The present disclosure relates to method of modulating autophagy by modulators of autophagy, wherein the autophagy includes but not limiting to macroautophagy, chaperone mediated autophagy and microautophagy. The present disclosure further relates to modulators of autophagy for increasing or decreasing the autophagic flux. The disclosure also relates to modulator per se in modulating autophagy.
Autophagy is a natural degradation pathway that ensures orderly degradation of damaged or dysfunctional cellular components. It is an evolutionarily conserved process in which cell's own components are degraded by the lysosomal machinery. The process involves isolation of the targeted cytoplasmic constituents within a vesicle known as an autophagosome which is surrounded by a double-membrane. This is followed by fusion of the autophagosome with a lysosome to form an autolysosome, where the engulfed contents referred to as ‘cargo’ are subjected to enzymatic degradation. The degradation products, like amino acids and other basic building blocks, are recycled back to the cytoplasm and are used up by the cell (Rabinowitz and White 2010). This conserved pathway from yeast to humans play critical role in cell survival during nutritional deprivation, clearance of damaged/superfluous organelles, protein aggregates and intracellular pathogens. Dysfunction of autophagy leads to cell death, cancer, neurodegenerative and other diseases. Therefore, there is a need for proper function and balance in the action of the autophagy to preserve homeostasis, and present invention aims at doing so in a manner as described herein below.
Accordingly, the present disclosure relates to a method of modulating autophagy in a cell comprising step of contacting cell with at least one autophagy modulator, wherein the modulator is mTOR dependent or mTOR independent and wherein the modulator enhances autophagosome lysosome fusion or inhibits autophagosome biogenesis autophagosome maturation or degradation of autophagy proteins, degradation of autophagic cargo following authophagosome lysosome fusion.
In an embodiment, the present disclosure relates to a modulator of autophagy for enhancing formation of autolysosome by promoting autophagosome and lysosome fusion, or inhibits autophagosome biogenesis, autophagosome maturation, degradation of autophagic cargo following autophagosome-lysosome fusion, or any combination thereof, thereby decreasing autophagic flux.
In another embodiment, the present disclosure relates to a modulator of autophagy, wherein the modulator enhances formation of autolysosome by promoting fusion of autophagosome and lysosome, thereby increasing autophagic flux or inhibits at least one of autophagosome biogenesis, autophagosome maturation, degradation of autophagic cargo inside vacuole after autophagosome-lysosome fusion, or any combination thereof, thereby decreasing autophagic flux.
In order that the invention may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure wherein:
(A) illustrates the degradation of luciferase assay, wherein S. cerevisiae shuttle vectors pRS306 (URA) and pRS305 (LEU) are used to clone the POT1 promoter and the firefly and Renilla luciferase genes, respectively.
(B) illustrates gradual decrease in luciferase counts upon induction of autophagy in wild type cells, whereas cells carrying core autophagy mutants atg1 and atg5 and selective autophagy mutant atg36 (adaptor protein for pexophagy) did not show any drop in the luciferase activity over time.
(A) illustrates screening two small molecule libraries for their effect on autophagy using luciferase based assay for monitoring autophagy.
(B) illustrates dose dependent effect on the rates of degradation of firefly luciferase by Bay11-7082 and ZPCK.
(A) illustrates box plot (representative plot for 100 compounds) demonstrating hits from small molecule library of pharmacologically active compounds, LOPAC1280, screened in S. cerevisiae toxicity model of α-synuclein. In the box plot, compounds that rescued the growth lag due to α-synuclein toxicity (denoted by absorbance, A600) of WT α-synuclein-EGFP strains≥3 SD units (grey box) are considered hits (blue) and the ones that did not rescue the growth lag due to α-synuclein toxicity are in green. WT EGFP (black) and untreated WT α-synuclein-EGFP (red) represent the positive and negative controls.
(B) illustrates growth curve of WT EGFP cells with or without 6-Bio (50 μM) treatment.
(C) illustrates western blot of GFP-Atg8 processing assay under growth condition, wherein fusion protein GFP-Atg8 accumulation and free GFP release is monitored across time course (0 h and 6 h) with or without 6-Bio (50 μM) treatment, respectively.
(D) illustrates western blot of GFP-Atg8 processing assay under starvation condition, wherein fusion protein GFP-Atg8 accumulation and free GFP release is monitored across time course (0 h, 2 h, 4 h and 6 h) with or without 6-Bio (50 μM) treatment, respectively.
(E) illustrates microscopy images of ptf LC3 transfected HeLa cells treated with 6-Bio (5 μm) and quantification of autophagosome and autolysosome indicating fold change over its untreated counterpart (n=25), scale bar=15 μm.
(A) Illustrates microscopy images of WT α-synuclein-EGFP yeast cells treated with or without 6-Bio (50 μM) for 16 h, vacuole stained with CMAC-Blue (100 nM), scale bar=2 μm.
(B) illustrates quantification plot for α-synuclein-EGFP degradation assay in wild-type (WT) yeast strain under growth condition upon treatment with 6-Bio (50 μM).
(C) illustrates quantification plot for α-synuclein-EGFP degradation assay in wild-type (WT) yeast strain under starvation condition upon treatment with 6-Bio (50 μM).
(D) illustrates quantification plot for α-synuclein-EGFP degradation assay in autophagy mutant (atg1Δ) strain under growth condition upon treatment with 6-Bio (50 μM).
(E) illustrates quantification plot for α-synuclein-EGFP degradation assay in autophagy mutant (atg1Δ) strain under starvation condition upon treatment with 6-Bio (50 μM).
(F) illustrates western blot (below) and graph (above) indicating fold change in EGFP-α-synuclein degradation in SH-SY5Y cells upon treatment with 6-Bio (5 μM), 3-MA (5 mM) and both, respectively.
(A) illustrates western blots indicating dose-dependent modulation of autophagy related proteins (LC3, P70S6 kinase and 4E-BPI) by 6-Bio in HeLa cells.
(B) Illustrates photomicrographs of TH+ immunostained dopaminergic neurons in SNpc (arrow) of mouse midbrain in Control group, MPTP treated (23 mg/kg body weight) mouse, 6-Bio (5 mg/kg body weight) treated mouse and both [Prophylaxis (MPTP+Pro) and Co-administration (MPTP+Co)], scale bar=300 μm.
(C) illustrates stereological quantification indicating the number of TH+ DA and its intensity in SNpc neurons.
(D)illustrates densitometric quantification indicating the number of TH+ DA and its intensity in SNpc neurons.
(A) illustrates a plot indicating the percent growth of WT α-synuclein-EGFP strain in presence of Agk2 (50 μM) and 6-Bio (50 μM).
(B) illustrates growth of autophagy mutants (atg1Δ, atg5Δ, atg8Δ, atg11Δ and atg15Δ) expressing α-synuclein-EGFP observed with or without 6-Bio (50 μM).
(A) illustrates a schematic representation of α-synuclein-EGFP degradation assay conditions.
(B) illustrates western blots for α-synuclein-EGFP degradation upon 6-Bio (50 μM) administration in wild type cell under growth condition.
(B) illustrates western blots for α-synuclein-EGFP degradation upon 6-Bio (50 μM) administration in wild type cell under starvation condition.
(C) illustrates western blots for α-synuclein-EGFP degradation upon 6-Bio (50 μM) administration in autophagy mutant (atg1Δ) cells under growth condition.
(D) illustrates western blots for α-synuclein-EGFP degradation upon 6-Bio (50 μM) administration in autophagy mutant (atg1Δ) cells under starvation condition.
(A) illustrates the schedule of dosage administration of MPTP (23 mg/kg) and 6-Bio (5 mg/kg) in mice groups.
(B) illustrates photomicrographs of TH+ immunostained DA neurons in SNpc of mouse midbrain of control, MPTP, 6-Bio and both [Prophylaxis (MPTP+Pro) and Co-administration (MPTP+Co)]groups.
(C) illustrates quantitative plot of SNpc volume of mouse brains for all the groups (control, MPTP, 6-Bio and both [Prophylaxis (MPTP+Pro) and Co-administration (MPTP+Co)]).
(A) illustrates the effect of acacetin on U1752 cells infected with Salmonella typhimurium SL1344.
(B) illustrates the effect of acacetin on HeLa cells infected with Salmonella typhimurium SL1344,
(A) illustrates the imageslive cell microscopic images of GFP-LC.3 transfected HeLa cells infected with mcherry-Salmonella typhimuriumSL1344 and treated with gentamycin.
(B) illustrates the imageslive cell microscopic images of GFP-LC3 transfected HeLa cells infected with mcherry-Salmonella typhimuriumSL1344 and treated with gentamycin, followed by acacetin.
(C) illustrates intensity of the red channel featured in live cell microscopic images measured using image J-Stacks T function.
(A) illustrates ptf-LC3 transfected HeLa cells treated with Acacetin for 2 hours.
(B) illustrates number of autophagosomes and autolysosomes counted using image J-cell counter function.
(A) illustrates POT1-GFP processing assay for accessing the effect of Bay11-7082 and ZPCK pexophagy under starvation condition.
(B) illustrates GFP-Atg8 assay for accessing the effect of Bay11-7082 and ZPCK on general autophagy.
(C) illustrates pexophagy as monitored via fluorescence microscopy.
(D) illustrates protease protection assay depicting conversion of precursor to matured form of aminopeptidase on treatment with proteinase K in Bay11-7082 treated cells.
Example 40 illustrates exertion of cellular neuroprotection by XCT 790 in an autophagy dependent mechanism. (a) Representative western blot of LC3 processing assay in SHSY-5Y cells treated with XCT 790 (2 h) under growth condition and normalized LC3-II levels were quantified. β-tubulin was used as a loading control. (b) Representative microscopy images of tandem RFP-EGFP-LC3 assay in HeLa cells treated with XCT 790 for 2 h. Yellow puncta was autophagosomes and red was autolysosomes. Fold change in autophagosomes and autolysosomes by XCT 790 were quantified. Scale bar was 15 μm. (c) Graph indicating the cell viability read out of SHSY-5Y overexpressing EGFP-α-synuclein treated with XCT 790 in presence of pharmacological autophagy inhibitor 3-MA. Cell viability was analysed using CellTitre Glo (Promega) assay. More RLU readout was indicative of more cell viability and vice-versa. (d) Representative western blots of mTOR substrates like P70S6K (phospho and total form) and 4EBP1 (phospho and total form) regulation by various treatments like XCT 790, EBSS and LiCl. β-tubulin was used as a loading control. (e) Representative western blots of signaling pathway proteins like AMPK (phospho and total form) and ULK1 (phospho and total form) regulation by XCT 790 and EBSS. β-tubulin was used as a loading control. Concentrations of XCT 790, 3-MA and LiCl used were 5 μM, 100 nM and 10 mM. Statistical analysis was performed. using one-way ANOVA and post-hoc Bonferroni test, Error bars, mean±SEM. ns-non significant, **-P<0.01, ***-P<0.001.
The present disclosure relates to a method of modulating autophagy in a cell comprising step of contacting cell with at least one autophagy modulator, wherein the modulator is mTOR dependent or mTOR independent and wherein the modulator enhances autophagosome lysosome fusion or inhibits autophagosome biogenesis autophagosome maturation or degradation of autophagy proteins, degradation of autophagic cargo following authophagosome lysosome fusion
In an embodiment, the modulator of autophagy is selected from a group comprising (2′Z,3′E)-6-Bromoindirubin-3′-oxime (6-Bio), acacetin, 7-dihydroxy-2-(4-methoxyphenyl)chromen-4-oneN6-(4-Aminobenzyl)-9-[5-(methylcarbonyl)-β-D-ribofuranosyl]adenine(AB-MECA, Lapidine; (3S,3aR,4R,8aR)-3-hydroxy-6,8a-dimethyl-8-oxo-3-propan-2-yl-2,3a,4,5-tetrahydro-1H-azulen-4-yl] (E)-2-methylbut-2-enoate, Senecionine, 12-Hydroxysenecionan-11,16-dione, XCT790; (3-[4-(2,4Bis-trifluorormethylbenzyloxy)-3-methoxyphenyl]-2-cyano-N-(5-trifluoromethyl-1,3,4-thiadiazol-2-yl)acrylamide), PD180970 (6-(2,6-Dichlorophenyl)-2-[(4fluoro-3-methylphenyl)amino]-8-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one), Ritodrine hydrochloride; (N-(p-Hydroxyphenethyl)-4-hydroxynorephedrine hydrochloride) and SB 242084 dihydrochloride hydrate; (6-Chloro-2,3-dihydro-5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1H-indole-1-carboxyamide dihydrochloride hydrate), N-Carbobenzyloxy-L-phenylalanylchloromethyl ketone (ZPCK), 3-[(4-methylphenyl)sulfonyl]-(2E)-propenenitrile (Bay11-7082), Elaidylphosphocholine, N-[4-(1H-Benzimidazol-2-yl)phenyl]-5-nitro-2-thiophenecarboxamide, Ethyl [(2-{[(5-nitro-2-thienyl)carbonyl]amino}-3-thienyl)carbonyl]carbamate, 2-Phenyl-N-[5-(3-thienyl)-1,3,4-oxadiazol-2-yl]-2H-1,2,3-triazole-4-carboxamide, 3-Bromo-N-[5-(5,6-dihydro-1,4-dioxin-2-yl)-1,3,4-oxadiazol-2-yl]benzamide, N-(6-Chloro-1,3-benzothiazol-2-yl)-2-(4-fluorophenyl)-N-(3-pyridinylmethyl)acetamide. N-(2-Methoxybenzyl)-5-nitro-4,6-pyrimidinediamine, N-[5-(2,5-Dichloro-3-thienyl)-1,3,4oxadiazol-2-yl]-5-nitro-2-furamide, N-2,5-Dimethylphenyl)-2-{[3-(3-methoxyphenyl)-4-oxo-3,4,6,7-tetrahydrothieno[3,2-d]pyrimidin-2-yl]sulfanyl}acetamide, 4-Fluoro-N-[2-(4-fluorophenyl)-2-(4-methyl-1-piperazinyl)ethyl]-3-methylbenzenesulfonamide, N-[5-(3,4-Dimethylphenyl)-1,3,4-oxadiazol-2-yl]-5-nitro-2-furamide, N-[3-(1,3-Benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl]-4-cyanobenzamide hydrochloride (1:1), N-(4,5-Diphenyl-1,3-thiazol-2-yl)-1,2-oxazole-5-carboxamide, 2-[5-(2,4-Difluorophenyl)-1,2-oxazol-3-yl]-N-(1,2-oxazol-3-yl)acetamide, 1-(2,4-Dihydroxy-3-methylphenyl)-2-4-propylphenoxy)ethanone, N-[5-(2,4-Dimethylphenyl)-1,3,4-oxadiazol-2-yl]-3,5-dimethoxybenzamide, N-[3-(1,3-Benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl]-1-methyl-1H-pyrazole-3-carboxamide hydrochloride (1:1), 2-(2-Bromobenzyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide, 2-[(5,7-Dibromo-8-quinolinyl)oxy]propanamide, 3-Amino-N-[4-(imidazo[1,2-a]pyridin-2-yl)phenyl]-2-pyrazinecarboxamide, 1-Benzyl-N-[5-chloro-2-(1-piperidinyl)phenyl]-1H-1,2,3-triazole-4-carboxamide, (4-Bromophenyl){4[(5-chloro-2-thienyl)sulfonyl]-1-piperazinyl}methanone, 1-Phenyl-N-(1,3,4-thiadiazol-2-yl)-3-(2-thienyl)-1H-pyrazole-5-carboxamide, 5-{[(7-Bromo-2,3-dihydro-1,4-benzodioxin-6-yl)methyl]sulfanyl}-N-cyclopropyl-1,3,4-thiadiazol-2-amine, 1-Ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid, Pentachlorophenol, N,N,N-Trimethyl-1-hexadecanaminium bromide, 1,4-Dihydroxy-5,8-bis({2-[(2-hydroxyethyl)amino]ethyl}amino)-9,10-anthraquinone dihydrochloride, (9E)-9-Octadecen-1-yl 2-(trimethylammonio)ethyl phosphate, 4-(1H-Indazol-3-yl)-N-(4-piperidinyl)-1H-pyrrolo[2,3-b]pyridin-6-amine, 2-(4-Methoxybenzyl)-6-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylmethyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidine, [(E)-octadec-9-enyl] 2-(trimethylazaniumyl)ethyl phosphate), TNP (N6-[(4-nitrophenyl)methyl]-N2-[[3-(trifluoromethyl)phenyl]methyl]-9H-Purine-2,6-diamine), Cinchonidine; (R)-[(2S,4S,5R)-5-ethenyl-1-azabicyclo[2,2,2]octan-2-yl]-quinolin-4-ylmethanol, Trioxsalen; (2,5,9-Trimethylfuro[3,2-g]benzopyran-7-one) or any combination thereof.
In another embodiment, the modulator of autophagy is 6-Bio, XCT-790, ZPCK., acacetin or Bay-11.
