Compounds for treating diseases and their screening methods

Abstract

The present invention relates to a compound for use in the prevention or treatment of neurodegenerative diseases associated with the formation of stress granules. The compound is selected from lipoic acid, lipoamide, dihydrolipoic acid, and dihydrolipoamide. The present invention further relates to a method for identifying a compound that modulates a property associated with one or more condensates comprising condensate-associated molecules, comprising: (a) contacting the compound with a cell composition comprising one or more condensates or a cell composition capable of forming one or more condensates, and (b) measuring a property associated with the one or more condensates. Modulation of the property as compared to a reference indicates that the compound modulates a property associated with the one or more condensates.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to EP18200401.0, filed on October 15, 2018, and EP19189772.7, filed on August 2, 2019, the entire contents of each of which are hereby incorporated by reference. Background Art

[0003] Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a poor prognosis and limited treatment options. Currently, only two FDA-approved drugs, riluzole and edaravone, are available, but both can only slow disease progression by a few months. The mechanisms of action of riluzole and edaravone are not fully understood, and they are likely not directly targeting the underlying disease mechanisms. Thus, there is an urgent need for new approaches. Most forms of ALS are sporadic, but about 10% are monogenic disorders. Familial ALS-related mutations often occur in RNA-binding proteins (RBPs) such as FUS and TDP-43. These RBPs have characteristic low-complexity domains (LCDs).

[0004] The exact mechanism of ALS pathogenesis is not clear; it is not known whether aggregates or oligomers are toxic, or whether they cause loss of protein function, and how this leads to downstream effects. However, mutant TDP-43 and FUS are often mislocalized to the cytoplasm, promoting the formation of disease-related stress granules and abnormal cytoplasmic aggregates. One hypothesis specifically suggests that the pathological stability of stress granules may be related to the disease. This suggests that dissolving stress granules and / or aggregates may alleviate the disease, regardless of whether the pathogenesis is through a toxic gain of function or a loss of function by protein sequestration.

[0005] Stress granules are liquid, membrane-less compartments, and recent studies have shown that proteins containing LCDs form these compartments through liquid-liquid phase separation. Thus, in principle, it may be targeted to this physicochemically-driven stress granule formation. There are known compounds that disrupt this phase separation, particularly 1,6-hexanediol and similar alcohols. However, these compounds have two problems: first, they require extremely high concentrations (1 - 10%) and are toxic. Second, their effects are not stress granule-specific, and they also affect other liquid, membrane-less compartments. Many other compartments are also liquid, particularly in the nucleus.

[0006] There is a need for a method of identifying a compound that more specifically regulates the phase separation of certain compartments to effectively and specifically treat ALS.

[0007] All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety. SUMMARY OF THE INVENTION

[0008] In some aspects, the present disclosure provides a method of identifying a compound that modulates a property associated with one or more condensates comprising condensate-associated molecules, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or a cell composition capable of forming one or more condensates, and (b) determining a property associated with the one or more condensates, wherein modulation of the property as compared to a reference indicates that the compound modulates a property associated with the one or more condensates.

[0009] In some embodiments, determining a property associated with the one or more condensates is based on any one or more of the following: (i) the number of condensates comprising and / or not comprising condensate-associated molecules; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the liquidity of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fibril formation; (xi) the location of the condensate-associated molecules; (xii) the partitioning of the condensate-associated molecules into the one or more condensates; and (xiii) the aggregation of the condensate-associated molecules.

[0010] In some embodiments, the one or more condensates are within one or more cells of the cell composition.

[0011] In some embodiments, the method described herein further comprises: subjecting the cell composition to condensate-forming conditions prior to determining a property associated with one or more condensates.

[0012] In some embodiments, the method described herein further comprises: subjecting the cell composition to condensate-forming conditions prior to contacting the compound with the cell composition.

[0013] In some embodiments, the condensate-forming conditions are any one or more of the following: (i) an oxidative stressor; (ii) a mitochondrial electron transport chain inhibitor; (iii) a heat stressor; (iv) an osmotic stressor; (v) a hyperosmotic stressor; and (vi) glycolysis inhibition.

[0014] In some embodiments, the condensate-associated molecule is a polypeptide. In some embodiments, the condensate-associated molecule is a wild-type polypeptide. In some embodiments, the condensate-associated molecule is a mutant polypeptide. In some embodiments, the condensate-associated molecule is selected from: FUS, EWSR1, TIAL1, PABPC1, and G3BP1.

[0015] In some embodiments, the cells in the cell composition express the condensate-associated molecule. In some embodiments, the cell composition comprises HeLA, iPSC, or iPSC MN cells.

[0016] In some embodiments, the methods described herein further comprise imaging at least a portion of the cell composition.

[0017] In some embodiments, the methods described herein further comprise contacting at least a portion of the cell composition with a fixative.

[0018] In some embodiments, the methods described herein further comprise contacting at least a portion of the cell composition with a stain.

[0019] In some embodiments, the methods described herein further comprise contacting at least a portion of the cell composition with DNA damage conditions. In some embodiments, the DNA damage condition is laser irradiation.

[0020] In some embodiments, the reference is a second condensate. In some embodiments, the reference is a second cell composition.

[0021] In some embodiments, the methods described herein further comprise using a second cell-based assay to evaluate the identified compound.

[0022] In some embodiments, the methods described herein further comprise using a biochemical assay to evaluate the identified compound.

[0023] In some embodiments, the methods described herein further comprise using an in vivo assay to evaluate the identified compound.

[0024] In another aspect, the present disclosure provides a method of identifying a compound useful for treating a disease, the method comprising identifying a compound according to any of the methods described herein. In some embodiments, the disease is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is ALS.

[0025] The present invention relates to compounds for treating neurodegenerative diseases associated with stress granule formation (particularly for treating amyotrophic lateral sclerosis).

[0026] One aspect of the present invention relates to a compound for use in a method for preventing or treating a neurodegenerative disease associated with stress granule formation, the compound being selected from lipoic acid (5-(1,2-dithiolan-3-yl)pentanoic acid; CAS No. 1200-22-2; 1077-27-6; 1077-28-7), lipoamide (5-(1,2-dithiolan-3-yl)pentanamide; CAS No. 940-69-2), dihydrolipoic acid (6,8-dimercaptooctanoic acid; CAS No. 462-20-4) and dihydrolipoamide (6,8-bis(thioalkyl)octanamide; CAS No. 3884-47-7).

[0027] In particular, the neurodegenerative disease is associated with stress granule formation in the cytosol of cells contained in the diseased tissue.

[0028] In particular, lipoic acid and / or lipoamide can be used in the form of the R-type, S-type or racemate.

[0029] Those skilled in the art will appreciate that any specifically mentioned compound can exist as a pharmaceutically acceptable salt of the compound. Pharmaceutically acceptable salts comprise an ionized drug and a counterion of opposite charge. Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, benzenesulfonate, bitartrate, bromide, carbonate, chloride, citrate, edetate, edisylate, pamoate, ethoate, fumarate, glucoheptonate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylbromide, methylsulfate, mucate, naphthalenesulfonate, nitrate, pectinate, phosphate, diphosphate, salicylate, bisalicylate, stearate, succinate, sulfate, tartrate, toluenesulfonate, triethiodide and valerate. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminum, benzathine, calcium, ethylenediamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine and zinc.

[0030] In some embodiments, the compound of the present invention is administered according to the following dosage regimen: administering a daily dose of 600 mg to 1,600 mg of the compound.

[0031] Advantageously, a daily dose of 600 mg can produce a plasma concentration of the compound of the present invention of 8 μM to 30 μM.

[0032] In some embodiments, the neurodegenerative diseases associated with stress granule formation are selected from amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease and Huntington's disease.

[0033] In some embodiments, a pharmaceutical composition for use in a method of preventing or treating a neurodegenerative disease associated with stress granule formation comprises a compound of the invention as described in paragraph 0025.

[0034] In some embodiments, the pharmaceutical composition is formulated for oral administration.

[0035] In some embodiments, the neurodegenerative disease associated with stress granule formation is amyotrophic lateral sclerosis.

[0036] Alternatively, a dosage form for use in a method of preventing or treating a neurodegenerative disease associated with stress granule formation is provided, wherein the dosage form comprises a compound of the invention, particularly a compound as described in paragraph 0025.

[0037] The dosage form can be used for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or in the form of an inhalation or suppository. Alternatively, parenteral administration can be used, such as in the form of subcutaneous, intravenous, intrahepatic or intramuscular injection. Optionally, a pharmaceutically acceptable carrier and / or excipient may be present.

[0038] In some embodiments, the dosage form is formulated for oral administration.

[0039] In some embodiments, the neurodegenerative disease associated with stress granule formation is selected from amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease and Huntington's disease.

[0040] Alternatively, the therapeutic aspect according to the invention can be formulated as a method for preventing or treating a neurodegenerative disease associated with stress granule formation, wherein the method comprises administering a compound of the invention, particularly a compound as described in paragraph 0025, to a patient in need thereof.

[0041] In some embodiments, the compound is administered at a daily dose of 600 mg to 1,600 mg.

[0042] In some embodiments, the compound is administered orally.

[0043] In some embodiments, the neurodegenerative disease associated with stress granule formation is selected from amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease and Huntington's disease.

[0044] In some embodiments, the neurodegenerative disease associated with stress granule formation is amyotrophic lateral sclerosis.

[0045] In certain aspects, the invention relates to a method for reducing or inhibiting stress granule formation in cells, wherein the method comprises using a compound selected from:

[0046] - lipoic acid, lipoamide, dihydrolipoic acid, dihydrolipoamide,

[0047] - heterotricyclic compounds, especially anthraquinones or anthraquinone derivatives, such as 1,4-dihydroxyanthraquinone,

[0048] - acridines or acridine derivatives, such as quinacrine or aminoacridine or mitoxantrone;

[0049] - tetracyclic compounds, and

[0050] - surfactants, especially cetylpyridinium chloride.

[0051] In some embodiments, the compound is provided in the culture medium of the cultured cells.

[0052] Those skilled in the art will also understand that changes may be made in the form and details of the implementations described herein without departing from the scope of the disclosure. Additionally, although various advantages, aspects, and objects have been described with reference to various implementations, the scope of the disclosure should not be limited by reference to such advantages, aspects, and objects. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1Shown is a screen for small molecule compounds that affect the formation of FUS-containing stress granules in isolated HeLa cells. a) Subcellular localization of FUS GFP in unstressed HeLa cells, cells stressed with a negative control of the compound solvent (DMSO), and cells stressed with positive controls of dimercaprol (an arsenic chelating drug) and emetine (which prevents stress granule formation by stabilizing polyribosomes). Stress causes nuclear export of FUS and the formation of stress granules (cytoplasmic liquid droplets containing FUS). b) Screening workflow for small molecules that have an effect on FUS GFP localization in isolated HeLa cells. c) Mahalanobis distance rankings (averages from six fields of view) for all 1600 screened compounds, where high values ​​mean compounds have greater effects. Several automated measures of FUS localization were combined into a single Mahalanobis distance score; the largest contributors were the number and area of ​​cytoplasmic FUS droplets. 47 compounds were selected for further analysis using a cutoff of 130. Dimercaprol and emetine are highlighted. d) Screening workflow for small molecules that have an effect on in vitro FUS liquid-liquid phase separation of purified FUS GFP. e) Z-score ranking of changes in droplet number and signal partitioning to FUS GFP droplets (formed under low salt conditions), where larger positive or negative values ​​mean greater effects of the compound. Scores were calculated at the maximum concentration at which the compound solvent (DMSO) negative control had no significant effect; 100 μM. Lipoic acid amide, surfactants, and heterotri / tetracyclic compounds are indicated by data point color, see f). f) Examples of three selected classes; lipoic acid amide, cetylpyridinium chloride (surfactant), mitoxantrone (heterotri / tetracyclic compound). g) Appearance of droplets for the compound solvent (DMSO) negative control or examples of the following compound classes: cetylpyridinium chloride (surfactant), lipoic acid amide, or mitoxantrone (heterotricyclic). Note that the droplets for cetylpyridinium chloride and lipoic acid amide are larger, while those for mitoxantrone are smaller.

[0054] Figure 2 Structure activity relationships of lipoamide-related compounds are shown, which reveal possible mechanisms of action. a) Dose response of HeLa cell FUS GFP droplet number (●, left axis) and nuclear / cytoplasmic signal ratio (○, right axis) after pretreatment with lipoamide for 1 h, followed by arsenate stress for 1 h, and continued treatment with lipoamide. b) Dose response of iPS cell FUS GFP droplet number and nuclear / cytoplasm ratio, as described in a). c) Dose response of HeLa cell FUS GFP droplet number and nuclear / cytoplasm ratio for a series of lipoamide-related compounds, using the same stress / treatment protocol as a). d) Summary of compound structures and effects on cytoplasmic FUS droplet number and FUS nuclear compartmentalization.

[0055] Figure 3Shows the mode of action of lipoamide and lipoic acid on different stresses under different scenarios and reports the possible mechanisms of action. a) The loss kinetics of cytoplasmic FUS GFP droplets in HeLa cells pre-stressed with arsenate for 1 hour, then treated with 10 μM lipoamide (or DMSO vehicle control), and then continuously stressed with arsenate. Representative images at 2 minutes and 100 minutes after addition of the compound are shown on the right. Lipoamide can reverse the effects of existing arsenate stress. b) Images of HeLa cells expressing FUS GFP that have been pretreated with 10 μM lipoamide (or DMSO control) for 1 hour, then washed 3 times, and then stressed with arsenate for 1 hour. Pretreatment with lipoamide has no lasting effect on the cell's ability to prevent response to arsenate stress. c) Images of HeLa cells expressing GFP-tagged stress granule markers (EWSR1, TIAL1, PABC1, or G3BP1) 1 hour after arsenate stress and 10 μM lipoamide or an isomer of lipoic acid, arsenate stress with DMSO vehicle control, or DMSO without arsenate. Lipoamide and lipoic acid affect many stress granule components. d) Images of HeLa cells expressing FUS GFP after being subjected to different stresses - rotenone (mitochondrial), serum-free, sorbitol (osmotic), heat, arsenate, or 6-deoxyglucose (glycolytic) - while being treated with 10 μM lipoamide or an isomer of lipoic acid. Lipoamide and lipoic acid are active against multiple stresses, including mitochondrial, osmotic, and oxidative stress.

[0056] Figure 4 Shows that lipoamide does not dissolve the nuclear FUS compartment and other nuclear compartments. a) Images of HeLa cells expressing GFP markers of other membrane-less compartments after treatment with 10 μM compound (or DMSO control) for 1 hour. The position of the nucleus is indicated by a dashed outline where unclear. Lipoamide does not disrupt P-bodies (DCP1A), Cajal bodies (COIL), DNA damage foci (TRP53BP1), or nucleoli (NCB1), while mitoxantrone does have non-stress granule-specific effects. b) After treatment with the compound for 1 hour followed by arsenate stress for 1 hour, FUS GFP recruitment to sites of UV laser-induced DNA damage in iPS cells. The top row shows FUS GFP fluorescence before laser ablation, with stress granules indicated by arrows. The bottom row shows the average FUS GFP signal intensity response to DNA damage and one standard deviation above and below the mean. Mitoxantrone prevents the formation of nuclear FUS droplets at DNA damage sites below the EC 50 of the number of cytoplasmic droplets, while lipoamide does prevent the formation of nuclear FUS droplets.

[0057] Figure 5Shown are the effects of lipoamide and lipoic acid on the properties of FUS condensates and the aggregation of ALS-associated mutant FUS in vitro. a) Schematic illustration of the quantification of condensate droplet fluidity using optical tweezers. The time taken for two droplets to contact and start to fuse and relax into a single spherical droplet (once adjusted for droplet size) is a measure of the ratio of the viscosity to the surface tension of the droplet - a measure of fluidity. Representative of two independent replicates. b) Droplet size-corrected relaxation times for droplet fusion with 300 μM lipoamide or equivalent DMSO solvent control (0.3%). Boxes denote the 25th, 50th, and 75th percentiles, whiskers denote the 5th and 95th percentiles. Lipoamide reduces the fusion time, indicating lower viscosity and / or greater surface tension. c - e) Effects of 10 μM lipoamide on G156E FUS GFP condensates (formed under dextran crowding) under "aging" while oscillating relative to equivalent DMSO solvent control (0.1%). These conditions match those previously reported 7) conditions. c) Representative images after 30 h of aging showing fiber formation in the DMSO sample. d) Representative fluorescence recovery after photobleaching (FRAP) time series of FUS condensates and fibers during aging. e) Quantification of FRAP in c). Error bars represent standard deviation. Relative to equivalent DMSO solvent control (0.3%), aged condensates treated with lipoamide maintain large FUS GFP mobile fractions and short FRAP half-lives, while untreated condensates harden. Effects of lipoamide and lipoic acid on the "aging" of G156E FUS GFP condensates during oscillation. Both compounds delay fiber formation.

[0058] Figure 6 Shown is that lipoamide and lipoic acid directly alter FUS phase separation and FUS G156E aggregation in vitro. a) Droplets formed by 2.8 μM FUS GFP in vitro with different concentrations of KCl (which inhibits droplet formation) with 100 μM lipoamide, lipoic acid, or equivalent DMSO solvent control (1%). Lipoamide and lipoic acid subtly promote droplet formation. b) Effects of lipoamide and lipoic acid on the "aging" of G156E FUS GFP droplets during oscillation relative to equivalent DMSO solvent control (0.3%). Both compounds delay fiber formation. c) Representative fluorescence recovery after photobleaching (FRAP) of FUS droplets and fibers formed during the aging shown in b). Aged droplets treated with lipoamide or lipoic acid retain rapid FRAP, indicating fluidity. d) Quantification of the fluorescence signal intensity shown in c). Error bars represent standard deviation. While untreated droplets harden, treatment with lipoamide and lipoic acid enables droplets to maintain large FUS GFP mobile fractions and short FRAP half-lives.

[0059] Figure 7 It was shown that lipoamide accumulates to high concentrations in cells without being metabolized. a) Synthesized and characterized 15 NR-(+) and (±)-lipoic acid amide. Use 15 N-edited 1H-detected 1D HSQC NMR experiments allow selective detection of both amide protons in biological media and HeLa cell pellets. The trans-amide protons (resonating at 6.9 ppm) can be quantified by NMR. The signal intensity is proportional to the lipoamide concentration below pH 8.5 and 10 °C. See Supplementary Methods for details. b) By exposing cells to 15 N-lipoamide samples were then separated and the NMR signal intensity from the trans-amide protons was measured. 15 N-lipoamide concentration was used to measure the uptake of lipoamide by HeLa cells. 15 After the culture medium was removed, the cells were washed with culture medium (without arsenate) and detached with EDTA-trypsin. The solution or cell pellet / intracellular NMR was used to determine 15 N-lipoamide concentration. Example spectra of cells stressed with 3 mM arsenate and incubated with R-(+)-lipoamide are shown on the same y-axis scale. c) Cellular uptake was determined by subtracting the signal from the medium incubated with cells (red) from the signal from the cell-free medium (cyan). This is a comparison of stressed (3 mM arsenate) or unstressed cells with 15 All four combinations of NR-(+) or (±)-lipoamide were performed. 15 NR-(+)-lipoamide treated stressed cells, high signal intensity from washed cell samples (green) consistent with substantial uptake from the culture medium calculated from signal intensity with (red) and without cells (cyan). d) Quantification of c), showing percent uptake and calculated intracellular concentration, assuming that lipoamide is uniformly distributed within the cell (see Supplementary Methods). The uncertainty of the measurements was approximately 30%, and there were no significant differences in uptake between conditions. All measurements showed substantial uptake of lipoamide, and the cellular concentration was >1 mM.

[0060] Figure 8Shown, lipoamide and lipoic acid have beneficial effects on ALS models in vitro and in vivo. a, b) In Caenorhabditis elegans, lipoic acid reduces the aggregation of aging-induced stress granules but not stress granule proteins. a) Toxicity and effects on protein aggregation of R-(+)- or S-(-)-lipoic acid in worms overexpressing aggregation-prone fluorescently labeled proteins. The incidence of PAB-1 aggregation in pharyngeal muscles was scored from the proportion of cells with >10 aggregates. The incidences of RHO-1 and KIN-19 were scored in low, medium, and high grades - see Methods. Toxicity was evaluated from the proportion of abnormally small or dead animals. Both isomers of lipoic acid caused a strong dose-dependent reduction in PAB-1 (but not RHO-1 or KIN-19) aggregation. Indicated a significant change compared to the DMSO control, Fisher's exact test. ***p < 0.0001, **p < 0.001, *P < 0.01. Error bars represent the standard error of the proportion, n > 100 for each sample. b) Z-projection stacks of confocal microscopy through the pharynx of worms expressing fluorescently labeled PAB-1 with or without lipoic acid treatment, showing a reduced number of aggregates. c-e) On neurons derived from iPS cells expressing FUS P525L, lipoic acid and lipoamide are associated with familial ALS. c) Schematic of neuron culture, showing the channels where axons grow from the cell bodies on the right. The regions shown in the micrographs in d, e) are indicated. d) iPS-derived neurons cultured with 0.02% DMSO for 60 days. Neurons expressing wild-type FUS have stable axons, while neurons expressing FUS P525L have unstable axons that leave material around the exit points of the axons from the channels after dying. e) iPS-derived neurons expressing FUS P525L in culture after 60 days in the presence of 2 μM lipoamide or racemic R-(+)- or S-(-)-lipoic acid. Representative images from a blinded experiment, which also included neurons treated with DMSO (solvent control) shown in c). f) Lipoic acid restores the motor function defects of Drosophila melanogaster overexpressing human wild-type FUS or ALS-related FUS mutations. Overexpression of FUS leads to motor defects and the animals cannot climb. Lipoic acid treatment shows a dose-dependent increase in the climbing ability of animals expressing wild-type FUS, FUS P525L, or FUS R512C. **p < 0.005, p < 0.05, one-way ANOVA. g) Lipoamide also restores the motor function defects of Drosophila melanogaster. Using lipoamide treatment instead of lipoic acid, conditions equivalent to f) were shown. *P < 0.05, **p < 0.005, Student's t-test. h) Lipoic acid restores the motor function defects of Drosophila melanogaster overexpressing human wild-type FUS or ALS-related FUS mutations. Overexpression of FUS leads to motor defects and the animals cannot climb.**p < 0.005, p < 0.05, one-way ANOVA.

[0061] Figure 9 iPS-derived neurons cultured with 0.02% DMSO for 5 days and 60 days are shown. Neurons expressing wild-type FUS have stable axons, while neurons expressing FUS P525L have unstable axons, which leave material around the exit point of the axon from the channel after necrosis.

[0062] Figure 10 Shows that the structure-activity relationship of heterotricyclic compounds implies that the tricyclic nucleus is the cause of activity. a) Dose responses of the number of FUS GFP droplets (●, left axis) and the nuclear / cytoplasmic signal ratio (○, right axis) in HeLa cells after pretreatment with mitoxantrone for 1 hour, then arsenate stress for 1 hour, and continued treatment with mitoxantrone. b) Dose responses of the number of FUS GFP droplets and the nuclear / cytoplasmic ratio in HeLa cells for a series of compounds related to mitoxantrone and other heterotricyclic extracts, quinacrine, and the tetracycline antibiotic family, using the same stress / treatment protocol as in a). b) Summary of compound structures and their effects on cytoplasmic FUS droplet number and FUS nuclear compartmentalization.

[0063] Figure 11 Shows that lipoamide reduces wild-type and P525L FUS intracellular aggregates in iPS cells. a) FUS GFP localization in isogenic iPS cells expressing wild-type or P525L FUS GFP under a combination of 1-hour arsenate stress followed by 1-hour stress with 30 μM lipoamide or DMSO negative control. FUS P525L leads to the formation of larger cytoplasmic FUS droplets, which remain sensitive to lipoamide. b) Relative optical density of aggregated FUS was evaluated by filter retardation of iPS cells in a) after pretreatment with 100 μM thioamide or DMSO vehicle control for 1 hour, followed by 1-hour arsenate stress or no stress. Significant changes are shown (Student's t-test), n = 5. c) After cell treatment in b), immunoblotting with anti-FUS and anti-GAPDH was performed. No significant change in FUS expression level was detected relative to GAPDH (Student's t-test, n = 3). ***anova***

[0064] Figure 12Lipoic acid amide is shown to restore axonal transport defects caused by expression of FUS P525L in motor neurons. a) Example kymograph of lysosomal movement in the distal part of FUS P525L GFP motor neuron axons after 3 days of treatment with compound solvent (DMSO) or 2 μM lipoic acid amide. Lysosomal probe fluorescence. b) For motor neurons expressing P525L or wild-type FUS, the proportion of lysosomes labeled with lysosomal probe that moved at an average speed greater than 2 μM / s after 3 days of treatment with 2 μM lipoic acid amide or equivalent DMSO concentration solvent control. n=5 (P525L) or n=3 (wild-type) biological replicates, 5 axonal tracts analyzed per replicate. Lipoic acid amide significantly increases lysosomal trafficking (Student's T-test). ***anova***

[0065] Figure 13 shows the 1 H NMR 15 Characterization of N-lipoamide. a) 15 Chemical structure of N-lipoamide. b) 1 The resonances in the HNMR spectrum can be unambiguously assigned to CDCl3 15 c) Obtain a single proton of N-lipoamide. 15 N filtered NMR experiment showing the cis-amide and trans-amide protons of lipoamide (environments 13 and 14, respectively). The relative signal intensities are sensitive to local solution conditions, indicating chemical exchange at 37°C. d) At pH 8.3, the intensities of both resonances decrease with increasing temperature. This is indicative of chemical exchange, where local molecular dynamics and / or interactions with H2O on the ms to μs time scale reduce the signal. Below 15°C, the intensity of the trans-amide resonance (14) approaches a plateau, indicating a slow exchange regime, where signal intensity is an unambiguous measure of concentration. e) At 10°C, the intensities of the cis- and trans-amide proton resonances increase with decreasing pH, indicating the presence of secondary dynamics on the ms to μs scale. Below pH 8.6, the intensity of the trans-amide proton is constant, indicating a slow exchange regime. In summary, d) and f) show that at 10°C and below pH 8.6, the intensity of the trans-amide proton resonances is constant, indicating a slow exchange regime. 15 In the 1H NMR experiments edited by N, the integrated signal intensity of the trans-amide protons of lipoamide is a reliable proxy for concentration. f) When dissolved in growth medium, the signal intensity of the trans-amide protons of lipoamide decreases over time at 37°C, but not at 10°C. At 10°C, the signal intensity is stable for >10h experiments. g) Signal intensity of cis- and trans-amide protons under different experimental conditions. This is Figure 13BAn extended version that only shows trans - amide protons and includes an additional condition: v) cells disrupted with Triton X - 100 and DNaseI (from iii). In summary, i) to iv) imply that lipoamide is taken up by HeLa cells in a mobile form, while most molecules are unmodified ( Figure 13C -D, the uptake was quantified). h) An extended plot of the spectra in g), i), and v). After disrupting the cells with Triton X - 100 and DNaseI, significant changes occurred in the spectra, indicating chemical modification of lipoamide when the cell compartments were disrupted. Detailed implementation

[0066] In some aspects, the present invention includes methods for identifying compounds that regulate condensates and their applications, such as the usefulness of such compounds in treating diseases.

