissolved by heating at 95°C for 5 min and separated by electrophoresis on a 4-20% polyacrylamide gradient gel containing 1% (v / v) SDS. Gels were stained with SYPRO Ruby (ThermoFisher) or Coomassie Blue to visualize proteins. Gel lanes were robotically cut into 1 mm slices and digested to completion with trypsin. Proteins in each slice were identified by mass spectrometry as previously reported (Ayyadevara et al., 2015;Ayyadevara et al., 2016a;Ayyadevara et al., 2016b;Ayyadevara et al., 2016c;Ayyadevara et al., 2016d;Balasubramaniam et al., 2018).

[0033] Modeling and MD simulation of GFAP structure. The 3D structure of GFAP was modeled using fold recognition and ab initio molecular orbital structure prediction implemented by the I-TASSER server-based algorithm. Among five different models generated by I-TASSER, the lowest energy conformer was chosen for further processing. For molecular dynamics (MD) simulations, the protein creation wizard of the Schrodinger Desmond simulation suite was used to create the model structure. To approximate physiological conditions during the simulations, an orthorhombic simulation box filled with simple point charge (SPC) water was created and the locally charged sites were filled with appropriate counterions (Na + , Cl - ) and then 0.15 M NaCl was added to achieve physiological isotonicity. For equilibration, the temperature and pressure were kept at 300°K and 110.23 kPa (1.1023 bar), respectively. The random sampling input seed was changed for each run, and the simulation was repeated at least three times, each for 200 to 500 nanoseconds. Phosphorylation was incorporated using the Maestro "Mutate residue" plugin to convert the specified residues to their phosphorylated forms. Trajectories were visualized with VMD and analyzed using BIOVIA Discovery Studio (Dassault Systèmes).

[0034] Virtual screening of target proteins against molecular structure libraries. High-throughput virtual screening of the ChemBridge molecular structure library for docking to GFAP was first performed using the Schrodinger Suite Glide module. Acquisition and preparation of the ChemBridge structures in 2D format was performed using the LigPrep Wizard (Schrodinger Suite). To improve the efficiency of virtual drug screening, a three-step strategy was adopted: (i.) the entire library of approximately 750,000 molecular structures was virtually docked to the GFAP protein in the high-throughput mode of Glide; (ii.) the top 1% of structures obtained in the high-throughput screening were redocked to GFAP in the standard accuracy mode of Glide; and (iii.) the binding free energies of the top 1% of structures obtained in the standard accuracy docking were predicted under MM-GBSA conditions using the Schrodinger Suite Prime module. The structures with the highest avidity (ΔG binding We performed simulations using the Schrodinger Desmond module for protein-ligand complexes (the lowest in terms of molecular weight) to evaluate their stability over time.

[0035] Statistical analysis. For replicate assays of protein aggregation, chemotaxis, and paralysis, the significance of differences between control and experimental groups was assessed by Fisher-Behrens heteroscedastic t-test (as appropriate for samples of unequal or unknown variances) with each experiment treated as one point. Within experiments, differences in rates (rate of paralysis or chemotaxis) were assessed by chi-square test or Fisher's exact test, as appropriate, based on sample size.

[0036] result Aggregates formed in the Alzheimer's disease hippocampus are enriched in glial fibrillary acidic protein, hyperphosphorylated, and oxidized. Glial fibrillary acidic protein (GFAP), a largely unstructured protein, is 2- to 2.5-fold more abundant in three subclasses of detergent-insoluble aggregates from AD hippocampi compared with those from age-matched controls (AMC) (Figure 1A). GFAP in control aggregates has no prevalent post-translational modifications (PTMs), whereas GFAP in AD aggregates is phosphorylated on three to five serine or threonine residues (Figure 1B). Notably, GFAP phosphorylation signatures differed reproducibly between individuals carrying the ApoE alleles ε3,ε3 or ε4,ε4 (abbreviated as "3,3" and "4,4", respectively). Western blots of AD(3,3) and AD(4,4) samples confirmed that AD tissues contained significantly more hyperphosphorylated GFAP (hP-GFAP) compared with AMC (Fig. 1C). Each genotype group differed from AMC(3,3) controls with P < 0.0001.