In yet another embodiment, 6-Bio or XCT-790 enhances autolysosome formation in the cell and causes degradation of α-synuclein (SNCA).
In still another embodiment, the 6-Bio enhances fusion of autophagosome and lysosome in the cell by about 8 fold to 10 fold.
In still another embodiment, the 6-Bio modulates autophagy by passive diffusion and the 6-Bio is mTOR dependent and GSK3B dependent.
In still another embodiment, the XCT-790 is mTOR independent and ERRα dependent.
In still another embodiment, the Bay-11 inhibits autophagosome lysosome fusion, autophagosome biogenesis or autophagosome maturation.
In still another embodiment, the ZPCK inhibits degradation of autophagic cargo inside the vacuole after fusion of autophagosome and lysosome.
In still another embodiment, the acacetin induces formation of autophagolysosome in the cell infected with intracellular microorganism.
In still another embodiment, the intracellular microorganism is selected from a group comprising Salmonella typhimurium Legionella pneumophila, Listeria monooytogenes, Shigella flexneri, Streptococcus pyrogenes, Mycobacterium tuberculosis, or any combination thereof.
In still another embodiment, the autophagy is selected from a group comprising macroautophagy, chaperone mediated autophagy, microautophagy, mitophagy, pexophagy, liphophagy, reticulophagy, ribophagy, zymophagy, Aggrephagy, xenophagy, or any combinations thereof.
In still another embodiment, the cell is eukaryotic cell selected from a group comprising yeast cell, plant cell and mammalian cell, or a combination thereof.
In still another embodiment, the concentration of the modulator is ranging from about 1 μM to about 150 μM.
The present disclosure further relates to a modulator of autophagy for enhancing formation of autolysosome by promoting autophagosome and lysosome fusion, or inhibits autophagosome biogenesis, autophagosome maturation, degradation of autophagic cargo following autophagosome-lysosome fusion, or any combination thereof, thereby decreasing autophagic flux.
In an embodiment, the modulator of autophagy is selected from a group comprising (2′Z,3′E)-6-Bromoindirubin-3′-oxime (6-Bio), acacetin, 7-dihydroxy-2-(4-methoxyphenyl)chromen-4-oneN6-(4-Aminobenzyl)-9-[5-(methylcarbonyl)-β-D-ribofuranosyl]adenine(AB-MECA, Lapidine; (3S,3aR,4R,8aR)-3-hydroxy-6,8a-dimethyl-8-oxo-3-propan-2-yl-2,3a,4,5-tetrahydro-1H-azulen-4-yl](E)-2-methylbut-2-enoate, Senecionine, 12-Hydroxysenecionan-11,16-dione, XCT790; (3-[4-(2,4-Bis-trifluoromethylbenzyloxy)-3-methoxyphenyl]-2-cyano-N-(5-trifluoromethyl-1,3,4-thiadiazol-2-yl)acrylamide), PD180970 (6-(2,6-Dichlorophenyl)-2-[(4fluoro-3-methylphenyl)amino]-8-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one), Ritodrine hydrochloride; (N-(p-Hydroxyphenethyl)-4-hydroxynorephedrine hydrochloride) and SB 242084 dihydrochloride hydrate; (6-Chloro-2,3-dihydro-5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1H-indole-1-carboxyamide dihydrochloride hydrate), N-Carbobenzyloxy-L-phenylalanylchloromethyl ketone (ZPCK), 3-[(4-methylphenyl)sulfonyl]-(2E)-propenenitrile (Bay11-7082), Elaidylphosphocholine, N-[4-(1H-1-Benzimidazol-2-yl)phenyl]-5-nitro-2-thiophenecarboxamide, Ethyl [(2-{[(5-nitro-2-thienyl)carbonyl]amino}-3-thienyl)carbonyl]carbamate, 2-Phenyl-N-[5-(3-thienyl)-1,3,4-oxadiazol-2-yl]-2H-1,2,3-triazole-4-carboxamide, 3-Bromo-N-[5-(5,6-dihydro-1,4-dioxin-2-yl)-1,3,4-oxadiazol-2-yl]benzamide, N-(6-Chloro-1,3-benzothiazol-2-yl)-2-(4-fluorophenyl)-N-(3-pyridinylmethyl)acetamide, N-(2-Methoxybenzyl)-5-nitro-4,6-pyrimidinediamine, N-[5-(2,5-Dichloro-3-thienyl)-1,3,4-oxadiazol-2-yl]-5-nitro-2-furamide, N-(2,5-Dimethylphenyl)-2-{[3-(3-methoxyphenyl)-4-oxo-3,4,6,7-tetrahydrothieno[3,2-d]pyrimidin-2-yl]sulfanyl}acetamide, 4-Fluoro-N-[2-(4-fluorophenyl)-2-(4-methyl-1-piperazinyl)ethyl]-3-methylbenzenesulfonamide, N-[5-(3,4-Dimethylphenyl)-1,3,4-oxadiazol-2-yl]-5-nitro-2-furamide, N-[3-(1,3-Benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl]-4-cyanobenzamide hydrochloride (1:1), N-(4,5-Diphenyl-1,3-thiazol-2-yl)-1,2-oxazole-5-carboxamide, 2-[5-(2,4-Difluorophenyl)-1,2-oxazol-3-yl]-N-(1,2-oxazol-3-yl)acetamide, 1-(2,4-Dihydroxy-3-methylphenyl)-2-(4-propylphenoxy)ethanone, N-[5-(2,4-Dimethylphenyl)-1,3,4-oxadiazol-2-yl]-3,5-dimethoxybenzamide, N-[3-(1,3-Benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl]-1-methyl-1H-pyrazole-3-carboxamide hydrochloride (1:1), 2-(2-Bromobenzyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide, 2-[(5,7-Dibromo-8-quinolinyl)oxy]propanamide, 3-Amino-N-[4-(imidazo[1,2-a]pyridin-2-yl)phenyl]-2-pyrazinecarboxamide, 1-Benzyl-N-[5-chloro-2-(1-piperidinyl)phenyl]-1H-1,2,3-triazole-4-carboxamide, (4-Bromophenyl){4-[(5-chloro-2-thienyl)sulfonyl]-1-piperazinyl}methanone, 1-Phenyl-N-(1,3,4-thiadiazol-2-yl)-3-(2-thienyl)-1H-pyrazole-5-carboxamide, 5-{[(7-Bromo-2,3-dihydro-1,4-benzodioxin-6-yl)methyl]sulfanyl}-N-cyclopropyl-1,3,4-thiadiazol-2-amine, 1-Ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-napthyridine-3-carboxylic acid, Pentachlorophenol, N,N,N-Trimethyl-1-hexadecanaminium bromide, 1,4-Dihydroxy-5,8-bis({2-[(2-hydroxyethyl)amino]ethyl}amino)-9,10-anthraquinone dihydrochloride, (9E)-9-Octadecen-1-yl 2-(trimethylammonio)ethyl phosphate, 4-(1H-Indazol-3-yl)-N-(4-piperidinyl)-1H-pyrrolo[2,3-b]pyridin-6-amine, 2-(4-Methoxybenzyl)-6-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylmethyl)-6,7-dihydro-5H-pyrrolo [3,4-d] pyrimidine, [(E)-octadec-9-enyl] 2-(trimethylazaniumyl)ethyl phosphate), TNP (N6-[(4-nitrophenyl)methyl]-N2-[[3-(trifluoromethyl)phenyl]methyl]-9H-Purine-2,6-diamine), Cinchonidine; (R)-[(2S,4S,5R)-5-ethenyl-1-azabicyclo[2.2.2]octan-2-yl]-quinolin-4-ylmethanol, Trioxsalen; (2,5,9-Trimethylfuro[3,2-g]benzopyran-7-one) or any combination thereof.
In another embodiment, the 6-Bio or XCT-790 enhances autolysosome formation in the cell and causes degradation of ax-synuclein (SNCA).
In yet another embodiment, the 6-Bio modulates autophagy by passive diffusion and wherein the 6-Bio is mTOR dependent and GSK3B dependent; wherein the XCT-790 is mTOR independent and ERRα dependent while modulating the autophagy and wherein the XCT-790 is inverse agonist of ERRα.
The present disclosure further relates to a modulator of autophagy, wherein the modulator enhances formation of autolysosome by promoting fusion of autophagosome and lysosome, thereby increasing autophagic flux or inhibits at least one of autophagosome biogenesis, autophagosome maturation, degradation of autophagic cargo inside vacuole after autophagosome-lysosome fusion, or any combination thereof, thereby decreasing autophagic flux.
In an embodiment, the modulator is selected from a group comprising (2′Z,3′E)-6-Bromoindirubin-3′-oxime (6-Bio), acacetin, 7-dihydroxy-2-(4-methoxyphenyl)chromen-4-oneN6-(4-Aminobenzyl)-9-[5-(methylcarbonyl)-β-D-ribofuranosyl]adenine(AB-MECA, Lapidine; (3S,3aR,4R,8aR)-3-hydroxy-6,8a-dimethyl-8-oxo-3-propan-2-yl-2,3a,4,5-tetrahydro-1H-azulen-4-yl](E)-2-methylbut-2-enoate, Senecionine, 12-Hydroxysenecionan-11,16-dione, XCT790; (3-[4-(2,4-Bis-trifluoromethylbenzyloxy)-3-methoxyphenyl]-2-cyano-N-(5-trifluoromethyl-1,3,4-thiadiazol-2-yl)acrylamide), PD180970 (6-(2,6-Dichlorophenyl)-2-[(4-fluoro-3-methylphenyl)amino]-8-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one), Ritodrine hydrochloride; (N-(p-Hydroxyphenethyl)-4-hydroxynorephedrine hydrochloride) and SB 242084 dihydrochloride hydrate; (6-Chloro-2,3-dihydro-5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1H-indole-1-carboxyamide dihydrochloride hydrate), N-Carbobenzyloxy-L-phenylalanylchloromethyl ketone (ZPCK), 3-[(4-methylphenyl)sulfonyl]-2E)-propenenitrile (Bay11-7082),Elaidylphosphocholine, N-[4-(1H-Benzimidazol-2-yl)phenyl]-5-nitro-2-thiophenecarboxamide, Ethyl [(2-{[(5-nitro-2-thienyl)carbonyl]amino}-3-thienyl)carbonyl]carbamate, 2-Phenyl-N-[5-(3-thienyl)-1,3,4-oxadiazol-2-yl]-2H-1,2,3-triazole-4-carboxamide, 3-Bromo-N-[5-(5,6-dihydro-1,3,4-oxadiazol-2-yl]benzamide, N-(6-Chloro-1,3-benzothiazol-2-yl)-2-(4-fluorophenyl)-N-(3-pyridinylmethyl)acetamide, N-(2-Methoxybenzyl)-5-nitro-4,6-pyrimidinediamine, N-[5-(2,5-Dichloro-3-thienyl)-1,3,4-oxadiazol-2-yl]-5-nitro-2-furamide, N-(2,5-Dimethylphenyl)-2-{[3-(3-methoxyphenyl)-4-oxo-3,4,6,7-tetrahydrothieno[3,2-d]pyrimidin-2-yl]sulfanyl}acetamide, 4-Fluoro-N-[2-(4-fluorophenyl)-2-4-methyl-1-piperazinyl)ethyl]-3-methylbenzenesulfonamide, N-[5-(3,4-Dimethylphenyl)-1,3,4-oxadiazol-2-yl]-5-nitro-2-furamide, N-[3-(1,3-Benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl]-4-cyanobenzamide hydrochloride (1.1), N-(4,5-Diphenyl-1,3-thiazol-2-yl)-1,2-oxazole-5-carboxamide, 2-[5-(2,4,-Difluorophenyl)-1,2-oxazol-3-yl]-N-(1,2-oxazol-3-yl)acetamide, 1-(2,4-Dihydroxy-3-methylphenyl)-2-(4-propylphenoxy)ethanone, N-[5-(2,4-Dimethylphenyl)-1,3,4-oxadiazol-2-yl]-3,5-dimethoxybenzamide, N-[3-(1,3-Benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl]-1-methyl-1H-pyrazole-3-carboxamide hydrochloride (1:1), 2-(2-Bromobenzyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide, 2-[(5,7-Dibromo-8-quinolinyl)oxy]propanamide, 3-Amino-N-[4-(imidazo[1,2-a]pyridin-2-yl)phenyl]-2-pyrazinecarboxamide, 1-Benzyl-N-[5-chloro-2(1-piperidinyl)phenyl]-1H-1,2,3-triazole-4-carboxamide, (4-Bromophenyl){4-[(5-chloro-2-thienyl)sulfonyl]-1-piperazinyl}methanone, 1-Phenyl-N-(1,3,4-thiadiazol-2-yl)-3-2-thienyl)-1H-pyrazole-5-carboxamide, 5-{[(7-Bromo-2,3-dihydro-1,4-benzodioxin-6-yl)methyl]sulfanyl}-N-cyclopropyl-1,3,4-thiadiazol-2-amine, 1-Ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid, Pentachlorophenol, N,N,N-Trimethyl-1-hexadecanaminium bromide, 1,4-Dihydroxy-5,8-bis({2-[(2-hydroxyethyl)amino]ethyl}amino)-9,10-anthraquinone dihydrochloride, (9E)-9-Octadecen-1-yl 2-(trimethylammonio)ethyl phosphate, 4-(1H-Indazol-3-yl)-N-(4-piperidinyl)-1H-pyrrolo[2,3-b]pyridin-6-amine, 2-(4-Methoxybenzyl)-6-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylmethyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidine, [(E)-octadec-9-enyl] 2-(trimethylazaniumyl)ethyl phosphate), TNP (N6-[(4-nitrophenyl)methyl]-N2-[[3-trifluoromethyl)phenyl]methyl]-9H-Purine-2,6-diamine), Cinchonidine; (R)-[(2S,4S,5R)-5-ethenyl-1-azabicyclo[2.2.2]octan-2-yl]-quinolin-4-ylmethanol, Trioxsalen; (2,5,9-Trimethylfuro[3,2-g]benzopyran-7-one) or any combination thereof.
In another embodiment, the 6-Bio or XCT-790 enhances autolysosome formation in the cell and causes degradation of +-synuclein (SNCA); wherein the 6-Bio modulates autophagy by passive diffusion and wherein the 6-Bio is mTOR dependent and GSK3B dependent; and wherein the XCT-790 is mTOR independent and ERRα dependent while modulating the autophagy and wherein the XCT-790 is inverse agonist of ERRα.
The present disclosure relates to a method for modulating autophagy.
In an embodiment of the present disclosure, the method of modulating autophagy involves treating cells with modulators of autophagy.
In an embodiment, the modulator of autophagy is mTOR dependent or mTOR independent, wherein the modulator in the method enhances formation of autolysosome by promoting fusion of autophagosome and lysosome.
In another embodiment, the modulator of autophagy employed in the method is mTOR dependent and GSK3B dependent.
In another embodiment, the modulator of autophagy employed in the method is mTOR independent but ERRα dependent while modulating the autophagy.
In an embodiment, the method of the present disclosure modulates autophagy including but not limiting to macroautophagy, chaperone mediated autophagy and microautophagy
In an embodiment, in the method of the present disclosure the modulator includes but not limited to activator, wherein the modulator which is an activator enhances formation of autolysosomes by promoting fusion of autophagosome and lysosome, thereby increasing autophagic flux.
In an embodiment, the method of modulating autophagy comprises the step of treating cells with modulator of autophagy which is mTOR dependent or mTOR independent, wherein the modulator enhances formation of autolysosome by promoting fusion of autophagosome and lysosome, thereby increasing autophagic flux.
In an embodiment, the modulator which is an activator of autophagy is selected from a group comprising (2′Z,3′E)-6-Bromoindirubin-3′-oxime (6-Bio), acacetin 5,7-dihydroxy-2-(4 methoxyphenyl)chromen-4-one N6-(4-Aminobenzyl)-9-[5-(methylcarbonyl)-β-D-ribofuranosyl]adenine(AB-MECA), Lapidine; [(3S,3aR,4R,8aR)-3-hydroxy-6,8a-dimethyl-8-oxo-3-propan-2-yl-2,3a,4,5-tetrahydro-1H-azulen-4-yl](E)-2-methylbut-2-enoate, Senecionine; 12-Hydroxysenecionan-11,16-dione, XCT790; (3-[4(2,4-Bis-trifluoromethylbenzyloxy)-3-methoxyphenyl]-2-cyano-N-(5-trifluoromethyl-1,3,4-thiadiazol-2-yl)acrylamide), PD180970; (6-(2,6-Dichlorophenyl)-2-[(4-fluoro-3-methylphenyl)amino]-8-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one), Ritodrine hydrochloride; (N-(p-Hydroxyphenethyl)-4-hydroxynorephedrine hydrochloride) and SB 242084 dihydrochloride hydrate; (6-Chloro-2,3-dihydro-5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1H-indole-1-carboxyamide dihydrochloride hydrate), or any combination thereof.