[0067] Traditionally, the various membrane - bound compartments (organelles) of cells were thought to be a jumbled mass of macromolecules and metabolites that all freely mixed with each other and diffused randomly within the boundaries of the membrane that outlined that particular compartment (nucleus, cytosol, mitochondria, endoplasmic reticulum, etc.). Cellular processes were thought to be regulated by the interactions of these molecules, which were driven by specific binding sites that could lead to biochemical reactions between molecules (enzyme activities, such as transcription, RNA processing, metabolic reactions, signal molecule reactions, such as phosphorylation, etc.). For example, a kinase diffuses until it happens to bind to a protein that it recognizes as its target, binds to it, and phosphorylates it.

[0068] From recent work, it is now clear that based on the intrinsic physical properties of proteins, RNA, and / or DNA, macromolecules are sorted into condensates within membrane - bound organelles. Macromolecules assemble or condense into liquid droplets in a phase - separation reaction, resulting in specific proteins and / or nucleic acid molecules being enriched within the condensate, while other specific proteins and / or nucleic acids are excluded.

[0069] The process of forming condensates is not driven by specific simple stoichiometric binding events as previously thought, but by phase - separation reactions. This is a layered structure within the cell that arranges the macromolecules of the entire cell into sub - compartments to increase the specificity of reactions and drive reactions through higher local concentrations.

[0070] Notably, condensates are liquid and reversible, such that upon changes in cell physiology (such as signal events, changes in the concentration of one of the macromolecules, or other changes in the local environment), the different condensates within the cell will change, sometimes dissolving completely and sometimes more subtly altering their molecular composition. Thus, this is a major mechanism for regulating almost all reactions within the cell, working in concert with the mechanism of specific binding events as previously understood.

[0071] Some diseases, including neurodegenerative diseases such as ALS, appear to be caused by abnormal protein aggregates that accumulate in cells. Previous attempts to screen for drugs that prevent or disrupt these aggregates were not based on the new understanding of condensates. The disclosure of the present application is at least partially based on the inventors' unique insight that in such cases, the phase separation of macromolecules into condensates is an intermediate step that occurs before the subsequent formation of abnormal protein aggregates, and thus, by using assays that directly monitor the effects of compounds on condensate formation and properties, new useful compounds for treating such diseases can be discovered.

[0072] In some aspects, the methods disclosed herein allow for the identification of compounds that modulate certain aspects of condensate behavior. For example, some aspects of the methods disclosed herein allow for the identification of compounds that modulate fluidity but not condensate formation. Additionally, using the methods described herein, the inventors have identified compounds that do not simply disrupt all condensate formation, but rather exhibit specificity for target condensates. This specificity can potentially enhance efficacy and reduce side effects such as toxicity.

[0073] The present application provides new methods for identifying compounds that modulate one or more properties of cellular and extracellular condensates and can thus be used to treat condensate-related diseases. The methods focus on changes in the behavior or properties of one or more condensates, thereby allowing for the identification of compounds without the need to know about the specific molecular targets of each compound. This allows one to rapidly screen for potentially useful compounds in a simple and elegant behavioral assay. Exemplary properties of condensates include, but are not limited to: (i) the number of condensates that contain and / or do not contain condensate-related molecules; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the fluidity of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-related molecules; (xii) the partitioning of the condensate-related molecules into the one or more condensates; and (xiii) the aggregation of condensate-related molecules. Compounds with desired properties can be identified by evaluating the ability to modulate some (e.g., any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) or all of the properties of one or more condensates. Additionally, compounds with desired properties can be identified by evaluating the ability to modulate some but not other features of condensates, or the ability to modulate some but not other condensates. Using the methods described herein, compounds that target the physical chemistry of condensates formed by liquid-liquid phase separation have been identified.

[0074] Thus, in some aspects, the present disclosure provides methods for screening compounds that modulate properties associated with one or more condensates and / or are useful in methods for treating diseases. In some aspects, the present disclosure provides methods for high-throughput screening of compounds that modulate properties associated with one or more condensates and / or are useful in methods for treating diseases.

[0075] In some embodiments, the method comprises: (a) contacting the compound with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining a property associated with the one or more condensates. In some embodiments, modulation of the property indicates that the compound modulates a property associated with one or more condensates. In some embodiments, modulation of the property relative to a reference indicates that the compound modulates a property associated with one or more condensates. In some embodiments, the method comprises determining multiple properties associated with one or more condensates.

[0076] In some embodiments, the method comprises: (a) contacting a plurality of candidate compounds with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining a property associated with the one or more condensates, wherein modulation of the property relative to a reference indicates that the candidate compound modulates a property associated with the one or more condensates, thereby obtaining a compound that modulates a property of one or more condensates.

[0077] In some aspects, the present disclosure provides methods for identifying a compound that modulates a property associated with one or more condensates comprising a first set of condensate-associated molecules, the method comprising: (a) contacting the compound with a cell composition comprising the first set and a second set of one or more condensates or with a cell composition capable of forming the first set and a second set of one or more condensates, and (b) determining a property associated with the first set of one or more condensates, and (c) determining a property associated with the second set of one or more condensates.

[0078] In some aspects, the present disclosure provides methods for identifying a compound that modulates a property associated with one or more condensates comprising a first set of condensate-associated molecules, the method comprising: (a) contacting the compound with a cell composition comprising the first set and a second set of one or more condensates or with a cell composition capable of forming the first set and a second set of one or more condensates, and (b) determining a property associated with the first set of one or more condensates, and (c) determining a property associated with the second set of one or more condensates.

[0079] In some aspects, provided herein are methods of screening (e.g., high-throughput screening) compounds that modulate a property associated with one or more condensates of a first set of condensate-associated molecules, the methods comprising: (a) contacting a plurality of candidate compounds with a cell composition comprising the one or more condensates of the first set or with a cell composition capable of forming the one or more condensates of the first set, and (b) determining a property associated with the one or more condensates of the first set, (c) contacting the plurality of candidate compounds with a cell composition comprising a second set of one or more condensates or with a cell composition capable of forming the second set of one or more condensates, the second set of one or more condensates comprising second condensate-associated molecules, and (d) determining a property associated with the second set of one or more condensates.

[0080] In some embodiments, modulation of the property associated with the first set is different from modulation of the property associated with the second set, indicating that the candidate compound modulates a property associated with one or more condensates of the first set, thereby obtaining a compound that modulates a property associated with one or more condensates of the first set. In some embodiments, modulation of the property associated with the second set is different from modulation of the property associated with the first set as compared to a reference, indicating that the compound modulates a property associated with one or more condensates of the second set, thereby obtaining a compound that modulates a property associated with one or more condensates of the second set.

[0081] Definitions

[0082] For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural and vice versa. In the event of any conflict between any of the definitions set forth below and any document incorporated herein by reference, the definition set forth shall prevail.

[0083] The terms “polypeptide” and “protein” are used interchangeably and refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues can contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers of amino acid residues, trimers of amino acid residues, and polymers of amino acid residues. Full-length proteins and fragments thereof are included in this definition. The term also includes post-translational modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, and the like.

[0084] As used herein, the terms "polynucleotide" or "nucleic acid" refer to nucleotides in polymeric form of any length, including ribonucleotides and deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, mRNA, DNA-RNA hybrids or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleobases. The backbone of a polynucleotide can include sugar and phosphate groups (such as can typically be found in RNA or DNA), or modified or substituted sugars or phosphate groups. The backbone of a polynucleotide can include repeating units linked by peptide bonds (such as peptide nucleic acids), such as N-(2-aminoethyl)-glycine. Alternatively, the backbone of a polynucleotide can include a polymer of synthetic subunits, such as phosphoramidates, and can thus be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate-phosphodiester oligomer.

[0085] As used herein, the terms "comprising", "having", "containing" and "including" and other similar forms and their grammatical equivalents are intended to be equivalent in meaning and are open-ended, since the one or more items following any of these words do not mean an exhaustive listing of such one or more items, or are not meant to be limited to the listed one or more items. For example, an article "comprising" components A, B and C can consist of components A, B and C (i.e., contain only components A, B and C), or can contain not only components A, B and C, but also one or more other components. Thus, it is intended and should be understood that "comprising" and its similar forms and their grammatical equivalents include embodiments that disclose "consisting essentially of" or "consisting of".

[0086] Where a range of values is provided, it is to be understood that, unless the context clearly dictates otherwise, each intermediate value between the upper and lower limits of the stated range and to the tenth of the unit of the lower limit and any other stated or intermediate value within the stated range is included in the present disclosure, subject to any explicit exclusions within the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present disclosure.

[0087] References herein to "about" a value or parameter include (and describe) variations that relate to that value or parameter per se. For example, a description of "about X" includes a description of "X".

[0088] As used herein, unless the context clearly dictates otherwise, including in the appended claims, the singular forms "a", "or" and "the" include plural referents.

[0089] As used herein, "condensate" refers to a membrane-less enclosed compartment formed by phase separation (including all stages of phase separation) of one or more proteins and / or other macromolecules.

[0090] "Condensate-associated molecule" as used herein refers to a molecule that can be found in or on a condensate under physiological or pathological conditions.

[0091] In the context of this specification, the term stress granule refers to a liquid-like, membrane-less bounded compartment that is located within the nucleus or cytosol of a cell and contains proteins and RNA, and appears when the cell is under stress.

[0092] In the context of this specification, the term neurodegenerative disease refers to a medical condition characterized by a progressive loss of the structure or function of neurons. Non-limiting examples of neurodegenerative diseases include amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease.

[0093] As used herein, the term treating or treatment of any disease or disorder (e.g., ALS) in one embodiment refers to ameliorating the disease or disorder (e.g., slowing or arresting or reducing the development of the disease or at least one of its clinical symptoms). In another embodiment, "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter, including those that may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. As used herein, the term prevent or preventing or prevention of any disease or disorder (e.g., ALS) refers to inhibiting (including completely inhibiting) the development or onset of the disease or disorder (e.g., delaying the development or onset of the disease or disorder). Unless specifically described below, methods for assessing the treatment and / or prevention of a disease are generally known in the art.

[0094] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0095] Methods for screening and identifying compounds

[0096] In some aspects of the present application, methods for identifying a compound that modulates a property associated with one or more condensates and / or is useful in a method of treating a disease are provided. In some aspects, methods for screening a compound that modulates a property associated with one or more condensates and / or is useful in a method of treating a disease are provided herein. In some aspects, methods for high-throughput screening of a compound that modulates a property associated with one or more condensates and / or is useful in a method of treating a disease are provided herein.

[0097] The techniques for identifying a compound are described in more detail below. Those skilled in the art will recognize that, given the provided description, multiple embodiments are possible within the scope and spirit of the disclosure of the present application.

[0098] In some aspects, methods for identifying a compound that modulates a property associated with one or more condensates comprising condensate-associated molecules are provided, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining a property associated with the one or more condensates, wherein modulation of the property as compared to a reference indicates that the compound modulates a property associated with the one or more condensates. In some embodiments, the method comprises determining a single property associated with one or more condensates. In some embodiments, the method comprises determining multiple properties associated with the one or more condensates.

[0099] In some aspects, provided herein are methods of screening (e.g., high-throughput screening) compounds that modulate a property associated with one or more condensates, the methods comprising evaluating each of a plurality of compounds in the screening using any of the methods described herein. In some embodiments, a method of screening (e.g., high-throughput screening) compounds that modulate a property associated with one or more condensates comprising condensate-associated molecules, the method comprising: (a) contacting each of a plurality of candidate compounds with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining a property associated with the one or more condensates, wherein modulation of the property, compared to a reference, indicates that the candidate compound modulates a property associated with the one or more condensates, thereby obtaining a compound that modulates a property of one or more condensates. In some embodiments, the method comprises determining a plurality of properties associated with the one or more condensates. In some embodiments, the plurality of compounds comprises at least any one of 100, 200, 300, 500, 800, 1000, 1500, 3,000, 5,000, 10,000 or more different candidate compounds. In some embodiments, the plurality of candidate compounds are contacted with the cell composition in separate reactions (e.g., separate wells or vessels). In some embodiments, the plurality of candidate compounds are contacted with the cell composition simultaneously in separate reactions.

[0100] In some aspects, provided herein are methods of identifying a compound that modulates a property associated with a first set of one or more condensates comprising a first condensate-associated molecule, the method comprising: (a) contacting the compound with a cell composition comprising the first and second sets of one or more condensates or with a cell composition capable of forming the first and second sets of one or more condensates, (b) determining a property associated with the first set of one or more condensates, and (c) determining a property associated with the second set of one or more condensates, wherein modulation of the property associated with the first set, compared to a reference, is different from modulation of the property associated with the second set, indicating that the compound modulates a property associated with the first set of one or more condensates.

[0101] In some aspects, provided herein are methods of identifying a compound that modulates a property associated with a first set of one or more condensates comprising a first condensate-associated molecule, the method comprising: (a) contacting the compound with a cell composition comprising the first set of one or more condensates, or with a cell composition capable of forming the first set of one or more condensates, (b) determining a property associated with the first set of one or more condensates, (c) contacting the compound with a cell composition comprising a second set of one or more condensates, or with a cell composition capable of forming the second set of one or more condensates, the second set of one or more condensates comprising a second condensate-associated molecule, and (d) determining a property associated with the second set of one or more condensates, wherein modulation of the property associated with the first set is different from modulation of the property associated with the second set, as compared to a reference, indicating that the compound modulates a property associated with the first set of one or more condensates.

[0102] In some aspects, provided herein are methods of screening (e.g., high-throughput screening) for a compound that modulates a property associated with a first set of one or more condensates comprising a first condensate-associated molecule, the method comprising: (a) contacting a plurality of candidate compounds with a cell composition comprising a first and a second set of one or more condensates, or with a cell composition capable of forming the first and second sets of one or more condensates, (b) determining a property associated with the first set of one or more condensates, and (c) determining a property associated with the second set of one or more condensates, wherein modulation of the property associated with the first set is different from modulation of the property associated with the second set, as compared to a reference, indicating that the compound modulates a property associated with the first set of one or more condensates, thereby obtaining a compound that modulates a property associated with the first set of one or more condensates.

[0103] In some aspects, provided herein are methods of screening (e.g., high-throughput screening) compounds that modulate a property related to a first set of one or more condensates comprising a first condensate-associated molecule, the methods comprising: (a) contacting a plurality of candidate compounds with a cell composition comprising the first set of one or more condensates or with a cell composition capable of forming the first set of one or more condensates, (b) determining a property related to the first set of one or more condensates, (c) contacting the plurality of candidate compounds with a cell composition comprising a second set of one or more condensates or with a cell composition capable of forming the second set of one or more condensates, the second set of one or more condensates comprising a second condensate-associated molecule, and (d) determining a property related to the second set of one or more condensates, wherein a modulation of the property related to the first set is different from a modulation of the property related to the second set as compared to a reference, indicating that the compound modulates a property related to the first set of one or more condensates, thereby obtaining a compound that modulates a property related to the first set of one or more condensates. In some embodiments, the first and second condensate-associated molecules are the same. In some embodiments, the first and second condensate-associated molecules are different. In some embodiments, the first set of one or more condensates is stress granules and the second set of one or more condensates is paraspeckles.

[0104] In some aspects, provided herein are methods of identifying a compound that modulates a property related to one or more stress granules, the methods comprising: (a) contacting the compound with a cell composition comprising one or more stress granules or with a cell composition capable of forming one or more stress granules, and (b) determining a property related to the one or more stress granules, wherein a modulation of the property as compared to a reference indicates that the compound modulates a property related to the one or more stress granules. In some embodiments, the method includes determining a single property related to the one or more stress granules. In some embodiments, the method comprises determining a plurality of properties related to the one or more stress granules.

[0105] In some aspects, provided herein are methods of screening (e.g., high-throughput screening) compounds that modulate a property associated with one or more stress granules, the methods comprising: (a) contacting a plurality of candidate compounds with a cell composition comprising one or more stress granules or with a cell composition capable of forming one or more stress granules, and (b) determining a property associated with the one or more stress granules, wherein modulation of the property as compared to a reference indicates that the candidate compound modulates a property associated with the one or more stress granules, thereby obtaining a compound that modulates a property of one or more stress granules. In some embodiments, the methods include determining a plurality of properties associated with the one or more stress granules. In some embodiments, the plurality of compounds includes at least any one of 100, 200, 300, 500, 800, 1000, 1500, 3,000, 5,000, 10,000 or more different candidate compounds. In some embodiments, the plurality of candidate compounds are contacted with the cell composition in separate reactions (e.g., separate wells or vessels). In some embodiments, the plurality of candidate compounds are contacted with the cell composition simultaneously in separate reactions.

[0106] In some aspects, provided herein are methods of identifying a compound that modulates a property associated with one or more stress granules, the methods comprising: (a) contacting the compound with a cell composition comprising one or more stress granules and one or more paraspeckles, condensates formed around DNA damage sites, P-bodies, Cajal bodies, and / or PML bodies, or with a cell composition capable of forming one or more stress granules and one or more paraspeckles, condensates formed around DNA damage sites, P-bodies, Cajal bodies, and / or PML bodies, (b) determining a property associated with the one or more stress granules, and (c) determining a property associated with the one or more paraspeckles, condensates formed around DNA damage sites, P-bodies, Cajal bodies, and / or PML bodies, wherein modulation of the property associated with the one or more stress granules that is different from modulation of the property associated with the one or more paraspeckles, condensates formed around DNA damage sites, P-bodies, Cajal bodies, and / or PML bodies as compared to a reference indicates that the compound modulates a property associated with the one or more stress granules.

[0107] In some aspects, provided herein are methods of identifying a compound that modulates a feature associated with one or more stress granules, the methods comprising: (a) contacting the compound with a cell composition comprising the one or more stress granules or with a cell composition capable of forming the one or more stress granules, (b) determining a feature associated with the one or more stress granules, (c) contacting the compound with a cell composition comprising one or more paraspeckles, condensates formed around DNA damage sites, P bodies, Cajal bodies, and / or PML bodies, or with a cell composition capable of forming one or more paraspeckles, condensates formed around DNA damage sites, P bodies, Cajal bodies, and / or PML bodies, and (d) determining a property associated with the one or more paraspeckles, condensates formed around DNA damage sites, P bodies, Cajal bodies, and / or PML bodies, wherein a modulation of a property associated with the one or more stress granules is different from a modulation of a property associated with the one or more paraspeckles, condensates formed around DNA damage sites, P bodies, Cajal bodies, and / or PML bodies as compared to a reference, indicating that the compound modulates a property associated with the one or more stress granules.

[0108] In some aspects, provided herein are methods of screening (e.g., high-throughput screening) compounds that modulate a property associated with one or more paraspeckles, condensates formed around DNA damage sites, P bodies, Cajal bodies, and / or PML bodies, the methods comprising: (a) contacting a plurality of candidate compounds with a cell composition comprising the one or more stress granules and one or more paraspeckles, condensates formed around DNA damage sites, P bodies, Cajal bodies, and / or PML bodies, or with a cell composition capable of forming the one or more stress granules and one or more paraspeckles, condensates formed around DNA damage sites, P bodies, Cajal bodies, and / or PML bodies, (b) determining a property associated with the one or more stress granules, and (c) determining a property associated with the one or more paraspeckles, condensates formed around DNA damage sites, P bodies, Cajal bodies, and / or PML bodies, wherein a modulation of a property associated with the one or more stress granules is different from a modulation of a property associated with the one or more paraspeckles, condensates formed around DNA damage sites, P bodies, Cajal bodies, and / or PML bodies as compared to a reference, indicating that the compound modulates a property associated with the one or more stress granules, thereby obtaining a compound that modulates a property associated with the one or more stress granules.

[0109] In some aspects, provided herein are methods of screening (e.g., high-throughput screening) compounds that modulate a property associated with one or more stress granules, the methods comprising: (a) contacting a plurality of candidate compounds with a cell composition comprising the one or more stress granules or with a cell composition capable of forming the one or more stress granules, (b) determining a property associated with the one or more stress granules, (c) contacting the plurality of candidate compounds with a cell composition comprising one or more paraspeckles, condensates formed around DNA damage sites, P-bodies, Cajal bodies, and / or PML bodies, or with a cell composition capable of forming the one or more paraspeckles, condensates formed around DNA damage sites, P-bodies, Cajal bodies, and / or PML bodies, and (d) determining a property associated with the one or more paraspeckles, condensates formed around DNA damage sites, P-bodies, Cajal bodies, and / or PML bodies, wherein a modulation of a property associated with the one or more stress granules is different, compared to a reference, from a modulation of a property associated with the one or more paraspeckles, condensates formed around DNA damage sites, P-bodies, Cajal bodies, and / or PML bodies, indicating that the compound modulates a property associated with the one or more stress granules, thereby obtaining a compound that modulates a property associated with the one or more stress granules.

[0110] In some embodiments, the method further comprises repeating the steps of the method for a plurality of compounds. For example, in some embodiments, the method includes repeating the steps of the method for any of at least about 2, 3, 4, 5, 10, 15, 20, 25, 40, 50, 75, 100, 250, 500, 1,000, 10,000, 100,000 or more compounds. In some embodiments, the method further comprises repeating the steps of the method with a plurality of concentrations of the compound.

[0111] In some embodiments, the methods described herein comprise contacting the compound with a cell composition comprising one or more condensates or a cell composition capable of forming one or more condensates. One of ordinary skill in the art will readily recognize that cellular processes, including the state of condensates and their components, are dynamic. Accordingly, the methods described herein include contacting a composition, such as a cell composition, with a compound at any point in the life cycle of one or more condensates. For example, the methods comprise contacting a cell composition with a compound when the condensate-associated molecule is present at any location, in any amount, or in any post-translational modification state (such as the presence, absence, or level of phosphorylated residues) in the cell. In some aspects, the methods may further comprise, for example, contacting a cell with a compound when one or more condensates are present (including absent) in any amount, at any location in the cell, and are undergoing morphological changes (such as changes in size or fluidity, or composition).

[0112] In some embodiments, the cell composition comprises one or more condensates prior to contacting with the compound. In some embodiments, the method further comprises subjecting the cell composition to condensate-forming conditions prior to contacting the compound with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates. In some embodiments, the cell composition does not comprise one or more condensates prior to contacting with the compound, and the method comprises subjecting the cell composition to condensate-forming conditions to form the one or more condensates. In some embodiments, the cell composition does not comprise one or more condensates prior to contacting with the compound, and the method comprises subjecting the cell composition to condensate-forming conditions after contacting the cell composition with the compound to form the one or more condensates. In some embodiments, the cell composition does not comprise one or more condensates prior to contacting with the compound, and one or more condensates are formed simultaneously with contacting the cell composition with the compound. In some embodiments, the condensates are formed simultaneously with and after addition of the compound. In some embodiments, the cell composition is subjected to condensate-forming conditions prior to determining a property associated with the one or more condensates. In some embodiments, the cell composition comprises one or more condensates, and additional condensates of the one or more condensates are formed simultaneously with contacting the cell composition with the compound. In some embodiments, the cell composition comprises one or more condensates, and additional condensates of the one or more condensates are formed after contacting the cell composition with the compound. In some embodiments, the cell composition comprises one or more condensates, and additional condensates of the one or more condensates are formed simultaneously with and after contacting the cell composition and the compound.

[0113] Accordingly, the methods described herein include contacting a cell composition with a compound, wherein (i) the cell composition comprises one or more target condensates; and / or (ii) the one or more target condensates are formed while and / or after contacting the cell composition with the compound. In some embodiments, the methods described herein include contacting a cell composition with a compound, wherein the cell composition (i) comprises one or more target condensates; and / or (ii) is capable of forming one or more condensates, wherein the one or more condensates are formed while and / or after contacting the cell composition with the compound. In some embodiments, the methods described herein include contacting a compound with a cell composition comprising one or more condensates or a cell composition capable of forming one or more condensates.

[0114] The condensate formation conditions can include the addition of a condensate inducer. In some embodiments, the method comprises subjecting the cell composition to any one or more of the following: (i) an oxidative stressor; (ii) a mitochondrial electron transport chain inhibitor; (iii) a heat stressor; (iv) an osmotic stressor; (v) a hyperosmotic stressor; and (vi) glycolysis inhibition. In some embodiments, the oxidative stressor is arsenate. In some embodiments, the mitochondrial electron transport chain inhibitor is rotenone. In some embodiments, the osmotic stressor is sorbitol. In some embodiments, the glycolysis inhibition is 6-deoxyglucose in the absence of glucose. In some embodiments, the heat stressor subjects the cell composition to a temperature of about 40-45 °C, such as about 42 °C.