[0037] Several other PTMs were screened using the PEAKS software (PTM module), but none were useful in distinguishing AD from AMC. In all groups, methylated arginine and lysine were observed at R88 and K95, and K107 was dimethylated, both at >90%. Deamidation was never observed in >10% of the spectral counts in any GFAP peptide, and pyroglutamine never exceeded 25% (data not shown).

[0038] Molecular dynamics simulations predict GFAP unfolding and identify druggable pockets. Molecular dynamics simulations of the hP-GFAP structure observed in AD aggregates (rendered with phosphomimetic substitutions) predict a more malleable GFAP structure in ApoE(3,3) individuals, whereas a higher degree of structural rigidity is predicted in AD(4,4) compared to the unphosphorylated GFAP seen in AMC aggregates (Figures 2A-2E). Because GFAP is a largely disordered protein, its full-length structure has not been experimentally determined. Therefore, we predicted its three-dimensional structure using I-TASSER fold recognition and ab initio molecular orbital procedures (Yang et al., 2015). The resulting hypothetical structure contains helices and loops (Figure 2A), forming a small pocket or cavity near the internal groove of the protein.

[0039] Given its disordered nature, the predicted protein structure is expected to be unstable and to unfold spontaneously, altering the pocket orientation and drug accessibility. To investigate the protein unfolding trajectory, the predicted structure of fully solvated GFAP was simulated for 0.5 μs (500 ns). The volume of the druggable pocket (Figure 2B) and several measures of the fluctuations in atomic positions (see below) provide useful descriptors of the structural changes. These analyses support the predicted unfolding of the initial structure of GFAP, which expands the druggable pocket (Figures 2A and 2C) over the course of the simulation. An intermediate metastable structure (200 ns into the simulation; Figure 2C) was selected for screening small molecule binding.

[0040] It is anticipated that the differences in phosphorylation of GFAP observed in AD aggregates (Figures 1A-1C) may affect the structural dynamics of GFAP. To assess this possibility, we "mutated" the observed phosphorylation sites to glutamic acid (phosphomimetic substitutions) and simulated the resulting structures for 0.5 microseconds. The results support the expectation that hyperphosphorylation of GFAP in AD brains likely alters the structural stability of GFAP. The root mean square deviation (RMSD) of GFAP atomic coordinates over this time interval is consistently lower for the unmodified molecule, "AMC(3,3) GFAP," than for "AD(3,3) GFAP," a phosphorylation-mimetic structure similar to that observed in AD(3,3) aggregates (Figure 2D). "AD(4,4) GFAP," a phosphorylation-mimetic of GFAP observed in AD(4,4) aggregates, is somewhat more fluctuating initially than AMC(3,3) GFAP, but achieves a relatively stable structure that is maintained from about 235 nanoseconds onwards (Figure 2D).

[0041] Plotting the average root mean square fluctuations (RMSFs) of individual residues over time indicates that both the AMC(3,3) and AD(3,3) structures exhibit moderate to high positional variability across the GFAP molecule (Figure 2E), in contrast to the relatively rigid AD(4,4) phosphomimetic structure. Together, these data (Figures 2D and 2E) support the prediction that AD-associated phosphorylation differences can alter the structure of GFAP, producing a somewhat unstable conformation in ApoE(3,3) but a relatively invariant conformation in ApoE(4,4).

[0042] Identification of kinases that may mediate GFAP phosphorylation. Using the phosphorylation prediction software NetPhos (http: / / www.cbs.dtu.dk / services / NetPhos) and GPS (http: / / gps.biocuckoo.org / online.php), we predicted the upstream kinases of each putative GFAP target residue (Table 1 and Figure 3A). Related kinases predicted to phosphorylate GFAP at the modification sites (AKT2, ROCK1, BARK / GRK, and PKA) have all been previously implicated in the onset or progression of AD (Banerjee et al., 2021;Henderson et al., 2016;Ko et al., 2019;Obrenovich et al., 2009a;Obrenovich et al., 2009b;Obrenovich et al., 2006;Russo, 2019;Taylor et al., 2021;Zhang et al., 2020). Sw To examine the effect of knockdown of individual kinases on protein aggregation in SH-SY5Y-APP (Figures 3B and 3C) and human glioblastoma cells (T98G; Figures 3D and 3E), tests were performed with or without siRNA-mediated knockdown. Sw Cellular aggregates were reduced by 60-70%, which was similar to (or even greater than) the effect of GFAP siRNA (Figure 3C). In T98G cells, only AKT2 siRNA reduced aggregation as efficiently as GFAP siRNA (Figure 3E). [Table 1]