In an exemplary embodiment, the modulator which is an activator of macroautophagy, an activator of chaperone mediated autophagy and an activator of microautophagy, is selected from a group comprising (2′Z,3′E)-6-Bromoindirubin-3′-oxime (6-Bio), acacetin 5,7-dihydroxy-2-(4-methoxyphenyl)chromen-4-oneN6-(4-Aminobenzyl)-9-[5-(m ethylcarbonyl)-β-D-ribofuranosyl]adenine(AB-MECA, Lapidine; [(3S,3aR,4R,8aR)-3-hydroxy-6,8a-dimethyl-8-oxo-3-propan-2-yl-2,3a,4,5-tetrahydro-1H-azulen-4-yl](E)-2-methylbut-2-enoate, Senecionine; 12-Hydroxysenecionan-11,16-dione, XCT790; (3-[4-(2,4-Bis-trifluoromethylbenzyloxy)-3-methoxyphenyl]-2-cyano-N-(5-trifluoromethyl-1,3,4-thiadiazol-2-yl)acrylamide), PD180970; (6-(2,6-Dichlorophenyl)-2-[(4-fluoro-3-methylphenyl)amino]-8-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one), Ritodrine hydrochloride; (N-(p-Hydroxyphenethyl)-4-hydroxynorephedrine hydrochloride) and SB 242084 dihydrochloride hydrate; (6-Chloro-2,3-dihydro-5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1H-indole-1-carboxyamide dihydrochloride hydrate), or any combination thereof.
In an embodiment, the modulator in the method of the present disclosure induces autophagy including but not limiting to macroautophagy, chaperone mediated autophagy and microautophagy, thereby enhances or increases autophagic flux
In another embodiment, the modulator in the method of the present disclosure while modulating autophagy restores homeostasis, particularly restores cellular homeostasis.
In an embodiment, the method enhances starvation induced autophagy including but not limiting to macroautophagy, chaperone mediated autophagy and microautophagy.
In an embodiment, the method enhances autolysosme formation in autophagy including but not limiting to macroautophagy, chaperon mediated autophagy and microautophagy.
In an embodiment, the method increases autolysosome number(s) by about 8 fold to about 12 fold in autophagy when compared to an autophagy devoid of modulator of the present disclosure, indicating enhanced fusion of autophagosomes with lysosomes by the modulator of autophagy in the method of the of the present disclosure.
In an embodiment, the method causes about 2 fold to about 4 fold increase in the autophagic flux when compared to the autophagy which is not driven by the method of the present disclosure.
In an embodiment, the method of the present disclosure enhances formation of autolysosome and increases autophagic flux during starvation or growth or both.
In a non-limiting embodiment, the method of the present disclosure modulates autophagy selected from a group comprising mitophagy (degradation of mitochondria), pexophagy (degradation of peroxisomes), lipophagy (degradation of lipid), reticulophagy (degradation of endoplasmic reticulum), ribophagy (degradation of ribosome), zymophagy (degradation of secretory granules), aggrephagy (degradation of protein aggregates), nucleophagy (degradation of nuclear parts) and xerophagy (degradation of pathogens), or any combinations thereof.
In an embodiment, the method of the present disclosure leads to about 2 fold to about 4 fold increase in aggrephagy (protein degradation) when compared to the autophagy not driven by the method of the present disclosure.
In another embodiment, the method of the present disclosure leads to about 2 fold increase in mitophagy (degradation of mitochondria), pexophagy (degradation of peroxisomes), lipophagy (degradation of lipid), reticulophagy (degradation of endoplasmic reticulum), ribophagy (degradation of ribosome), zymophagy (degradation of secretory granules), nucleophagy (degradation of nuclear parts) or xenophagy (degradation of pathogens)when compared to the autophagy not driven by the method of the present disclosure.
In an embodiment, the method of the present disclosure while modulating does not affect the normal functioning of the cell or tissue or combination thereof.
In another embodiment, the method of the present disclosure while modulating autophagy does not affect cell viability and growth of cell or tissue or combination thereof.
In an embodiment, the method of the present disclosure enhances basal autophagy and induced autophagy, independently or in combination.
In an embodiment, in the method of the present disclosure, the modulator, 6-Bio enhances starvation induced autophagy or growth.
In an exemplary embodiment, in the method of the present disclosure, (2′Z,3′E)-6-Bromoindirubin-3′-oxime (6-Bio) modulates autophagy including but not limiting to mitophagy (degradation of mitochondria), pexophagy (degradation of peroxisomes), lipophagy (degradation of lipid), reticulophagy (degradation of endoplasmic reticulum), ribophagy (degradation of ribosome), zymophagy (degradation of secretory granules), aggrephagy (degradation of protein aggregates) and xenophagy.
In another exemplary embodiment, in the method of the present disclosure acacetin modulates autophagy including but not limiting to mitophagy (degradation of mitochondria), pexophagy (degradation of peroxisomes), lipophagy (degradation of lipid), reticulophagy (degradation of endoplasmic reticulum), ribophagy (degradation of ribosome), zymophagy (degradation of secretory granules), Aggrephagy (degradation of protein aggregates) and xenophagy.
In an embodiment, in the method of the present disclosure the 6-Bio induces macroautophagy leading to increased autophagic flux resulting in aggregate degradation or clearance of protein aggregate including but not limiting to α-synuclein within a cell or tissue or both.
In an embodiment, in the method of the present disclosure, the 6-Bio rescues growth lag due to α-synuclein toxicity in yeast as opposed to other GSK-3 inhibitors known in the art.
In another embodiment, in the method of the present disclosure, the 6-Bio shows increased efficiency in activation of autophagy compared to known neuro protective compounds such as Agk2.
In a further embodiment, in the method of the present disclosure, the 6-Bio enhances starvation induced autophagy more efficiently as compared to other known neuro-protective compounds such as Agk2.
In exemplary embodiment, in the method of the present disclosure the 6-Bio activates autophagy including but not limiting to aggrephagy, wherein the 6-Bio clears or leads to degradation of α-synuclein aggregates and restores cellular homeostasis.
In exemplary embodiment, the method of the present disclosure manages neurodegenerative disorder including but not limiting to Alzheimer's disease, Amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease and Spinal muscular atrophy.
In another exemplary embodiment, the method of the present disclosure treats neurodegenerative disorder including but not limiting to Alzheimer's disease, Amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease and Spinal muscular atrophy.
In a non-limiting embodiment, in the method of the present disclosure the 6-Bio treats neurodegenerative disorder including but not limiting to Alzheimer's disease, Amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease and Spinal muscular atrophy.
In another non-limiting embodiment, in the method of the present disclosure, the 6-Bio halts neurodegeneration, unlike commonly administered drugs such as L-DOPA for Parkinson's disease.
In another embodiment, the 6-Bio in the method of the present disclosure inhibits Glycogen synthase kinase 3 beta (GSK3B) function.
In a further embodiment, the 6-Bio in the method of the present disclosure shows increased autophagy induction than GSK3B inhibitors.
In another embodiment, in the method of the present disclosure 6-Bio modulates GSK3B, PDK1 and Jak/STAT3 signaling pathways.
In a further embodiment, in the method of the present disclosure 6-Bio prevents cytotoxicity by restoring cellular proteostasis.
In an alternate embodiment, the method of the present disclosure inhibits intracellular growth of the microorganisms including but not limiting to Salmonella typhimurium, Legionella pneumophila, Listeria monocytogenes, Shigella flexneri, Streptococcus pyrogenes and Mycobacterium tuberculosis, while modulating autophagy.
In another alternate embodiment, in the method of the present disclosure acacetin inhibits the intracellular growth of the microorganism including but not limiting to Salmonella typhimurium Legionella pneumophila, Listeria monocytogenes, Shigella flexneri, Streptococcus pyrogenes and Mycobacterium tuberculosis.
In another alternate embodiment, the acacetin enhances pexophagy.
In an embodiment, in the method of the present disclosure the concentration of 6-bio that modulates autophagy including but not limiting to macroautophagy, chaperone mediated autophagy and microautophagy, ranges from about 5 μM to about 150 μM.
In an embodiment, in the method of the present disclosure the concentration of 6-bio that modulates autophagy including but not limiting to macroautophagy, chaperone mediated autophagy and microautophagy, ranges from about 40 μM to about 150 μM, preferably about 50 μM in yeast cells.
In another embodiment, in the method of the present disclosure the concentration of Acacetin that modulates autophagy including but not limiting to macroautophagy, chaperone mediated autophagy and microautophagy, ranges from about 25 μl to about 50 μM.
In another embodiment, in the method of the present disclosure the concentration of 6-bio that modulates autophagy in mammalian cells ranges from about 5 μM to about 50 μM, preferably about 5 μM.
In a further embodiment, in the method of the present disclosure the concentration of Acacetin that modulates autophagy in yeast cells ranges from about 25 μM to about 50 μM, preferably about 50 μM.
In another embodiment, in the method of the present disclosure the concentration of Acacetin that modulates autophagy including but not limiting to macroautophagy, chaperone mediated autophagy and microautophagy in yeast cells is 25 μM, 25.5 μM, 30 μM, 30.5 μM, 31 μM, 31.5 μM, 32 μM, 32.5 μM, 33 μM, 33.5 μM, 34 μM, 34.5 μM, 35 μM, 35.5 μM, 36 μM, 36.5 μM, 37 μM, 37.5 μM, 38 μM, 38.5 μM, 39 μM, 39.5 μM, 40 μM, 40.5 μM, 41 μM, 41.5 μM, 42 μM, 42.5 μM, 43 μM, 43.5 μM, 44 μM, 44.5 μM, 45 μM, 45.5 μM, 46 μM, 46.5 μM, 47 μM, 47.5 μM, 48 μM, 48.5 μM, 49 μM, 49.5 or 50 μM.
In another embodiment, in the method of the present disclosure the concentration of Acacetin that modulates autophagy in mammalian cells ranges from about 25 μM to about 50 μM, preferably about 50 μM in mammalian cells.
In another embodiment, Acacetin can be used in the management of bacterial infections.
In an exemplary embodiment, in the method of the present disclosure, 3-[4-(2,4-Bis-trifluoromethylbenzyloxy)-3-methoxyphenyl]-2-cyano-N-(5-trifluoromethyl-1,3,4-thiadiazol-2-yl)acrylamide (XCT790) modulates autophagy including but not limiting to mitophagy (degradation of mitochondria), pexophagy (degradation of peroxisomes), lipophagy (degradation of lipid), reticulophagy (degradation of endoplasmic reticulum), ribophagy (degradation of ribosome), zymophagy (degradation of secretory granules), aggrephagy (degradation of protein aggregates) and xenophagy.
In an embodiment, in the method of the present disclosure the XCT-790 induces macroautophagy leading to increased autophagic flux resulting in aggregate degradation or clearance of protein aggregate including but not limiting to α-synuclein within a cell or tissue or both.
In an embodiment, in the method of the present disclosure, the XCT-790 rescues growth lag due to α-synuclein toxicity in yeast as opposed to untreated condition.
In another embodiment, in the method of the present disclosure, the XCT-790 shows increased efficiency in activation of autophagy compared to known neuro-protective compounds such as Agk2.
In exemplary embodiment, in the method of the present disclosure the XCT-790 activates autophagy including but not limiting to aggrephagy, wherein the XCT-790 clears or leads to degradation of a-,synuclein aggregates and restores cellular homeostasis.
In a non-limiting embodiment, in the method of the present disclosure the XCT-790 treats neurodegenerative disorder including but not limiting to Alzheimer's disease, Amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease and Spinal muscular atrophy.
In an embodiment, in the method of the present disclosure the concentration of XCT-790 that modulates autophagy including but not limiting to macroautophagy, chaperone mediated autophagy and microautophagy, ranges from about 5 μM to about 150 μM.
In an embodiment, in the method of the present disclosure the concentration of XCT-790 that modulates autophagy including but not limiting to macroautophagy, chaperone mediated autophagy and microautophagy, ranges from about 40 μM to about 150 μM, preferably about 50 μM in yeast cells.
In another embodiment, in the method of the present disclosure the concentration of XCT-790 that modulates autophagy in mammalian cells ranges from about 5 μM to about 50 μM, preferably about 5 μM.
In an embodiment, in the method of the present disclosure, XCT-790 modulates autophagosome formation in an ERRα dependent manner.
In an embodiment, in the method of the present disclosure, the XCT-790 modulates fusion of autophagaosome to lysosome in an ERRα dependent manner.
In an embodiment, the ERRα is localized on to the autophagosomes and upon autophagy induction by XCT-790 by the method of the present disclosure, localization is lost and it is accompanied with an increase in autophagosome biogenesis.
In an embodiment, in the method of the present disclosure, XCT-790 clears α-synuclein (SNCA) aggregates in an autophagy-dependent manner in both yeast and human neuronal cells. The XCT-790 significantly induces autophagy through an mTOR-independent mechanism and ERRα-dependent mechanism.
In an embodiment, the present disclosure relates to a method of modulating autophagy, wherein the method comprising step of contacting cell with modulator of autophagy, wherein the modulator is an inhibitor of autophagy including but not limiting to macroautophagy, chaperon mediated autophagy and microautophagy.
In an exemplary embodiment, in the method of the present disclosure, the modulator which is an inhibitor of autophagy is selected from a group comprising N-Carbobenzyloxy-L-phenylalanylchloromethyl ketone (ZPCK), 3-[(4-methylphenyl)sulfonyl]-(2E)-propenenitrile (Bay11-7082), Elaidylphosphocholine, N-[4-(1H-Benzimidazol-2-yl)phenyl]-5-nitro-2-thiophenecarboxamide, Ethyl [(2-{[(5-nitro-2-thienyl)carbonyl]amino}-3-thienyl)carbonyl]carbamate, 2-Phenyl-N-[5-(3-thienyl)-1,3,4-oxadiazol-2-yl]-2H-1,2,3-triazole-4-carboxamide, 3-Bromo-N-[5-(5,6-dihydro-1,4-dioxin-2-yl)-1,3,4-oxadiazobenzamide, N-(6-Chloro-1,3-benzothiazol-2-yl)-2-(4-fluorophenyl)-N-(3-pyridinylmethyl)acetamide, N-(2-Methoxybenzyl)-5-nitro-4,6-pyrimidinediamine, N-[5-(2,5-Dichloro-3-thienyl-1,3,4-oxadiazol-2-yl]-5-nitro-2-furamide, N-(2,5-Dimethylphenyl)-2-{[3-(3-methoxyphenyl)-4-oxo-3,4,6,7-tetrahydrothieno[3,2-d]pyrimidin-2-yl]sulfanyl}acetamide, 4-Fluoro-N-[2-(4-fluorophenyl)-2-(4-methyl-1-piperazinyl)ethyl]-3-methylbenzenesulfonamide, N-[5-(3,4-Dimethylphenyl)-1,3,4-oxadiazol-2-yl]-5-nitro-2-furamide, N[3-(1,3-Benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl]-4-cyanobenzamide hydrochloride (1:1), N-(4,5-Diphenyl-1,3-thiazol-2-yl)-1,2-oxazole-5-carboxamide, 2-(5-(2,4-Difluorophenyl)-1,2-oxazol-3-yl]-N-(1,2-oxazol-3-yl)acetamide, 1-(2,4-Dihydroxy-3-methylphenyl)-2-(4-propylphenoxy)ethanone, N-[5-(2,4-Dimethylphenyl)-1,3,4-oxadiazol-2-yl]-3,5-dimethoxybenzamide. N-[3-(1,3-Benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl]-1-methyl-1H-pyrazole-3-carboxamide hydrochloride (1:1), 2-(2-Bromobenzyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide, 2-[(5,7-Dibromo-8-quinolinyl)oxy]propanamide, 3-Amino-N-[4-(imidazo[1,2-a]pyridin-2-yl)phenyl]-2-pyrazinecarboxamide, 1-Benzyl-N-[5-chloro-2-(1-piperidinyl)phenyl]-1H-1,2,3-triazole-4-carboxamide, (4-Bromophenyl){4-[(5-chloro-2-thienyl)sulfonyl]-1-piperazinyl}methanone, 1-Phenyl-N-(1,3,4-thiadiazol-2-yl)-3-(2-thienyl)-1H-pyrazole-5-carboxamide, 5-{-[(7-Bromo-2,3-dihydro-1,4-benzodioxin-6-yl)methyl]sulfanyl}-N-cyclopropyl-1,3,4-thiadiazol-2-amine, 1-Ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid, Pentachlorophenol, N,N,N-Trimethyl-1-hexadecanaminium bromide, 1,4-Dihydroxy-5,8-bis({2-[(2-hydroxyethyl)amino]ethyl}amino)-9,10-anthraquinone dihydrochloride, (9E)-9-Octadecen-1-yl 2-(trimethylammonio)ethyl phosphate, 4-(1H-Indazol-3-yl)-N-(4-piperidinyl)-1H-pyrrolo[2,3-b]pyridin-6-amine, 2-(4-Methoxybenzyl)-6-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylmethyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidine, [(E)-octadec-9-enyl] 2-(trimethylazaniumyl)ethyl phosphate), TNP (N6-[(4-nitrophenyl)methyl]-N2-[[3-(trifluoromethyl)phenyl]methyl]-9H-Purine-2,6-diamine), Cinchonidine; (R)-[(2S,4S,5R)-5-ethenyl-1-azabicyclo[2.2.2]octan-2-yl]-quinolin-4-ylmethanol, Trioxsalen and (2,5,9-Trimethylfuro[3,2-g]benzopyran-7-one), or any combinations thereof.