[0115] In some embodiments, the reference is an experimental control. In some embodiments, the condensate is a first condensate and the reference is a second condensate. In some embodiments, the second condensate is a condensate that does not contain condensate-related molecules. In some embodiments, the second condensate is a condensate that contains condensate-related molecules. In some embodiments, the first condensate and the second condensate are in different parts of the cell composition. In some embodiments, the first condensate and the second condensate are in different parts of the cell. In some embodiments, the first condensate and the second condensate are in different cell compositions. In some embodiments, the cell composition is a first composition and the reference is a second cell composition. In some embodiments, the reference is a cell composition that is not contacted with the compound. In some embodiments, the reference is a cell composition that is not treated with the condensate formation conditions. In some embodiments, the reference is a cell composition treated with a reference compound.

[0116] In some embodiments, a reference is prepared in a manner such that meaningful results of a compound can be evaluated. For example, in some embodiments, the reference is a cell composition, wherein the cell composition is prepared in a manner similar to the cell composition that contacts the compound, except that the reference cell composition is not subjected to the test compound or the same steps of contacting with the compound. In some embodiments, the reference is a cell composition that contacts a reference compound (e.g., a positive or negative control compound).

[0117] Properties related to condensates

[0118] In some embodiments, the properties related to one or more condensates are determined based on any one or more of the following: (i) the number of condensates that contain and / or do not contain condensate-related molecules; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the fluidity of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-related molecules; (xii) the partitioning of the condensate-related molecules into the condensates; and (xiii) the aggregation of the condensate-related molecules. In some embodiments, the method includes determining a first property related to one or more condensates and a second property related to one or more condensates. In some embodiments, the method includes determining a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth property related to the one or more condensates. Exemplary techniques that can be used to determine properties are disclosed in the examples.

[0119] In some embodiments, the method includes determining a first property related to one or more condensates and a second property related to one or more condensates. In some embodiments, the first property is (i) the number of condensates that contain and / or do not contain condensate-related molecules, and the second property is (ii) the size of one or more condensates, (iii) the location of one or more condensates, (iv) the distribution of one or more condensates, (v) the surface area of one or more condensates, (vi) the composition of one or more condensates, (vii) the mobility of one or more condensates, (viii) the solidification of one or more condensates, (ix) the dissolution of one or more condensates, (x) the presence and / or amount of fiber formation, (xi) the location of condensate-related molecules, (xii) the partitioning of the condensate-related molecules into the condensates, and (xiii) the aggregation of the condensate-related molecules, any one (or more, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) thereof. In some embodiments, the first property is (i) the number of condensates that contain and / or do not contain condensate-related molecules, and the second property is (ii) the size of one or more condensates. In some embodiments, the first property is (i) the number of condensates that contain and / or do not contain condensate-related molecules, and the second property is (iii) the location of the one or more condensates. In some embodiments, the first property is (i) the number of condensates that contain and / or do not contain condensate-related molecules, and the second property is (iv) the distribution of one or more condensates. In some embodiments, the first property is (i) the number of condensates that contain and / or do not contain condensate-related molecules, and the second property is (v) the surface area of the one or more condensates. In some embodiments, the first property is (i) the number of condensates that contain and / or do not contain condensate-related molecules, and the second property is (vi) the composition of the one or more condensates. In some embodiments, the first property is (i) the number of condensates that contain and / or do not contain condensate-related molecules, and the second property is (vii) the mobility of the one or more condensates. In some embodiments, the first property is (i) the number of condensates that contain and / or do not contain condensate-related molecules, and the second property is (viii) the solidification of the one or more condensates. In some embodiments, the first property is (i) the number of condensates that contain and / or do not contain condensate-related molecules, and the second property is (ix) the dissolution of the one or more condensates. In some embodiments, the first property is (i) the number of condensates that contain and / or do not contain condensate-related molecules, and the second property is (x) the presence and / or amount of fiber formation.In some embodiments, the first property is (i) the number of condensates that contain and / or do not contain condensate-associated molecules, and the second property is (xi) the location of the condensate-associated molecules. In some embodiments, the first property is (i) the number of condensates that contain and / or do not contain condensate-associated molecules, and the second property is (xii) the partitioning of the condensate-associated molecules into the condensates. In some embodiments, the first property is (i) the number of condensates that contain and / or do not contain condensate-associated molecules, and the second property is (xiii) the aggregation of the condensate-associated molecules.

[0120] In some embodiments, the method includes determining a first property associated with the one or more condensates and a second property associated with the one or more condensates. In some embodiments, the first property is (ii) the size of the one or more condensates, and the second property is any one (or more, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) of (iii) the location of the one or more condensates, (iv) the distribution of the one or more condensates, (v) the surface area of the one or more condensates, (vi) the composition of the one or more condensates, (vii) the mobility of the one or more condensates, (viii) the solidification of the one or more condensates, (ix) the dissolution of the one or more condensates, (x) the presence and / or amount of fiber formation, (xi) the location of the condensate-associated molecules, (xii) the partitioning of the condensate-associated molecules into the condensates, and (xiii) the aggregation of the condensate-associated molecules. In some embodiments, the first property is (ii) the size of the one or more condensates, and the second property is (iii) the location of the one or more condensates. In some embodiments, the first property is (ii) the size of the one or more condensates, and the second property is (iv) the distribution of the one or more condensates. In some embodiments, the first property is (ii) the size of the one or more condensates, and the second property is (v) the surface area of the one or more condensates. In some embodiments, the first property is (ii) the size of the one or more condensates, and the second property is (vi) the composition of the one or more condensates. In some embodiments, the first property is (ii) the size of the one or more condensates, and the second property is (vii) the mobility of the one or more condensates. In some embodiments, the first property is (ii) the size of the one or more condensates, and the second property is (viii) the solidification of the one or more condensates. In some embodiments, the first property is (ii) the size of the one or more condensates, and the second property is (ix) the dissolution of the one or more condensates. In some embodiments, the first property is (ii) the size of the one or more condensates, and the second property is (x) the presence and / or amount of fiber formation. In some embodiments, the first property is (ii) the size of the one or more condensates, and the second property is (xi) the location of the condensate-associated molecules. In some embodiments, the first property is (ii) the size of the one or more condensates, and the second property is (xii) the partitioning of the condensate-associated molecules into the condensates. In some embodiments, the first property is (ii) the size of the one or more condensates, and the second property is (xiii) the aggregation of the condensate-associated molecules.

[0121] In some embodiments, the method includes determining a first property associated with the one or more condensates and a second property associated with the one or more condensates. In some embodiments, the first property is (iii) the location of the one or more condensates, and the second property is any one (or more, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10) of (iv) the distribution of the one or more condensates, (v) the surface area of the one or more condensates, (vi) the composition of the one or more condensates, (vii) the mobility of the one or more condensates, (viii) the solidification of the one or more condensates, (ix) the dissolution of the one or more condensates, (x) the presence and / or amount of fiber formation, (xi) the location of the condensate-associated molecules, (xii) the partitioning of the condensate-associated molecules into the condensates, and (xiii) the aggregation of the condensate-associated molecules. In some embodiments, the first property is (iii) the location of the one or more condensates, and the second property is (iv) the distribution of the one or more condensates. In some embodiments, the first property is (iii) the location of the one or more condensates, and the second property is (v) the surface area of the one or more condensates. In some embodiments, the first property is (iii) the location of the one or more condensates, and the second property is (vi) the composition of the one or more condensates. In some embodiments, the first property is (iii) the location of the one or more condensates, and the second property is (vii) the mobility of the one or more condensates. In some embodiments, the first property is (iii) the location of the one or more condensates, and the second property is (viii) the solidification of the one or more condensates. In some embodiments, the first property is (iii) the location of the one or more condensates, and the second property is (ix) the dissolution of the one or more condensates. In some embodiments, the first property is (iii) the location of the one or more condensates, and the second property is (x) the presence and / or amount of fiber formation. In some embodiments, the first property is (iii) the location of the one or more condensates, and the second property is (xi) the location of the condensate-associated molecules. In some embodiments, the first property is (iii) the location of the one or more condensates, and the second property is (xii) the partitioning of the condensate-associated molecules into the condensates. In some embodiments, the first property is (iii) the location of the one or more condensates, and the second property is (xiii) the aggregation of the condensate-associated molecules.

[0122] In some embodiments, the method includes determining a first property related to the one or more condensates and a second property related to the one or more condensates. In some embodiments, the first property is (iv) the distribution of the one or more condensates, and the second property is any one (or more, such as 2, 3, 4, 5, 6, 7, 8, or 9) of (v) the surface area of the one or more condensates, (vi) the composition of the one or more condensates, (vii) the fluidity of the one or more condensates, (viii) the solidification of the one or more condensates, (ix) the dissolution of the one or more condensates, (x) the presence and / or amount of fiber formation, (xi) the location of the condensate-related molecules, (xii) the partitioning of the condensate-related molecules into the condensates, and (xiii) the aggregation of the condensate-related molecules. In some embodiments, the first property is (iv) the distribution of the one or more condensates, and the second property is (v) the surface area of the one or more condensates. In some embodiments, the first property is (iv) the distribution of the one or more condensates, and the second property is (vi) the composition of the one or more condensates. In some embodiments, the first property is (iv) the distribution of the one or more condensates, and the second property is (vii) the fluidity of the one or more condensates. In some embodiments, the first property is (iv) the distribution of the one or more condensates, and the second property is (viii) the solidification of the one or more condensates. In some embodiments, the first property is (iv) the distribution of the one or more condensates, and the second property is (ix) the dissolution of the one or more condensates. In some embodiments, the first property is (iv) the distribution of the one or more condensates, and the second property is (x) the presence and / or amount of fiber formation. In some embodiments, the first property is (iv) the distribution of the one or more condensates, and the second property is (xi) the location of the condensate-related molecules. In some embodiments, the first property is (iv) the distribution of the one or more condensates, and the second property is (xii) the partitioning of the condensate-related molecules into the condensates. In some embodiments, the first property is (iv) the distribution of the one or more condensates, and the second property is (xiii) the aggregation of the condensate-related molecules.

[0123] In some embodiments, the method includes determining a first property related to the one or more condensates and a second property related to the one or more condensates. In some embodiments, the first property is (v) the surface area of the one or more condensates, and the second property is any one (or more, such as 2, 3, 4, 5, 6, 7, or 8) of (vi) the composition of the one or more condensates, (vii) the fluidity of the one or more condensates, (viii) the solidification of the one or more condensates, (ix) the dissolution of the one or more condensates, (x) the presence and / or amount of fiber formation, (xi) the location of the condensate-related molecules, (xii) the partitioning of the condensate-related molecules into the condensates, and (xiii) the aggregation of the condensate-related molecules. In some embodiments, the first property is (v) the surface area of the one or more condensates, and the second property is (vi) the composition of the one or more condensates. In some embodiments, the first property is (v) the surface area of the one or more condensates, and the second property is (vii) the fluidity of the one or more condensates. In some embodiments, the first property is (v) the surface area of the one or more condensates, and the second property is (viii) the solidification of the one or more condensates. In some embodiments, the first property is (v) the surface area of the one or more condensates, and the second property is (ix) the dissolution of the one or more condensates. In some embodiments, the first property is (v) the surface area of the one or more condensates, and the second property is (x) the presence and / or amount of fiber formation. In some embodiments, the first property is (v) the surface area of the one or more condensates, and the second property is (xi) the location of the condensate-related molecules. In some embodiments, the first property is (v) the surface area of the one or more condensates, and the second property is (xii) the partitioning of the condensate-related molecules into the condensates. In some embodiments, the first property is (v) the surface area of the one or more condensates, and the second property is (xiii) the aggregation of the condensate-related molecules.

[0124] In some embodiments, the method includes determining a first property associated with the one or more condensates and a second property associated with the one or more condensates. In some embodiments, the first property is (vi) the composition of the one or more condensates, and the second property is any one (or more, such as 2, 3, 4, 5, 6, or 7) of (vii) the fluidity of the one or more condensates, (viii) the solidification of the one or more condensates, (ix) the dissolution of the one or more condensates, (x) the presence and / or amount of fiber formation, (xi) the location of the condensate-associated molecules, (xii) the partitioning of the condensate-associated molecules into the condensates, and (xiii) the aggregation of the condensate-associated molecules. In some embodiments, the first property is (vi) the composition of the one or more condensates, and the second property is (vii) the fluidity of the one or more condensates. In some embodiments, the first property is (vi) the composition of the one or more condensates, and the second property is (viii) the solidification of the one or more condensates. In some embodiments, the first property is (vi) the composition of the one or more condensates, and the second property is (ix) the dissolution of the one or more condensates. In some embodiments, the first property is (vi) the composition of the one or more condensates, and the second property is (x) the presence and / or amount of fiber formation. In some embodiments, the first property is (vi) the composition of the one or more condensates, and the second property is (xi) the location of the condensate-associated molecules. In some embodiments, the first property is (vi) the composition of the one or more condensates, and the second property is (xii) the partitioning of the condensate-associated molecules into the condensates. In some embodiments, the first property is (vi) the composition of the one or more condensates, and the second property is (xiii) the aggregation of the condensate-associated molecules.

[0125] In some embodiments, the method comprises determining a first property related to the one or more condensates and a second property related to the one or more condensates. In some embodiments, the first property is (vii) the fluidity of the one or more condensates and the second property is (viii) the solidification of the one or more condensates, (ix) the dissolution of the one or more condensates, (x) the presence and / or amount of fiber formation, (xi) the location of the condensate-related molecules, (xii) the partitioning of the condensate-related molecules into the condensates, and (xiii) the aggregation of the condensate-related molecules, any one (or more, such as 2, 3, 4, 5, or 6) thereof. In some embodiments, the first property is (vii) the fluidity of the one or more condensates and the second property is (viii) the solidification of the one or more condensates. In some embodiments, the first property is (vii) the fluidity of the one or more condensates and the second property is (ix) the dissolution of the one or more condensates. In some embodiments, the first property is (vii) the fluidity of the one or more condensates and the second property is (x) the presence and / or amount of fiber formation. In some embodiments, the first property is (vii) the fluidity of the one or more condensates and the second property is (xi) the location of the condensate-related molecules. In some embodiments, the first property is (vii) the fluidity of the one or more condensates and the second property is (xii) the partitioning of the condensate-related molecules into the condensates. In some embodiments, the first property is (vii) the fluidity of the one or more condensates and the second property is (xiii) the aggregation of the condensate-related molecules.

[0126] In some embodiments, the method comprises determining a first property related to the one or more condensates and a second property related to the one or more condensates. In some embodiments, the first property is (viii) the solidification of the one or more condensates, and the second property is any one (or more, such as 2, 3, 4, or 5) of (ix) the dissolution of the one or more condensates, (x) the presence and / or amount of fiber formation, (xi) the location of the condensate-related molecules, (xii) the partitioning of the condensate-related molecules into the condensates, and (xiii) the aggregation of the condensate-related molecules. In some embodiments, the first property is (viii) the solidification of the one or more condensates, and the second property is (ix) the dissolution of the one or more condensates. In some embodiments, the first property is (viii) the solidification of the one or more condensates, and the second property is (x) the presence and / or amount of fiber formation. In some embodiments, the first property is (viii) the solidification of the one or more condensates, and the second property is (xi) the location of the condensate-related molecules. In some embodiments, the first property is (viii) the solidification of the one or more condensates, and the second property is (xii) the partitioning of the condensate-related molecules into the condensates. In some embodiments, the first property is (viii) the solidification of the one or more condensates, and the second property is (xiii) the aggregation of the condensate-related molecules.

[0127] In some embodiments, the method comprises determining a first property related to the one or more condensates and a second property related to the one or more condensates. In some embodiments, the first property is (ix) the dissolution of the one or more condensates, and the second property is any one (or more, such as 2, 3, or 4) of (x) the presence and / or amount of fiber formation, (xi) the location of the condensate-related molecules, (xii) the partitioning of the condensate-related molecules into the condensates, and (xiii) the aggregation of the condensate-related molecules. In some embodiments, the first property is (ix) the dissolution of the one or more condensates, and the second property is (x) the presence and / or amount of fiber formation. In some embodiments, the first property is (ix) the dissolution of the one or more condensates, and the second property is (xi) the location of the condensate-related molecules. In some embodiments, the first property is (ix) the dissolution of the one or more condensates, and the second property is (xii) the partitioning of the condensate-related molecules into the condensates. In some embodiments, the first property is (ix) the dissolution of the one or more condensates, and the second property is (xiii) the aggregation of the condensate-related molecules.

[0128] In some embodiments, the method comprises determining a first property associated with the one or more condensates and a second property associated with the one or more condensates. In some embodiments, the first property is (x) the presence and / or amount of fiber formation, and the second property is any one (or more, such as two or three) of (xi) the location of the condensate-associated molecules, (xii) the partitioning of the condensate-associated molecules into the condensates, and (xiii) the aggregation of the condensate-associated molecules. In some embodiments, the first property is (x) the presence and / or amount of fiber formation, and the second property is (xi) the location of the condensate-associated molecules. In some embodiments, the first property is (x) the presence and / or amount of fiber formation, and the second property is (xii) the partitioning of the condensate-associated molecules into the condensates. In some embodiments, the first property is (x) the presence and / or amount of fiber formation, and the second property is (xiii) the aggregation of the condensate-associated molecules.

[0129] In some embodiments, the method comprises determining a first property associated with the one or more condensates and a second property associated with the one or more condensates. In some embodiments, the first property is (xi) the location of the condensate-associated molecules, and the second property is any one or both of (xii) the partitioning of the condensate-associated molecules into the condensates and / or (xiii) the aggregation of the condensate-associated molecules. In some embodiments, the first property is (xi) the location of the condensate-associated molecules, and the second property is (xii) the partitioning of the condensate-associated molecules into the condensates.

[0130] In some embodiments, the method comprises determining a first property associated with the one or more condensates and a second property associated with the one or more condensates. In some embodiments, the first property is (xii) the partitioning of the condensate-associated molecules into the condensates, and the second property is (xiii) the aggregation of the condensate-associated molecules.

[0131] In some embodiments, the compound modulates one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or all) of the following properties: (i) the number of condensates with and / or without the condensate-associated molecules; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecules; (xii) the partitioning of the condensate-associated molecules into the condensates; and (xiii) the aggregation of the condensate-associated molecules.

[0132] In some embodiments, the compound modulates (i) the number of condensates with and / or without the condensate-associated molecules. In some embodiments, the compound modulates (ii) the size of the one or more condensates. In some embodiments, the compound modulates (iii) the location of the one or more condensates. In some embodiments, the compound modulates (iv) the distribution of the one or more condensates. In some embodiments, the compound modulates (v) the surface area of the one or more condensates. In some embodiments, the compound modulates (vi) the composition of the one or more condensates. In some embodiments, the compound modulates (vii) the mobility of the one or more condensates. In some embodiments, the compound modulates (viii) the solidification of the one or more condensates. In some embodiments, the compound modulates (ix) the dissolution of the one or more condensates. In some embodiments, the compound modulates (x) the presence and / or amount of fiber formation. In some embodiments, the compound modulates (xi) the location of the condensate-associated molecules. In some embodiments, the compound modulates (xii) the partitioning of the condensate-associated molecules into the condensates. In some embodiments, the compound modulates (xiii) the aggregation of the condensate-associated molecules.

[0133] In some embodiments, the compound does not modulate one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or all) of the following properties: (i) the number of condensates that contain and / or do not contain the condensate-associated molecule; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecule; (xii) the partitioning of the condensate-associated molecule into the condensate; and (xiii) the aggregation of the condensate-associated molecule.

[0134] In some embodiments, the compound does not modulate (i) the number of condensates that contain and / or do not contain the condensate-associated molecule. In some embodiments, the compound does not modulate (ii) the size of the one or more condensates. In some embodiments, the compound does not modulate (iii) the location of the one or more condensates. In some embodiments, the compound does not modulate (iv) the distribution of the one or more condensates. In some embodiments, the compound does not modulate (v) the surface area of the one or more condensates. In some embodiments, the compound does not modulate (vi) the composition of the one or more condensates. In some embodiments, the compound does not modulate (vii) the mobility of the one or more condensates. In some embodiments, the compound does not modulate (viii) the solidification of the one or more condensates. In some embodiments, the compound does not modulate (ix) the dissolution of the one or more condensates. In some embodiments, the compound does not modulate (x) the presence and / or amount of fiber formation. In some embodiments, the compound does not modulate (xi) the location of the condensate-associated molecule. In some embodiments, the compound does not modulate (xii) the partitioning of the condensate-associated molecule into the condensate. In some embodiments, the compound does not modulate (xiii) the aggregation of the condensate-associated molecule.

[0135] In some embodiments, the compound modulates one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) of the following properties: (i) the number of condensates with and / or without condensate-associated molecules; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the fluidity of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecules; (xii) the partitioning of the condensate-associated molecules into the condensates; and (xiii) the aggregation of the condensate-associated molecules, and does not modulate another of the thirteen properties.

[0136] In some embodiments, the compound modulates a first property related to one or more condensates without modulating a second property related to the one or more condensates, where the first property is different from the second property, and where the properties are selected from: (i) the number of condensates with and / or without condensate-associated molecules; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the fluidity of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecules; (xii) the partitioning of the condensate-associated molecules into the condensates; and (xiii) the aggregation of the condensate-associated molecules, and does not modulate another of the thirteen properties.

[0137] In some embodiments, the compound modulates one or more of the following: one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) properties selected from: (i) the number of condensates with and / or without the condensate-associated molecule; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecule; (xii) the partitioning of the condensate-associated molecule into condensates in a first portion of the cell composition (e.g., a first portion of the cell), and does not modulate one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) properties selected from: (i) the number of condensates with and / or without the condensate-associated molecule; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecule; (xii) the partitioning of the condensate-associated molecule into condensates in a second portion of the cell composition (e.g., a second portion of the cell).

[0138] In some embodiments, the compound modulates one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) of the following properties: (i) the number of condensates with and / or without condensate-associated molecules; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecules; (xii) the partitioning of the condensate-associated molecules into condensates in the cytoplasm, and does not modulate one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) of the following properties: (i) the number of condensates with and / or without condensate-associated molecules; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecules; (xii) the partitioning of the condensate-associated molecules into condensates in the nucleus.

[0139] In some embodiments, the compound modulates one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) of the following properties: (i) the number of condensates with and / or without condensate-associated molecules; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecules; (xii) the partitioning of the condensate-associated molecules into condensates for a first set of one or more condensates and does not modulate one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) of the following properties: (i) the number of condensates with and / or without condensate-associated molecules; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecules; (xii) the partitioning of the condensate-associated molecules into condensates for a second set of one or more condensates.

[0140] In some embodiments, the compound modulates one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) of the following properties: (i) the number of condensates with and / or without the condensate-associated molecule; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fibril formation; (xi) the location of the condensate-associated molecule; (xii) the partitioning of the condensate-associated molecule into condensates in one or more stress granules, and does not modulate one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) of the following properties: (i) the number of condensates with and / or without the condensate-associated molecule; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fibril formation; (xi) the location of the condensate-associated molecule; (xii) the partitioning of the condensate-associated molecule into condensates in one or more paraspeckles, condensates formed around DNA damage sites, P bodies, Cajal bodies, and PML bodies.

[0141] In some embodiments, the method is a method of identifying a compound that modulates one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or all) of the following properties: (i) the number of condensates that contain and / or do not contain the condensate-associated molecules; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecules; (xii) the partitioning of the condensate-associated molecules into the condensates; and (xiii) the aggregation of the condensate-associated molecules.

[0142] In some embodiments, the method is a method of identifying a compound that modulates (i) the number of condensates that contain and / or do not contain the condensate-associated molecules. In some embodiments, the method is a method of identifying a compound that modulates (ii) the size of the one or more condensates. In some embodiments, the method is a method of identifying a compound that modulates (iii) the location of the one or more condensates. In some embodiments, the method is a method of identifying a compound that modulates (iv) the distribution of the one or more condensates. In some embodiments, the method is a method of identifying a compound that modulates (v) the surface area of the one or more condensates. In some embodiments, the method is a method of identifying a compound that modulates (vi) the composition of the one or more condensates. In some embodiments, the method is a method of identifying a compound that modulates (vii) the mobility of the one or more condensates. In some embodiments, the method is a method of identifying a compound that modulates (viii) the solidification of the one or more condensates. In some embodiments, the method is a method of identifying a compound that modulates (ix) the dissolution of the one or more condensates. In some embodiments, the method is a method of identifying a compound that modulates (x) the presence and / or amount of fiber formation. In some embodiments, the method is a method of identifying a compound that modulates (xi) the location of the condensate-associated molecules. In some embodiments, the method is a method of identifying a compound that modulates (xii) the partitioning of the condensate-associated molecules into the condensates. In some embodiments, the method is a method of identifying a compound that modulates (xiii) the aggregation of the condensate-associated molecules.

[0143] In some embodiments, the compound does not modulate one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or all) of the following properties: (i) the number of condensates that contain and / or do not contain the condensate-associated molecule; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecule; (xii) the partitioning of the condensate-associated molecule into the condensate; and (xiii) the aggregation of the condensate-associated molecule.

[0144] In some embodiments, the compound does not modulate (i) the number of condensates that contain and / or do not contain the condensate-associated molecule. In some embodiments, the compound does not modulate (ii) the size of the one or more condensates. In some embodiments, the compound does not modulate (iii) the location of the one or more condensates. In some embodiments, the compound does not modulate (iv) the distribution of the one or more condensates. In some embodiments, the compound does not modulate (v) the surface area of the one or more condensates. In some embodiments, the compound does not modulate (vi) the composition of the one or more condensates. In some embodiments, the compound does not modulate (vii) the mobility of the one or more condensates. In some embodiments, the compound does not modulate (viii) the solidification of the one or more condensates. In some embodiments, the compound does not modulate (ix) the dissolution of the one or more condensates. In some embodiments, the compound does not modulate (x) the presence and / or amount of fiber formation. In some embodiments, the compound does not modulate (xi) the location of the condensate-associated molecule. In some embodiments, the compound does not modulate (xii) the partitioning of the condensate-associated molecule into the condensate. In some embodiments, the compound does not modulate (xiii) the aggregation of the condensate-associated molecule.