[0043] Furthermore, we used RNAi constructs silencing these human kinases and their closest worm orthologues to compare the effects of targeted kinase knockdown in C. elegans models of protein aggregation, including the aggregation model of Huntington's disease (strain AM141) by aggregation of polyglutamine arrays, and the amyloid-forming Aβ1-42 We used aggregation models that mimic Alzheimer's disease using neuronal expression of the peptide (CL2355 strain) or expression of human tau in muscle (VH255 strain), which forms toxic aggregates that lead to paralysis. In the Huntington's model, the total aggregate intensity in each worm (Figure 7A), i.e., the product of the number of aggregates in each worm and the average YFP fluorescence per aggregate, was reduced by 50-60% by knockdown of C. elegans genes orthologous to BARK / GRK or ROCK1 (P<0.00005 for each), similar to the effect of RNAi against ifp-1 (a partial homolog of GFAP). In a C. elegans model of AD-like neuronal amyloidosis (CL2355), induction of an Aβ transgene in neurons reduced chemotaxis and reduced worm migration toward n-butanol (a chemoattractant). RNAi knockdown of ROCK1, AKT2, or BARK / GRK orthologues (let-502, akt-2, and grk-2, respectively) rescued the chemotaxis defect at least as well as KD of ifp-1, an intermediate filament protein with homology to human GFP (Figure 7B). These results in C. elegans are similar to those observed in the human glioblastoma cell line T98G, where siRNA KD of ROCK1, AKT2, or PKA reduced total aggregated protein by 50-60% (Figure 7C).

[0044] ROCK1 is increased in ApoE-expressing glioblastoma cells. Protein levels of Rho-associated protein kinase 1 (ROCK1) are elevated in mild cognitive impairment and AD, and reducing ROCK1 by hemizygous knockout mitigated the high amyloid levels seen in mouse models of AD (Henderson et al., 2016). Having observed a significant effect of reducing ROCK1 levels in both cultured human cells and intact C. elegans aggregate models, we measured its levels in T98G glioblastoma cells overexpressing either the APOE3 or APOE4 alleles. Human glial cells overexpressing the APOE4 allele had at least six-fold higher ROCK1 protein levels than the same cells expressing the APOE3 transgene (P < 0.0001; Figures 4A and 4B), which may explain the additional GFAP phosphorylation sites observed when AD aggregates from ApoE(4,4) compared with ApoE(3,3) individuals (Figures 1B and 1C).

[0045] Identification of novel small molecules predicted to stably bind to GFAP by computational screening. To identify novel GFAP-specific inhibitors, structures were screened by in silico docking simulations from the ChemBridge library containing approximately 750,000 small molecules. A predicted druggable pocket in the transient structure of GFAP (Figure 2C, 200 ns) was selected for target-based docking. To increase the prediction throughput, three stages of computational screening were performed in the Schrodinger Glide docking module (Balasubramaniam et al., 2020), increasing the docking stringency at each stage (see Methods for details). First, virtual docking of the entire ChemBridge library was performed in high-throughput mode, followed by redocking of the top 1% of lead molecules in standard accuracy mode. The top 1% of molecules obtained in the second stage (74 structures) were analyzed for the free energy of implicit solvent-based interactions using Schrodinger's MM-GBSA module (Balasubramaniam et al., 2020). Next, ΔG binding The molecules predicted to have the lowest ΔG were then examined for their effect on protein aggregation in vivo. This three-step procedure of simulating docking generated a set of molecules predicted to bind stably with the highest affinity to the target pocket of GFAP. The best three candidates (termed MSR1, MSR2, and MSR3) had ΔG values ​​of >-46 kcal / mol. binding and were predicted to fit well within the modeled GFAP pocket (Figure 2C). In counterscreens for binding to tubulin, these drugs had negligible affinity for α or β tubulin, or oligomers of α and β tubulin (ΔG binding ≥-7 kcal / mol), whereas most of the top-ranked drugs were predicted to bind tubulin with similar affinity as they bind GFAP (data not shown).