In an embodiment, in the method of the present disclosure wherein the modulator is inhibitor is ZPCK or Bay 11-7082 (Bay-11) or both.
In an embodiment, in the method of the present disclosure the modulator inhibits at least one of autophagosome biogenesis, autophagosome maturation, autophagosome-lysosome fusion, degradation of autophagic cargo inside vacuole after autophagosome lysosome fusion, or any combinations thereof, thereby decreasing autophagic flux.
In an exemplary embodiment, in the method of the present disclosure, wherein the modulator is an inhibitor of autophagy, inhibits autophagy at a step prior to the fusion of autophagosomes to the vacuole or inhibits autophagy at the step of degradation of autophagic bodies inside the vacuole, or a combination thereof.
In a non-limiting embodiment, in the method of the present disclosure the N-Benzyloxycarbonyl-Lphenylalaninylchloromethyl ketone (ZPCK) inhibits the degradation of autophagic bodies inside the vacuole in autophagy including but not limited to macroautophagy, chaperone mediated autophagy and microautophagy.
In another non-limiting embodiment, in the method of the present disclosure, the 3-[4-methylphenyl)sulfonyl]-(2E)-propenenitrile (Bay11-7082) inhibits at a step prior to fusion of autophagosomes to the vacuole in autophagy including but not limiting to macroautophagy, chaperone mediated autophagy and microautophagy.
In an exemplary embodiment, in the method of the present disclosure, the 3-[(4-methylphenyl)sulfonyl]-(2E)-propenenitrile (Bay11-7082) acts during the autophagosome biogenesis step in autophagy including but not limiting to macroautophagy, chaperone mediated autophagy and microautophagy.
In an exemplary embodiment, in the method of the present disclosure, the inhibitor of autophagy including but not limiting to 3-[(4-methylphenyl)sulfonyl]-(2E)-propenenitrile (Bay11-7082) inhibits autophagy including but not limiting to macroautophagy, chaperone mediated autophagy and microautophagy in a dose dependent manner.
In a non-limiting embodiment, the method of the present disclosure, wherein the modulator inhibits autophagy, manages cancer or treats cancer, or both.
In another embodiment, the method of the present disclosure, wherein the modulator inhibits autophagy, acts on the tumour cells that are highly dependent on autophagy for survival, ultimately leading to cell death.
In another embodiment, the method of the present disclosure, wherein the modulator inhibits autophagy acts on pathogens that use autophagy machinery for their survival,
In an embodiment, in the method of the present disclosure the concentration of ZPCK that modulates autophagy including but not limiting to macroautophagy, chaperone mediated autophagy and microautophagy ranges from about 25 μM to about 50 μM.
In an embodiment, in the method of the present disclosure the concentration of ZPCK that modulates autophagy in yeast cells ranges from about 25 μM to 50 μM, preferably about 50 μM in yeast cells.
In another embodiment, in the method of the present disclosure the concentration of ZPCK that modulates autophagy in yeast cells is 25 μM, 25.5 μM, 30 μM, 30.5 μM, 31 μM, 31.5 μM, 32 μM, 32.5 μM, 33 μM, 33.5 μM, 34 μM, 34.5 μM, 35 μM, 35.5 μM, 36 μM, 36.5 μM, 37 μM, 37.5 μM, 38 μM, 38.5 μM, 39 μM, 39.5 μM, 40 μM, 40.5 μM, 41 μM, 41.5 μM, 42 μM, 42.5 μM, 43 μM, 43.5 μM, 44 μM, 44.5 μM, 45 μM, 45.5 μM, 46 μM, 46.5 μM, 47 μM, 47.5 μM, 48 μM, 48.5 μM, 49 μM, 49.5 μM or 50 μM.
In an embodiment, in the method of the present disclosure the concentration of ZPCK that modulates autophagy in mammalian cells ranges from about 25 μM to 50 μM, preferably about 25 μM.
In another embodiment, in the method of the present disclosure the concentration of ZPCK that modulates autophagy in mammalian cells is 25 μM, 25.5 μM, 30 μM, 30.5 μM, 31 μM, 31.5 μM, 32 μM, 32.5 μM, 33 μM, 33.5 μM, 34 μM, 34.5 μM, 35 μM, 35.5 μM, 36 μM, 36.5 μM, 37 μM, 37.5 μM, 38 μM, 38.5 μM, 39 μM, 39.5 μM, 40 μM, 40.5 μM, 41 μM, 41.5 μM, 42 μM, 42.5 μM, 43 μM, 43.5 μM, 44 μM, 44.5 μM, 45 μM, 45.5 μM, 46 μM, 46.5 μM, 47 μM, 47.5 μM, 48 μM, 48.5 μM, 49 μM, 49.5 μM or 50 μM.
In an embodiment, in the method of the present disclosure the concentration of Bay-11 that modulates autophagy including but not limiting to macroautophagy, chaperone mediated autophagy and microautophagy ranges from about 1 μM to 25 μM.
In an embodiment, in the method of the present disclosure the concentration of Bay-11 that modulates autophagy in yeast cells ranges from about 1 μM to 25 μM preferably about 25 μM.
In another embodiment, in the method of the present disclosure the concentration of Bay-11 that modulates autophagy in yeast cells is 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, 10 μM, 10.5 μM, 11 μM, 11.5 μM, 12 μM, 12.5 μM, 13 μM, 13.5 μM, 14 μM, 14.5 μM, 15 μM, 15.5 μM, 16 μM, 16.5 μM, 17 μM, 17.5 μM, 18 μM, 18.5 μM, 19 μM, 19.5 μM, 20 μM, 20.5 μM, 21 μM, 21.5 μM, 22 μM, 22.5 μM, 23 μM, 23.5 μM, 24 μM, 24.5 or 25 μM.
In an embodiment, in the method of the present disclosure the concentration of Bay-11 that modulates autophagy in mammalian cells ranges from about 1 M to 10 μM, preferably about 2.5 μM in mammalian cells.
In another embodiment, in the method of the present disclosure the concentration of Bay-11 that modulates autophagy in mammalian cells is 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM or 10 μM.
In another embodiment, in the method of the present disclosure, the ZPCK manages or treats a condition including but not limiting to cancer/tumour, condition caused by tumour cells and pathogens, in a subject in need thereof.
In another embodiment, in the method of the present disclosure, the ZPCK manages or treats a condition including but not limiting to cancer/tumour, condition caused by tumour cells and pathogens in a subject in need thereof.
In another embodiment, in the method of the present disclosure, the Bay11-7082 manages or treats a condition including but not limiting to cancer/tumour, condition caused by tumour cells and pathogen in a subject in need thereof.
In an exemplary embodiment, microscopic studies in S. cerevisiae for the degradation of peroxisomes through autophagy shows a decrease in the degradation of peroxisomes an presence of both the inhibitors of autophagy such as ZPCK and Bay11-7082 employed in the method of the present disclosure as observed through accumulation of GFP positive punctate structures (peroxisomes) inside or outside of the vacuole (labelled with FM4-64).
In another embodiment, in cells treated with autophagy inhibitor Bay11-7082 by the method of the present disclosure, peroxisomes get accumulated outside the vacuole even in starvation in the cell.
In another embodiment, ZPCK treated cells by the method of the present disclosure show build-up of peroxisomes inside the vacuole.
In a further embodiment, to elucidate the step of action of Bay 11-7082 in the method of the present disclosure, a protease protection assay is performed using aminopeptidase as a marker, which is also a substrate for autophagy on starvation; wherein the principle of the assay is that a cargo protected by a membrane is resistant to the action of proteases; with the help of a detergent like Triton X-100, the membrane is dissolved, and the cargo is made available for degradation by proteinase K treatment; untreated cells show both precursor as well as the matured form, due to both the membrane protected cargo sequestered within the autophagosome and the free form present in the cytosol respectively, when treated with only proteinase K (
In another embodiment, traffic light assay used for studying autophagic flux is employed to assess the effect of both autophagy inhibitors Bay11-7082 and ZPCK employed in the method of the present disclosure in HeLa cells (
The present disclosure further relates to modulators of autophagy for, enhancing formation of autolysosome by promoting fusion of autophagosome and lysosome, thereby increasing autophagic flux or for inhibiting at least one of autophagosome biogenesis, autophagosome maturation, autophagosome-lysosome fusion, degradation of autophagic cargo inside vacuole after autophagosome-lysosome fusion, or any combination thereof, thereby decreasing autophagic flux.
In an embodiment, the modulator is (2′Z,3′E)-6-Bromoindirubin-3′-oxime (6-Bio), acacetin, 7-dihydroxy-2-(4-methoxyphenyl)chromen-4-oneN6-(4-Aminobenzyl)-9-[5-(methylcarbonyl)-β-D-ribofuranosyl]adenine(AB-MECA, Lapidine; (3S,3aR,4R,8aR)-3-hydroxy-6,8a-dimethyl-8-oxo-3-propan-2-yl-2,3a,4,5-tetrahydro-1H-azulen-4-yl](E)-2-methylbut-2-enoate, Senecionine, 12-Hydroxysenecionan-11,16-dione, XCT790; (3-[4-(2,4-Bis-trifluoromethylbenzyloxy)-3-methoxyphenyl]-2-cyano-N-(5-trifluoromethyl-1,3,4-thiadiazol-2-yl)acrylamide), PD180970 (6-(2,6-Dichlorophenyl)-2-[(4-fluoro-3-methylphenyl)amino]-8-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one), Ritodrine hydrochloride; (N-(p-Hydroxyphenethyl)-4-hydroxynorephedrine hydrochloride) and SB 242084 dihydrochloride hydrate; (6-Chloro-2,3-dihydro-5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1H-indole-1-carboxyamide dihydrochloride hydrate), N-Carbobenzyloxy-L-phenylalanylchloromethyl ketone (ZPCK), 3-[(4-methylphenyl)sulfonyl]-(2E)-propenenitrile (Bay11-7082), Elaidylphosphocholine, N-[4-(1H-Benzimidazol-2-yl)phenyl]-5-nitro-2-thiophenecarboxamide, Ethyl [(2-{[(5-nitro-2-thienyl)carbonyl]amino}-3-thienyl)carbonyl]carbamate, 2-Phenyl-N-[5-(3-thienyl)-1,3,4-oxadiazol-2-yl]-2H-1,2,3-triazole-4-carboxamide, 3-Bromo-N-[5-(5,6-dihydro-1,4-dioxin-2-yl)-1,3,4-oxadiazol-2-yl]benzamide, N-(6-Chloro-1,3-benzothiazol-2-yl)-2-(4-fluorophenyl)-N-(3-pyridinylmethyl)acetamide, N-(2-Methoxybenzyl)-5-nitro-4,6-pyrimidinediamine, N-[5-(2,5-Dichloro-3-thienyl)-1,3,4-oxadiazol-2-yl]-5-nitro-2-furamide, N-(2,5-Dimethylphenyl)-2-{[3-(3-methoxyphenyl)-4-oxo-3,4,6,7-tetrahydrothieno[3,2-d]pyrimidin-2-yl]sulfanyl}acetamide, 4-Fluoro-N-[2-(4-fluorophenyl)-2-(4-methyl-1-piperazinyl)ethyl]-3-methylbenzenesulfonamide, N-[5-3,4-Dimethylphenyl)-1,3,4-oxadiazol-2-yl]-5-nitro-2-furamide, N-[3-(1,3-Benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl]-4-cyanobenzamide hydrochloride (1:1), N-(4,5-Diphenyl-1,3-thiazol-2-yl)-1,2-oxazole-5-carboxamide, 2-[5-(2,4-Difluorophenyl)-1,2-oxazol-3-yl]-N-(1,2-oxazol-3-yl)acetamide, 1-(2,4-Dihydroxy-3-methylphenyl)-2-(4-propylphenoxy)ethanone, N-[5-(2,4-Dimethylphenyl)-1,3,4-oxadiazol-2-yl]-3,5-dimethoxybenzamide, N-[3-(1,3-Benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl]-1-methyl-1H-pyrazole-3-carboxamide hydrochloride (1:1), 2-(2Bromobenzyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide, 2-[(5,7-Dibromo-8-quinolinyl)oxy]propanamide, 3-Amino-N -[4-(imidazo[1,2-a]pyridin-2-yl)phenyl]-2-pyrazinecarboxamide, 1-Benzyl-N-[5-chloro-2-(1-piperidinyl)phenyl]-1H-1,2,3-triazole-4-carboxamide, (4-Bromophenyl){4-[(5-chloro-2-thienyl)sulfonyl]-1-piperazinyl}methanone, 1-Phenyl-N-(1,3,4-thiadiazol-2-yl)-3-(2-thienyl)-1H-pyrazole-5-carboxamide, 5-{[(7-Bromo-2,3-dihydro-1,4-benzodioxin-6-yl)methyl]sulfanyl}-N-cyclopropyl-1,3,4-thiadiazol-2-amine, 1-Ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid, Pentachlorophenol, N,N,N-Trimethyl-1-hexadecanaminium bromide, 1,4-Dihydroxy-5,8-bis({2-[(2-hydroxyethyl)amino]ethyl}amino)-9,10-anthraquinone dihydrochloride, (9E)-9-Octadecen-1-yl 2-(trimethylammonio)ethyl phosphate, 4-(1H-Indazol-3-yl)-N-(4-piperidinyl)-1H-pyrrolo[2,3-b]pyridin-6-amine, 2-(4-Methoxybenzyl)-6-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylmethyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidine, [(E)-octadec-9-enyl] 2-(trimethylazaniumyl)ethyl phosphate), TNP (N6-[(4-nitrophenyl)methyl]-N2-[[3-trifluoromethyl)phenyl]methyl]-9H-Purine-2,6-diamine), Cinchonidine; (R)-[(2S,4S,5R)-5-ethenyl-1-azabicyclo[2.2.2]octan-2-yl]-quinolin-4-ylmethanol, Trioxsalen; (2,5,9-Trimethylfuro[3,2-g]benzopyran-7-one) or any combination thereof.
In an embodiment, the modulator of autophagy for enhancing autolysosmes formation in the cell is 6-Bio or XCT-790 or both, wherein the 6-Bio or the XCT-790 or both causes degradation of protein selected from a group comprising α-synuclein (SNCA). During modulation of autophagy, the 6-Bio is mTOR dependent and GSK3B dependent, the XCT-790 is mTOR independent and ERRα dependent and the XCT-790 is an inverse agonist of ERRα.
In an embodiment, the modulator of autophagy increases autolysosomes numbers by about 8 fold to 12 fold. In another embodiment, the modulator of autophagy causes about 2 fold to 4 fold increase in the autophagic flux.
The present disclosure further relates to modulators of autophagy wherein the modulator enhances formation of autolysosome by promoting fusion of autophagosome and lysosome, thereby increasing autophagic flux or inhibits at least one of autophagosome biogenesis, autophagosome maturation, autophagosome-lysosome fusion, degradation of autophagic cargo inside vacuole after autophagosome-lysosome fusion, or any combination thereof, thereby decreasing autophagic flux.