[0145] In some embodiments, the method is a method of identifying a compound that modulates one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) of the following properties: (i) the number of condensates that contain and / or do not contain the condensate-associated molecule; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecule; (xii) the partitioning of the condensate-associated molecule into the condensate; and (xiii) the aggregation of the condensate-associated molecule, and does not modulate another of the thirteen properties.

[0146] In some embodiments, the method is a method of identifying a compound that modulates one or more of the following: one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) properties selected from the following: (i) the number of condensates that contain and / or do not contain the condensate-associated molecule; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecule; (xii) the partitioning of the condensate-associated molecule into condensates in a first portion of the cell composition (e.g., a first portion of the cell), and does not modulate one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) properties selected from the following: (i) the number of condensates that contain and / or do not contain the condensate-associated molecule; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecule; (xii) the partitioning of the condensate-associated molecule into condensates in a second portion of the cell composition (e.g., a second portion of the cell).

[0147] In some embodiments, the method is a method of identifying a compound that modulates one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) properties selected from: (i) the number of condensates with and / or without the condensate-associated molecule; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecule; (xii) the partitioning of the condensate-associated molecule into condensates in the cytoplasm, and does not modulate one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) properties selected from: (i) the number of condensates with and / or without the condensate-associated molecule; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecule; (xii) the partitioning of the condensate-associated molecule into condensates in the nucleus.

[0148] In some embodiments, the method is a method of identifying a compound that modulates one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) of the following properties: (i) the number of condensates that contain and / or do not contain the condensate-associated molecule; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecule; (xii) the partitioning of the condensate-associated molecule into condensates for a first set of one or more condensates, and does not modulate one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) of the following properties: (i) the number of condensates that contain and / or do not contain the condensate-associated molecule; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecule; (xii) the partitioning of the condensate-associated molecule into condensates for a second set of one or more condensates.

[0149] In some embodiments, the method is a method of identifying a compound that modulates one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) properties selected from: (i) the number of condensates that contain and / or do not contain the condensate-associated molecule; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fibril formation; (xi) the location of the condensate-associated molecule; (xii) the partitioning of the condensate-associated molecule into the condensates of one or more of stress granules, and does not modulate one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) properties selected from: (i) the number of condensates that contain and / or do not contain the condensate-associated molecule; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fibril formation; (xi) the location of the condensate-associated molecule; (xii) the partitioning of the condensate-associated molecule into the condensates of one or more of paraspeckles, condensates formed around DNA damage sites, P-bodies, Cajal bodies, and PML bodies.

[0150] In some embodiments, the assay is performed within about 60 days of contact with the compound, such as within about 35 days, about 28 days, about 21 days, about 14 days, about 10 days, about 7 days, about 5 days, about 3 days, about 2 days, about 1 day, about 12 hours, about 5 hours, about 2 hours, about 1 hour, about 45 minutes, about 30 minutes, about 15 minutes, about 5 minutes, about 1 minute, or about 30 seconds. In some embodiments, the assay is performed about 5 seconds after contact with the compound, such as about 15 seconds, about 30 seconds, about 1 minute, about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 5 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 5 days, about 7 days, about 10 days, about 14 days, about 21 days, about 28 days, about 35 days, or about 60 days after contact.

[0151] In some embodiments, the method includes determining the property before and after contacting the compound. In some embodiments, the method further includes comparing the property before and after contacting the compound.

[0152] In some embodiments, the method further includes repeating the determination step of the method. For example, in some embodiments, the method includes repeating the determination step of the method at least about 2, 3, 4, 5, 10 or more times. In some embodiments, the method includes performing the determination step on a first portion of the cell composition and a second portion of the cell composition (such as a first cell and a second cell in the cell composition). In some embodiments, the method includes performing the determination step on a third, fourth, fifth, sixth or more portions of the cell composition (such as the third, fourth, fifth, sixth or more cells in the cell composition). In some embodiments, the method includes performing the determination step on a first portion of the cells in the cell composition and a second portion of the cells in the cell composition, such as in the cytoplasm and the nucleus, or in a first organelle and a second organelle. In some embodiments, the method includes performing the determination step on a third, fourth, fifth, sixth or more portions of the cells in the cell composition.

[0153] In some embodiments, the determination step of the method is repeated after a certain time interval, such as about 30 seconds, about 1 minute, about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 5 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 5 days, about 7 days, about 10 days, about 14 days, about 21 days, about 28 days, about 35 days, about 60 days or longer. In some embodiments, the determination step, when repeated, is based on the same property. In some embodiments, the method further includes comparing the property over time, such as comparing the number of condensates between two determinations spaced one day apart.

[0154] Visualizing condensates can help determine properties associated with one or more condensates. Condensates can be visualized by a variety of methods, such as microscopy, including, for example, stereomicroscopy, bright-field microscopy, polarized light microscopy, phase-contrast microscopy, differential interference contrast microscopy, fluorescence microscopy, total internal reflection fluorescence microscopy, confocal microscopy, or multiphoton excitation microscopy. Analysis, such as counting condensates or measuring the size of condensates, can be determined by various methods, including manually or automatically, and can be, for example, from an image or directly from the microscope. For simplicity, in some embodiments, condensates and / or condensate-associated molecules can be labeled, for example, with a fluorophore. In some embodiments, the condensate-associated molecules comprise a fluorescent label, such as a fluorescent protein (e.g., GFP, RFP, YFP, etc.). In some embodiments, the method comprises contacting at least a portion of the cell composition with a label. In some embodiments, the label is a labeled binding molecule, such as an antibody (e.g., a labeled secondary antibody) or a streptavidin. In some embodiments, the label is a stain, such as a stain specific for an organelle.

[0155] In some embodiments, the method further comprises imaging at least a portion of the cell composition, such as a field of view. In some embodiments, the method further comprises contacting at least a portion of the cell composition with a fixative. In some embodiments, the method further comprises contacting at least a portion of the cell composition with a stain. In some embodiments, the method further comprises contacting at least a portion of the cell composition with DNA damage conditions. In some embodiments, the DNA damage condition is laser irradiation.

[0156] Properties of a portion or all of the cell composition can be determined. Thus, in some embodiments, the method comprises determining the properties of a portion of the cell composition. In some embodiments, the method comprises determining the properties of the entire cell composition. In some embodiments, the method comprises determining the properties of one or more cells in the cell composition. In some embodiments, the method comprises determining the properties of a single cell in the cell composition.

[0157] Properties of some or all of the cells in the cell composition can also be determined. Thus, in some embodiments, the method comprises determining the properties of a portion of one or more cells in the cell composition. In some embodiments, the method comprises determining the properties of a portion of a single cell in the cell composition. In some embodiments, the method comprises determining the properties of the cytoplasm in the cell composition. In some embodiments, the method comprises determining the properties of the nucleus in the cell composition. In some embodiments, the method comprises determining the properties of an organelle.

[0158] Number of condensates

[0159] In some embodiments, the properties associated with the one or more condensates are based on the number of condensates that contain and / or do not contain condensate-associated molecules. In some embodiments, the properties associated with one or more condensates are based on the number of condensates that contain condensate-associated molecules. In some embodiments, the properties associated with one or more condensates are based on the number of condensates that do not contain condensate-associated molecules. Thus, in some embodiments, provided herein are methods of identifying a compound that modulates a property associated with one or more condensates that contain condensate-associated molecules, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining a property associated with the one or more condensates, wherein modulation of the property, compared to a reference, indicates that the compound modulates a property associated with the one or more condensates, wherein the property is modulation of the number of condensates that contain and / or do not contain the condensate-associated molecules. In some embodiments, provided herein are methods of identifying a compound that modulates the number of condensates that contain and / or do not contain the condensate-associated molecules, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining the number of condensates that contain and / or do not contain the condensate-associated molecules, wherein modulation of the number of condensates, compared to a reference, indicates that the compound modulates the number of condensates that contain and / or do not contain the condensate-associated molecules.

[0160] The number of condensates in a portion or all of the cell composition can be determined. Thus, in some embodiments, the method comprises determining the number of condensates that contain and / or do not contain condensate-associated molecules in a portion of the cell composition. In some embodiments, the method comprises determining the number of condensates that contain and / or do not contain condensate-associated molecules in the entire cell composition. In some embodiments, the method comprises determining the number of condensates that contain and / or do not contain condensate-associated molecules in one or more cells in the cell composition. In some embodiments, the method comprises determining the number of condensates that contain and / or do not contain condensate-associated molecules in a single cell in the cell composition.

[0161] It is also possible to determine the number of condensates of some or all of the cells in the cell composition. Thus, in some embodiments, the method comprises determining the number of condensates that contain and / or do not contain condensate-related molecules in a portion of one or more cells in the cell composition. In some embodiments, the method comprises determining the number of condensates that contain and / or do not contain condensate-related molecules in a portion of a single cell in the cell composition. In some embodiments, the method comprises determining the number of condensates that contain and / or do not contain condensate-related molecules in the cytoplasm. In some embodiments, the method comprises determining the number of condensates that contain and / or do not contain condensate-related molecules in the nucleus. In some embodiments, the method comprises determining the number of condensates that contain and / or do not contain condensate-related molecules in an organelle.

[0162] In some embodiments, the method comprises determining the number of condensates that contain condensate-related molecules; determining the number of condensates that do not contain condensate-related molecules; and comparing the number of condensates that contain condensate-related molecules with the number of the condensate-related molecules. In some embodiments, the method comprises determining the number of condensates that contain and / or do not contain condensate-related molecules in a first portion of the cell composition (e.g., any portion of the cell composition disclosed herein); determining the number of condensates that contain and / or do not contain condensate-related molecules in a second portion of the cell composition (e.g., any portion of the cell composition disclosed herein); and comparing the number of condensates that contain and / or do not contain condensate-related molecules in the first portion with the number of condensates that contain and / or do not contain condensate-related molecules in the second portion. In some embodiments, the method comprises determining the number of condensates that contain condensate-related molecules in the cytoplasm of the cell composition; determining the number of condensates that contain condensate-related molecules in the nucleus of the cell composition; and comparing the number of condensates that contain condensate-related molecules in the cytoplasm with the number of condensates that contain condensate-related molecules in the nucleus.

[0163] In some embodiments, the number of condensates that contain condensate-related molecules increases compared to a reference. In some embodiments, the number of condensates that contain condensate-related molecules decreases compared to a reference. In some embodiments, the number of condensates that do not contain condensate-related molecules increases compared to a reference. In some embodiments, the number of condensates that do not contain condensate-related molecules decreases compared to a reference.

[0164] In some embodiments, compared to a reference, the number of condensates containing and / or not containing condensate-associated molecules in the first part of the cell composition is reduced, and compared to the reference, the number of condensates containing and / or not containing condensate-associated molecules in the second part of the cell composition is not reduced. In some embodiments, compared to a reference, the number of condensates containing and / or not containing condensate-associated molecules in the first part of the cell composition is increased, and compared to the reference, the number of condensates containing and / or not containing condensate-associated molecules in the second part of the cell composition is not increased. In some embodiments, the first part of the cell composition is the cytoplasm and the second part of the cell composition is the nucleus, or the first part of the cell composition is the nucleus and the second part of the cell composition is the cytoplasm. In some embodiments, compared to a reference, the number of condensates containing condensate-associated molecules in the cytoplasm is reduced, and compared to the reference, the number of condensates containing condensate-associated molecules in the nucleus is not reduced. In some embodiments, compared to a reference, the number of condensates containing condensate-associated molecules in the cytoplasm is reduced, and compared to the reference, the number of condensates containing condensate-associated molecules in the nucleus is increased.

[0165] In some embodiments, compared to the second part of the cell composition, the number of condensates containing and / or not containing condensate-associated molecules in the first part of the cell composition is increased, or compared to the first part of the cell composition, the number of condensates containing and / or not containing condensate-associated molecules in the second part of the cell composition is increased. In some embodiments, the first part of the cell composition is the cytoplasm and the second part of the cell composition is the nucleus, or the first part of the cell composition is the nucleus and the second part of the cell composition is the cytoplasm.

[0166] Size of condensates

[0167] In some embodiments, the property associated with the one or more condensates is based on the size of the one or more condensates. Thus, in some embodiments, provided herein are methods of identifying a compound that modulates a property associated with one or more condensates comprising condensate-associated molecules, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining a property associated with the one or more condensates, wherein modulation of the property as compared to a reference indicates that the compound modulates a property associated with the one or more condensates, wherein the property is modulation of the size of the one or more condensates. In some embodiments, provided herein are methods of identifying a compound that modulates the size of the one or more condensates, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining the size of the one or more condensates, wherein modulation of the size as compared to a reference indicates that the compound modulates the size of the one or more condensates. In some embodiments, the size is the average of two or more condensates.

[0168] The size of the condensates in a part or all of the cell composition can be determined. Thus, in some embodiments, the method comprises determining the size of one or more condensates in a part of the cell composition. In some embodiments, the method comprises determining the size of one or more condensates in the entire cell composition. In some embodiments, the method comprises determining the size of one or more condensates in one or more cells of the cell composition. In some embodiments, the method comprises determining the size of one or more condensates in a single cell of the cell composition.

[0169] The size of the condensates in a part or all of the cells in the cell composition can also be determined. Thus, in some embodiments, the method comprises determining the size of one or more condensates in a part of one or more cells in the cell composition. In some embodiments, the method comprises determining the size of one or more condensates in a part of a single cell in the cell composition. In some embodiments, the method comprises determining the size of one or more condensates in the cytoplasm. In some embodiments, the method comprises determining the size of one or more condensates in the nucleus. In some embodiments, the method comprises determining the size of one or more condensates in an organelle.

[0170] In some embodiments, the size of the one or more condensates increases as compared to a reference. In some embodiments, the size of the one or more condensates decreases as compared to a reference.

[0171] In some embodiments, the size of one or more condensates in the first portion of the cell composition is reduced compared to a reference, and the size of one or more condensates in the second portion of the cell composition is not reduced compared to the reference. In some embodiments, the size of one or more condensates in the first portion of the cell composition is increased compared to a reference, and the size of one or more condensates in the second portion of the cell composition is not increased compared to the reference. In some embodiments, the first portion of the cell composition is the cytoplasm and the second portion of the cell composition is the nucleus, or the first portion of the cell composition is the nucleus and the second portion of the cell composition is the cytoplasm. In some embodiments, the size of one or more condensates in the cytoplasm is reduced compared to a reference, and the size of one or more condensates in the nucleus is not reduced compared to the reference. In some embodiments, the size of one or more condensates in the cytoplasm is reduced compared to a reference, and the size of one or more condensates in the nucleus is increased compared to the reference.

[0172] In some embodiments, the size of one or more condensates in the first portion of the cell composition is increased compared to the second portion of the cell composition, or the size of one or more condensates in the second portion of the cell composition is increased compared to the first portion of the cell composition. In some embodiments, the first portion of the cell composition is the cytoplasm and the second portion of the cell composition is the nucleus, or the first portion of the cell composition is the nucleus and the second portion of the cell composition is the cytoplasm.

[0173] Location of condensates

[0174] In some embodiments, the properties associated with the one or more condensates are based on the location of the one or more condensates. Thus, in some embodiments, provided herein are methods of identifying a compound that modulates a property associated with one or more condensates comprising condensate-associated molecules, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or a cell composition capable of forming one or more condensates, and (b) determining a property associated with the one or more condensates, wherein modulation of the property, compared to a reference, indicates that the compound modulates a property associated with the one or more condensates, wherein the property is modulation of the location of the one or more condensates. In some embodiments, provided herein are methods of identifying a compound that modulates the location of the one or more condensates, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or a cell composition capable of forming one or more condensates, and (b) determining the location of the one or more condensates, wherein modulation of the location, compared to a reference, indicates that the compound modulates the location of the one or more condensates.

[0175] The location of condensates in a part or all of the cell composition can be determined. Thus, in some embodiments, the method comprises determining the location of the one or more condensates in a part of the cell composition. In some embodiments, the method comprises determining the location of the one or more condensates in the entire cell composition. In some embodiments, the method comprises determining the location of the one or more condensates in one or more cells of the cell composition. In some embodiments, the method comprises determining the location of the one or more condensates in a single cell of the cell composition.

[0176] The location of condensates in a part or all of the cells in the cell composition can also be determined. Thus, in some embodiments, the method comprises determining the location of the one or more condensates in a part of one or more cells in the cell composition. In some embodiments, the method comprises determining the location of the one or more condensates in a part of a single cell in the cell composition. In some embodiments, the method comprises determining the location of the one or more condensates in the cytoplasm. In some embodiments, the method comprises determining the location of the one or more condensates in the nucleus. In some embodiments, the method comprises determining the location of the one or more condensates in an organelle.

[0177] Distribution of condensates

[0178] In some embodiments, the properties associated with the one or more condensates are based on the distribution of the one or more condensates, such as the spatial distribution of the one or more condensates within a cell. Thus, in some embodiments, provided herein are methods of identifying a compound that modulates a property associated with one or more condensates comprising condensate-associated molecules, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining a property associated with the one or more condensates, wherein modulation of the property, compared to a reference, indicates that the compound modulates a property associated with the one or more condensates, wherein the property is modulation of the distribution of the one or more condensates. In some embodiments, provided herein are methods of identifying a compound that modulates the distribution of one or more condensates, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining the distribution of the one or more condensates, wherein modulation of the distribution, compared to a reference, indicates that the compound modulates the distribution of the one or more condensates.

[0179] In some embodiments, the distribution of the one or more condensates is based on the number of condensates comprising condensate-associated molecules in a cellular location (such as any one or more of the cytosol, nucleus, organelles, or any part thereof). In some embodiments, the distribution of the one or more condensates is determined to be modulated by the compound when the number of condensates comprising condensate-associated molecules in a cellular location (such as the cytosol or nucleus) is decreased compared to the number of condensates comprising condensate-associated molecules in the cellular location of a reference cell composition (such as a cell composition not contacted with the compound or a cell composition contacted with a control compound). In some embodiments, the distribution of the one or more condensates is determined to be modulated by the compound when the number of condensates comprising condensate-associated molecules in a cellular location (such as the cytosol or nucleus) is increased compared to the number of condensates comprising condensate-associated molecules in the cellular location of a reference cell composition (such as a cell composition not contacted with the compound or a cell composition contacted with a control compound).

[0180] In some embodiments, the distribution of one or more condensates is based on the number of condensates containing condensate-associated molecules in one or more portions of the cell composition (e.g., in one or more fields of view or one or more portions of an image of the cell composition). In some embodiments, the distribution of one or more condensates is determined to be modulated by the compound when the number of condensates containing condensate-associated molecules in one or more portions of the cell composition (such as one or more portions of the cytosol or nucleus) is decreased compared to the number of condensates containing condensate-associated molecules in one or more portions of a reference cell composition (such as one or more portions of a cell composition not contacted with the compound, or one or more portions of a cell composition contacted with a control compound). In some embodiments, the distribution of one or more condensates is determined to be modulated by the compound when the number of condensates containing condensate-associated molecules in one or more portions of the cell composition (such as one or more portions of the cytosol or nucleus) is increased compared to the number of condensates containing condensate-associated molecules in one or more portions of a reference cell composition (such as one or more portions of a cell composition not contacted with the compound, or one or more portions of a cell composition contacted with a control compound).

[0181] In some embodiments, the distribution of condensates is determined by calculating the ratio (e.g., percentage, per mille, etc.) of condensates in a first portion of the cell composition to condensates in a second portion of the cell composition. In some embodiments, the method comprises determining the number of condensates containing condensate-related molecules in a first portion of the cell composition, determining the number of condensates containing condensate-related molecules in a second portion of the cell composition, and calculating the ratio of the number of condensates containing condensate-related molecules in the first and second portions of the cell composition. In some embodiments, the first portion of the cell composition is a first portion of the cell and a second portion of the cell, such as the cytoplasm, nucleus, or organelles. In some embodiments, the first portion of the cell composition is the cytoplasm and the second portion is the nucleus. In some embodiments, the ratio increases compared to a reference (e.g., a cell composition not contacted with the compound or a cell composition contacted with a control compound). In some embodiments, the ratio decreases compared to the reference. In some embodiments, the ratio of nuclear to cytoplasmic condensates increases compared to the reference. In some embodiments, the ratio of nuclear to cytoplasmic condensates decreases compared to the reference. In some embodiments, when the ratio of condensates in a first portion (such as the nucleus) of the cell composition to condensates in a second portion (such as the cytosol) of the cell composition increases compared to the reference, it is determined that the distribution of one or more condensates is modulated by the compound. In some embodiments, when the ratio of condensates in a first portion (such as the nucleus) of the cell composition to condensates in a second portion (such as the cytosol) of the cell composition decreases compared to the reference, it is determined that the distribution of one or more condensates is modulated by the compound.

[0182] In some embodiments, imaging techniques are used to determine the distribution of one or more condensates.

[0183] Surface area of condensates

[0184] In some embodiments, the property associated with the one or more condensates is based on the surface area of the one or more condensates. Thus, in some embodiments, provided herein are methods of identifying a compound that modulates a property associated with one or more condensates comprising condensate-associated molecules, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or a cell composition capable of forming one or more condensates, and (b) determining a property associated with the one or more condensates, wherein modulation of the property as compared to a reference indicates that the compound modulates a property associated with the one or more condensates, wherein the property is modulation of the surface area of the one or more condensates. In some embodiments, provided herein are methods of identifying a compound that modulates the surface area of one or more condensates, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or a cell composition capable of forming one or more condensates, and (b) determining the surface area of the one or more condensates, wherein modulation of the surface area as compared to a reference indicates that the compound modulates the surface area of the one or more condensates. In some embodiments, the surface area is an average of two or more condensates.

[0185] The surface area of condensates in a part or all of the cell composition can be determined. Thus, in some embodiments, the method comprises determining the surface area of one or more condensates in a part of the cell composition. In some embodiments, the method comprises determining the surface area of one or more condensates in the entire cell composition. In some embodiments, the method comprises determining the surface area of one or more condensates in one or more cells of the cell composition. In some embodiments, the method comprises determining the surface area of one or more condensates in a single cell of the cell composition.

[0186] The surface area of condensates in a part or all of the cells in the cell composition can also be determined. Thus, in some embodiments, the method comprises determining the surface area of one or more condensates in a part of one or more cells in the cell composition. In some embodiments, the method comprises determining the surface area of one or more condensates in a part of a single cell in the cell composition. In some embodiments, the method comprises determining the surface area of one or more condensates in the cytoplasm. In some embodiments, the method comprises determining the surface area of one or more condensates in the nucleus. In some embodiments, the method comprises determining the surface area of one or more condensates in an organelle.

[0187] In some embodiments, the surface area of the one or more condensates is increased as compared to a reference. In some embodiments, the surface area of the one or more condensates is decreased as compared to a reference.

[0188] In some embodiments, the surface area of the one or more condensates in the first portion of the cell composition is decreased compared to a reference, and the surface area of the one or more condensates in the second portion of the cell composition is not decreased compared to the reference. In some embodiments, the surface area of the one or more condensates in the first portion of the cell composition is increased compared to a reference, and the surface area of the one or more condensates in the second portion of the cell composition is not increased compared to the reference. In some embodiments, the first portion of the cell composition is the cytoplasm and the second portion of the cell composition is the nucleus, or the first portion of the cell composition is the nucleus and the second portion of the cell composition is the cytoplasm. In some embodiments, the surface area of the one or more condensates in the cytoplasm is decreased compared to a reference, and the surface area of the one or more condensates in the nucleus is not decreased compared to the reference. In some embodiments, the surface area of the one or more condensates in the cytoplasm is decreased compared to a reference, and the surface area of the one or more condensates in the nucleus is increased compared to the reference.

[0189] In some embodiments, the surface area of the one or more condensates in the first portion of the cell composition is increased compared to the second portion of the cell composition, or the surface area of the one or more condensates in the second portion of the cell composition is increased compared to the first portion of the cell composition. In some embodiments, the first portion of the cell composition is the cytoplasm and the second portion of the cell composition is the nucleus, or the first portion of the cell composition is the nucleus and the second portion of the cell composition is the cytoplasm.

[0190] Composition of condensates

[0191] In some embodiments, the properties associated with the one or more condensates are based on the composition of the one or more condensates. Thus, in some embodiments, provided herein are methods of identifying a compound that modulates a property associated with one or more condensates comprising condensate-associated molecules, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or a cell composition capable of forming one or more condensates, and (b) determining a property associated with the one or more condensates, wherein modulation of the property as compared to a reference indicates that the compound modulates a property associated with the one or more condensates, wherein the property is modulation of the composition of the one or more condensates. In some embodiments, provided herein are methods of identifying a compound that modulates the composition of the one or more condensates, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or a cell composition capable of forming one or more condensates, and (b) determining the composition of the one or more condensates, wherein modulation of the composition as compared to a reference indicates that the compound modulates the composition of the one or more condensates.