[0046] The top ranked drugs inhibit aggregation in vitro and in vivo as effectively as GFAP knockdown and inhibit the same coaggregation components. ΔG binding The three lead compounds with the lowest predictive values ​​(Figure 5A) were then experimentally validated in a human cell culture model of neurodegenerative amyloidosis and in a C. elegans whole animal model of AD-like aggregation. MSR3 was not tested because initial testing showed that it was cytotoxic to neuroblastoma cells and retarded C. elegans growth at all doses tested (data not shown). MSR1 and MSR2 were first validated in SH-SY5Y-APP Sw The in vivo efficacy in neuroblastoma cells was tested. Aggregation in this model was particularly well suppressed by MSR1 (Figures 5B and 5C). In all assays, the efficacy of MSR1 was superior or comparable to that of MSR2. Figure 5B shows that SH-SY5Y-APP Sw Neuroblastoma cells were stained for amyloid with thioflavin T after exposure to vehicle (control) or MSR1. Thioflavin fluorescence was reduced by nearly half in MSR1-treated cells in multiple experiments. Total sarkosyl-insoluble aggregated proteins were isolated and separated on an acrylamide-SDS gel, after which proteins were stained with SYPRO Ruby. Gel lanes (Figure 5D, left to right) represent vehicle-treated control cells, cells treated for 48 hours with small interfering RNA (siRNA) targeting GFAP, or cells treated for 48 hours with MSR1 or MSR2, agents predicted to bind stably and selectively to GFAP. GFAP siRNA inhibited aggregated protein by 65-80%, MSR1 by 60-75%, whereas MSR2 did not significantly reduce the amount of aggregated protein. SH-SY5Y-APP SwProteomic identification of proteins in sarkosyl-insoluble aggregates of cells revealed that proteins present in sarkosyl-insoluble aggregates of control cells (spectral count ≥ 7) and completely eliminated by MSR1 treatment (0 hits) were remarkably identical (87% identity) to proteins eliminated by siRNA knockdown of GFAP (Fig. 5E). On the other hand, four proteins (CBX8, TSPYL5, CDK2, and KRT33B) were found to be substantially upregulated by both treatments. The excluded group includes several proteins involved in microtubule assembly and / or interaction: plectin, dynactin-1, synapsin-1, ankyrin B, MAP1A, MAP2, and α-tubulin. MSR1 and GFAP siRNA correlated well with the effect of treatment (R = 0.77, P < 3E-280; Fig. 5F). The observation that MSR1 and GFAP RNAi had such highly concordant and correlated effects on aggregate composition provides compelling evidence that GFAP is the primary functional target through which MSR1 reduces aggregation. The drugs MSR1 and MSR2 were also tested in several C. elegans models of human neurodegenerative disease. In a tauopathy model strain (VH255) expressing normal human tau in C. elegans muscles, tau aggregation caused paralysis (inability to move in response to stimuli) that was alleviated to a similar extent by 1 μM MSR1 or by treatment with siRNA against ifp-1, the closest worm homologue of GFAP (Figure 7D). MSR1 also inhibited human Aβ 42It also resulted in significant restoration of chemotaxis in a C. elegans model of neuronal amyloidosis (CL2355), in which migration towards a chemoattractant is impaired by pan-neuronal expression of MSR1. Addition of MSR1 at 0.1 μM restored chemotaxis to approximately 90% (Figure 7e), comparable to wild-type or uninduced worms (not shown). Fluorescent muscle aggregates accumulate with age in strains expressing Q40::YFP in muscle. This strain mimics the expansion of the polyglutamine array in the huntingtin protein to a threshold sufficient to cause Huntington's disease in humans and paralysis in C. elegans. Figure 7F shows the intensity of aggregates in individual worms at 5 days after hatching. 10 μM MSR1 resulted in an approximately 50% reduction, whereas 0.1 μM resulted in a 35% reduction (P<0.005 different from vehicle-only controls for each treatment).

[0047] Consideration Aging is the most influential non-genetic risk factor for dementia, as well as many other lifestyle-related diseases that impose a significant burden on the elderly and on the healthcare system (Niccoli and Partridge, 2012). A wide range of diseases, including neurodegenerative diseases such as AD, Parkinson's disease, and amyotrophic lateral sclerosis (ALS), as well as hypertension (Ayyadevara et al., 2016d), sarcopenia (Ayyadevara et al., 2016c), and even some cancers (Ano Bom et al., 2012), all show the formation of unique aggregates that feature disease-specific "diagnostic" proteins. We have identified many proteins within immunopurified aggregate subsets (Ayyadevara et al., 2016b) and identified intraaggregate protein-protein interfaces by cross-linking (Balasubramaniam et al., 2019).