In an embodiment, the modulator is selected from a group comprising (2′Z,3′E)-6-Bromoindirubin-3′-oxime (6-Bio), acacetin, 7-dihydroxy-2-(4-methoxyphenyl)chromen-4-oneN6-(4-Aminobenzyl)-9-[5-(methylcarbonyl)-β-D-ribofuranosyl]adenine(AB-MECA, Lapidine; (3S,3aR,4R, 8aR)-3-hydroxy-6,8a-dimethyl-8-oxo-3-propan-2-yl-2,3a,4,5-tetrahydro-1H-azulen-4-yl](E)-2-methylbut-2-enoate, Senecionine, 12-Hydroxysenecionan-11,16-dione, XCT790; (3-[4-(2,4-Bis-trifluoromethylbenzyloxy)-3-methoxyphenyl]-2-cyano-N-(5-trifluoromethyl-1,3,4-thiadiazol-2-yl)acrylamide), PD180970 (6-(2,6-Dichlorophenyl)-2-[(4-fluoro-3-methylphenyl)amino]-8-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one), Ritodrine hydrochloride; (N-(p-Hydroxyphenethyl)-4-hydroxynorephedrine hydrochloride) and SB 242084 dihydrochloride hydrate; (6-Chloro-2,3-dihydro-5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1H-indole-1-carboxyamide dihydrochloride hydrate), N-Carbobenzyloxy-L-phenylalanylchloromethyl ketone (ZPCK), 3-[(4-methylphenyl)sulfonyl]-(2E)-propenenitrile (Bay11-7082), Elaidylphosphocholine, N-[4-(1H-Benzimidazol-2-yl)phenyl]-5-nitro-2-thiophenecarboxamide, Ethyl [(2-{[(5-nitro-2-thienyl)carbonyl]amino}-3-thienyl)carbonyl]carbamate, 2-Phenyl-N-[5-(3-thienyl)-1,3,4-oxadiazol-2-yl]-2H-1,2,3-triazole-4-carboxamide, 3-Bromo-N-[5-(5,6-dihydro-1,4-dioxin-2-yl)-1,3,4-oxadiazol-2-yl]benzamide, N-(6-Chloro-1,3-benzothiazol-2-yl)-2-(4-fluorophenyl)-N-(3-pyridinylmethyl)acetamide, N-(2-Methoxybenzyl)-5-nitro-4,6-pyrimidinediamine, N-[5-(2,5-Dichloro-3-thienyl)-1,3,4-oxadiazol-2-yl]-5-nitro-2-furamide, N-(2,5-Dimethylphenyl)-2-{[3-(3-methoxyphenyl)-4-oxo-3,4,6,7-tetrahydrothieno[3,2-d]pyrimidin-2-yl]sulfanyl}acetamide, 4-Fluoro-N-[2-(4-fluorophenyl)-2-(4-methyl-1-piperazinyl)ethyl]-3-methylbenzenesulfonamide, N-[5-(3,4-Dimethylphenyl)-1,3,4-oxadiazol-2-yl]-5-nitro-2-furamide, N-[3-(1,3-Benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl]-4-cyanobenzamide hydrochloride (1:1), N-(4,5-Diphenyl-1,3-thiazol-2-yl)-1,2-oxazole-5-carboxamide, 2-[5-(2,4-Difluorophenyl)-1,2-oxazol-3-yl]-N-(1,2-oxazol-3-yl)acetamide, 1-(2,4-)Dihydroxy-3-methylphenyl)-2-(4-propylphenoxy)ethanone, N-[5-(2,4-Dimethylphenyl)-1,3,4-oxadiazol-2-yl]-3,5-dimethoxybenzamide, N-[3-(1,3-Benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl]-1-methyl-1H-pyrazole-3-carboxamide hydrochloride (1:1), 2-(2-Bromobenzyl-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide, 2-[(5,7-Dibromo-8-quinolinyl)oxy]propanamide, 3-Amino-N-[4-(imidazo[1,2-a]pyridin-2-yl)phenyl]-2-pyrazinecarboxamide, 1-Benzyl-N-[5-chloro-2-(1-piperidinyl)phenyl]-1H-1,2,3-triazole-4-carboxamide, (4-Bromophenyl){4-[(5-chloro-2-thienyl)sulfonyl]-1-piperazinyl}methanone, 1-Phenyl-N-(1,3,4-thiadiazol-2-yl)-3-(2-thienyl)-1H-pyrazole-5-carboxamide, 5-{[(7-Bromo-2,3-dihydro-1,4-benzodioxin-6-yl)methyl]sulfanyl}-N-cyclopropyl-1,3,4-thiadiazol-2-amine, 1-Ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid, Pentachlorophenol, N,N,N-Trimethyl-1-hexadecanaminium bromide, 1,4-Dihydroxy-5,8-bis({2-[(2-hydroxyethyl)amino]ethyl}amino)-9,10-anthraquinone dihydrochloride, (9E)-9-Octadecen-1-yl 2-(trimethylammonio)ethyl phosphate, 4-(1H-Indazol-3-yl)-N-(4-piperidinyl)-1H-pyrrolo[2,3-b]pyridin-6-amine, 2-(4-Methoxybenzyl)-6-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylmethyl)-6,7-dihyrdro-5H-pyrrolo[3,4-d]pyrimidine, [(E)-octadec-9-enyl] 2-(trimethylazaniumyl)ethyl phosphate), TNT (N6-[(4-nitrophenyl)methyl]-N2-[[3-(trifluoromethyl)phenyl]methyl]-9H-Purine-2,6-diamine), Cinchonidine; (R)-[(2S,4S,5R)-5-ethenyl-1-azabicyclo[2.2.2]octan-2-yl]-quinolin-4-ylmethanol, Trioxsalen; (2,5,9-Trimethylfuro[3,2-g]benzopyran-7-one) or any combination thereof.
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In another embodiment of the present disclosure, figure illustrates effect of Bay11 treatment on maturation of autophagosomes, wherein (A) GFP-Atg8 fluorescence microscopy showed an accumulation of GFP-Atg8 positive puncta on treatment with Bay11 under starvation condition. Graphs showing diffused GFP inside the vacuole (B) and number of puncta in the cytosol at 4 hours of starvation (C) in wild type, Δypt7 and wild type cells treated with Bay11. (D) To elucidate the step of action of Bay11, a protease protection assay was performed using aminopeptidase as a marker, which is also a substrate for autophagy on starvation. Conversion of precursor to matured form of aminopeptidase on treatment with proteinase K (PK) in Bay11 treated cells indicated that the cargo is not protected by the autophagosome. (E) Quantitation showing relative precursor and mature form of aminopeptidase levels for different treatment groups. Y-axis shows the total aminopeptidase levels. TX—Triton X-100; PK—Proteinase K, (F) Co-localization of genomically tagged GFP-Atg8 and Atg5-RedStar* proteins in untreated and Bay11 treated conditions. (G) Quantitation showing percentage number of cells with more than one Atg5 puncta. (H) Quantitation showing number of co-localization events per 100 cells in untreated and Bay11 treated cells. Scale bar=5 μm. Data shown represent a minimum of 100 cells from 3 independent experiments and are expressed as the mean±SD. ***P<0.001 (individual means compared using two-tailed unpaired t-test).
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The present disclosure is further described with reference to the following examples, which are only illustrative in nature and should not be construed to limit the scope of the present disclosure in any manner.
Materials and Methods Employed in the Examples of the Present Disclosure
Chemicals and Antibodies
Yeast extract, peptone, dextrose, galactose and amino acids (leucine, lysine, methionine, histidine and uracil) are purchased from HiMedia.
3-MA (M9281), 6-Bio (B1686), LOPAC (LO1280), anti LC3 antibody (L7543), MPTP (methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine, M0896), DMEM (D5648), DMEM F-12 (D8900), Penicillin and Streptomycin (P4333), DAB (3, 3′-Diaminobenzidine, D3939), Atto 663 (41176) and Trypsin EDTA (59418C) are purchased from Sigma-Aldrich. Anti phospho P70S6K T389 antibody (9239) and total P70S6K antibody (9202), anti phospho GSK3β S9 antibody (5558) and total GSK3β antibody (9315), anti phospho4E-BP1T37/46 antibody (2855) and total 4E-BP1 antibody (9452), anti LAMP1 antibody (9091) and anti rabbitIgG, HRP (7074) antibody are purchased from Cell Signaling Technology. Anti β-Tubulin (MA5-16308) and anti GAPDH (MA5-15738) antibodies are purchased from Thermo Scientific.
Anti PGK1 (ab 38007) antibody is purchased from Abcam.
Anti GFP (11 814 460 001) antibody is purchased from Roche.
Anti Tyrosine Hydroxylase (N196) antibody is purchased from Santa Cruz Biotechnology.
Anti mouseIgG, HRP (172-1011) antibody is purchased from Bio-Rad. CMAC-Blue (C2110) is purchased from Life Technologies.
Bafilomycin A1. (11038) is purchased from Cayman chemical.
VECTASTAIN Elite ABC Kit (PK-6200) is purchased from VECTOR laboratories.
Plasmid Constructs and Yeast Strains
Plasmids used are pRS 316 GFP-Atg8, pRS 306 (α-synuclein-EGFP) under galactose promoter and pRS 306 (EGFP-βA1-42), ptfLC3 (Addgene number. 21074), pRS 306 (EGFP-synuclein). Yeast strains employed in the instant disclosure are listed in Table 1 and the said yeast strains are obtained from EUROSCARF, Europe.
Culture and Culture Conditions
Techniques Employed:
TCA Precipitation
All samples are collected in 12.5% TCA final concentration and stored at −80° C. for at least half an hour. Later, the samples are thawed on ice and centrifuged for 10 minutes at 16000 g and the pellet is washed with 250 μl of ice cold 80% acetone twice and air dried. This pellet is resuspended in 40 μl of 1% SDS-0.1N NaOH solution. Sample buffer (5×, 10 μl) is added to the lysate and boiled for 10 minutes before loading.
Immunoblotting
Total cell lysates are electrophoresed on different 1 percentages of SDS-PAGE based on the desired protein size and transferred onto PVDF membrane at constant current of 2 Ampere for 30 minutes (Transblot turbo, Bio-Rad Inc, USA). Transfer is confirmed by Ponceau S staining of blot and the blots scanned are used as loading controls. Blots are incubated overnight with 5% skim milk in primary antibody (Anti-GFP, Roche #11814460001, Anti-MAP1LC3B, CST #L7543). Secondary antibody used at 1:10.000 is goat anti-mouse (Bio-7 Rad #172-1011) or goat anti-rabbit antibody (Bio-Rad #172-1019) conjugated to HRP. Blots are developed by using ECL substrate (Thermo Scientific #34087 or Bio-Rad #170-5061) and images captured using auto capture program in Syngene G-Box, UK. image J (NIH) are used for quantitation of band intensities.
For mammalian cells, following appropriate treatments, cells are washed with ice cold PBS. Cells are then lysed in 100 μl of sample buffer (10% w/v SDS, 10 mM DTT, 20% v/v glycerol, 0.2 Tris-HCL pH 6.8, 0.05% w/v bromophenol blue) and then collected using a rubber cell scraper. The lysates are boiled at 99° C. for 15 minutes and stored at −20° C.
Western Blotting
Western blotting is performed using standard methods. Immunoblotting in MEFs is carried out as described previously. Dilutions of primary antibodies used are as follows: Anti-p62 1:1000 (Progen Biotechnik, GP62-C), Anti-MAP1LC3B 1:3000 (Nevus Biologicals, NB100-2220) and Anti-GAPDH (Cell Signalling Technologies, 2118S). Secondary antibodies conjugated to HRP are used at 1:10000 dilution as follows: Anti-Guinea-Pig-HRP (Abeam, ab50210) and Anti-Rabbit-HRP (Calbiochem, 401393).
Pexophagy Assay
Pot1-GFP positive strains are allowed to grow till the A600 reached 0.8-1 in YPD. Peroxisome biogenesis is induced by growing these cells in oleate medium (0.1% oleate, 0.5% Tween-40, 0.25% yeast extract, 0.5% peptone, and 5 mM phosphate buffer) for 12hours. Cells are harvested, washed twice to remove traces of oleate and transferred to starvation medium without nitrogen, at inoculum density A600 3, to induce pexophagy. Cells are collected at various time intervals after pexophagy induction and processed by TCA method.
Immunofluorescence
Appropriate number of cells are plated on top of coverslips placed in 65 mm cell culture dishes for transfection. Transfected cells are divided into different treatment groups. Post treatment, cells are washed with PBS and fixed in 4% paraformaldehyde and permeabilized using 0.25% Triton X-100, Overnight incubation with Anti-p62/SQSTM1 (rabbit polyclonal, #PM045), Anti-EEA1 (rabbit polyclonal, CST #3288) is done at 4° C. Excess antibody is washed with PBS and coverslips are incubated with Atto-633 (goat anti-rabbit IgG, Sigma #41176). The coverslips are mounted with VECTASHIELD antifade reagent (H-1000/H-1200, Vector laboratories). Imaging for HeLa cells is carried out using Delta vision microscope (Olympus 60×/1.42, Plan ApoN, excitation and emission filter Cy5, FITC and TRITC, polychroic Quad).
Immunofluorescence analysis in MEFs is carried out by fixing the cells with 4% methanol free paraformaldehyde for 15 minutes, permeabilised with 0.5% TritonX-100 in PBS for 10 minutes, and then blocking with 5% FES in PBS for 30 minutes at room temperature, along with PBS washes in between every steps. Anti-p62 antibody (Progen Biotechnik, GP62-C) is used at 1:250 dilution in 5% PBS in PBS and incubated overnight at 4° C. Cells are then washed and incubated with goat anti-guinea pig Alexa 594 (ThermoFisher Scientific, A-15 11076) secondary antibody at 1:1000 dilution for 1 hour at room temperature. Cells are washed, counterstained with DAPI (ThermoFisher Scientific, D1306) in PBS for 5 minutes, washed again and then mounted using Prolong diamond anti-fade reagent (ThermoFisher Scientific, P36970). Slides are imaged using a Zeiss LSM 510 Meta Confocal Microscope using 100× objective. Analysis is performed by assessing for the percentage of cells displaying an accumulation of endogenous p62-positive aggregates.
Analysis of Autophagosome Maturation Using mRFP-GFP-MAP1LC3B Reporter
Transfection is done on a 60 mm dish with HeLa cells at 60-70% confluency. Cells were transfected with tandem RFP-GFP-MAP1LC3B construct (Addgene plasmid #21074) using 5 μl of Lipofectamine 2000 (11668-019, Invitrogen) and 2.5 μg of DNA (2:1 ratio) diluted in 100 μl of OPTI-MEM (31985-070, Invitrogen) separately. 72 hours after transfection cells are either left untreated or treatment with various concentrations of Bay11-7082 ZPCK is done for 2 hours. Starvation is induced by treating cells with Earle's balanced salt solution (EBBS). After treatment, cells are fixed in 4% paraformaldehyde and permeabilized using 0.25% Triton X-100, The coverslip is mounted with VECTASHIELD antifade reagent (H-1000, Vector laboratories). Imaging for HeLa cells is carried out using Delta vision microscope (Olympus 60×/1.4, Plan ApoN, excitation and emission filter Cy5, FITC and TRITC, polychroic Quad).
EGFR Trafficking
HeLa cells are plated on 6 well plates and allowed to attach on the surface. The cells are washed with PBS and then starved in DMEM (serum free media) for 3 hours. Pre-treatment with compounds is carried out for 1 hour, following which they are pulsed with 100 ng/ml of EGF and samples are collected at 0, 1, 2 and 3 hours.
Quantification of Cells with Increased p62+ Aggregates
Analysis of p62 aggregates is done as described previously. Briefly, immunofluorescence analysis with anti-p62 antibody is performed for assessing endogenous p62+ aggregates using confocal microscope. The percentage of cells with increased p62+ aggregates is quantified by assessing 200 cells per condition from independent experiments, in which a cell with an accumulation of p62+ aggregates was given a score of 1 whereas a cell having basal (low) levels of p62+ aggregates was given a score of 0.
Mean Intensity Calculation
ImageJ software (NIH) is used to calculate the mean intensity. Images are opened using the split channel plugin. Co-localization plugin in the analysis tools is used to obtain the colocalized area between two channels as a separate window. The intensity is calculated using the measure plugin in analysis tools.
Statistical Analysis Employed in the Instant Disclosure
Statistical analyses are performed using unpaired Student's t-test and ANOVA (one-way or two-way or both) followed by post-hoc Bonferroni test in GraphPad Prism. Error bars are expressed as mean±SEM.
Image Preparation
Yeast and mammalian images were prepared using Softworx software (GE healthare). Lace plant MIP images were prepared using NIS elements software (Nikon, Canada). Images were plated using Adobe Photoshop CC. Fluorescent MIP images had their brightness and contrast modified equally using Adobe Photoshop CC.
The S. cerevisiae shuttle vectors pRS306 (URA) and pRS305 (LEU) are used to clone the POT1 promoter and the Firefly and Renilla luciferase genes, respectively. The oleate responsive region of the POT1 promoter is amplified from yeast genomic DNA and along with the firefly and Renilla luciferase genes (firefly gene from pMY30 and Renilla gene from pRL-TK) is cloned into these vectors to obtain the constructs pPM3 and pPM5. These plasmid constructs are linearized using suitable restriction enzymes in the selection marker and transformed into wild type strains of S. cerevisiae and P. pastoris by standard transformation methods-Lithium acetate or PEG based transformation.
The transformed colonies of S. cerevisiae and P. pastoris are then tested for firefly luciferase activities. The colonies positive for firefly are then co-transformed with Renilla luciferase vector and tested for its activity,
The vectors are transformed into S. cerevisiae and P. Pastoris haploid strains including wild-type (WT), and the atg1. (systematic gene name, YGL180W), atg5 (YPL149W) and atg36 deletion strains (Gietz and Woods 2002). The wild type and the deletion mutants are from the MATa collection, created by the. Saccharomyces Genome Deletion Project. These strains are blocked in all autophagy related-pathways, including pexophagy.
To validate the luciferase assay developed in the laboratory, it is compared to the conventional Pot1-GFP processing assay, Both pexophagy and general autophagy are measured by following the rates of degradation of firefly and Renilla activities (
In the luciferase assay, the activity for the wild type cells goes down with time whereas the mutant shows no decrease in the activity which resembles the western for Pot1-GFP processing assay (
Z-factor is calculated for 5 assays done in triplicates in 384 well format for both firefly as well as Renilla luciferase activities. Z-factor for Fluc=0.8628±0.03481 and Z-factor for Rluc=0.8224±0.03879, which suggests a very good assay which when scaled up to millions of compounds would give very less false positives and better reliability.