[0192] In some embodiments, the composition of the condensate is determined by detecting the presence of one or more macromolecules contained in the condensate, measuring the amount of one or more macromolecules contained in the condensate, calculating the ratio of one macromolecule contained in the condensate to a second macromolecule, and / or comparing the amount of one or more macromolecules contained in the condensate to the size and / or surface area of the condensate. In some embodiments, the composition of the condensate is determined by detecting the presence of one or more macromolecules (such as 1, 2, 3, 4, 5, 6, 7 or more macromolecules) contained in the condensate. In some embodiments, the composition of the condensate is determined by measuring the amount of one or more macromolecules (such as 1, 2, 3, 4, 5, 6, 7 or more macromolecules) contained in the condensate. In some embodiments, the composition of the condensate is determined by calculating the ratio of one macromolecule contained in the condensate to a second macromolecule. In some embodiments, the composition of the condensate is determined by comparing the amount of one or more macromolecules contained in the condensate to the size and / or surface area of the condensate, such as 1, 2, 3, 4, 5, 6, 7 or more macromolecules. In some embodiments, the macromolecule is a condensate-associated molecule. In some embodiments, the macromolecule is a polynucleotide or a polypeptide. In some embodiments, the macromolecule is a polypeptide. In some embodiments, the macromolecule is a wild-type polypeptide. In some embodiments, the macromolecule is a mutant polypeptide. In some embodiments, the macromolecule is FUS, EWSR1, TIAL1, PABPC1 or G3BP1 or a mutant thereof.

[0193] The composition of condensates of a part or all of the cellular composition can be determined. Thus, in some embodiments, the method comprises determining the composition of one or more condensates in a part of the cellular composition. In some embodiments, the method comprises determining the composition of one or more condensates in the entire cellular composition. In some embodiments, the method comprises determining the composition of one or more condensates in one or more cells of the cellular composition. In some embodiments, the method comprises determining the composition of one or more condensates in a single cell of the cellular composition.

[0194] The composition of condensates of a part or all of the cells in the cellular composition can also be determined. Thus, in some embodiments, the method comprises determining the composition of one or more condensates in a part of one or more cells in the cellular composition. In some embodiments, the method comprises determining the composition of one or more condensates in a part of a single cell in the cellular composition. In some embodiments, the method comprises determining the composition of one or more condensates in the cytoplasm. In some embodiments, the method comprises determining the composition of one or more condensates in the nucleus. In some embodiments, the method comprises determining the composition of one or more condensates in the organelles.

[0195] Mobility of condensates

[0196] In some embodiments, the property related to the one or more condensates is based on the mobility of the one or more condensates. Thus, in some embodiments, provided herein is a method of identifying a compound that modulates a property related to one or more condensates comprising condensate-associated molecules, the method comprising: (a) contacting the compound with a cellular composition comprising one or more condensates or with a cellular composition capable of forming one or more condensates, and (b) determining a property related to the one or more condensates, wherein modulation of the property as compared to a reference indicates that the compound modulates a property related to the one or more condensates, wherein the property is modulation of the mobility of the one or more condensates. In some embodiments, provided herein is a method of identifying a compound that modulates the mobility of the one or more condensates, the method comprising: (a) contacting the compound with a cellular composition comprising one or more condensates or with a cellular composition capable of forming one or more condensates, and (b) determining the mobility of the one or more condensates, wherein modulation of the mobility as compared to a reference indicates that the compound modulates the mobility of the one or more condensates. In some embodiments, the mobility is based on measurements considering two or more condensates, such as the average or distribution of the mobilities of two or more condensates.

[0197] It is possible to measure the condensate mobility of one or more condensates in a part or all of the cell composition. Thus, in some embodiments, the method includes measuring the mobility of one or more condensates in a part of the cell composition, such as one or more fields of view or one or more portions of an image of the cell composition. In some embodiments, the method includes measuring the mobility of one or more condensates in the entire cell composition. In some embodiments, the method includes measuring the mobility of the one or more condensates in one or more cells of the cell composition. In some embodiments, the method includes measuring the mobility of the one or more condensates in a single cell of the cell composition. In some embodiments, the method includes measuring the mobility of the one or more condensates in a part of one or more cells of the cell composition. In some embodiments, the method includes measuring the mobility of the one or more condensates in a part of a single cell of the cell composition. In some embodiments, the method includes measuring the mobility of one or more condensates in the cytoplasm. In some embodiments, the method includes measuring the mobility of one or more condensates in the nucleus. In some embodiments, the method includes measuring the mobility of one or more condensates in an organelle.

[0198] In some embodiments, when the mobility of one or more condensates is increased compared to a reference (including a reference condensate), it is determined that the mobility of the one or more condensates is regulated by the compound. In some embodiments, when the mobility of one or more condensates is decreased compared to a reference (including a reference condensate), it is determined that the mobility of the one or more condensates is regulated by the compound. In some embodiments, compared to the reference, the mobility of one or more condensates in the first part of the cell composition is decreased, and compared to the reference, the mobility of one or more condensates in the second part of the cell composition is not decreased. In some embodiments, compared to the reference, the mobility of one or more condensates in the first part of the cell composition is increased, and compared to the reference, the mobility of one or more condensates in the second part of the cell composition is not increased. In some embodiments, the first part of the cell composition is the cytoplasm and the second part of the cell composition is the nucleus, or the first part of the cell composition is the nucleus and the second part of the cell composition is the cytoplasm. In some embodiments, compared to the reference, the mobility of one or more condensates in the cytoplasm is decreased, and compared to the reference, the mobility of one or more condensates in the nucleus is not decreased. In some embodiments, compared to the reference, the mobility of one or more condensates in the cytoplasm is decreased, and compared to the reference, the mobility of one or more condensates in the nucleus is increased.

[0199] In some embodiments, the mobility of one or more condensates in the first portion of the cell composition is increased compared to the second portion of the cell composition, or the mobility of one or more condensates in the second portion of the cell composition is increased compared to the first portion of the cell composition. In some embodiments, the first portion of the cell composition is the cytoplasm and the second portion of the cell composition is the nucleus, or the first portion of the cell composition is the nucleus and the second portion of the cell composition is the cytoplasm.

[0200] In some embodiments, the mobility of one or more condensates is evaluated by one or more of the following: droplet size, surface tension, phase diagram, equilibrium state, droplet coarsening, and hardening.

[0201] In some embodiments, imaging techniques are used to determine the mobility of one or more condensates.

[0202] Solidification of condensates

[0203] In some embodiments, the properties associated with the one or more condensates are based on the solidification of the one or more condensates. Thus, in some embodiments, provided herein are methods of identifying a compound that modulates a property associated with one or more condensates comprising condensate-associated molecules, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining a property associated with the one or more condensates, wherein modulation of the property relative to a reference indicates that the compound modulates a property associated with the one or more condensates, wherein the property is modulation of the solidification of the one or more condensates. In some embodiments, provided herein are methods of identifying a compound that modulates the solidification of one or more condensates, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining the solidification of the one or more condensates, wherein modulation of the solidification relative to a reference indicates that the compound modulates the solidification of the one or more condensates. In some embodiments, the solidification is based on measurements that account for two or more condensates, such as the average or distribution of the solidification of two or more condensates.

[0204] It is possible to measure the condensation of one or more condensates or their products in part or all of the cell composition. Thus, in some embodiments, the method comprises measuring the condensation of one or more condensates in a portion of the cell composition, such as one or more fields of view or one or more portions of an image of the cell composition. In some embodiments, the method comprises measuring the condensation of one or more condensates in the entire cell composition. In some embodiments, the method comprises measuring the condensation of one or more condensates in one or more cells in the cell composition. In some embodiments, the method comprises measuring the condensation of one or more condensates in a single cell in the cell composition. In some embodiments, the method comprises measuring the condensation of one or more condensates in a portion of one or more cells in the cell composition. In some embodiments, the method comprises measuring the condensation of one or more condensates in a portion of a single cell in the cell composition. In some embodiments, the method comprises measuring the condensation of one or more condensates in the cytoplasm. In some embodiments, the method comprises measuring the condensation of one or more condensates in the nucleus. In some embodiments, the method comprises measuring the condensation of one or more condensates in an organelle.

[0205] In some embodiments, when the condensation of one or more condensates increases compared to a reference (including a reference condensate or its product), it is determined that the condensation of the one or more condensates or their products is modulated by the compound. In some embodiments, when the condensation of one or more condensates decreases compared to a reference (including a reference condensate), it is determined that the condensation of the one or more condensates is modulated by the compound. In some embodiments, compared to the reference, the condensation of one or more condensates in the first portion of the cell composition is reduced, and compared to the reference, the condensation of one or more condensates in the second portion of the cell composition is not reduced. In some embodiments, compared to the reference, the condensation of one or more condensates in the first portion of the cell composition increases, and compared to the reference, the condensation of one or more condensates in the second portion of the cell composition does not increase. In some embodiments, the first portion of the cell composition is the cytoplasm and the second portion of the cell composition is the nucleus, or the first portion of the cell composition is the nucleus and the second portion of the cell composition is the cytoplasm. In some embodiments, compared to the reference, the condensation of one or more condensates in the cytoplasm is reduced, and compared to the reference, the condensation of one or more condensates in the nucleus is not reduced. In some embodiments, compared to the reference, the condensation of one or more condensates in the cytoplasm is reduced, and compared to the reference, the condensation of one or more condensates in the nucleus increases.

[0206] In some embodiments, the solidification of one or more condensates in the first portion of the cell composition is increased compared to the second portion of the cell composition, or the solidification of one or more condensates in the second portion of the cell composition is increased compared to the first portion of the cell composition. In some embodiments, the first portion of the cell composition is the cytoplasm and the second portion of the cell composition is the nucleus, or the first portion of the cell composition is the nucleus and the second portion of the cell composition is the cytoplasm.

[0207] In some embodiments, the compound modulates the liquid-to-gel transition of the one or more condensates compared to a reference. In some embodiments, the compound modulates the liquid-to-gel transition of the one or more condensates compared to a reference without modulating the gel-to-solid transition of the one or more condensates. In some embodiments, the compound modulates the gel-to-solid transition of the one or more condensates compared to a reference. In some embodiments, the compound modulates the gel-to-solid transition of the one or more condensates compared to a reference without modulating the liquid-to-gel transition of the one or more condensates.

[0208] In some embodiments, the solidification of the one or more condensates is evaluated by one or more of the following: droplet size, surface tension, phase diagram, equilibrium state, droplet coarsening, hardening, and fiber and / or aggregate formation.

[0209] In some embodiments, imaging techniques are used to determine the solidification of one or more condensates. In some embodiments, the solidification of one or more condensates is determined by measuring the formation of aggregates or fibers. In some embodiments, filtration-based techniques are used to determine the solidification of one or more condensates.

[0210] Dissolution of condensates

[0211] In some embodiments, the properties associated with the one or more condensates are based on the dissolution of the one or more condensates. Thus, in some embodiments, provided herein are methods of identifying a compound that modulates a property associated with one or more condensates comprising condensate-associated molecules, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or a cell composition capable of forming one or more condensates, and (b) determining a property associated with the one or more condensates, wherein modulation of the property, compared to a reference, indicates that the compound modulates a property associated with the one or more condensates, wherein the property is modulation of the dissolution of the one or more condensates. In some embodiments, provided herein are methods of identifying a compound that modulates the dissolution of one or more condensates, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or a cell composition capable of forming one or more condensates, and (b) determining the dissolution of the one or more condensates, wherein modulation of the dissolution, compared to a reference, indicates that the compound modulates the dissolution of the one or more condensates. In some embodiments, the dissolution is based on a measurement taking into account two or more condensates, such as an average or distribution of the dissolution of two or more condensates.

[0212] The dissolution of some or all of the one or more condensates in the cell composition can be determined. Thus, in some embodiments, the method comprises determining the dissolution of one or more condensates in a portion of the cell composition. In some embodiments, the method comprises determining the dissolution of one or more condensates in the entire cell composition. In some embodiments, the method comprises determining the dissolution of one or more condensates in one or more cells of the cell composition. In some embodiments, the method comprises determining the dissolution of one or more condensates in a single cell of the cell composition. In some embodiments, the method comprises determining the dissolution of one or more condensates in a portion of one or more cells of the cell composition. In some embodiments, the method comprises determining the dissolution of one or more condensates in a portion of a single cell of the cell composition. In some embodiments, the method comprises determining the dissolution of one or more condensates in the cytoplasm. In some embodiments, the method comprises determining the dissolution of the one or more condensates in the nucleus. In some embodiments, the method comprises determining the dissolution of the one or more condensates in an organelle.

[0213] In some embodiments, it is determined that the dissolution of one or more condensates is modulated by the compound when the dissolution of the one or more condensates is increased compared to a reference. In some embodiments, it is determined that the dissolution of the one or more condensates is modulated by the compound when the dissolution of the one or more condensates is decreased compared to a reference. In some embodiments, compared to a reference, the dissolution of one or more condensates in a first portion of the cell composition is reduced and, compared to a reference, the dissolution of one or more condensates in a second portion of the cell composition is not reduced. In some embodiments, compared to a reference, the dissolution of one or more condensates in a first portion of the cell composition is increased and, compared to a reference, the dissolution of one or more condensates in a second portion of the cell composition is not increased. In some embodiments, the first portion of the cell composition is the cytoplasm and the second portion of the cell composition is the nucleus, or the first portion of the cell composition is the nucleus and the second portion of the cell composition is the cytoplasm. In some embodiments, compared to a reference, the dissolution of one or more condensates in the cytoplasm is reduced and, compared to a reference, the dissolution of one or more condensates in the nucleus is not reduced. In some embodiments, compared to a reference, the dissolution of one or more condensates in the cytoplasm is reduced and, compared to a reference, the dissolution of one or more condensates in the nucleus is increased.

[0214] In some embodiments, the dissolution of one or more condensates in a first portion of the cell composition is increased compared to a second portion of the cell composition, or the dissolution of one or more condensates in a second portion of the cell composition is increased compared to a first portion of the cell composition. In some embodiments, the first portion of the cell composition is the cytoplasm and the second portion of the cell composition is the nucleus, or the first portion of the cell composition is the nucleus and the second portion of the cell composition is the cytoplasm.

[0215] In some embodiments, the method comprises subjecting the cell composition to condensate-forming conditions prior to contacting the compound with the cell composition. In some embodiments, the dissolution is evaluated by one or more of the number of condensates, the size of the condensates (such as a decrease in size), the disappearance of the condensates.

[0216] In some embodiments, imaging techniques are used to determine the dissolution of one or more condensates.

[0217] Presence and / or amount of fiber formation

[0218] In some embodiments, the properties associated with the one or more condensates are based on the presence and / or amount of fiber formation. In some embodiments, the properties associated with the one or more condensates are based on the presence of fiber formation. In some embodiments, the properties associated with the one or more condensates are based on the amount of fiber formation. Thus, in some embodiments, provided herein are methods of identifying a compound that modulates a property associated with one or more condensates comprising condensate-associated molecules, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining a property associated with the one or more condensates, wherein modulation of the property as compared to a reference indicates that the compound modulates a property associated with the one or more condensates, wherein the property is modulation of the presence and / or amount of fiber formation. In some embodiments, provided herein are methods of identifying a compound that modulates the presence and / or amount of fiber formation, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining the presence and / or amount of fiber formation, wherein modulation of the presence and / or amount of fiber formation as compared to a reference indicates that the compound modulates the presence and / or amount of fiber formation.

[0219] Fiber formation can be detected and measured by a variety of methods, such as visualizing the fibers using a microscope and / or a fiber binding assay, such as the fiber binding assay described in the Examples.

[0220] The presence and / or amount of fiber formation can be determined in a part or all of the cell composition. Thus, in some embodiments, the method comprises determining the presence and / or amount of fiber formation in a part of the cell composition. In some embodiments, the method comprises determining the presence and / or amount of fiber formation in the whole cell composition. In some embodiments, the method comprises determining the presence and / or amount of fiber formation in one or more cells in the cell composition. In some embodiments, the method comprises determining the presence and / or amount of fiber formation in a single cell in the cell composition.

[0221] It is also possible to determine the presence and / or amount of fibril formation in some or all of the cells in the cell composition. Thus, in some embodiments, the method comprises determining the presence and / or amount of fibril formation in a portion of one or more cells in the cell composition. In some embodiments, the method comprises determining the presence and / or amount of fibril formation in a portion of a single cell in the cell composition. In some embodiments, the method comprises determining the presence and / or amount of fibril formation in the cytoplasm. In some embodiments, the method comprises determining the presence and / or amount of fibril formation in the cell nucleus. In some embodiments, the method comprises determining the presence and / or amount of fibril formation in the cell organelles.

[0222] In some embodiments, the amount of fibril formation is increased compared to a reference. In some embodiments, the amount of fibril formation is decreased compared to a reference.

[0223] In some embodiments, the amount of fibril formation in a first portion of the cell composition is decreased compared to a reference, and the amount of fibril formation in a second portion of the cell composition is not decreased compared to the reference. In some embodiments, the amount of fibril formation in a first portion of the cell composition is increased compared to a reference, and the amount of fibril formation in a second portion of the cell composition is not increased compared to the reference. In some embodiments, the first portion of the cell composition is the cytoplasm and the second portion of the cell composition is the cell nucleus, or the first portion of the cell composition is the cell nucleus and the second portion of the cell composition is the cytoplasm. In some embodiments, the amount of fibril formation in the cytoplasm is decreased compared to a reference, and the amount of fibril formation in the cell nucleus is not decreased compared to the reference. In some embodiments, the amount of fibril formation in the cytoplasm is decreased compared to a reference, and the amount of fibril formation in the cell nucleus is increased compared to the reference.

[0224] In some embodiments, the amount of fibril formation in a first portion of the cell composition is increased compared to a second portion of the cell composition, or the amount of fibril formation in a second portion of the cell composition is increased compared to a first portion of the cell composition. In some embodiments, the first portion of the cell composition is the cytoplasm and the second portion of the cell composition is the cell nucleus, or the first portion of the cell composition is the cell nucleus and the second portion of the cell composition is the cytoplasm.

[0225] Location of condensate - related molecules

[0226] In some embodiments, the properties associated with the one or more condensates are based on the location of the one or more condensates. Thus, in some embodiments, provided herein are methods of identifying a compound that modulates a property associated with one or more condensates comprising condensate-associated molecules, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining a property associated with the one or more condensates, wherein modulation of the property relative to a reference indicates that the compound modulates a property associated with the one or more condensates, wherein the property is modulation of the location of the one or more condensates. In some embodiments, provided herein are methods of identifying a compound that modulates the location of one or more condensates, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining the location of the one or more condensates, wherein modulation of the location relative to a reference indicates that the compound modulates the location of the one or more condensates.

[0227] The location of the condensates can be determined for a portion or all of the cell composition. Thus, in some embodiments, the method comprises determining the location of one or more condensates in a portion of the cell composition. In some embodiments, the method comprises determining the location of one or more condensates in the entire cell composition. In some embodiments, the method comprises determining the location of one or more condensates in one or more of the one or more cells of the cell composition. In some embodiments, the method comprises determining the location of one or more condensates in a single cell of the cell composition.

[0228] The location of the condensates can also be determined for a portion or all of the cells in the cell composition. Thus, in some embodiments, the method comprises determining the location of one or more condensates in a portion of one or more cells of the cell composition. In some embodiments, the method comprises determining the location of one or more condensates in a portion of a single cell of the cell composition. In some embodiments, the method comprises determining the location of one or more condensates in the cytoplasm. In some embodiments, the method comprises determining the location of one or more condensates in the nucleus. In some embodiments, the method comprises determining the location of one or more condensates in an organelle.

[0229] In some embodiments, imaging techniques are used to determine the location of the one or more condensates.

[0230] Partitioning of condensate - related molecules into condensates

[0231] In some embodiments, the properties associated with the one or more condensates are based on the partitioning of condensate-associated molecules into the one or more condensates. Thus, in some embodiments, provided herein are methods of identifying a compound that modulates a property associated with one or more condensates comprising condensate-associated molecules, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining a property associated with the one or more condensates, wherein modulation of the property, compared to a reference, indicates that the compound modulates a property associated with the one or more condensates, wherein the property is modulation of the partitioning of the condensate-associated molecules into the one or more condensates. In some embodiments, provided herein are methods of identifying a compound that modulates the partitioning of the condensate-associated molecules into one or more condensates, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining the partitioning of the condensate-associated molecules into the one or more condensates, wherein modulation of the partitioning, compared to a reference, indicates that the compound modulates the partitioning of the condensate-associated molecules into the one or more condensates.

[0232] The partitioning of condensate-associated molecules into condensates can be determined for a part or all of the cell composition. Thus, in some embodiments, the method comprises determining the partitioning of condensate-associated molecules into one or more condensates in a part of the cell composition. In some embodiments, the method comprises determining the partitioning of condensate-associated molecules into one or more condensates in the entire cell composition. In some embodiments, the method comprises determining the partitioning of condensate-associated molecules into one or more condensates in one or more cells in the cell composition. In some embodiments, the method comprises determining the partitioning of condensate-associated molecules into one or more condensates in a single cell in the cell composition.

[0233] It is also possible to determine the partitioning of condensate-related molecules into condensates in some or all of the cells in the cell composition. Thus, in some embodiments, the method comprises determining the partitioning of condensate-related molecules in a portion of one or more cells in the cell composition into one or more condensates. In some embodiments, the method comprises determining the partitioning of the condensate-related molecules in a portion of a single cell in the cell composition into the one or more condensates. In some embodiments, the method comprises determining the partitioning of the condensate-related molecules in the cytoplasm into one or more condensates. In some embodiments, the method comprises determining the partitioning of the condensate-related molecules in the nucleus into the one or more condensates. In some embodiments, the method comprises determining the partitioning of the condensate-related molecules in an organelle into the one or more condensates.

[0234] In some embodiments, the partitioning of condensate-related molecules into one or more condensates is increased compared to a reference. In some embodiments, the partitioning of condensate-related molecules into one or more condensates is decreased compared to a reference.

[0235] In some embodiments, the partitioning of condensate-related molecules in a first portion of the cell composition into one or more condensates is decreased compared to a reference, and the partitioning of condensate-related molecules in a second portion of the cell composition into one or more condensates is not decreased compared to the reference. In some embodiments, the partitioning of condensate-related molecules in a first portion of the cell composition into one or more condensates is increased compared to a reference, and the partitioning of condensate-related molecules in a second portion of the cell composition into one or more condensates is not increased compared to the reference. In some embodiments, the first portion of the cell composition is the cytoplasm and the second portion of the cell composition is the nucleus, or the first portion of the cell composition is the nucleus and the second portion of the cell composition is the cytoplasm. In some embodiments, the partitioning of condensate-related molecules in the cytoplasm into one or more condensates is decreased compared to a reference, and the partitioning of condensate-related molecules in the nucleus into one or more condensates is not decreased compared to the reference. In some embodiments, the partitioning of condensate-related molecules in the cytoplasm into one or more condensates is decreased compared to a reference, and the partitioning of condensate-related molecules in the nucleus into one or more condensates is increased compared to the reference.

[0236] In some embodiments, the partitioning of condensate-associated molecules into one or more condensates is increased in the first part of the cell composition compared to the second part of the cell composition, or the partitioning of condensate-associated molecules into one or more condensates is increased in the second part of the cell composition compared to the first part of the cell composition. In some embodiments, the first part of the cell composition is the cytoplasm and the second part of the cell composition is the nucleus, or the first part of the cell composition is the nucleus and the second part of the cell composition is the cytoplasm.

[0237] Aggregation of condensate - related molecules

[0238] In some embodiments, the properties associated with the one or more condensates are based on the aggregation of condensate-associated molecules. Thus, in some embodiments, provided herein is a method of identifying a compound that modulates a property associated with one or more condensates comprising condensate-associated molecules, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining a property associated with the one or more condensates, wherein modulation of the property relative to a reference indicates that the compound modulates a property associated with the one or more condensates, wherein the property is modulation of the aggregation of the condensate-associated molecules. In some embodiments, provided herein is a method of identifying a compound that modulates the aggregation of condensate-associated molecules, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or with a cell composition capable of forming one or more condensates, and (b) determining the aggregation of the condensate-associated molecules, wherein modulation of the aggregation relative to a reference indicates that the compound modulates the aggregation of the condensate-associated molecules.

[0239] The aggregation of condensate-associated molecules in a portion or all of the cell composition can be determined. Thus, in some embodiments, the method comprises determining the aggregation of condensate-associated molecules in a portion of the cell composition. In some embodiments, the method comprises determining the aggregation of condensate-associated molecules in the entire cell composition. In some embodiments, the method comprises determining the aggregation of condensate-associated molecules in one or more cells of the cell composition. In some embodiments, the method comprises determining the aggregation of condensate-associated molecules in a single cell of the cell composition.

[0240] It is also possible to measure the aggregation of condensate-related molecules in some or all of the cells in the cell composition. Thus, in some embodiments, the method comprises measuring the aggregation of condensate-related molecules in a portion of one or more cells in the cell composition. In some embodiments, the method comprises measuring the aggregation of condensate-related molecules in a portion of a single cell in the cell composition. In some embodiments, the method comprises measuring the aggregation of condensate-related molecules in the cytoplasm. In some embodiments, the method comprises measuring the aggregation of condensate-related molecules in the nucleus. In some embodiments, the method comprises measuring the aggregation of condensate-related molecules in the organelles.

[0241] In some embodiments, the aggregation of the condensate-related molecules is increased compared to a reference. In some embodiments, the aggregation of the condensate-related molecules is decreased compared to a reference.

[0242] In some embodiments, the aggregation of the condensate-related molecules in the first portion of the cell composition is decreased compared to a reference, and the aggregation of the condensate-related molecules in the second portion of the cell composition is not decreased compared to the reference. In some embodiments, the aggregation of the condensate-related molecules in the first portion of the cell composition is increased compared to a reference, and the aggregation of the condensate-related molecules in the second portion of the cell composition is not increased compared to the reference. In some embodiments, the first portion of the cell composition is the cytoplasm and the second portion of the cell composition is the nucleus, or the first portion of the cell composition is the nucleus and the second portion of the cell composition is the cytoplasm. In some embodiments, the aggregation of the condensate-related molecules in the cytoplasm is decreased compared to a reference, and the aggregation of the condensate-related molecules in the nucleus is not decreased compared to the reference. In some embodiments, the aggregation of the condensate-related molecules in the cytoplasm is decreased compared to a reference, and the aggregation of the condensate-related molecules in the nucleus is increased compared to the reference.