[0048] GFAP is one of many proteins that are abundant in AD aggregates compared to age-matched controls (Ayyadevara et al., 2016b), but it now joins a small set of neuropathology-associated proteins that exhibit disease-specific hyperphosphorylation. These include tau (AD, PD, ALS), Aβ 1-42 These include AD, TDP43 (ALS), and α-synuclein (PD) (Ayyadevara et al., 2016b; Bai et al., 2021; Ferrer et al., 2021; Mavroudis et al., 2020; Sternburg et al., 2021; Xu et al., 2015; Zhang et al., 2019). We identified three GFAP serines that are highly phosphorylated in aggregates isolated from the hippocampus of AD(3,3) individuals, and further identified additional serines and threonines near the N- and C-termini that are phosphorylated only in AD(4,4) aggregates (Figure 1C). Although only APOE3 or APOE4 homozygous tissues were investigated, it is reasonable to expect intermediate results for APOE3 / E4 heterozygotes as well.

[0049] The observed sites of GFAP phosphorylation in AD coincide with known targets of several kinases that have been predicted to play a role in the pathogenesis of AD. These include AKT2 (one of two mammalian AKT paralogs), Rho-associated kinase 1 (ROCK1), G protein-coupled receptor kinase 2 (BARK / GRK2), and protein kinase A (PKA). Alterations in insulin signaling have been implicated in AD, and AKT is a key downstream effector of the kinase cascade that transmits insulin / insulin-like signals (Chen et al., 2012;Yang et al., 2018;Zheng and Wang, 2021). GRK2 (also known as BARK) has also been shown to contribute to the pathogenesis of cardiovascular disease. PKA, a cAMP-dependent kinase, is involved in multiple signaling pathways, influences tau hyperphosphorylation, and has been implicated in the progression of several neurodegenerative diseases, including AD, PD, and HD (Carlyle et al., 2014;Dagda et al., 2011;Greggio et al., 2017;Li et al., 2018;Taylor et al., 2021). PKA has also been shown to play a role in diabetes (Li et al., 2018) and anxiety-related behaviors (Keil et al., 2016).

[0050] Knockdown of several kinases that may be responsible for the observed hyperphosphorylation of GFAP mitigated protein aggregation and associated physiological decline in C. elegans and in human neuroblastoma cells expressing amyloid precursor protein. Of note, some of these same kinases have putative target sites in other AD-related proteins, such as the microtubule-associated protein tau, which would be expected to further increase their impact. That these kinases have multiple targets that promote AD is suggested by the results shown in Figures 3C and 3E, where knockdown of some kinases resulted in more effective rescue from AD-like traits compared to knockdown of GFAP itself. However, it should be noted that these experiments did not monitor the efficacy of siRNA knockdown in neurons, which are typically less effective in neurons than in other target cells, making the above comparison misleading.

[0051] Based on our data, GFAP is a novel target to reduce the aggregation burden in AD and other aggregation-related diseases. Therefore, starting from the 3D structure of GFAP predicted by robust fold recognition and ab initio molecular orbital procedures, we screened for small molecules that specifically target GFAP (Yang et al., 2015). The initial lowest ΔG structure has a small, druggable pocket (Figure 2A). Time-dependent molecular dynamics simulations of the GFAP structure revealed that the binding cavity expands to about three times its initial volume upon transition to a metastable state. The molecular structure of GFAP at 200 ns (Figure 2C) was selected as a target for drug screening. Several descriptors (RMSD, RMSF, and pocket volume) were used to monitor the changes in the GFAP structure during the simulation.

[0052] Proteomics of brain aggregates showed striking differences in phosphorylation of GFAP from AD tissue. Although the phosphomimetic substitutions do not perfectly mimic actual protein phosphorylation, computer simulations of the predicted structures of AD(3,3) and AD(4,4) incorporating the observed pseudophosphorylation sites were consistent with the hypothesis that phosphorylation states can alter the structural dynamics of GFAP. Simulation data do not allow us to infer the extent to which phosphorylation of individual kinase targets contributes to destabilizing GFAP structure or to what extent it may facilitate access to kinase sites for subsequent modification.