The wild type cells show a gradual decrease in luciferase counts upon induction of autophagy whereas core autophagy mutants atg1 and atg5 and selective autophagy mutant atg36 (adaptor protein for pexophagy) do not show any drop in the luciferase activity over time (
Screening of Small Molecule Libraries Identified Several Putative Modulators of Autophagy
After validation of the luciferase assay, two small molecule libraries are screened for their effect on autophagy. The library from Sigma contains 1280 FDA approved drugs and Enzo library of natural compounds has 502 small molecules.
To screen small molecule library LOPAC1280 in S. cerevisiae SNCA toxicity model, working plates containing 50 μM drugs in 1.5% DMSO (190 compounds/plate) are prepared in a 384 well format. WT SNCA-GFP with or without small molecules and untreated WT GFP are grown under optimized conditions (80 μl, 30° C. and 420 rpm) for 36 h in a plate reader (Varioskan Flash, Thermo Scientific) in duplicates with automatic absorbance (A600) recording every 20 min. Growth curves of untreated WT GFP and WT SNCA-GFP strains are plotted and mid to late exponential phase time point of untreated WT GFP strain is chosen as reference for data analysis. Its corresponding time point of untreated and drug treated WT SNCA-GFP strains are plotted separately in a box plot. Small molecules that rescued the growth lag by ≥3 SD units of untreated WT SNCA-GFP strain are considered as ‘Hits’.
Thus, a 3 standard deviation (SD) parameter is used as a criterion to obtain the hits from the primary screen. Primary screening also identifies several known autophagy modulators as hits (
Small Molecule Screening Reveals 6-Bio as a Potent Inducer of Autophagy
In Yeast Cells:
The occurrence of protein aggregates and cytotoxicity by SNCA overexpression is recapitulated in the budding yeast, Saccharomyces cerevisiae.
The yeast model is employed to screen for small molecules that prevent cytotoxicity by aggregate degradation (
In Mammalian Cells:
MAP1LC3B/LC3B (Microtubule-associated protein 1A/1B light chain 3B, a mammalian autophagosome marker) processing and tandem RFP-GFP-LC3B assays are employed. In HeLa cells, 6-Bio increases LC3B-II (processed form of LC3B-I) levels in a dose dependent manner suggesting autophagy modulation (
Effect of 6-Bio on Lysosomal Function
HeLa cells are treated with 6-Bio and/or E64D and Pepstatin A for 2 h, followed by treatment with lysotracker for 20 min. Lysotracker fluorescence intensity is reduced in presence of protease inhibitor like E64D and Pepstatin A. The fluorescence intensity of lysotracker is found to be comparable between untreated and 6-Bio treated cells. Thus, no difference in both E64D +Pep A only and 6-Bio+E64D and Pep A treatments is found. Lysotracker staining indicates that there was no change in lysosome acidification. LAMP1 (Lysosomal-associated membrane protein 1) positive vesicle intensities and distribution also are unaltered upon 6-Bio treatment suggesting that perhaps lysosomal functions are not perturbed by 6-Bio.
To address if the molecule enters into cell via endocytosis, we carried out the tandem RFP-GFP LC3B assay at 16° C. At this temperature, endocytosis pathway is highly reduced as evident by the significantly decreased cellular uptake of FITC-Dextran (70 kDa) as compared at 37° C. However, the effect of 6-Bio in increasing fusion between autophagosomes and lysosomes at 37° C. (˜10 fold, control vs 6-Bio treated, P<0.001,
These results suggest that 6-Bio affects autophagy independent of endocytosis perhaps by passive diffusion. From these two model systems, we noticed that 6-Bio not only induces autophagy but also enhances starvation induced autophagy and strikingly promotes autolysosome formation without perturbing the lysosomal function.
Growth assays: In a 384 well plate, appropriate yeast strains are seeded (A600˜0.07) with and without drugs and incubated (80 μl, 30° C. and 420 rpm) in a multiplate reader (Varioskan Flash, Thermo Scientific) for 48 h with automatic absorbance (A600) recording every 20 min. Growth curves are plotted and analyzed using GraphPad Prism.
Induction of α-synuclein-EGFP aggregates: To induce α-synuclein-EGFP aggregates, the corresponding strains are inoculated in SD-Ura medium, Secondary culture is inoculated from primary inoculum and incubated till A600 reaches 0.8/ml. Cells are washed twice with sterile water and aggregates are induced by adding SG-Ura for 12-16 h.
To screen small molecule library LOPAC1280 in S. cerevisiae α-synuclein toxicity model, working plates containing 50 μM drugs (small molecule) in 1.5% DMSO (190 compounds/plate) are prepared in a 384 well format. WT α-synuclein-EGFP with and without small molecules and untreated WT EGFP are grown under optimized conditions (80 μl, 30° C. and 420 rpm) for 36 h in a plate reader (Varioskan Flash, Thermo Scientific) in duplicates with automatic absorbance (A600) recording every 20 min.
Growth curves of untreated WT EGFP and WI α-synuclein-EGFP strains are plotted and mid to late exponential phase time point of untreated WT EGFP strain is chosen as reference for data analysis. Its corresponding time point of untreated and drug treated WT α-synuclein-EGFP strains are plotted separately in a box plot. Small molecules that rescue growth lag due to α-synuclein toxicity by ≥3 SD units of untreated WT α-synuclein-EGFP strain are considered as ‘Hits’.
Of the hits that rescue growth lag due to α-synuclein toxicity in this model are Agk2 which is known to rescue growth lag due to α-synuclein toxicity (T. F. Outeiro et al.) affirming the reliability of the assay and the 6-Bio [(2′Z,3′E)-6-Bromoindirubin-3′-oxime](P. Polychronopoulos et al.) (
In order to understand the involvement of 6-Bio in autophagy, GFP-Atg8 (an autophagosome marker) processing assay under both growth and starvation conditions is employed.
GFP-ATG8 Processing Assay Protocol:
Saccharomyces cerevisiae strain containing the GFP-Atg8 (pRS 316 vector backbone) plasmid is grown in synthetic complete medium lacking uracil (SC-URA) under appropriate conditions (30° C. 250 rpm). From this, a secondary culture is inoculated at A600 nm=0.2 and grown as above until A600 nm reached ˜0.65. The cultures are transferred to SD-N (nitrogen starvation) medium at A600 nm=3, separately with and without the compounds, and samples are collected at different time intervals. Sample preparation is done by the TCA precipitation method and immunoblotting is performed using standard methods.
Preparation of Yeast Lysates for Immunoblot Analysis:
Yeast strains (A600=3) are resuspended in trichloroacetic acid (12.5%) and stored at −80° C. for at least half an hour. Samples are thawed on ice, centrifuged (16000×g, 10 min) and pellet is washed twice with ice-cold acetone (80%). Pellets are air dried, resuspended in lysis (1% SDS and 0.1 N NaOH) solution and Laemmii buffer and boiled for 10 min.
To assess α-synuclein-EGFP degradation efficacy by 6-Bio, after inducing α-synuclein-EGFP aggregate, galactose promoter is turned-off by adding dextrose. Cells are treated with 6-Bio (50 μM) for 0, 6, 12 and 24 h. Subsequent degradation of α-synuclein-EGFP levels are analyzed using immunoblotting (
Yeast cultures after respective treatments are washed, mounted on agarose (2%) pad and imaged. Images are acquired using DeltaVision Elite widefield microscope (API, GE) with following filters: DAPI (390/18 and 435/48), FITC (490/20 and 529/38), TRITC (542/27 and 594/45) and Cy5 (632/22 and 676/34). Images are processed using DV SoftWoRX software. Autophagosome (yellow) and autolysosome (red) are counted using Cell Counter plug-in in imager software (NIH) and graphically represented as fold difference versus untreated (
Assays carried out to monitor degradation of α-synuclein-EGFP aggregates in presence of 6-Bio under non-starvation and starvation conditions revealed a time-dependent and significant decrease in the α-synuclein-EGFP levels in wild type cells (
During growth conditions where autophagy is barely detectable, 6-Bio dramatically induces autophagy (6 h time point, P<0.001 versus untreated;
HeLa cells are maintained in DMEM containing 10% FBS (Pan-Biotech), SH-SY5Y cells are maintained in DMEM-F12 containing 10% FBS (Life technologies). Cell lines are cultured in presence of 5% CO2 and 37° C.
To perform autophagy assays, equal numbers of sub-confluent HeLa cells are seeded in 6 well dishes, allowed to attach for 24 h, treated with 6-Bio μM) and/or 3-MA (5 mM) in growth medium for 2 h. For EGFP-α-synuclein degradation assay, equal numbers of sub-confluent SH-SY5Y cells are seeded in 6 well dishes and allowed to attach for 24 h. Cells are transfected with EGFP-α-synuclein plasmid using Lipofectamine 2000 (Life technologies) and allowed to express for 24 h. Cells are treated with 6-Bio (5 μM) for 24 h and fold EGFP-α-synuclein levels are analyzed using immunoblotting.
Tandem RFP-EGFP-LC3 Assay
For tandem RFP-EGFP-LC3 assay, sub-confluent cells are seeded in 60 mm dishes, transfected with ptf LC3 construct and allowed to express for 24 h. Later, cells are trypsinized, reseeded (105 cells) and allowed to attach on cover slips in a 12 well plate. Cells are treated with and without 6-Bio (5 μM) for 2 h and cover slips are processed for imaging.
It is observed that in HeLa cells, 6-Bio increases LC3-II (processed form of LC3-I) levels in a dose dependent manner suggesting autophagy activation (
Preparation of Mammalian Cell Lysates for Immunoblot Analysis
After treatments, cells are collected in Laemmli buffer to perform LC3 processing assay, P70S6K, GSK3β and 4E-BP1 immunoblotting. To validate EGFP-α-synuclein degradation by 6-Bio, treated cells are scraped and collected in growth medium. After washing with phosphate buffer, pellets are lysed in Laemmli buffer and boiled for 10 min.
Samples are electrophoresed on SDS-PAGE (8-15%) and then transferred onto PVDF (Bio-Rad) membrane using Transblot turbo (Bio-Rad). Transferred blots are stained with Ponceau S, probed with appropriate primary antibodies overnight and subsequently horseradish peroxidase conjugated secondary antibody. Signals are developed using enhanced chemiluminescence substrate (Clarity, Bio-Rad), imaged using a gel documentation system (G-Box, Syngene) and bands are quantified using image software (NIH). (
It is observed that 6-Bio significantly reduces EGFP-α-synuclein levels (˜2 fold, P<0.001versus untreated).
Effect of 3-MA on EGFP-α-Synuclein Clearance by 6-Bio
In the presence of autophagy inhibitor 3-methyl adenine (3-MA) (5 mM for 2 hours), the EGFP-α-synuclein levels does not change upon 6-Bio co-treatment suggesting that autophagy is the primary mechanism for degradation (
Effect of 6-Bio on mTOR Signalling
As mTOR negatively controls autophagy, it is tested if 6-Bio affected mTOR signaling. 6-Bio decreases phosphorylation levels of P70S6 kinase and 4E-BPI in a dose dependant manner (
Microscopy
For mammalian cell microscopy, after 2 h of treatment, coverslips are fixed using 4% paraformaldehyde (PFA) (Sigma) and permeabilized using Triton X-100 (0.2%, HiMedia). Coverslips are mounted using antifade, Vectashield (Vector laboratories). For antibody staining, coverslips are blocked in 5% BSA for 1 h, incubated in primary antibody overnight and subsequent probing with fluorescent conjugated antibody. Coverslips are mounted using antifade, Vectashield (Vector laboratories).
Images are acquired using DeltaVisionElitewidefield microscope (API, GE) with following filters: DAPI (390/18 and 435/48), FITC (490/20 and 529/38), TRITC (542/27 and 594/45) and Cy5 (632/22 and 676/34). Images are processed using DV SoftWoRX software. Autophagosome (yellow) and autolysosome (red) are counted using Cell Counter plug-in in ImageJ software (NIH) and graphically represented as fold difference versus untreated (
Cell Viability Assay
SH-SY5Y cells are seeded on a 96 well plate and transfected with GFP-SNCA only and/or is cotransfected with shRNA GSK3B and GFP-SNCA. To cells, the drugs were added (24 h) after 48 h of transfection. Then, the cell viability was measured using the CellTitre-Glo® (Promega) and luminescence was measured using Varioskan Flash (Thermo Scientific).
Autophagy Dependent GSK3B Mediated Neuro(Cyto) Protection by 6-Bio
Significant reduction of GSK3B activity upon 6-Bio treatment is observed as revealed by the reduced p-GSK3B levels (
Mice are randomly allocated to 5 different study groups, viz., Control (n=5), 6-Bio-only (n=5), MPTP only (n=5), MPTP+Prophylactic (2 days prior) administration of 6-Bio (n=3) and MPTP+Co-administration of 6-Bio (n=5).
As described by Vernice Jackson-Lewis & Serge Przedborski (2007), 23.4 mg/kg MPTP.HCl (equivalent to 20 mg/kg free base) in 10 ml/kg body wt. of saline is administered intraperitoneally (i.p.) for 4 times at 2 h intervals.
Further, 5 mg/kg body wt. of 6-Bio in 100 μl of saline is administered i.p. to the MPTP-injected animals by following either of the two different regimen; first regimen comprised of a prophylactic/pre-treatment which is begun two days prior to MPTP administration (MPTP+Pro); while the second involved treatment given alongside the MPTP injection (MPTP+Co). In both the cases, 6-Bio is administered for 7 days post-MPTP administration daily (
Tyrosine Hydroxylase (TH) Immunohistochemistry:
Mice are anaesthetized using Halothane BP (Piramal Healthcare) inhalation and perfused intracardially with normal saline followed by 4% PFA in 0.1 M phosphate buffer, pH 7.4. Brains are removed quickly and post-fixed with 4% PFA for 24 h-48 h at 4° C. Following cryoprotection in 15% and 30% sucrose, 40 μm thick coronal cryosections of midbrain are collected serially on gelatinized slides. Immunoperoxidase labelling protocol as identical to that reported. Briefly, endogenous peroxidase is quenched using H2O2 (0.1%) in methanol (70%), followed by blocking of non-specific staining by buffered solution (3%) of bovine serum albumin for 4 h at room temperature. Sections are incubated with TH primary antibody (1:500) followed by biotin conjugated secondary antibody (1:200 dilution, Vector Laboratories). Tertiary labelling is performed with avidin-biotin complex solution (1:100, Vector Laboratories). Staining is visualized using 3′-3′-diaminobenzidine (0.05%) as a chromogen in a solution of 0.1M acetate imidazole buffer (pH 7.4) and H2O2 (0.1%). Negative controls are processed identically, except that primary antibody is replaced with dilution buffer.
Stereological Quantification of TH+ Dopaminergic Neurons in SNpc:
As described by Y. Fu et al., SNpcis delineated using the 4× objective of Olympus BX61 Microscope (Olympus) equipped with Stereo Investigator Software Version 7.2 (MicroBrightField). Stereological quantification is performed using optical fractionator probe with slight modifications(P. A. Alladi et al.). Briefly, every sixth midbrain section containing SNpc is chosen and TH+ cells are counted under 100× objective, with a regular grid interval of 22500 μm2 (x=150 μm, y=150 μm) and counting frame with an area of 3600 μm2 (x=60 μm, y=60 μm) (
Densitometry Based Image Analysis:
High magnification images of TH stained nigral DAergic neurons, captured for offline assessment of Tyrosine Hydroxylase (TH) enzyme expression levels=used. Expression intensity is measured using a Windows based image analysis system (Q Win V3, Leica Systems). Cumulative mean is derived from the values obtained from sampling, approximately 200 DAergic neurons per animal. Intensity output is measured on a grey scale of 0-255, where 0 equals intense staining and 255 means absence of staining. Thus, lower grey values suggest higher protein expression and vice-versa.
From the above assays, it is observed that number and health of DA neurons (as revealed by tyrosine hydroxylase (TH) staining intensities) are significantly reduced in MPTP treated mice (˜3 fold, P<0.001 compared to control;
In Vivo Blood Brain Barrier Assay:
Animals are randomly allocated for placebo control and drug treatment cohorts, C57BL/6 mice are injected with placebo control or 6-Bio (5 mg/kg of body weight, intraperitoneally) twice with 24 h time interval. After second injection, the brains are harvested at 15 min, 30 min, 60 min, 6 h, 12 h and 24 h by cervical dislocation.
Mice brains are immediately homogenized with RIPA buffer (with protease inhibitor cocktail, Roche) for both treatment cohorts, Homogenously macerated mouse brain sample (100 μl) is mixed with acetonitrile (ACN, 400 μl), formic acid (0.2%) and aceclofenac (100 ng/ml, an internal standard), vortexed for 10 min at 2000 rpm Orbital shaker. Then, samples are centrifuged and supernatant is injected to LCMS/MS.
Standard samples are prepared by spiking 6-Bio standard in to blank control brain sample.