[0243] In some embodiments, the aggregation of the condensate-related molecules in the first portion of the cell composition is increased compared to the second portion of the cell composition, or the aggregation of the condensate-related molecules in the second portion of the cell composition is increased compared to the first portion of the cell composition. In some embodiments, the first portion of the cell composition is the cytoplasm and the second portion of the cell composition is the nucleus, or the first portion of the cell composition is the nucleus and the second portion of the cell composition is the cytoplasm.

[0244] Compound

[0245] As used herein, "compound" refers to any reagent. In some embodiments, the compound is a small molecule, polypeptide, lipid, or nucleic acid. In some embodiments, the compound is an approved compound, such as a compound approved by the US Food and Drug Administration for medical treatment. In some embodiments, the compound is a novel compound. In some embodiments, the compound is charged. In some embodiments, the compound is hydrophobic. In some embodiments, the compound is hydrophilic. In some embodiments, the compound is a small molecule. In some embodiments, the small molecule is an alkaloid, glycoside, phenazine, phenol, polyketide, terpene, or tetrapyrrole. In some embodiments, the compound is an antibody. In some embodiments, the compound is a nucleic acid. In some embodiments, the compound is RNA, such as siRNA, miRNA, or mRNA. In some embodiments, the compound is a non-naturally occurring compound. In some embodiments, the compound is a naturally occurring compound. When multiple candidate compounds are used for screening, the multiple candidate compounds can be of the same type or different types.

[0246] In some embodiments, the compound is capable of selectively non-covalently interacting with a biomolecule (especially a protein or nucleic acid) under conditions prevalent in living cells, wherein the compound and the biomolecule form a complex having a dissociation constant Kd of 10 -4 mol / l or less. In some embodiments, the compound has a molecular mass greater than 160 Da but less than 1000 Da, particularly less than 700 Da, more particularly less than 500 Da, and the compound contains up to five hydrogen bond donors, up to ten hydrogen bond acceptors, and is characterized by an octanol-water partition coefficient logP below 5.6. These are the so-called "Lipinski" rule of five for drug-like compounds (originally referring to molecules between 160 and 500 Da). It should be understood that any of these embodiments can be applied and combined with any compound described herein (including the foregoing paragraphs).

[0247] Cells and condensates

[0248] In some embodiments, the cell composition comprises microbial or animal cells. In some embodiments, the cell composition comprises human cells. In some embodiments, the cell composition comprises neurons. In some embodiments, the cell composition includes cancer cells. In some embodiments, the cell composition comprises cells that are or are derived from induced pluripotent stem cells (iPS cells), HeLa cells, or HEK293 cells. In some embodiments, the cell composition comprises dysregulated condensates. In some embodiments, the cell composition comprises cells containing mutations associated with a disease. In some embodiments, the cell composition comprises cells having one or more characteristics of a neurodegenerative or proliferative disease. In some embodiments, the cell composition comprises cells that express a protein labeled with a fluorescent protein. In some embodiments, the protein is a protein known to be enriched in condensates. In some embodiments, the condensate-associated molecule is labeled, for example, by linkage or fusion with a fluorescent protein.

[0249] In some embodiments, the cells in the cell composition express the condensate-associated molecule. In some embodiments, expression can include any one of gene replication, transcription, and translation. In some embodiments, the condensate-associated molecule is a polynucleotide, such as RNA, wherein the condensate-associated molecule is transcribed in the cells of the cell composition. In some embodiments, the condensate-associated molecule is a polypeptide, such as a protein, wherein the condensate-associated molecule is translated in the cells of the cell composition. In some embodiments, the condensate-associated molecule is heterologous to the cell.

[0250] Many condensates are well known in the art. Examples of known condensates include the spliceosome, P-bodies, histone locus bodies, multivesicular bodies, neuronal RNA granules, nuclear gems, nuclear pores, nuclear speckles, nuclear stress bodies, nucleoli, Oct1 / PTF / transcription (OPT) domains, paraspeckles, perinuclear compartments, PML nuclear bodies, PML oncogenic domains, polycomb bodies, processing bodies, Sam68 nuclear bodies, stress granules, or splicing speckles. Many more condensates are known to form but have not been described. Many condensates can be identified using microscopy. In some embodiments, the method further comprises identifying one or more condensates. In some embodiments, the one or more condensates are cellular condensates. In some embodiments, the one or more condensates are within one or more cells of the cell composition. In some embodiments, the one or more condensates are one or more stress granules. In some embodiments, a first group of one or more condensates is one or more stress granules. In some embodiments, a second group of one or more condensates is one or more nuclear paraspeckles, condensates formed around DNA damage sites, P-bodies, Cajal bodies, and PML bodies.

[0251] In some embodiments, the condensates are selected from stress granules, P bodies, Cajal bodies, PML bodies, paraspeckles (e.g., nuclear paraspeckles), DNA damage focus condensates, mitotic spindles, P granules, histone locus bodies, multivesicular bodies, neuronal RNA granules, nuclear gems, nuclear pores, nuclear stress bodies, nucleoli, Oct1 / PTF / transcription (OPT) domains, perinuclear compartments, PML oncogenic domains, polycomb bodies, processing bodies, Sam68 nuclear bodies, and splicing speckles. Exemplary condensates are discussed, for example, in Banani et al., Nat Rev Mol Cell Biol, 18, 2017, “Biomolecular condensates: organizers of cellular biochemistry”; Brangwynne et al., Science, 324, 2009, “Germline P granules are liquid droplets that localize by controlled dissolution / condensation”; Patel et al., Cell, 162, 2015, “A Liquid-to-Solid Phase Transition of the ALS Protein Accelerated by Disease Mutation”; Alberti, S., Current Biology, 27, R1089–R1107, 2017, “Phase Separation in Biology.”

[0252] Condensate-related molecules

[0253] In some embodiments, the condensate-related molecule is a polynucleotide or a polypeptide. In some embodiments, the condensate-related molecule is a polypeptide. In some embodiments, the condensate-related molecule is a wild-type polypeptide. In some embodiments, the condensate-related molecule is a mutant polypeptide. In some embodiments, the condensate-related molecule is FUS, EWSR1, TIAL1, PABPC1, or G3BP1, or a mutant thereof. In some embodiments, the condensate-related molecule is FUS.

[0254] Additional assays

[0255] In some embodiments, the method further comprises evaluating the compound by one or more additional assays, such as one or more of a second cell-level assay, a biochemical assay, and an in vivo assay.

[0256] In some embodiments, the biochemical assay method is a method for identifying a compound that modulates a property related to condensates.

[0257] In some embodiments, the biochemical assay method comprises: (a) mixing the compound with a precursor molecule to form a reaction composition, (b) subjecting the reaction composition to condensate formation conditions, wherein (if formed) the condensate comprises the precursor molecule; and (c) measuring a property related to the condensate, wherein modulation of the property as compared to a reference identifies the compound as a compound that modulates the property. In some embodiments, the condensate formation conditions are a reduced salt concentration. In some embodiments, the method is repeated using different condensate formation conditions.

[0258] In some embodiments, the biochemical assay method comprises: (a) mixing the compound with a condensate comprising a precursor molecule to form a reaction composition; and (b) measuring a property related to the condensate, wherein modulation of the property as compared to a reference identifies the compound as a compound that modulates the property. In some embodiments, the biochemical assay method further comprises subjecting the reaction composition to aging conditions after mixing the compound and the condensate to form the reaction composition. In some embodiments, the aging conditions are: incubation, shaking, and / or heating.

[0259] In some embodiments, measuring a property related to a condensate is based on any one or more of the following: (i) the number of condensates; (ii) the size of the condensates; (iii) the partitioning of the precursor molecule into the condensates; (iv) the surface area of the condensates; (v) the composition of the condensates; (vii) the mobility of the condensates; (viii) the solidification of the condensates; (ix) the aggregation of the precursor molecule; (x) the dissolution of the condensates; and (xi) the presence and / or amount of fiber formation.

[0260] In some embodiments, the precursor molecule is a polypeptide. In some embodiments, the precursor molecule is a wild-type polypeptide. In some embodiments, the precursor molecule is a mutant polypeptide. In some embodiments, the polypeptide is selected from: FUS, EWSR1, TIAL1, PABPC1, and G3BP1.

[0261] In some embodiments, the biochemical assay method further comprises imaging the reaction composition after subjecting the reaction composition to condensate formation conditions. In some embodiments, the reaction composition comprises a fixed ratio of the compound and the precursor molecule. In some embodiments, the biochemical assay method is repeated with two or more different ratios of the compound and the precursor molecule.

[0262] In some embodiments, the reference is a reaction composition that does not contain the compound.

[0263] Exemplary embodiments

[0264] The embodiments provided are:

[0265] Embodiment 1. A compound for use in a method of preventing or treating a neurodegenerative disease associated with stress granule formation, wherein the compound is selected from:

[0266] - lipoic acid

[0267] - lipoamide,

[0268] - dihydrolipoic acid, and

[0269] - dihydrolipoamide.

[0270] Embodiment 2. The compound for use in a method of preventing or treating a neurodegenerative disease associated with stress granule formation according to Embodiment 1, comprising a dosing regimen:

[0271] - administering a daily dose of 600 mg to 1,600 mg of the compound.

[0272] Embodiment 3. The compound for use in a method of preventing or treating a neurodegenerative disease associated with stress granule formation according to Embodiment 1 or 2, wherein the neurodegenerative disease associated with stress granule formation is amyotrophic lateral sclerosis.

[0273] Embodiment 4. A pharmaceutical composition for use in a method of preventing or treating neurodegeneration associated with stress granule formation, comprising the compound according to any one of Embodiments 1 to 3.

[0274] Embodiment 5. The pharmaceutical composition for use in a method of preventing or treating amyotrophic lateral sclerosis according to Embodiment 4, wherein the pharmaceutical composition is formulated for oral administration.

[0275] Embodiment 6. The pharmaceutical composition according to Embodiment 4 or 5, wherein the neurodegenerative disease associated with stress granule formation is amyotrophic lateral sclerosis.

[0276] Embodiment 7. A dosage form for use in a method of preventing or treating amyotrophic lateral sclerosis, comprising the compound according to any one of Embodiments 1, especially administered at the dose specified in Embodiment 2.

[0277] Embodiment 8. The dosage form according to Embodiment 7, wherein the dosage form is formulated for oral administration.

[0278] Embodiment 9. The dosage form according to Embodiment 7 or 8, wherein the neurodegenerative disease associated with stress granule formation is amyotrophic lateral sclerosis.

[0279] Embodiment 10. A method for treating or preventing a neurodegenerative disease associated with stress granule formation, comprising administering to a patient in need thereof a compound according to Embodiment 1.

[0280] Embodiment 11. The method for treating or preventing a neurodegenerative disease according to Embodiment 10, wherein the compound is administered at a daily dose of 600 mg to 1,600 mg.

[0281] Embodiment 12. The method for treating or preventing a neurodegenerative disease according to Embodiment 10 or 11, wherein the compound is administered orally.

[0282] Embodiment 13. The method for treating or preventing a neurodegenerative disease according to any one of Embodiments 10 to 12, wherein the neurodegenerative disease associated with stress granule formation is amyotrophic lateral sclerosis.

[0283] Embodiment 14. A method for reducing or inhibiting the formation of stress granules in cells, comprising using a compound selected from:

[0284] - lipoic acid

[0285] - lipoamide,

[0286] - dihydrolipoic acid, and

[0287] - dihydrolipoamide,

[0288] - hetero-tricyclic compounds, especially anthraquinones or anthraquinone derivatives (such as 1,4-dihydroxyanthraquinone), acridines or acridine derivatives (such as quinacrine or aminoacridine or mitoxantrone);

[0289] - tetracyclic compounds, and

[0290] - surfactants, especially cetylpyridinium chloride.

[0291] Further exemplary embodiments

[0292] Wherein the provided embodiments also include:

[0293] E1. A method for identifying a compound that modulates a property related to one or more condensates comprising condensate-related molecules, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or a cell composition capable of forming one or more condensates, and (b) determining a property related to the one or more condensates, wherein modulation of the property, compared to a reference, indicates that the compound modulates a property related to the one or more condensates.

[0294] E2. The method according to embodiment E1, wherein the property related to the one or more condensates is determined based on any one or more of the following: (i) the number of condensates comprising and / or not comprising the condensate-related molecules; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of one or more condensates; (v) the surface area of the one or more condensates; (vi) the composition of the one or more condensates; (vii) the mobility of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-related molecules; (xii) the partitioning of the condensate-related molecules into the one or more condensates; and (xiii) the aggregation of the condensate-related molecules.

[0295] E3. The method according to embodiment E1 or E2, wherein the one or more condensates are within one or more cells of the cell composition.

[0296] E4. The method according to any one of embodiments E1-E3, further comprising subjecting the cell composition to condensate formation conditions before step (b).

[0297] E5. The method according to any one of embodiments E1-E3, further comprising subjecting the cell composition to condensate formation conditions before step (a).

[0298] E6. The method according to embodiment E4 or E5, wherein the condensate formation conditions are any one or more of the following: (i) an oxidative stressor; (ii) a mitochondrial electron transport chain inhibitor; (iii) a heat stressor; (iv) an osmotic stressor; (v) a hyperosmotic stressor; and (vi) glycolysis inhibition.

[0299] E7. The method according to any one of embodiments E1-E6, wherein the condensate-related molecule is a polypeptide.

[0300] E8. The method according to any one of embodiments E1 - E7, wherein the condensate - associated molecule is a wild - type polypeptide.

[0301] E9. The method according to any one of embodiments E1 - E7, wherein the condensate - associated molecule is a mutant polypeptide.

[0302] E10. The method according to any one of embodiments E1 - E9, wherein the condensate - associated molecule is selected from: FUS, EWSR1, TIAL1, PABPC1, and G3BP1.

[0303] E11. The method according to any one of embodiments E1 - E10, wherein the cells in the cell composition express the condensate - associated molecule.

[0304] E12. The method according to any one of embodiments E1 - E11, wherein the cell composition comprises HeLA, iPSC, or iPSC MN cells.

[0305] E13. The method according to any one of embodiments E1 - E12, further comprising imaging at least a portion of the cell composition.

[0306] E14. The method according to any one of embodiments E1 - E13, further comprising contacting at least a portion of the cell composition with a fixative.

[0307] E15. The method according to any one of embodiments E1 - E14, further comprising contacting at least a portion of the cell composition with a stain.

[0308] E16. The method according to any one of embodiments E1 - E15, further comprising contacting at least a portion of the cell composition with DNA - damaging conditions.

[0309] E17. The method according to embodiment E16, wherein the DNA - damaging condition is laser irradiation.

[0310] E18. The method according to any one of embodiments E1 - E17, wherein the reference is a second condensate.

[0311] E19. The method according to any one of embodiments E1 - E17, wherein the reference is a second cell composition.

[0312] E20. The method according to any one of embodiments E1 - E19, further comprising using a second cell - based assay to evaluate the identified compound.

[0313] E21. The method according to any one of embodiments E1 - E20 further comprises evaluating the identified compound using a biochemical assay.

[0314] E22. The method according to any one of embodiments E1 - E21 further comprises evaluating the identified compound using an in vivo assay.

[0315] E23. A method of identifying a compound useful for treating a disease, the method comprising identifying a compound according to any one of the methods of embodiments E1 - E22.

[0316] E24. The method according to embodiment E23, wherein the disease is a neurodegenerative disease.

[0317] E25. The method according to embodiment E24, wherein the neurodegenerative disease is ALS.

[0318] E26. The method according to any one of the foregoing embodiments E1 to E25, wherein the condensate is a membrane - less encapsulated compartment formed by phase separation of a protein with one or more of other macromolecules, particularly DNA and / or RNA.

[0319] E27. The method according to E27, wherein the condensate is selected from: stress granules, P - bodies, Cajal bodies, promyelocytic leukemia protein (PML) bodies, paraspeckles (such as nuclear paraspeckles), DNA damage foci condensates, divisomes, P - granules, histone locus bodies, multivesicular bodies, neuronal RNA granules, nuclear gems, nuclear pores, nuclear stress bodies, nucleoli, Oct1 / PTF / transcription (OPT) domains, perinuclear compartments, PML oncogenic domains, polycomb bodies, processing bodies, Sam68 nuclear bodies, and splicing speckles.

[0320] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the disclosure of this application. The present invention is further illustrated by the following examples and drawings, from which further embodiments and advantages can be obtained. These examples are for illustrative purposes and do not limit the scope of the invention.

[0321] Examples

[0322] Example 1

[0323] Screening compounds using FUS as a model LCD / RBP stress granule protein

[0324] The present inventors developed a cell-level screening using a HeLa cell line stably expressing GFP-tagged FUS at near-endogenous levels. Although not essential for stress granule formation, FUS is a well-characterized protein with a domain structure typical of stress granule proteins. In the absence of cellular stress, FUS-GFP is mainly localized in the nucleus, where FUS-GFP is partially excluded from the nucleolus and localized in small puncta called paraspeckles ( Figure 1 A). These are nuclear droplets involved in retaining RNA in the nucleus for rapid stress response. Cells were pretreated with 10 μM of a compound from a compound library for 1 hour and then stressed with 1 mM potassium arsenate (still in the presence of the compound) ( Figure 1 B), and the localization of FUS to stress granules was monitored. Arsenate disrupts the antioxidant response by reacting with thiol groups, blocks the tricarboxylic acid (TCA) cycle by reacting with the thiol in the important lipoyl moiety, and causes general oxidative damage. In untreated stressed cells, FUS is partially exported to the cytoplasm, where it binds to other proteins and mRNAs, which phase-separate to form liquid-like stress granules surrounded by cytoplasm depleted of FUS ( Figure 1 A).

[0325] Many compounds affect stress granule formation. Multivariate image analysis (including the number of cytoplasmic puncta, the number of nuclear puncta, and the nuclear / cytoplasmic compartmentalization) was used to rank compounds by the intensity of their effect on FUS localization in stressed cells ( Figure 1 C). Two compounds in the library were predicted to reduce stress granules. These are the polyribosome-stabilizing compound emetine, which prevents mRNA release, and the heavy metal chelating compound dimercaprol (which should chelate the stressor). Edaravone, which is considered an antioxidant and is used as an ALS therapeutic agent in Japan and the United States, had no effect on FUS localization in arsenate-stressed cells. Newly selected classes of compounds that tend to have a large effect on FUS localization (usually reducing the number of stress granules) include cardiac glycosides, heterocyclic tricyclic and tetracyclic compounds (anthraquinones and acridines), surfactants, and benzimidazoles.

[0326] Compounds that can directly interact with FUS are the main targets, so the present inventors performed a follow-up screening of the effect of compounds on the in vitro formation of FUS droplets ( Figure 1 D). The inventors analyzed the top 47 strongest selected compounds from the cell-level screening. Selected compounds were identified by their effect on the number of FUS droplets formed and the partitioning of FUS into the formed droplets in low-salt conditions in vitro in the presence of 1 mM DTT to mimic the reducing intracellular environment ( Figure 1 E). Of these 47 compounds, 7 significantly affected FUS in vitro ( Figure 1E), and this identified three classes of compounds that directly affect FUS droplet formation. Surfactants, hetero- and tetracyclic compounds, and lipoic amide, where the latter two are seemingly plausible therapeutic agents( Figure 1 F).

[0327] In this non-equilibrium snapshot, hetero- and tetracyclic compounds tend to reduce condensate formation in a dose-dependent manner, and the resulting droplets are smaller. In contrast, surfactants and lipoic amide tend to increase the number of condensate droplets, increase partitioning into the droplets and the droplets are larger( Figure 1 E, G). Surfactants are not seemingly plausible therapeutic agents as they permeabilize cell membranes and they are present in the library due to their use as topical antimicrobials.

[0328] The formation of stress granules is associated with the export of FUS from the nucleus, typically stress granule proteins containing LCD / RBPs. Persistent stress granules or stress granule proteins may cause deleterious loss of nuclear function, including reduced FUS DNA damage response. Restoring the nuclear localization of this protein may be beneficial.

[0329] Therefore, the dose-dependent activities of lipoic amide, lipoic acid, and related compounds were analyzed. In HeLa cells expressing FUS GFP, dose-response analysis showed that lipoic amide reduced the number of stress granules and increased the nuclear / cytoplasmic partitioning of FUS, with a similar EC 50 ( Figure 2 A). Not all of the selected compounds had this behavior, for example mitoxantrone (a hetero-tetracyclic anthraquinone) reduced nuclear / cytoplasmic FUS partitioning while reducing the number of stress granules( Figure 10 ). The inventors confirmed these key activities of lipoic amide using an induced pluripotent stem cell (iPSC) line expressing FUS GFP under arsenite stress. This also showed that lipoic amide reduced the number of stress granules and restored the nuclear FUS location( Figure 2 B). Thus, lipoic amide appears to simultaneously reverse the export of FUS from the nucleus and its incorporation into stress granules in response to stress. It was later shown that lipoic acid has the same effect (see below). The return of FUS to the nucleus may be beneficial, mimicking the unstressed cell state, suggesting that lipoic amide is a more promising candidate.

[0330] Lipoamide is related to lipoic acid, a naturally occurring compound. Lipoic acid has two stereoisomers, where R-(+)-lipoic acid occurs naturally in cells and is synthesized in mitochondria, while S-(-)-lipoic acid does not. A mixture of the two isomers is racemic lipoic acid or (±)-lipoic acid. It exists in cells at very low free concentrations and is usually covalently bound to proteins as a lipoyl moiety via a secondary amide. This bound form is similar to lipoamide. These compounds are dithiols, and the thiols of the lipoyl moiety are used as hydrogen carriers by multiple enzymes, including one that enters the tricarboxylic acid (TCA) cycle and one in the TCA cycle. The R-(+) isomer can be interconverted in cells between the oxidized (dithiol) and reduced (cyclic disulfide) states by dihydrolipoamide dehydrogenase, and although evidence for direct antioxidant action is controversial, these compounds are thought to be antioxidants. Importantly, both lipoic acid and lipoamide are non-toxic, and lipoic acid has well-characterized pharmacokinetics: it is non-toxic and an oral dose of 1,600 mg gives a plasma concentration of 8 to 30 μM in humans. This is very promising as concentrations of around 10 μM used for in vitro characterization can be achieved in humans - lipoic acid has a long history of use at a dose of around 600 mg / day in the treatment of diabetic neuropathy. Overall, lipoamide and lipoic acid dissolve stress granules, return FUS to the nucleus of stressed cells, interact directly with FUS, and are of low toxicity. Thus, they plausibly have utility as therapeutic agents.

[0331] The action of lipoamide / lipoic acid seems to be unrelated to enzyme or antioxidant action

[0332] The activity of lipoamide in subsequent in vitro screens ( Figure 1 D, E) demonstrated that it has a direct effect on the phase separation of stress granule proteins. Thus, the inventors sought to rule out more common mechanisms of activity in cell-level screens, such as stressor chelation, enzymatic action, or antioxidant activity. The inventors tested this using compounds related to lipoamide and lipoic acid ( Figure 2 C, D).

[0333] Screens were performed with arsenite stress, which can cause general oxidative damage, disrupt the antioxidant response by reacting with thiol groups, and block the TCA cycle by reacting with thiol groups in the important lipoyl moiety. To determine the extent of direct arsenite reaction, antioxidant action, and the contribution of enzymatic / glycolytic action to the activity of lipoic acid compounds, the dose-dependent activities of (±)-lipoic acid, (±)-lipoamide, (±)-dihydrolipoic acid, 1,3-propanedithiol, and R-(+) and S-(-)-lipoic acid were analyzed in HeLa cells expressing FUS-GFP ( Figure 2C, D). Except for 1,3-propanedithiol, all had comparable ECs in reducing the number of stress granules after 1 h of 1 mM arsenate stress. 50 .

[0334] This suggests that lipoic acid is inactive by supporting glycolysis because free lipoamide is not a normal component of cellular metabolism and should be unable to serve as a coenzyme / to be added to the apoenzyme via lipoic acid-protein ligase. It also suggests that lipoic acid is not directly used as an antioxidant because the reduced (dihydrolipoic acid) form should be more reactive than the oxidized (lipoic acid) form, although a redox cycle involving dihydrolipoamide dehydrogenase can be supplemented (where only the naturally occurring R-(+)-isomer may be active). We also note that menadione (vitamin K precursor and a typical antioxidant) did not reduce the number of stress granules in the initial HeLa cell screen. Arsenate reacts with thiols, so 1,3-propanedithiol and dihydrolipoic acid should be equally able to directly chemically inactivate arsenate stress, but 1,3-propanedithiol had no effect at up to 100 μM. Additionally, the lipoic acid EC 50 suggests that arsenate must be inactivated at a stoichiometry of ~1:50. Finally, any enzymatic action in lipoic acid activity, as the lipoyl moiety supporting glycolysis or through the antioxidant action and recycling of dihydrolipoamide dehydrogenase, should be specific to the naturally occurring R-(+)-lipoic acid. However, S-(-)-lipoic acid has very similar activity.

[0335] Overall, this suggests some other mechanism of action; perhaps a stress signaling mechanism or a direct physicochemical effect on the phase separation of stress granule proteins.

[0336] Lipoamide / lipoic acid reverses stress granule formation induced by multiple types of cellular stress

[0337] Cell-level screening was performed by pre-treating cells with compounds before arsenate stress and only FUS was analyzed. To gain insight into the breadth of the actions of lipoamide and lipoic acid and to further understand the possible mechanisms, their effects on HeLa cells were comprehensively characterized.

[0338] To determine whether lipoic acid can cause the dissolution of existing stress granules, the inventors pre-stressed HeLa cells expressing FUS-GFP and then treated them with lipoamide. Time-lapse microscopy showed that adding fresh medium containing 10 μM lipoamide and 1 mM arsenate dissolved 80 - 90% of the cytoplasmic FUS droplets within 20 minutes, while fresh medium containing 1 mM arsenate slightly reduced the number of stress granules ( Figure 3 A). This response may be too fast to represent a transcriptional / translational response.