[0053] The top three candidates from three successive screens were tested for efficacy in various aggregation model systems, with counterscreens to exclude agents that bind tubulin. Of these, MSR1 was nearly as effective as GFAP knockdown (using RNAi against GFAP or its closest C. elegans homolog) in each assay, and proteomic analysis of the aggregates revealed a nearly identical set of proteins depleted or excluded from the aggregates.

[0054] These results demonstrate the influence of GFAP and its predicted kinases on protein aggregation in human cell and C. elegans models of neuropathic aggregation, and demonstrate an in vivo anti-aggregation effect.

[0055] References JPEG2025500850000003.jpg42170 JPEG2025500850000004.jpg124170 JPEG2025500850000005.jpg102170 JPEG2025500850000006.jpg109170 JPEG2025500850000007.jpg109170 JPEG2025500850000008.jpg125170 JPEG2025500850000009.jpg95170 JPEG2025500850000010.jpg103170 JPEG2025500850000011.jpg109170 JPEG2025500850000012.jpg117170 JPEG2025500850000013.jpg109170 JPEG2025500850000014.jpg110170 JPEG2025500850000015.jpg109170 JPEG2025500850000016.jpg80170

Claims

1. A pharmaceutical composition for reducing the amount of aggregated protein in protein aggregates in a target, comprising an effective amount of a compound that stably binds to glial fibrillary acidic protein (GFAP), wherein the aggregated protein comprises BSN, SYN1, MAP2, PLEC, RAB10, MAP1A, DCTN, TUBA4A, SPART, PRKDC, or a combination thereof.

2. The pharmaceutical composition according to claim 1, wherein the subject is suffering from a neurodegenerative disease.

3. The pharmaceutical composition according to claim 2, wherein the neurodegenerative disease is Alzheimer's disease (AD).

4. The pharmaceutical composition according to claim 1, wherein the compound is MSR1.

5. The pharmaceutical composition according to claim 1, wherein GFAP is phosphorylated GFAP.

6. The pharmaceutical composition according to claim 1, wherein the compound has negligible affinity for α-tubulin, β-tubulin, or their oligomers.

7. The ΔG of the above compound relative to GFAP binding The pharmaceutical composition according to claim 1, wherein the calorific value is less than -40 kcal / mol.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the aggregated protein comprises BSN, SYN1, MAP2, PLEC, RAB10, MAP1A, DCTN, TUBA4A, SPART, and PRKDC.

9. A pharmaceutical composition for the treatment of subjects suffering from neurodegenerative diseases, comprising an effective amount of a compound that stably binds to GFAP.

10. The pharmaceutical composition according to claim 9, wherein the compound is MSR1.

11. The pharmaceutical composition according to claim 9, wherein the neurodegenerative disease is AD.

12. The pharmaceutical composition according to claim 9, wherein the GFAP is phosphorylated GFAP.

13. The pharmaceutical composition according to claim 9, wherein the compound has negligible affinity for α-tubulin, β-tubulin, or their oligomers.

14. The aforementioned compound ΔG binding The pharmaceutical composition according to any one of claims 9 to 13, wherein the calorific value is less than -50 kcal / mol.

15. A pharmaceutical composition for increasing the amount of aggregated protein in protein aggregates in a target, comprising an effective amount of a compound that stably binds to glial fibrillary acidic protein (GFAP), wherein the aggregated protein comprises CBX8, TSPYL5, CDK2, KRT33B, or a combination thereof.

16. The pharmaceutical composition according to claim 15, wherein the subject is suffering from a neurodegenerative disease.

17. The pharmaceutical composition according to claim 16, wherein the neurodegenerative disease is Alzheimer's disease (AD).

18. The pharmaceutical composition according to claim 15, wherein the compound is MSR1.

19. The pharmaceutical composition according to claim 15, wherein GFAP is phosphorylated GFAP.

20. The pharmaceutical composition according to claim 15, wherein the compound has negligible affinity for α-tubulin, β-tubulin, or their oligomers.

21. The aforementioned compound ΔG binding The pharmaceutical composition according to claim 15, wherein the calorific value is less than -40 kcal / mol.

22. The pharmaceutical composition according to any one of claims 15 to 21, wherein the aggregated protein comprises CBX8, TSPYL5, CDK2, and KRT33B.

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