Standard spiking is performed such that resultant concentrations are 0, 10, 100, 500, 1000, 1500 and 2000 ng/ml of drug in blank brain matrix. For LC, PE 200 (Perkin Elmer) HPLC with Agilent Zorbax XDB C8 4,6×75 mm, 3.5 μm column is employed. The following conditions are used for LC: Mobile phases (0.1% formic acid in water (5%): Methanol (95%), Isocratic flow rate (0.7 ml/min), Run time (4 min), Injection volume (15 μl) and Needle wash solution (1:1 methanol:water mix containing 0.1% thrmic acid). The mass spectrometry (API3000, AB Sciex) is used with aceclofenac as an internal control and data were processed using Analyst Software V1.4.2, Drug injections of the various cohorts and preparations of the brain homogenates are performed at JNCASR. ACQUITY LABORATORIES performed the LC MS/MS analysis of the brain samples.
6-Bio Enhances Autophagy and Clears Toxic Protein Aggregates in Mice Brain:
The autophagy and toxic aggregate levels in the DAergic neurons in SNpc of midbrain are evaluated. A significant reduction of LC3B puncta per neuron in MPTP treated cohort than that of placebo (˜1.8 fold, placebo vs MPTP, P<0.001,
When 6-Bio is co administered along with MPTP, LC3B puncta per neuron increases (˜2,5 fold, MPTP vs MPTP+Co, P<0.001,
Behavioral Studies:
All the behavioural experiments are done on 3-4 month old, male C57/B16 mice. Experimenters are blind to the drug injected animals. Experimenters handle mice used for behavioral experiments for 3 consecutive days prior to the training paradigm. Behavioral experiments are designed in an order of low to high stress activity for mice. Therefore, Open Field Test is conducted in forenoon while rotarod is performed in the afternoon.
Mice were habituated to the behaviour room for 15 minutes every day before start of experiments. The light intensity is maintained at 100 lux throughout the experiment. Mice are weighed every day before training or test to ensure their good health. Mice are randomly allocated into three treatment cohorts: placebo control, MPTP and 6-Bio. Data is plotted using GraphPad prism 5 software.
Rotarod Trials:
The rotarod instrument is custom made at the Mechanical workshop, National Centre of Biological Sciences, Bengaluru, India. The rotating rod (diameter 3.3 cm) is made of Delrin and is textured to enhance the grip of mice. The rod is fixed at a height of 30 cm from the cushioned platform where mice fell on to during training and test. The rod is partitioned into three areas of 9.3 cm distance between each partition using discs (40 cm diameter) made of Teflon. Mice are trained in rotarod for five consecutive days prior to drug injection. Each mouse is trained in rotarod by gradually increasing the rotation on every day. On Day 1, mice are trained on 540 rpm (accelerated 612 at 1 rpm/5 seconds), 11-15 rpm (accelerated at 1 rpm/15 seconds) on second day, 16-20 rpm (accelerated at 1 rpm/5 seconds) on third day and at 20 rpm (fixed) for Day 4 and 5. Mice are trained at above specific rpm for 3 times with 5 minute interval between trials. The rod is rotated from 5 rpm to 20 rpm by manually changing the speed of motor (non-automated). During test (Day 13 post injection), the rotarod is started at 20 rpm. Mice are tested in a rotating rotarod for a maximum of 60 seconds and their latencies were noted down. At the end of each trial, the rotarod is wiped with 70% ethanol and left for drying before placing next set of mice. The entire trial is video recorded using a DSLR camera (Nikon D5100) and latencies are scored manually. The average time spend on rotating rotarod across three trials are plotted as mean latency to fall.
Open Field Test:
Open field arena (50 cm×50 cm×45 cm) is custom-made (JNCASR) using plywood and the internal surface was coated with white polish. Mice are trained in open field for 2 consecutive days prior to drug injection. During training or testing, one animal at a time is placed in zone periphery in open field arena and allowed to explore the arena for 5 minutes. The activity is video recorded (SONY® color video camera, Model no. SSC-G118) using a software (SMART v3.0.04 from Panlah, Harvard Apparatus, USA). After 5 minutes, the mouse is returned to its home cage. The open field arena is then wiped using 70% ethanol and allowed to dry before placing the next mouse. Distance travelled is analyzed offline by an experimenter who was not involved in performing the experiment.
6-Bio Ameliorates MPTP-Induced Behavioral Deficits: 1541
To study whether 6-Bio can combat the MPTP induced behavioral impairments in motor co-ordination, locomotion and exploration abilities, we two widely used behavior tests namely rotarod and Open Field Test are employed. Stereology is performed on day 7 post MPTP/placebo administrations, so behavior experiments are conducted on day 13 i.e., day 7 post MPTP/placebo administrations. In rotarod test, the latency to fall for MPTP cohort reduces significantly to that of placebo cohort on day-13 post-administration (MPTP versus placebo, P<0.001,
These impairments are improved after 6-Bio administration as the distance travelled by, mice increased dramatically (Co versus MPTP, P<0.001,
Since the 6-Bio treated cohort spent more time on the rotarod (as the placebo cohort) and also travelled more distance in open field unlike MPTP treated, it can therefore be can infer that 6-Bio rescued the MPTP induced motor, locomotion and exploratory impairments. It is observed that 6-Bio fails to protect the MPTP induced behavioral deficits when administered 48 h after MPTP dosage.
Standard autophagy assays are performed in S. cerevisiae for the degradation of autophagy markers, such as Pot1-GFP for pexophagy (
To elucidate the step of action of Bay11, a protease protection assay is performed using aminopeptidase as a marker, which is also a substrate for starvation-induced autophagy. Untreated cells in presence of proteinase K show both the precursor as well as the matured form due to the autophagosome-sequestered membrane-protected cargo and the cytosolic free form, respectively (
Owing to the conserved nature of autophagy, the putative inhibitors as obtained through the yeast screen are analysed in mammalian cells for their autophagy inhibitory effects.
The effects of Bay11 and ZPCK in mouse cells is assessed for their ability in impairing autophagic cargo degradation by analysing the clearance of the specific autophagy substrate, p62/SQSTM1. In mouse embryonic fibroblasts (MEFs), it is found that both the compounds cause significant accumulation of endogenous p62 aggregates at 24 h and 48 h (
Bay11 is further analysed with whether this accumulation of p62 is autophagy dependent by employing Atg5+/+ (wild-type) and Atg5−/− (autophagy-deficient) MEFs. As expected, while Bay11 significantly increases endogenous p62 levels in Atg5+/+ MEFs, it has no significant effect in Atg5−/− MEFs (
Bay11 is blocked at a step prior to BFA action, whereas ZPCK acts downstream of BFA (
To further assess the impact of Bay11 and ZPCK on autophagic degradation in HeLa cells, the fluorescence intensity of endogenous p62 and its co-localization with mRFP MAP1LC3B-positive autophagosomal compartments is studied. It is observed that p62 accumulates either outside or inside the mRFP-MAP1LC3B-positive compartments upon treatment with Bay11 and ZPCK, respectively (
ZPCK like BFA prevents the degradation of p62 once captured by the autophagosomes, and hence p62 accumulates in MAP1LC3B-positive structures. This result combined with the data using mRFP-GFP-MAP1LC3B reporter (
Next, the effects of Bay11 and ZPCK on other trafficking pathways such as endocytosis using the endocytosis-mediated EGFR degradation assay (
However, the fact that ZPCK caused accumulation of p62 suggests that it possibly affects some lysosomal protease specific for autophagic cargo.
Lace Plant Cultures and Experiments
Axenic lace plant cultures are grown in magenta boxes and prepared according to Gunawardena et al. Leaves in the window stage are removed from the corm and rinsed thoroughly with distilled water prior to being sectioned into 2 mm2 pieces. For starvation treatments, window stage leaves are removed from the plant, placed in distilled water and kept in the dark overnight.
Leaf sections are stained with monodansylcadaverine (MDC; 300 μM) (Sigma, D4008) and simultaneously treated with autophagy modulators for 2 hours in the dark. (1 hour vacuum infiltration at 15 psi). Treatment times, along with stain and modulator applications are optimized using concentration gradients followed by microscopy. The optimized concentrations are 5 μM rapamycin (Enzo Life Sciences (BML A275-0005), 5 μM wortmannin (Santa Cruz Biotechnology, sc-3505), 1 μM concanamycin A (Santa Cruz Biotechnology, sc-202111), 50 μM Bay 11 (Sigma, B5556) and 50 μM ZPCK (Sigma, 860794).
Tissue sections are then rinsed and mounted in distilled water prior to being scanned using a Nikon Eclipse Ti confocal microscope (Nikon 40X/1.30, Plan Fluor, 405 nm excitation and 450/30 nm emission). Areoles in the early phases of PCD are scanned to avoid cellular debris. The mean number of puncta are quantified for each treatment group with a minimum of four independent experiments using NIS Elements Advanced Research software. Additionally, starvation treatment leaves are also exposed to 5 μM, wortmannin, 50 μM Bay11 and 50 μM ZPCK treatments and then qualitatively assessed via confocal microscopy.
Immunostaining in Lace Plant
ATG8 immunolocalization in lace plant window stage leaves is achieved using a modified protocol from Pasternak et al, 2015.
Whole leaves are treated for two hours prior to fixation in 100% Methanol at 37° C. and then hydrophilized to 20% methanol by adding distilled water at 60° C. every two minutes for 32 minutes. Samples are then sectioned and placed on a multiwall slide and allowed to air dry for 10 minutes to facilitate membrane permeabilization. Blocking is done for 30 minutes at 37° C. with 4% low fat milk powder in 1× microtubule stabilization buffer MTSB (Modified 2× MTSB stock solution: 15 g PIPES, 1.9 g EDTA, 1.22 g MgSO4*7H2O and 2.5 g KOH—pH 7.0). Primary antibody incubation for anti-rabbit ATG8 (Agrisera, AS14 2769) is done at a 1:1000 dilution in 1× MTSB for 30 minutes at 37° C. Samples are then washed for 5 minutes, 3 times with 1× MTSB. Secondary antibody incubation with Goat anti-rabbit Dylight 488 (Agrisera AS09 633) at a 1:2000 dilution in 1× MTSB is done for 30 minutes at 37° C. and then samples are rinsed as above. Tissues are mounted in Mowiol 4-88 solution (Sigma, 81381) and scanned via confocal microscopy as mentioned above. The mean number of puncta per cell is determined using maximum intensity projections (MIPs) for each replicate. The total number of cells within a field of view are counted manually and the number of puncta are counted automatically using ImageJ.
The aquatic lace plant, Aponogeton madagascariensis, has leaves that are nearly transparent and ideal for live-cell imaging. Leaves taken from axenic cultures are sectioned and then assigned to treatment groups. Treatments included a control with no autophagy modulators (
In ZPCK treated cells the punctate structures appear to accumulate inside the vacuoles (Vid. S4). Additionally, overnight starvation leaves treated with either Bay11 or wortmannin showed fewer puncta compared to the overnight starvation group, as well as the overnight starvation combined with ZPCK, which have a similar appearance to the starvation group (
Pot1-GFP Assay
Pot1-GFP positive strains are allowed to grow in yeast extract peptone dextrose (YPD) (2% dextrose, 2% peptone and 1% yeast extract) till the Absorbance at 600 nm reaches 0.6-0.8. Peroxisome biogenesis is induced by growing these cells in YPG medium (1% yeast extract, 2% peptone, 3% glycerol) for 12 hours.
Cells are harvested, washed twice to remove traces of oleate and transferred to starvation medium with and without Acacetin, at. inoculum density Absorbance at 600 nm 3/ml, to induce pexophagy. Cells are collected at various time intervals after pexophagy induction and processed by TCA precipitation.
Cells treated with Acacetin show an enhanced accumulation of free GFP over time as compared to the untreated cells, which indicates an increase in the levels of pexophagy. (
Fold Change for Acacetin Using Burden Assay
U1752 cell line and HeLa cell line are infected with Salmonella typhimurium SL1344, and grown overnight in micro-aerophilic condition, at an MOI of 400 for one hour. The cells are treated with media containing Gentamycin at the concentration of 100 μg/ml for 2 hours to kill the extracellular bacteria. The cells are then treated with compounds and incubated for ?hours to 4 hours. At the end, the cells are lysed using lysis buffer (0.1% SDS, 1% Triton X-100, 1× PBS) and the intercellular Salmonella is plated and the CFU is counted. The CFU of Salmonella in the Acacetin treated cells is reduced by almost 2 fold compared to that of the untreated cells. Statistical analysis of the results is done using Graphpad prism—two tailed T test (
A single colony of Salmonella typhimurium WT strain SL1344 grown overnight at 37° C. is diluted in Luria Broth media to get an O.D of 0.2. The diluted culture is used for treatments with Acacetin and Acacetin with gentamycin (100 μg/ml). The growth curve of the culture is obtained by measuring the absorbance at 600 nm using varioskan Flash Multiplate Spectrophotometer at 300 rpm and O.D taken at every 30 minutes interval for 10 hours is plotted using GraphPad Prism.
HeLa cells are transfected with GFP-LC3 using lipofectamine 3000. After 24 hours, cells are infected with Salmonella typhimurium WT strain SL1344 with an MOI of 400 for 15 minutes followed by gentamycin treatment at the concentration of 100 μg/ml for 10 minutes to kill the extracellular bacteria.
The cells are treated with and without Acacetin and incubated for different time points (1, 2, 4 and 6 hours) at 37° C. Quantitation of LC3 co-localization with Salmonella typhimurium SL1344 is done using ImageJ-Cell counter option (
GFP-LC3 transfected HeLa cells is infected with mcherry-Salmonella typhimurium SL1344 for 15 minutes (MOI-400) and is treated with gentamycin for 10 minutes. The cells are then washed with 1× PBS and changed to either only media (a) and media containing Acacetin (b) and imaged by FV10i-olympus confocal live cell imaging microscope, using 60× water immersion lens, with confocality aperature set to 1.0. Images are taken at an interval of 15 minutes. (c) The intensity of the Red channel denoting the mcherry tagged S. typhimuriumis measured using image J—Stacks T function (intensity of red channel signifies the replication of the S. typhimurium over time). The replication of Salmonella in Acacetin treated samples is restricted as compared to that of untreated samples (
ptf-LC3 transfected HeLa cells are treated with the Acacetin for 2 hours. Following treatment, the number of autophagosomes and autolysosomes are counted using image J-cell counter function. The starvation medium (HBSS), is used as positive control which shows higher counts than the basal level of growth medium (GM). The compound treated sample shows an increase in the number of autolysosomes (red dots) (
Yeast media used for culturing is SD-Ura [Synthetic dextrose (2%) medium without uracil] for culturing α-synuclein-EGFP strains (wild type and atg1Δ) and EGFP-Atg8 processing assay, SG-Ura [Synthetic galactose (2%) medium without uracil] to induce α-synuclein-EGFP protein expression. Above mentioned strains are cultured at 250 rpm and 30° C.
Yeast Growth Assays:
Appropriate yeast strains are seeded (A600 ˜0.07) with or without drugs in a 384-well plate and incubated (420 rpm, 30° C. and 80 μl) in a multiplate reader (Varioskan Flash, Thermo Scientific) for 48 h that records absorbance (A600) automatically for every 20 min. Growth curves are plotted using GraphPad Prism.
α-Synuclein-EGFP Aggregates Induction in Yeast:
For inducing α-synuclein-EGFP aggregates in yeast, the appropriate strains are inoculated SD-Ura medium. Then, secondary cultures are inoculated from the primary culture and incubated till A600 reaches 0.8/ml. Cells are washed twice with sterile water and the aggregates are induced by incubating the cultures in SG-Ura for 12-16 h.
α-Synuclein-EGFP Degradation Assays:
After inducing α-synuclein-EGFP aggregates in the corresponding yeast strains driven by galactose promoter, the protein expression is turned off by adding dextrose in the medium. Then, α-synuclein-EGFP aggregates degradation by XCT 790 are assessed by collecting cells treated with and without XCT 790 (50 μM) for 0 and 24 h. Subsequently, the protein levels are analyzed using immunoblotting.
Immunoblot Analysis:
Yeast Lysates Preparation:
The appropriate yeast strains (A600=3) are mixed in trichloroacetic acid (12.5%) and then stored at −80° C. Then, the samples are thawed on ice, centrifuged (16000×g, 15 min) and the pellets are washed twice with ice-cold acetone (80%). Pellets are air dried, resuspended in lysis solution (0.1 N NaOH and 1% SDS) and Laemmli buffer and boiled for 15 min.
Microscopy:
After respective treatments, the yeast cultures were washed, seeded on agarose (2%) pad and then imaged.
A thiadiazoleacrylamide, XCT 790 is found to be one of the ‘Hits’ that showed significant rescue of growth in yeast cells overexpressing α-synuclein (
Toxic protein aggregates are known to be substrates of the autophagy pathway for their effective cellular degradation. Consistently, XCT 790 failed to rescue the growth lag in core autophagy mutant cells (atg1Δ) ascertaining its autophagy-mediated rescue of the cells from α-synuclein toxicity (atg1Δ α-syn cells; untreated vs XCT 790 treated, P>0.05,
The study, identifies XCT 790 as an autophagy inducer with a potential to clear toxic protein aggregates.