[0339] To test whether treatment of cells with lipoamide can confer durable resistance to stress granule formation in the absence of continuous lipoamide treatment, HeLa cells were pretreated with 10 μM lipoamide for 1 h and then tested for arsenite stress for 1 h in the absence of lipoamide. This did not prevent stress granule formation, indicating no durable lipoamide-induced cellular adaptation to resist stress( Figure 3 B).

[0340] A useful therapeutic agent is expected to prevent the localization of all PLD-containing stress granule proteins to stress granules. In the absence of treatment or in the presence of 10 μM lipoic acid or lipoamide, the inventors stressed a HeLa cell line expressing GFP fusions of stress granule proteins (EWSR1, TIAL1, PABPC1, G3BP1) with 1 mM arsenite. In the absence of treatment and in the absence of racemic, S-(−)- or R-(+)-lipoic acid or lipoamide, all proteins localized to stress granules( Figure 3 C). Since FUS is not required for stress granule formation, this means that FUS is neither the sole target of lipoamide or lipoic acid in cells nor necessarily the primary target. Instead, all LCD / RBP proteins are affected, and a protein that is essential for stress granule formation may be the target. This is a promising property for neurodegenerative disease therapies because many stress granule-associated proteins are associated with different diseases and suggests a therapeutic effect on pathologies arising from mutations in other stress granule proteins.

[0341] The stressor (arsenite) reacts with the thiol group of lipoamide. Therefore, to rule out the possibility that lipoamide acts only by removing the stressor, it was further tested whether lipoic acid could prevent stress granule formation triggered by stresses other than arsenite (oxidative) stress: mitochondrial electron transport chain inhibition (rotenone), heat stress (42 °C), osmotic stress (sorbitol, a non-metabolizable sugar), glycolysis inhibition (6-deoxyglucose in the absence of glucose) or serum starvation. 10 μM racemic lipoamide or racemic, R-(+)- or S-(−)-lipoic acid all reduced stress granule formation in HeLa cells using mitochondrial, osmotic or arsenite stress( Figure 3 D). Thus, these compounds do not affect stress granules formed after all stresses but are not specific for arsenite stress.

[0342] Collectively, these data show that lipoamide and lipoic acid completely inhibit stress granule formation under multiple stresses. Their mode of action on different proteins and different stresses is consistent with a combined effect on the phase separation of important LCD stress granule proteins (such as TIA1) or a step in the stress granule formation signaling shared by specific stresses.

[0343] Lipoamide does not affect other cytoplasmic or nuclear liquid - like compartments

[0344] Stress granules are one of a variety of similar cytoplasmic RNA-containing liquid compartments. Notably, this also includes processing bodies (P-bodies). A variety of stress granule proteins also form nuclear bodies: paraspeckles and foci at DNA damage sites. There are also many other nuclear liquid compartments. A panel of cell lines expressing GFP fusions was then used to ask whether lipoamide also affects these other bodies. For this analysis, mitoxantrone was included as a representative of the selected extracts of the hetero-tricyclic / tetracyclic compound class ( Figure 1 F). An overly broad effect on liquid compartments may cause side effects of treatment.

[0345] Under conditions that dissolve stress granules, lipoamide does not affect the localization of proteins in RNA processing bodies (P-bodies, cytoplasm), PML bodies (nucleus), or DNA damage foci (nucleus) ( Figure 4 A). In contrast, mitoxantrone affects all of these compartments to some extent ( Figure 4 B). Thus, the action of lipoamide is specific to the signaling and / or physical chemistry that drives stress granule formation, stress granule phase separation, and / or specific signaling pathways. Compared to many other compartments, stress granules typically form and dissolve rapidly.

[0346] FUS is recruited to DNA damage sites, and the potential mechanism underlying ALS pathogenesis is due to reduced DNA damage response caused by sequestration of FUS in the cytoplasm of stress granules. The strong association of FUS nuclear localization (NLS) mutations (e.g., P525L) with familial ALS mutations supports this hypothesis. Therefore, the present inventors used arsenite-stressed iPSCs expressing FUS GFP to test whether lipoamide affects the recruitment of FUS to DNA damage sites induced by focused UV laser irradiation sites ( Figure 4 B). 20 μM lipoamide, which dissolves stress granules, had no significant effect on the recruitment of FUS GFP to DNA damage sites. 1 μM lipoamide, which does not affect stress granules, increased the recruitment of FUS GFP to DNA damage sites, which may be beneficial. In contrast, mitoxantrone blocked the recruitment of FUS GFP to DNA damage sites at a concentration insufficient to dissolve cytoplasmic FUS droplets. Thus, the action of lipoamide is specific to the cytoplasmic FUS liquid compartment, which is a therapeutically useful feature.

[0347] Lipoamide / lipoic acid directly interacts with FUS to reduce fiber formation and hardening

[0348] The present inventors selected lipoic acid and lipoamide for further characterization because lipoamide affects FUS droplet formation in vitro ( Figure 1D, E). It results in the formation of larger droplets, similar to the effect of surfactants in the library. This indicates faster Ostwald ripening / coarsening, likely driven by an increase in surface tension. Continuing to use FUS as a well-characterized phase-separated stress granule protein, we tested whether lipoic acid or lipoamide has other effects on phase separation in vitro.

[0349] The effect of lipoamide on the mobility (surface tension and viscosity) of FUS eGFP condensates was determined. Here, condensate droplets were brought together by optical tweezers, and the rates of fusion and relaxation into spheres were measured ( Figure 5 A). Lipoamide tripled the mobility ( Figure 5 B). In summary, the combined results of in vitro analysis indicate that the observed ( Figure 1 E) increase in droplet size and number is caused by the faster generation of larger droplets, due to increased mobility, followed by the sedimentation of the larger droplets (see Materials and Methods for more details). Since lipoamide / lipoic acid does not cause the in vitro dissolution of FUS droplets, we were able to examine the effects of lipoamide and lipoic acid on fibril formation and hardening. First, FUS condensates formed under (dextran-induced) crowded conditions were analyzed ( Figure 5 C-E).

[0350] It was determined whether lipoic acid or lipoamide affects the conditions under which FUS undergoes phase separation in vitro. The inventors only saw a minor effect of 100 μM lipoic acid or lipoamide, slightly increasing the minimum KCl concentration at which phase separation can occur ( Figure 6 A). This indicates that lipoic acid or lipoamide has little or no effect on the chemical potential of the system, which can alter the phase diagram and equilibrium state. Instead, these compounds affect coarsening, which is a change in the kinetics of reaching the equilibrium state.

[0351] Therefore, hardening and fibril formation, which are another kinetic phenomenon, were analyzed. FUS droplets in vitro "age" over time, first hardening and then tending to form amyloid / prion-like fibrils. This is accelerated for G156E FUS (a mutation associated with familial ALS). Both 30 μM lipoic acid and lipoamide delay the fibril formation of G156E FUS ( Figure 6 B). In addition, both compounds delay the hardening of FUS droplets, and the droplets retain a large FUS mobile fraction ( Figure 6 C). This occurs in the presence of 1 mM DTT (an approximately 1:30 excess of reducing agent), strongly indicating a non-antioxidant mechanism for lipoic acid / lipoamide.

[0352] Prevention of hardening and faster coarsening indicate direct interaction of FUS to increase "mobility" at concentrations achieved in cells. This may allow stress granules to dissolve more readily and may reduce aggregation of FUS (and other PLD-containing stress granules) in vivo. The inventors first tested iPSCs in vitro using a filter-binding assay to detect insoluble FUS aggregates. This showed that lipoamide reduced the amount of insoluble FUS, wild-type or P525L, in arsenite-stressed cells ( Figure 11 B, C).

[0353] In vitro data indicate that the mechanism of action of lipoamide requires a transient and weak interaction between lipoamide and FUS within the granule. Capturing this interaction is a challenging prospect for conventional tools in structural biology. Due to this mechanism relevant inside cells, lipoamide must enter the interior of cells in an unmodified form at a high enough concentration to be effective. Lipoamide and lipoic acid have a physical effect on FUS, but large amounts of the compound are required relative to the protein, e.g., 100 μM lipoamide versus 2.8 μM FUS ( Figure 6 A). Since the cytoplasmic FUS concentration is low μM, the compound must accumulate in cells to make this plausible: it must enter the cell, become enriched, and not be metabolized. The signaling mechanism does not require any of these mechanisms.

[0354] Finally, mechanistic details were analyzed at the submolecular level by using NMR of the FUS prion-like N-terminal LCD to determine the putative sites of lipoamide interaction. The LCD can phase-separate in vitro to form condensates, and individual residues can be resolved and partitioned in 1 H- 15 N heteronuclear single quantum coherence spectra. 1 H- 15 N analysis revealed changes in the environment of various residues, as indicated by their corresponding chemical shifts. The magnitude of the chemical shift changes was compared in the presence and absence of lipoamide or mitoxantrone. Although there is no single clear site of interaction of lipoamide with the FUS LCD, mitoxantrone caused weak shifts on the LCD, consistent with weak interaction with tyrosine residues. This indicates that lipoamide does not act through direct high-affinity protein binding. This leaves multiple possibilities: perhaps lipoamide only interacts with the FUS LCD when phase-separated, potentially reducing viscosity, or interacts with the condensate phase interface, possibly increasing surface tension. This interaction could reduce the formation or swelling of the aggregated form of FUS involved in condensate hardening and reduce fluidity.

[0355] Lipoamide becomes highly enriched in cells

[0356] The concentration of lipoamide required to affect condensate mobility in vitro is higher than the EC for cells, relative to the protein 50 , ~100 μM lipoamide with 1 μM FUS( Figure 1 D, E, Figure 5 ). In cells, the FUS concentration is high for the protein (low μM) 41 and the cellular EC 50 is ~10 μM( Figure 2 A, Figure 3 D). We also saw a reversal of lipoamide on FUS-containing condensates in vitro (more, larger condensates) and in cells (fewer stress granules). To reconcile this difference and understand the relevance of the effects outside the cell body, we wanted to know the actual concentration of the compound in cells. In principle, isotope labeling allows direct monitoring of the isotope-labeled compound by appropriate spectroscopy, even in complex environments. To use this method, 15 N-labeled lipoamide( Figure 7 A) was synthesized. Then solution-state NMR experiments were used to quantitatively detect the NH2 protons covalently attached to 15 N in the cell to determine the lipoamide concentration from the complex mixture within the cell, while also revealing any chemical modifications of the amide group (manifested as chemical shift changes or spectral alterations) (Figure 13).

[0357] After culturing lipoamide for 1 h at 37 °C in the absence or presence of HeLa cells, the uptake of 15 N-lipoamide by cells from the medium was quantified by NMR( Figure 7 B). For combinations of R-(+)- or (±)-lipoamide and unstressed or stressed cells, the cellular uptake was determined by calculating the difference between the medium with or without cell culture( Figure 7 C). For one sample, stressed cells with R-(+)-lipoamide, we confirmed that the strong signal from the cell fraction was consistent with the uptake of a large proportion of lipoamide( Figure 7 C). Both R-(+)- and ±-lipoamide measurements showed that 35 ± 11% of the lipoamide present in the medium was taken up (n = 3 and 2, respectively). No significant difference in R-(+) uptake was observed compared to (±)-lipoamide, nor was a significant difference observed between stressed and unstressed cells( Figure 7 D).

[0358] There was no evidence of metabolism or any other chemical modification of lipoamide: NMR signals from cell samples indicated that lipoamide was present in the cell in an unmodified form( Figure 7 B).

[0359] Approximate intracellular 15 N-lipoamide concentrations can be calculated from the uptake percentage, cell number, and their volume (Supplementary Methods) - these indicate an average intracellular concentration of 5.0 ± 1.6 mM ( Figure 7 D), significantly higher than the concentration obtained in vitro under optimal conditions in H2O with 1% v / v DMSO. Thus, lipoamide is readily taken up by HeLa cells (to concentrations comparable to those of abundant cellular metabolites). It is present in an unchemically modified form ( Figure 13G , H). The concentrations are an order of magnitude higher than those that affect FUSGFP condensates in vitro, suggesting that the physico-chemical effects in cells seem reasonable.

[0360] Lipoamide / lipoic acid prevents the aggregation of stress granule proteins in vivo

[0361] Since lipoamide and lipoic acid have a large effect on FUS aggregation in vitro, we used a filter-trap retention assay (where aggregated proteins from cell lysates tend to be retained on the membrane) to test whether lipoamide has a beneficial effect on the spontaneous aggregation of wild-type or P525L FUS GFP in iPSCs ( Figure 11 ). Both cell lines had some evidence of FUS aggregation, which decreased after treatment with lipoamide ( Figure 11 B).

[0362] Lipoic acid prevents persistent stress granules / aggregates in vivo

[0363] To look for evidence of the in vivo role of lipoic acid in stress granule formation and protein aggregation, the inventors turned to Caenorhabditis elegans. In C. elegans, aging or chronic stress is associated with the aggregation of stress granule proteins, including orthologs of TIAL1 and PABC1. This may be similar to the pathogenesis of ALS. Thus, it was tested whether lipoic acid prevents stress granule condensation. C. elegans grown in liquid cultures containing R-(+)- or S-(-)-lipoic acid both showed a dose-dependent decrease in the proportion of animals with aggregates of the stress granule protein PAB-1, which has an LCD / RBP structure, and the PABC1 ortholog. At the highest test concentration (2 mM), there was some toxicity resulting in 6 to 8% worm death. It was not possible to test lipoamide because it precipitates in the worm medium.

[0364] To investigate whether the effect of lipoic acid is specific to RBPs with an LCD, the aggregation of two globular proteins, KIN-19 and RHO-1, which have previously been shown to aggregate with aging in C. elegans, was tested. Neither protein has an RNA-binding domain or an LCD. No effect of 1.5 mM lipoic acid on RHO-1 aggregation was found. KIN-19 aggregation was slightly decreased, but 1.5 mM lipoic acid was more toxic.

[0365] Thus, lipoic acid can affect the condensation of stress granules over a longer time scale of the organism's lifespan, related to its behavior in the short term. The behavior of lipoic acid is consistent with directly interacting with stress granule proteins to reduce stress granule formation and / or stress granule protein aggregation.

[0366] Lipoic acid and lipoamide restore FUS - related defects in neurons and organisms

[0367] In humans, one cause of familial ALS is FUS mutation, especially in the C-terminal NLS. P525L is one such mutation, and iPSC-derived motor neurons (iPSC MN) expressing P525L from ALS patients show defects consistent with those of motor neurons in patients. iPSC MN can grow through silicone channels, positioning the cell body on one side and the axon protruding through the channel to the distal side. Such cultures can be maintained for a long time (>60 days). However, when expressing P525L FUS, the axons die within that time period without an exogenous cellular stressor, although P525L FUS MNs have a greater tendency to form stress granules. This is similar to axonal retraction, leading to motor dysfunction in patients. Thus, it was tested whether lipoamide or lipoic acid could improve these phenotypes. FUS P525L neurons initially showed normal morphology ( Figure 8 D, Figure 9 ), but by 60 days, axonal material that had died in the culture had accumulated around the exit of the neurons from the silicone channels ( Figure 8 D, Figure 9 ). Inclusion of 2 μM lipoic acid or lipoamide in the medium prevented this death. It is thought that defective axonal transport leads to axonal death. To test this, the inventors analyzed the transport of lysosomes within the axons of iPS MNs expressing P525L FUS with or without thioamide compared to iPS MNs expressing WT FUS ( Figure 12 ). Lipoamide restored axonal transport in P525L FUS iPS MNs to the same level as WT neurons. In these assays, the inventors did not use a treatment to induce stress granule formation in iPSC MNs, although in iPSCs, P525L FUS has a greater tendency to form them ( Figure 8 A). Since no stressor was required to induce axonal death, it is suggested that lipoic acid either helps iPSC MNs handle random stress in the culture or restores an intrinsic defect caused by P525L.

[0368] The fly model was further used to test whether lipoic acid has a similar beneficial effect on motor neurons in vivo. Drosophila melanogaster has a FUS ortholog cabeza, which is required for normal neuronal development. Cabeza has a shorter N-terminal PLD than FUS, and the expression of human FUS in Drosophila results in motor defects, including reduced climbing ability. The expression of P525L or R521C human FUS (both are NLS mutants) leads to even more severe motor defects. Food supplements with lipoic acid restore motor defects in a dose-dependent manner, restoring the ability to climb 4 cm from ~50% to >80% within 30 s. This significantly restores the climbing ability of the flies. Thus, lipoic acid can restore FUS-induced motor neuron defects.

[0369] Discussion

[0370] Stress granules are considered key sites in the pathogenesis of ALS. They are formed by protein liquid-liquid phase separation, driven by multivalent weak interactions. This is different from the strong enzyme-substrate or protein-protein interactions that are usually targeted by drugs. Therefore, it is unclear whether it is possible to identify drug-like compounds that interfere with phase separation, although it seems conceptually reasonable. The inventors searched for compounds that affect stress granule formation by directly interacting with LCD-containing stress granule proteins, focusing on FUS as a well-characterized model protein. This approach identified lipoamide and the related compound lipoic acid. These compounds have a long and complex history as bioactive molecules and potential therapeutic agents for various conditions, but without a clear mechanism of action. Here, it has been shown that lipoamide can directly alter the properties of phase-separated FUS droplets, reduce the formation of stress granules in cells, reduce the aggregation of FUS in vitro and in cells, reduce the aggregation of stress granule proteins in animals, and restore the phenotypes caused by FUS mutants associated with familial ALS in neurons and animals.

[0371] The basic research of the present invention focuses on lipoamide rather than lipoic acid as a more effective extract in vitro. Evidence suggests that the physicochemical mechanism of action seems reasonable. Lipoamide accumulates at high concentrations in cells and affects the fluidity of phase-separated droplets at these concentrations (using FUS as an in vitro model). In cells, lipoamide completely dissolves stress granules formed under certain stresses ( Figure 3 D), but not other membraneless liquid-like compartments - this includes compartments formed by the same protein in different regions of the cell (perinuclear speckles and around DNA damage sites) or compartments formed by other proteins (P bodies, Cajal bodies, and PML bodies)( Figure 4 ). Lipoamide also reduces the condensation of these phase-separated compartments in vitro ( Figure 6 B-D), in cells, and on the surface of whole animals.Figure 8 A). Lipoyl amide affects the behavior of FUS in vitro, but FUS is not essential for the formation of stress granules in cells. Therefore, lipoyl amide may also interact with FUS-like proteins (other proteins containing LCD / RBP) that are crucial for the formation of stress granules in cells. However, the exact nature of the interaction of FUS or other stress granule proteins containing LCD remains unclear.

[0372] In cells, lipoyl amide causes the dissolution of stress granules formed under various cellular stresses ( Figure 3 ), while in vitro, lipoyl amide does not cause the dissolution of FUS condensate droplets ( Figure 5 ), seemingly affecting the kinetics rather than the thermodynamics of FUS phase separation. One possible reason is that lipoyl amide accumulates to a significantly high concentration in cells ( Figure 7 ), where it may be reduced but not metabolized (Figure 13). The estimated cellular concentration (about 5 mM) is an order of magnitude higher than the concentration that can be achieved in vitro (300 μM), and much higher than the concentration required to increase the mobility and reduce the hardening of phase-separated FUS in vitro ( Figure 5 ). It is possible that 5 mM lipoyl amide could dissolve FUS condensate droplets in vitro. There are also other possible explanations. First, the higher mobility of stress granules may make them more sensitive to cellular stress granule dissolution factors. Second, cells have a stress granule, cytoplasmic, and nuclear environment, making it a more complex three-phase system (possibly related to the effect of lipoyl amide on the nuclear / cytoplasmic partitioning of FUS, Figure 2 ). Finally, lipoyl amide has a strong effect on the condensation of FUS ( Figure 5 ), and it is thought that this is partly driven by LCD-LCD interactions that are not required for in vitro phase separation. If LCD-LCD interactions are more important for intracellular phase separation, then this could manifest as an increased sensitivity of stress granules to lipoyl amide relative to in vitro FUS.

[0373] The cellular targets of lipoyl amide or lipoic acid cannot be determined unambiguously. Although these compounds alter the properties of FUS condensates in vitro, FUS is not essential for the formation of stress granules. Therefore, FUS cannot be the only cellular target. Many stress granule proteins are FUS-like proteins with an LCD domain and are affected by treatment in cells, including G3BP1, which is thought to nucleate stress granules ( Figure 3 ).

[0374] Lipoamide and lipoic acid may affect the common properties of multiple FUS-like stress granule proteins or key vital stress granule proteins. If the physicochemical mechanism is correct, then theoretically, lipoamide regulates the transient and weak interactions between FUS molecules to regulate condensate properties rather than having a single binding site. Although there is an obvious effect on the physical properties of FUS condensates in vitro, it may not be surprising that the interaction cannot be detected by NMR ( Figure 5 , Figure 6 ). It should be noted that this assay only involves FUS LCD-lipoamide in a non-phase-separated phase and may interact with other regions of FUS (such as the RNA-binding domain not present in this assay).

[0375] The precise mechanism of ALS pathogenesis remains unclear. However, based on the hypothesis that stress granules are key to ALS pathogenesis, lipoamide and lipoic acid seem to be potential therapeutic agents. The usefulness of lipoamide / lipoic acid depends on whether the formation of stress granules promotes ALS pathogenesis or is part of an important cellular response to stress. Similarly, whether stress granule protein aggregation is a means of sequestering harmful proteins or whether it is inherently harmful. Therefore, the inventors characterized the potential of lipoamide and lipoic acid to treat ALS-like diseases, mainly lipoic acid because of its known pharmacokinetics and toxicology in humans, although it is slightly less potent in vitro. Lipoic acid has been shown to reduce the aggregation of stress granule proteins in Caenorhabditis elegans, which is a marker of aging in this organism and a phenomenon that shares general similarities with the aggregation of LCD-containing proteins in ALS pathology ( Figure 8 A). The inventors also tested two models of ALS caused by FUS mutations and saw that lipoic acid rescued axonal stability defects in vitro neurons and defects in motor control caused by the expression of FUS mutants associated with ALS pathogenesis in Drosophila melanogaster ( Figure 8 C). Lipoic acid has previously shown some efficacy in the SOD1 animal model of ALS, and our work shows that it exerts efficacy in a stress granule protein-driven ALS pathogenesis model through a novel mechanism of action. In humans, a daily dose of 600 mg of lipoic acid produces plasma concentrations of 8 to 30 μM, comparable to the concentrations used in our cell-based assays, meaning that lipoic acid has surprisingly reasonable potential as a therapeutic agent.

[0376] Lipoic acid is a naturally occurring metabolite, and lipoamide is closely related. Despite being related to a naturally occurring metabolite, there is no evidence of lipoamide metabolism. Both are regarded as antioxidants. Lipoamide is indeed simply considered an antioxidant in prion-related diseases, but its activity is not dependent on the redox state of lipoic acid in cells, and lipoic acid and lipoamide are active in vitro in the presence of excess reducing agents. This suggests a non-enzymatic / non-metabolic and non-redox effect, making alterations in phase separation a plausible mechanism; especially considering the high concentration of lipoamide that accumulates in cells. For example, this is different from preventing stress granule formation by inhibiting eIF2α phosphorylation with the small molecule ISRIB. Although the detailed mode of action is not clear, it may be a different mode of action from "conventional" compounds that target strong, specific enzyme-substrate or protein-protein interactions.

[0377] Generally speaking, the success of the screening used shows that physicochemical compounds targeting proteins can be discovered and that this overall screening method is feasible. This is important in diseases including ALS and other diseases; many different LCD-containing proteins are involved, and many different mutations are associated with familial ALS. It is not feasible to target each individual target separately. Instead, compounds can be found that keep protein species more liquid or more soluble in the cytoplasm. This points to a new class of drugs - those that affect the physicochemistry of membraneless compartments, or physicochemical drugs.

[0378] Stress granules form by the phase separation of proteins within the cytoplasm and some small molecules that regulate phase separation (notably 1,6-hexanediol) have been previously identified. However, lipoic acid and lipoamide seem far more reasonable as therapeutic agents. Concentrations of 1,6-hexanediol between 1 and 10% (hundreds of mM) are required for in vitro or cellular activity, and this is rapidly toxic. The molecules we identified in our screen (heterocyclic tricyclic compounds, tetracyclic compounds, and lipoamide and related compounds) are active in vitro ( Figure 1 , 5) and on cells ( Figure 1 , 2, 10) at significantly reduced concentrations (tens to hundreds of μM), three to four orders of magnitude lower than the concentration of 1,6-hexanediol. The effect of lipoamide on stress granules is also specific in the sense that lipoamide does not affect other membraneless liquid-like compartments in cells. It does not affect the nuclear compartments formed by FUS ( Figure 4 B) or the nuclear compartments formed by other proteins in the cytoplasm or nucleus ( Figure 4 A), and is well tolerated by HeLa, iPS, and motor neuron cells in culture.

[0379] In addition, the anticancer drug mitoxantrone can effectively disrupt many membrane-less compartments. Mitoxantrone induces DNA damage, which is attributed to its role as a topoisomerase inhibitor through DNA binding. However, it seems to disrupt rather than increase the number of membrane-less DNA damage foci - a physicochemical mechanism may also be at play here.

[0380] Lipoic acid, a natural metabolite, is of interest. Recent data have identified another metabolite, ATP, as a hydrotrope, which has the property of keeping proteins soluble. Solutions to prevent abnormal protein aggregation in disease compartments may support the cell's ability to maintain a soluble environment. Future screens may identify more such molecules.