SH-SY5Y cells are cultured in DMEM-F12 containing 10% FBS (Life 558 technologies). HeLa cells are cultured in DMEM containing 10% FBS 559 (Pan-Biotech). Cell lines are maintained in following conditions of 37° C. and 5% CO2.
The autophagy assays are performed by seeding equal numbers of sub-confluent 1-HeLa or SH-SY5Y cells in 6-well dishes and allowed to attach for 24 h, then treated with XCT 790 (5 μM) and/or 3-MA (5 mM) and/or lithium chloride (10 mM) in fed condition for 2 h. After treatments, the cell lysates are analyzed by immunoblotting.
RFP-EGFP-LC3 Assay:
Sub-confluent HeLa and/or SH-SY5Y cells are seeded into 60 mm cell culture dishes, then transfected with ptf LC3 construct and/or siRNA and allowed to express for 48 h. Cells are trypsinized, seeded again on poly-D-lysine coated cover slips in a 12 or 24 well plates and allowed to attach. After appropriate treatments, the coverslips containing cells are processed for imaging. For immunofluorescent antibody staining, the cover slips are incubated in primary antibody at 4° C. for overnight followed by secondary antibody incubation at room temperature.
Immunoblot Analysis:
Mammalian cell lysates preparation: After treatments, cells are collected in Laemmli buffer to perform LC3 processing assay, P70S6K, AMPK, ULK1 and 4E-BP1 immunoblotting. Samples are electrophoresed onto SDS-PAGE (8-15%) and then transferred onto PVDF (Bio-Rad) membrane through Transblot turbo (Bio-Rad). Blots are stained with Ponceau S, then probed with appropriate primary antibodies at 4° C. for overnight and subsequently HRP-conjugated secondary antibody. Signals are attained using enhanced chemiluminescence substrate (Clarity, Bio-Rad) and imaged using a gel documentation system (G Box, Syngene) and then bands are quantitated using Image software (NIH).
Microscopy:
For imaging the mammalian cells, after appropriate treatments, coverslips containing cells are fixed using 4% paraformaldehyde (PFA) (Sigma) and then permeabilized using Triton X-100 (0.2%, HiMedia). On slide, coverslips are mounted using antifade, Vectashield mounting medium (Vector laboratories). For antibody staining, coverslips are blocked using 5% BSA for 1 h at room temperature, then incubated with primary antibody at 4° C., overnight and then subsequently probed with corresponding fluorescent dye conjugated secondary antibody.
Images are acquired using DeltaVision Elite widefield microscope (API, GE) with following filters: FITC (490/20 and 529/38), TRITC (542/27 and 594/45) and Cy5 (632/22 and 676/34). Acquired images.
Cell Viability Assay:
SH-SY5Y cells are seeded onto tissue culture treated 96 well plate and then transfected with EGFP-α-synuclein only and/or co-transfected with siRNA. To cells, appropriate drugs are added (24 h) after 48 h of transfection. Using luminescence-based CellTitre-Glo® (Promega) kit, the cell viability is assayed using automated microtitre plate reader Varioskan Flash (Thermo Scientific) are processed using DV SoftWoRX software.
XCT 790 Modulates Autophagy through and mTOR Independent Pathway:
Autophagy is regulated by mTOR (mammalian target of rapamycin) dependent and mTOR-independent pathways that are amenable to chemical perturbations 12. To delineate the mechanism of autophagy modulation by XCT 790, the activity of mTOR through monitoring its substrates such as P70S6K and 4EBP1 is examined.
Upon XCT 790 treatment, mTOR activity is unaffected as revealed by its substrates such as phospho-P70S6K and phospho-4EBP1 protein levels which are comparable to that of nutrient rich condition (
It is further examined whether XCT 790 exerts its effects through AMPK pathway, one of the predominant mTOR-independent mechanisms known to regulate autophagy. It is observed that treatment of XCT 790 for 2 hours did not affect the activity of AMPK, as evident by the unchanged T172 phosphorylation of AMPK (
Whereas, under nutrient sufficiency, high mTOR activity inhibits Ulk1 activation by phosphorylating Ulk1 at Ser 757 and disrupting the interaction between Ulk1 and AMPK.
Therefore, it is further examined the regulation of levels of activating (S555) and inhibitory (S757) phosphorylation of ULK1 by XCT 790. Consistent with unchanged levels of phosphorylated AMPK after treatment with XCT 790 for 2 hours, the downstream phosphorylation of ULK1 at S555 is unaffected and comparable to the nutrient rich conditions (
XCT 790 Induces Autophagy through Regulation of estrogen-Related Receptor Alpha (ERRα):
XCT 790 is found to be the first potent and selective inverse agonist of ERRα 9. To elucidate the role of ERRα in contributing to the function of XCT 790 as autophagy inducer, the following two approaches are used:
a) siRNA-based silencing of ERRα,
b) over expression of ERRα
To evaluate the level at which the knockdown exerts its effects, cells are transfected with siRNAs targeting ERRα. A non-targeting pool is used as a control. Post 48 hours of transfection, the effect of knockdown on regulation of autophagy by ERRα is monitored by microscopy based tandem RFP-EGFP-LC3 assays.
Knockdown efficiency is confirmed by western blotting to be around 80% (Scrambled vs ERRα siRNA, P<0.001,
Autophagosome and autolysosome numbers in XCT 790 treated and ERRα downregulated cells are found to be comparable. These results suggest that XCT 790 modulated autophagy through ERRα.
This question is addressed through another approach to understand the autophagy modulation upon overexpression of ERRα. In ERRα over expressed cells, more autophagosomes (˜2 fold, P<0.01, ERRα overexpressed vs untreated) and less autolysosomes (˜2 fold, P<258 0.01, ERRα overexpressed vs untreated) are found than that of control (
When XCT 790 is treated in ERRα over expressed cells, more autophagosomes (˜2 fold, P<0.01, ERRα overexpressed vs untreated) and less autolysosomes (˜2 fold, P<0.01, overexpressed vs untreated) than that of untreated are found (
Collectively, these results suggest that XCT 790 modulates autophagy through ERRα.
ERRα Regulates Autophagy by Localizing onto Autophagosomes:
From knock down and over expression of ERRα studies, there is a clear indication that ERRα can modulate autophagy pathway. Autophagy is induced when ERRα is downregulated (
It is examined whether active transcription is required for autophagic function of XCT 790. Upon XCT 790 treatment in presence of actinomycin D, the autophagosomes and autolysosomes are similar to that of only XCT 790 (XCT 790+Act D vs XCT 790 only, P>0.05.
It is then attempted to assess whether ERRα localizes to autophagic related structures such as autophagosomes and autolysosomes. It is analysed if ERRα localizes with either autophagosomes autolysosomes. FCC of ERRα with autophagosomes (˜0.85) are found to be significantly more than that with autolysosomes (˜0.3) under nutrient rich condition (˜2.5 fold, autophagosomes vs autolysosomes, P>0.001,
Colocalization of ERRα with autolysosomes is not regulated when compared to that of control (control or scrambled siRNA vs ERRα siRNA or XCT 790 or ERRα over expressed, P>0.05,
Animal studies: All procedures in this study are approved by JNCASR Institute Animal Ethical Committee and conducted as per institutional guidelines. Inbred male C57BL/6 mice (3-4 months old) were used for all experimental groups (n=6). The animals are maintained under standard laboratory conditions i.e. temperature 25°±2° C., 12 h light: 12 h dark cycle and 50±5% relative humidity with ad libitum access to food and water.
MPTP.HCl and XCT 790 Treatment:
The mice are distributed into three groups' viz., vehicle, MPTP and MPTP+XCT 790 injected respectively. The vehicle group receives intraperitoneal injections of dimethyl sulfoxide (DMSO) injections i.e. the solvent. The MPTP group receives 23.4 mg/kg MPTP.HCl in 10 ml/kg body wt. of saline is administered intraperitoneally for 4 times at 2 h interval. The MPTP+XCT 790 group mice are injected with 5 mg/kg body wt, of XCT 790 dissolved in DMSO, alongside the first MPTP injection. The treatment is continued by administering XCT 790 in “an injection a day regime” for 6 days. All the mice are sacrificed 7 days after MPTP administration and the brains are processed for immunohistochemistry.
Tissue Processing for Immunohistochemistry:
The mice are anaesthetized using halothane inhalation and perfused intracardially with saline, followed by 4% buffered paraformaldehyde (pH 7.4), The brains are removed quickly and post fixed in the same buffer tier 24-48 h at 4° C. and cryoprotected in increasing grades of sucrose. Coronal midbrain cryosections of 40 μm thick are collected serially on gelatinized slides. Every sixth midbrain section is used for immunostaining.
Immunoperoxidase Staining of Tyrosine Hydroxylase (TH):
Briefly, the endogenous expression of peroxidase is quenched using 0.1% H2O2 in 70% methanol, followed by blocking of non-specific staining by 3% buffered solution of bovine serum albumin for 4 h at room temperature. The sections are then incubated with the rabbit polyclonal anti-TH antibody (1:800, Santacruz Biotechnology Inc, USA), followed by anti-rabbit secondary antibody (1:200 dilution; Vector Laboratories, Burlingame, USA). The tertiary labelling is performed using avidin-biotin complex solution (1:100, Elite ABC kits; Vector Laboratories, USA).
The staining is visualized using 0.05% solution of 3′-3′-diaminobenzidine, in 0.1 M acetate imidazole buffer (pH 7.4) with 0.1% H2O2. Phosphate buffered saline (0.01 M) containing 0.3% Triton X-100 (0.01M PBST, pH 7.4) is used as both diluent and washing buffer. Appropriate negative controls are processed identically.
Stereological Quantification of TH-Immunoreactive (TH-ir) Neurons at SNpc:
Stereological quantification of TH-ir dopaminergic neurons is performed using optical fractionator probe. The SNpc is delineated on every sixth. TH-ir midbrain section using 4× objective of the Olympus BX61 Microscope (Olympus Microscopes, Japan) equipped with StereoInvestigator (Software Version 7.2, Micro-brightfield Inc., 664 Colchester, USA). The cells are counted using oil immersion lens 665 (100×), with a regular grid interval of 22500 μm2 (x=150 μm, y=150 μm) and counting frame of 3600 μm2 (x=60 μm, y=60 μm).
The mounted thickness averages to 25 μm. A guard zone of 4 μm is implied on either side, thus providing 17 μm of z-dimension to the optical dissector. The quantification is performed starting with the first anterior appearance of TH-ir neurons in SNpc to the caudal most part in both hemispheres and added to arrive at the total number. The volume of SNpc is estimated by planimetry.
Densitometry Based Image Analysis:
The offline evaluation of TH expression is performed on high magnification images of TH immunostained nigral dopaminergic neurons using Q Win V3 (Leica Systems, Germany); a ‘Windows’ based image analysis system. A cumulative mean is derived from the values obtained from sampling approximately 200 dopaminergic neurons per animal, and expressed as grey values on a scale of 0-255, where ‘0’ means absence of staining and ‘255’ equals intense staining.
Immunofluorescence Based Double Staining of SNpc Dopaminergic Neurons:
The sequential immunolabeling procedure is used to co-label the TH and LC3 and/or A11. First, the midbrain sections are equilibrated with 0.1 M PBS (pH 7.4) for 10 min and then incubated with buffered bovine serum albumin (3%) for 4 h to block non-specific epitopes. Then, the sections are incubated in rabbit anti-LC3 antibody (1:1000) and/or anti-oligomer antibody (A11, 1:1000) for 72 h at 4° C. After subsequent washes, the sections are incubated in corresponding fluorescent secondary antibody (1:200) at 4° C., overnight.
Co-labeling with TH is performed on the same sections using rabbit anti-TH antibody (1:500), followed by secondary labeling. PBST (0.01 M, pH 7.4) is used as both working and washing buffer. Sections are then mounted using Vectashield hardset mounting medium.
Considering that the autophagic mechanism is highly conserved between the yeast and the mammalian system, the potential of XCT 790 to clear toxic α-synuclein protein aggregates through autophagy in mammalian cells such as human neuroblastoma SH-SY5Y and HeLa cell lines is validated.
To test the modulation of mammalian autophagy and its flux by XCT 790, western blot analysis based LC3 (autophagosome marker) and microscopy-based tandem RFP-EGFP-LC3 assays are employed. In tandem RFP-EGFP-LC3 assay, XCT 790 treatment significantly induces autophagosomes and autolysosome formation in both SH-SY5Y (control vs XCT 790 treated, autophagosomes, ˜2 fold, P<0.05; autolysosomes, ˜4 fold, P<0.01,
These results demonstrate that XCT 790 also modulates mammalian autophagy as in yeast.
The question whether XCT 790 protects SH-SY5Y cells from EGFP-α-synuclein mediated toxicity is then addresses. Overexpression of EGFP-α-synuclein SH-SY5Y cells is toxic and leads to its significant cell death as measured by cell viability assay (˜4 fold, vector control or untransfected vs a-syn transfected,
It is observed that potential of XCT 790 to protect from EGFP-α-synuclein toxicity is abrogated in presence of pharmacological autophagy inhibitor, 3-MA (α-syn over expressed cells, XCT 790 vs XCT 790+3-MA, ˜4 fold, P<0.001,
XCT 790 Alleviates the MPTP Induced Dopaminergic Neuronal Loss:
A significant proportion of dopaminergic neurons in Substantia Nigra pars compacta (SNpc) are lost after MPTP treatment (˜68%, MPTP vs Vehicle, P<0.001,
Cellular Tyrosin Hydroxylase (TH) Expression is preserved in XCT 790 Co-Treatment Group: The cellular TH expression of individual TH-immunoreactive (TH-ir) dopaminergic, as measured by densitometry, is significantly reduced in surviving neurons in MPTP group (MPTP vs Vehicle, P<0.001,
XCT 790 Enhances Autophagy and Clears Toxic Protein Aggregates in an In-Vivo Mouse Model of PD:
In neurons, the autophagy process is indispensable for clearing the misfolded toxic protein aggregates. During the neurodegenerative progression, autophagy would be defunct and becomes incompetent to maintain cellular proteostasis.
To delineate the mechanism of neuroprotective action of XCT 790, the autophagy status is examined in the various mice treatment cohorts. Yeast and cell lines results strongly indicate that XCT 790 might exert neuroprotection through modulating autophagy. In MPTP toxicity model, the LC3 puncta per neuron is reduced significantly than that of vehicle treated (˜0.8 fold, vehicle vs MPTP treated, P<0.01,
Interestingly, XCT 790 only cohort exhibits significantly increased LC3 puncta per cell compared to that of vehicle treated cohort (˜3 fold, vehicle vs XCT 790 only, P<0.001,
During protein aggregation, the toxic misfolded protein oligomeric species would be accumulated in the neurons. It is examined whether autophagy induction by XCT 790 could clear the toxic oligomeric intermediates in the neurons. In a vehicle treated cohort, the occurrences of aggregates are significantly less compared to that of MPTP treated cohort (˜6.5 fold, vehicle vs MPTP treated, P<0.001,
In addition, the presence of aggregates in the steady state level of cell in the XCT 790 only is comparable to that of vehicle cohort (vehicle vs XCT 790 only, ns, P>0.05,
Mechanistically XCT 790 exerts neuroprotection by clearing misfolded protein aggregates through inducing autophagy demonstrated in the in-vivo preclinical mouse model of PD.
XCT 790 Ameliorated MPTP-Induced Behavioral Impairments:
Parkinson's disease patients exert movement disorder symptoms such as motor co-ordination, exploration and locomotion disabilities that can be recapitulated in a MPTP mice toxicity model.
As data herein shows neuroprotective role of XCT 790 at both cellular and tissue level, it is tested whether its effect can be translated up to behavioral level. To address this, a set of well-known behavioral experiments is performe-Rotarod and Open Field tests—specific for assaying the movement disorders.
To test the exploratory ability of mice, the distance travelled in periphery zone of open field arena is compared across different cohorts. It is observed that distance travelled in the zone periphery is drastically reduced in MPTP treated cohort compared to that of vehicle treated cohort (MPTP versus vehicle control, P<0.001,
Upon co-administration of XCT 790 along with MPTP, the distance travelled is significantly more than that of MPTP cohort (Co versus MPTP cohort, P<0.001,
Importantly, exploratory behaviour of various cohorts is evident in the trajectory maps (
Also, the results of vehicle treated and co-treated cohorts are fairly comparable (Co versus vehicle control, P>0.05, 44a, b) on both days. Therefore, these results suggest restoration of exploratory and motor coordination abilities in MPTP toxicity model, upon administration of XCT 790. Therefore, XCT 790 ameliorates the behavioural disabilities of MPTP treated mouse model.
Number | Date | Country | Kind |
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6596/CHE/2015 | Dec 2015 | IN | national |
Filing Document | Filing Date | Country | Kind |
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PCT/IB2016/057498 | 12/9/2016 | WO | 00 |