[0381] Methods

[0382] A stable KyotoHeLa BAC cell line expressing a protein with a C-terminal GFP fluorescent marker was generated using BAC recombineering. This gives near-endogenous expression levels of the fusion protein. In these cell lines, GFP is part of a modified localization and affinity purification (LAP) tag, providing a short linker. HeLa cells were grown in high-glucose DMEM supplemented with 10% FCS. The cultures were supplemented with 1% penicillin-streptomycin and maintained at 37 °C under 5% CO2 in geneticin (Gibco, 400 μg / ml) selection.

[0383] Human iPS cell lines derived from three different donors were used, which express FUS with a C-terminal GFP fluorescent marker. All were generated using CRISPR / Cas9-assisted tagging and mutagenesis and have been previously described. In summary: JS-SL-C1 iPS cells expressing wild-type or P525L FUS GFP were generated ex vivo from healthy female donors. The JS-SL-C1 iPS cells were used for compound dose-response analysis. KOLF iPS cell lines expressing wild-type FUS GFP or P525L FUS GFP were generated ex vivo from the KOLF-C1 clonal iPS cell line, which was generated as part of the Human Induced Pluripotent Stem Cell Initiative (HipSci). The KOLF-C1 cells were from a healthy male donor. In these cell lines, GFP is part of a modified localization and affinity purification (LAP) tag, providing a short linker and giving the same fusion protein sequence as the Koyoto HeLa BAC cell line. The JS-SL-C1 and KOLF-C1 iPS cell lines were used as isogenic pairs to analyze the DNA damage response and the effect of lipoic acid on P525L FUS. AH-ALS1-F58 iPS cells expressing P525L FUS with a C-terminal GFP fluorescent marker were generated ex vivo from a clonal iPS cell line from a female ALS patient expressing P521C FUS. The P525L mutation and GFP tag were introduced, and the P521C mutation was corrected by simultaneous tagging and mutagenesis. The iPS cells were grown in TeSR E8 medium (StemCell Technologies) at 37 °C and 5% CO2.

[0384] As previously described, MNs were generated from AH-ALS1-F58 iPS cells expressing P525L FUS in Matrigel-coated plates with silicone channels for axons by induced differentiation. This produced a cluster of cell bodies on one side of the channel, with axons extending through the channel and protruding from the distal side of the channel. AH-ALS1-F58 was used to generate motor neurons (MNs) because they had previously been characterized in axonal transport assays. Unless otherwise stated, iPS MNs were used in assays within 4 weeks of completing differentiation.

[0385] All procedures using human cell samples were in accordance with the Helsinki Convention and approved by the Ethics Committee of Technische Dresden (EK45022009, EK393122012).

[0386] Recombinant proteins

[0387] For in vitro droplet formation screening and coagulation assays, exactly as previously described 7, the recombinant GFP FUS and GFP G156E FUS were purified using a baculovirus / insect cell expression system. Briefly, His MBP FUS GFP was purified from cell lysates by Ni-NTA affinity purification, His MBP was cleaved, then concentrated by dialysis and further purified by size exclusion chromatography. The storage buffer for the purified FUS consisted of 1 M KCl, 50 mM Tris·HCl pH 7.4, 5% glycerol, and 1 mM DTT. Before use, the concentration of FUS in the storage buffer was adjusted to 30 μM.

[0388] Compounds

[0389] For in vitro and ex vivo screening, the PHARMAKON 1600 library was used, prepared as a 10 mM stock solution in DMSO. For subsequent analysis, compounds were purchased again and prepared as 10 mM stock solutions in DMSO; lipoamide (T5875, Sigma Aldrich or sc-239160, Santa Cruz Biotechnology), lipoic acid (62320, Sigma Aldrich), R-(+)-lipoic acid (07039, Sigma Aldrich), S-(-)-lipoic acid (08561, Sigma Aldrich), dihydrolipoic acid (T8260, Sigma Aldrich), valeric acid (240370, Sigma Aldrich), 1,3-propanedithiol (P50609, Sigma Aldrich), mitoxantrone (M6545, Sigma Aldrich), N-(2-hydroxyethyl)ethylenediamine (127582, Sigma Aldrich), 1,4-dihydroxyanthraquinone (Q906, Sigma Aldrich), cetylpyridinium chloride (C9002, Sigma Aldrich), quinacrine (Q3251, Sigma Aldrich), 9-aminoacridine (A38401, Sigma Aldrich), 2-amino-5-diethylaminopentane (A48806, Aldrich), daunorubicin (30450, Sigma Aldrich), 8-acetyl-6,11-dihydroxy-7,8,9,10-tetrahydronaphtho[2,1-a]anthracene-5,12-dione (R162892, Sigma Aldrich). 15 N racemic and R-(+)-lipoamide (see below) were synthesized and characterized by 1 1H NMR, 13 13C NMR, mass spectrometry, infrared spectroscopy, and melting point. The yield was ~50%, 15 and the 15N labeling was ~99%.

[0390] In vitro HeLa cell screening

[0391] Screen for the effect of compounds on FUS GFP localization in stressed HeLa cells in 384-well format. Seed 4000 cells per well and incubate for 24 hours, then replace the medium with 40 μl of fresh medium and add compounds to a final concentration of 10 μM by acoustic dispensing (Labcyte Echo 550). The final concentration of DMSO in all samples is 0.1%. After 1 hour, add potassium arsenite from a 5× stock solution to a final concentration of 1 mM and incubate the cells for an additional 1 hour, then fix the cells with 7.4% formaldehyde and stain with 1 mg / ml Hoechst 33342 and 1:10,000 CellMask Blue (ThermoFisher). Use a CellVoyager CV7000 automated spinning disk confocal microscope (Yokogawa) to capture six fields of view per well using a 40× NA1.3 water immersion objective. Each plate includes 48 wells treated with 0.1% DMSO and stressed with arsenite (compound vehicle control), and 8 untreated unstressed wells and 4 unstressed untreated wells with parental Koyoto HeLa cells. All images are displayed with γ 0.7 to simultaneously show bright stress granules and blurred nuclei.

[0392] For the initial screen, analyze FUS GFP signals using KNIME. Identify the cytoplasm from weak (CellMask Blue) blue fluorescence signals and the nucleus from strong (Hoechst 33342) blue fluorescence signals. Measure the number and total area of granules in the nucleus and cytoplasm in the green (FUS GFP) channel, the granularity at 9, 10 & 11 px (cytoplasm) or 1, 5, 6, 7, 8 & 9 px (nucleus) scales, the texture at 10 px scale, and the integrated signal intensity. For each parameter, calculate the Z-score (z = (x - μ) / σ, where x is the observed value, μ is the control mean and σ is the control standard deviation) relative to the DMSO-treated control wells on each plate and combine them into a Mahalanobis distance.

[0393] In vitro purified FUS GFP screening

[0394] The effects of compounds on FUS GFP droplets in vitro were evaluated in 384-well plate format. By acoustic dispensing (Labcyte Echo 550), volumes of compounds (in DMSO) required for final concentrations of 1, 3, 10, 30 or 100 μM were added to wells of a 96-well plate containing 3 μl of FUS GFP diluted in 50 mM Tris·HCl pH 7.4, 1 mM DTT (for low-salt assays). The final DMSO concentration was 0.01 to 1%. Using a Freedom Evo 200 liquid handling workstation (TECAN), the FUS GFP / compound mixture was diluted in 7 μl of assay buffer containing 50 mM Tris·HCl pH 7.4, 1 mM DTT, 50 mM KCl and 0.7 μM FUS GFP. The compound / FUS GFP and assay buffer were mixed by a standardized pipetting procedure and then dispensed into four wells in a clear-bottom 384-well plate and immediately imaged using a CellVoyager CV7000 automated spinning disk confocal microscope (as above). Droplets in the suspension in six fields of view were imaged as maximum intensity projections of six focal planes at 2-μm steps for each sample. Droplet number and FUS GFP partitioned into droplets were analyzed using ImageJ with a fixed intensity threshold. Since droplet sedimentation made droplet number and partitioning weakly time-dependent, normalization was performed assuming linear change over time by reference to DMSO controls at the start and end of each plate row.

[0395] Compound characterization on HeLa and iPS cells

[0396] The effects of compounds were evaluated in live cell imaging in HeLa or iPS cells expressing wild-type or P525L FUS GFP under various conditions. Different combinations of 1-hour pre-treatment with compounds or treatment with compounds 1 hour after pre-stressing with arsenite and / or stressing with arsenite were employed. Unless otherwise stated, cells were pre-treated with 10 μM compound (from a 10 mM stock in DMSO) (or an equal volume of DMSO control) for 1 hour and then stressed with 1 mM potassium arsenite for 1 hour in the presence of the compound. GFP fluorescence was imaged by wide-field epifluorescence using an inverted Olympus IX71 microscope, an Olympus 100× NA 1.4 Plan Apo oil immersion objective and a CoolSNAP HQ CCD camera (Photometrics), using a DeltaVision climate control unit (37 °C, 5% CO2) (Applied Precision). Unless otherwise stated, for single time point images captured after 1 hour, the kinetics of post-pre-stress treatment were analyzed from images captured at 2-minute intervals for 100 minutes.

[0397] By replacing the 1 h 1 mM potassium arsenate treatment with other conditions, various cellular stresses were obtained: 100 μM rotenone (R8875, Sigma Aldrich), from a 1 M stock solution in DMSO, for 2 h (mitochondrial stress). Serum-free DMEM, for 2.5 h (serum starvation stress). Sorbitol (S1876, Sigma Aldrich), from a 4 M stock solution in H2O, for 1 h (osmotic stress). At 42 °C in normal growth medium, for 30 min (heat stress). 100 mM 6-deoxyglucose (D9761, Sigma Aldrich), from a 1 M stock solution in H2O, in glucose-free DMEM (11966025, ThermoFisher Scientific) supplemented with 10% FCS, for 1 h (glycolytic stress). Appropriate solvent controls were used.

[0398] For western blotting and analysis of intracellular FUS aggregates, iPS cells were lysed with RIPA buffer. Western blotting was performed using standard methods and the following antibodies: mouse anti-FUS (AMAB90549 Sigma Aldrich, 1:500 dilution), rabbit anti-GFP (sc-8334 Santa Cruz, 1:400), or rabbit anti-GAPDH (2118S NEB, 1:5000) primary antibodies with horseradish peroxidase-conjugated anti-mouse or anti-rabbit (Dianova 1:10,000) secondary antibodies. As previously described 56 A filtration retardation assay for intracellular FUS aggregates was performed. Briefly, protein extracts were loaded onto 0.2 μm cellulose acetate membranes, followed by microfiltration to retain the aggregated proteins on the membrane. Aggregated FUS was detected as described above.

[0399] Compound dose - response on HeLa cells

[0400] In addition to manually preparing serial compound dilutions in media from 80 μM to ~0.4 nM in 1.189× dilution steps, as in the in vitro HeLa cell screen, a 1 h 1 mM potassium arsenite stress was used to evaluate the dose-dependent effects of compounds on HeLa or iPS cells expressing FUS GFP. Small dilution steps were chosen instead of concentration replicates because they provide greater statistical power from a set number of samples. The final DMSO concentration in all samples was 0.08%, and each plate included at least 12 control wells containing 0.08% DMSO. The number of cytoplasmic FUS droplets and the nuclear / cytoplasmic partitioning of FUS were analyzed using a custom macro in ImageJ. Nuclei were identified by intensity thresholding of blue fluorescence images after a 5 px Gaussian blur. Cytoplasmic FUS droplets were obtained by intensity thresholding of the green image after a 10 px weight 0.9 unsharp mask filtered by the thresholded nuclei mask, and nuclear FUS droplets were obtained by intensity thresholding of the green image after a 5 px weight 0.9 unsharp mask and 10 px rolling ball background subtraction masked to include only the thresholded nuclei. The ratio of cytoplasmic FUS droplets to nuclei was taken as the cytoplasmic FUS droplets per cell per field of view, and the ratio p of FUS partitioning to the nucleus and cytoplasm was derived from a = v n / v t , the ratio of nuclei to total green signal per field of view, where p = a / (1 - a). These data were log-transformed and fit to a Rodbard sigmoidal curve to determine the EC 50 . Six fields of view were captured and analyzed for each condition.

[0401] In vitro FUS GFP solidification assay

[0402] For in vitro coagulation assays, FUS GFP in storage buffer was diluted in water to give 10 μM FUS, 50 mM Tris·HCl pH 7.4 and 1 mM DTT in a 20 μl volume in a non-binding clear-bottom 384-well plate (Greiner Bio-One, 781906). Then compounds or an equal volume of DMSO were added to give a final compound concentration of 30 μM and 0.3% DMSO. 'Aging' was induced by shaking at 800 rpm for 1 h at room temperature to cause fibrils. Fibre and droplet formation were analysed by wide-field epifluorescence using a DeltaVision Elite microscope (GE Healthcare Life Sciences), with a Plan ApoN 60× NA 1.4 oil immersion objective (Olympus) and an sCMOS camera (PCO). For the salt sensitivity of droplet formation, FUS GFP in storage buffer was diluted in KCl at the appropriate concentration pre-mixed with compounds or DMSO to give a final series of KCl concentrations with a compound concentration of 30 μM and 0.3% DMSO.

[0403] Fluorescence recovery after photobleaching (FRAP) of FUS GFP droplets and fibrils was performed on a Nikon TiE inverted microscope with a Nikon Apo 100× NA 1.49 oil immersion objective, using a Yokogawa CSU-X1 spinning disk head and an Andor iXon EM+ DU-897EMCCD camera. Using an Andor FRAPPA beam delivery unit, a 10×10 px area was bleached for 50 ns with a 6 mW 405 nm laser and then imaged at 5 Hz for 5 min. Recovery curve half-life and mobile fraction were calculated in ImageJ.

[0404] In vitro DNA cleavage assay

[0405] UV microirradiation of live cells was performed to induce DNA damage. KOLF iPS MN expressing wild-type GFP were stressed for 1 h by adding 1 mM potassium arsenite and then treated with lipoic acid, mitoxantrone or an equal volume of DMSO for 1 h. As described for FRAP, a single point in the nucleus was subjected to 3 UV pulses, but the laser power was 10%. GFP fluorescence was imaged at 1 Hz and the response intensity was analysed in ImageJ.

[0406] NMR

[0407] To analyse the interaction of compounds with FUS, the unlabelled FUS low complexity domain (residues 1 to 163) was expressed, purified and used 1 H- 1515N heteronuclear single quantum coherence NMR and the sample conditions described previously 14 Analysis was performed in the presence of 500 μM compound or equivalent DMSO solvent control (1%).

[0408] To analyze cellular uptake of 15N lipoamide, HeLa cells expressing FUS GFP were grown to 10 6 cells / well in 6-well plates in DMEM supplemented with 10% FCS. To simultaneously stress and treat the cells, the medium was replaced with 0.6 ml of medium supplemented with potassium arsenate and 100 μM 15 15N racemic or R-(+)-lipoamide for 1 h at 37 °C. A high concentration of the compound was used to maximize the signal. The medium was then removed and retained (medium sample), the cells were washed with ~2 ml PBS, and then the cells were removed by trypsinization: 0.3 ml of TrypLE Express (12604013, ThermoFisher) was added and incubated at 37 °C for 5 min, and then 0.3 ml of medium was added to quench the trypsin. The resuspended cells were retained (cell sample). All samples were frozen at -80 °C. Wells were prepared for all combinations of no compound (1% DMSO control), 15 15N(±)-lipoamide or 15 15NR-(+)-lipoamide with or without potassium arsenate and with or without cells. The 1 1H-detected 15 15N-edited 1 1H sensitivity enhanced HSQ was used to quantify 15 15N lipoamide concentration (see Supplementary Information). Solvent, pH, and temperature sensitivity of the primary amide proton chemical shift were determined using mock samples assembled from the appropriate solvent and added compound.

[0409] Neuronal death and axonal transport assay

[0410] To analyze axonal dystrophy transport, AH-ALS1-F58 iPS MNs expressing P525L FUS or isogenic controls expressing WT FUS were grown for 60 days with 2 μM compound or an equal volume of DMSO. During the length of the culture, neurons expressing WT FUS had stable axons, while neurons expressing P525L FUS did not. Axonal dystrophy was visible as cell debris accumulating at the channel exits of the axons. The experiments and analyses were performed blinded, and phase contrast images captured every 10 to 20 days were used to qualitatively score axonal dystrophy.

[0411] To analyze axonal transport, iPS MNs expressing P525L FUS were treated with 2 μM compound or an equal volume of DMSO for 3 days. A longer incubation time was chosen to ensure penetration and action of the compound along the length of the axonal channel. 2 μM was chosen as the highest concentration without toxic effects (qualitative assessment). Analysis of axonal transport of liposomes was performed as follows: Liposomes were labeled by adding 50 nM lysosomal red probe Red (ThermoFisher), and imaged for 120 s at the proximal or distal end of the silicone channel containing axons in an incubator (37 °C, 5% CO2) using a Leica DMI6000 inverted microscope with a 100× NA 1.46 oil immersion objective and an AndoriXON 897EMCCD camera at 3 1 / 3 Hz. For five video micrographs, particle tracking was used to determine the proportion of particles with a migration speed greater than 0.2 μm / s. Each video included a variable population of non-moving background particles; thus, for each biological replicate, the data were normalized to the mean proportion of fast-moving lysosomes in DMSO (solvent control)-treated samples.

[0412] Protein aggregation in Caenorhabditis elegans

[0413] The effect of lipoic acid on the in vivo aggregation of stress granule proteins was analyzed using a Caenorhabditis elegans model for stress granule formation and aggregation. As previously described, fluorescently labeled PAB-1 forms a large number of stress granules and large solid aggregates during aging or chronic stress. RHO-1 and KIN-19 also aggregate during aging, but are not RNA-binding or stress granule proteins. Three cell lines were used: fluorescently labeled PAB-1 (DCD214:N2; uqIs24[pmyo-2::tagrfp::pab1 gene]), KIN-19 (CF3649:N2; muIs209[pmyo-3::kin-19::tagrfp+ptph-1::GFP] and RHO-1 (DCD13:N2; uqIs9[pmyo-2::rho-1::tagrfp+ptph-1::gfp]). Each was analyzed as follows, except that DCD13 was maintained at 20 °C.

[0414] Starting from the L4 larval stage, animals were exposed to lipoic acid in liquid culture in 96-well plates, with a total volume of 50 μL per well of S-complete (100 mM NaCl, 50 mM potassium phosphate pH 6, 10 mM potassium citrate, 3 mM MgSO4, 3 mM CaCl2, 5 μg / mL cholesterol, 50 μM ethylenediaminetetraacetic acid (EDTA), 25 μM FeSO4, 10 μM MnCl2, 10 μM ZnSO4, 1 μM CuSO4), supplemented with heat-inactivated OP50 and 50 μg / mL carbenicillin. For each experiment, at least nine wells were treated with R-(+)- or S-(-)-lipoic acid or an equal volume of DMSO, with 13 animals per well. Toxicity was evaluated from the number of dead or abnormal small animals.

[0415] Forty-eight hours after transferring L4s from 20 °C to 25 °C (day 2 of adulthood), extensive aggregations of fluorescently labeled PAB-1 and RHO-1 appeared in the pharyngeal muscles and KIN-19 of the whole animals. After fixation with 2 mM levamisole, scoring was performed using a fluorescence stereomicroscope (Leica M165 FC, Plan Apo 2.0× objective). For PAB-1, aggregates mainly appeared in the terminal bulb of the pharynx, and the aggregation score was high (>10 aggregates / animal) or low (<10). For RHO-1, aggregates were scored in the isthmus of the pharynx, and the aggregation score was high (>50% of the isthmus), medium (<50%), or low (no aggregation). For KIN-19, aggregates appeared in the whole body wall muscles, and the aggregation score was high (aggregates in the head, midbody, and tail), medium (>15 aggregates in the head and midbody), or low (>15 aggregates in the head or midbody). High-magnification images were obtained using a Leica SP8 confocal microscope, using an HC Plan Apo CS2 63× NA 1.40 oil objective and a Leica HyD hybrid detector. tagRFP::PAB-1 was detected using 555 nm as the excitation light and an emission range of 565 - 650 nm. Representative confocal images are shown as maximum z-stack projections.

[0416] Motor defects in Drosophila melanogaster

[0417] All Drosophila stocks were maintained on standard cornmeal at 25 °C in a light / dark-controlled incubator. w1118, UAS-eGFP, and D42-GAL4 were obtained from the Blooming Stock Center. UAS-FUS WT, UAS-FUS P525L, and UAS-FUS R521C were previously described in Anderson et al. (Hum. Mol. Genet. 27, 1366 - 1381, 2018).

[0418] The climbing assay was performed as described above. Briefly, flies expressing FUS, eGFP or w1118 were grown in the presence or absence of (±)-α-lipoic acid (0.43 mM or 2.15 mM diluted in ethanol), (R)-(+)-α-lipoic acid (0.43 mM or 2.15 mM diluted in ethanol), (S)-(-)-α-lipoic acid (0.43 mM or 2.15 mM diluted in ethanol), (±)-α-lipoic amide (0.43 mM diluted in DMSO) or PP 242 / Torkinib (10 μM or 50 μM diluted in DMSO), then anesthetized, placed in vials and allowed to acclimate in new vials for 15 minutes. For each fly genotype, the vial was tapped three times on the bench on the substrate and the flies climbing up the vial wall were recorded using a camera. The percentage of flies climbing 4 cm in 30 seconds was recorded. Statistical analysis was performed using Student's T-test or one-way ANOVA with multiple comparison tests of Tukey or Dunnet in GraphPad Prism 6.

[0419] Racemic (±) and 15 N Synthesis and characterization of R-(+)-lipoamide

[0420] 15 N(±)-lipoamide

[0421]

[0422] (±)-Lipoic acid (1.08 g, 5.24 mmol), N-hydroxysuccinimide (660 mg, 5.60 mmol) and (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.1 g, 5.76 mmol) were stirred in DMF (20 ml) under an argon atmosphere at 25 °C for 4 h. The solution was diluted with EtOAc (100 ml) and washed with H2O (100 ml) and saturated aqueous NaHCO3 solution (100 ml). The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. The resulting NHS ester, trimethylamine (1.1 ml, 7.89 mmol) and 15 NH4Cl (500 mg, 9.36 mmol) were dissolved in DCM (20 ml) and the mixture was stirred for 20 h. The solution was diluted with DCM (100 ml) and washed with H2O (100 ml), saturated aqueous NaHCO3 solution (100 ml) and again with H2O (100 ml, 2 times). The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure to give the crude 15 N-lipoic amide, which was further purified by silica gel column chromatography (DCM:MeOH = 30:1). The solvent was removed under reduced pressure and yellow solid 15 N-lipoic amide was obtained. Yield: 4.2 mg (51%). 11H NMR (400 MHz, chloroform-d) δ 5.62 (d, J = 31.3 Hz, 1H, NH 顺 ), 5.37 (d, J = 31.0 Hz, 1H, NH 反 ), 3.60 (ddt, 1H, SSCH), 3.26–3.06 (m, 2H, SSCH2), 2.58–2.40 (m, 1H, SSCH2CH 反 ), 2.26 (t, J = 7.5 Hz, 2H, CH2CONH2), 2.02–1.84 (m, 1H, SSCH2CH 顺 ), 1.83–1.60 (m, 4H), 1.60–1.39 (m, 2H, CH2CH2CH2CONH2). 13 13C NMR (101 MHz, chloroform-d) δ 175.02 (d, J = 13.6 Hz, CONH2), 56.39, 40.26, 38.49, 35.61, 35.53, 28.84, 25.14. ESI-MS: m / z = 229.05 (M+Na)+. Electrospray (M+Na)+ ion detection. Data indicate that 15 the N is labeled at ~99%. IR: 3352 cm -1 , 3176 cm -1 (CONH2), 2937 cm -1 , 2898 cm -1 , 2865 cm -1 , 2783 cm -1 (C-H), 1746 cm -1 , 1650 cm -1 , 1629 cm -1 , 1464 cm -1 , 1413 cm -1 , 1367 cm -1 , 1342 cm -1 , 1321 cm -1 , 1292 cm -1 , 1281 cm -1 , 1252 cm -1 , 1226 cm -1 , 1203 cm -1 , 1144 cm -1 , 1125 cm -1 , 1078 cm -1 , 1034 cm -1 , 999 cm -1 , 950 cm -1 , 911 cm -1 , 868 cm -1 , 803 cm-1 , 734 cm -1 , 675 cm -1 , 629 cm -1 (C=O). Melting point: 130 °C, Rf = 0.60 (DCM:MeOH = 20:1).

[0423] 15 N R-(+)-lipoamide

[0424]

[0425] At 25 °C under an argon atmosphere, R-(+)-lipoic acid (1.08 g, 5.24 mmol), N-hydroxysuccinimide (660 mg, 5.60 mmol), and (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.1 g, 5.76 mmol) were stirred in DMF (20 ml) for 4 hours. The resulting solution was diluted with EtOAc (100 ml) and washed with H2O (100 ml) and saturated aqueous NaHCO3 solution (100 ml). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The obtained NHS ester, trimethylamine (1.1 ml, 7.89 mmol), and 15 NH4Cl (500 mg, 9.36 mmol) were dissolved in DCM (20 ml), and the mixture was stirred for 20 hours. The solution was diluted with DCM (100 ml) and washed with H2O (100 ml), saturated aqueous NaHCO3 solution (100 ml), and then again with H2O (100 ml, 2 times). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure to obtain crude 15 N-lipoamide, which was further purified by silica gel column chromatography (DCM:MeOH = 30:1). The solvent was removed under reduced pressure to obtain yellow solid 15 N(R)-lipoamide. Yield: 554.5 mg (51%). 1 H NMR (400 MHz, chloroform-d) δ 5.56 (d, J = 4.1 Hz, 1H, NH 顺 ), 5.34 (d, J = 4.7 Hz, 1H, NH 反 ), 3.60 (ddt, 1H, SSCH), 3.30–3.03 (m, 2H, SSCH2), 2.61–2.35 (m, 1H, SSCH2CH 反 ), 2.32–2.18 (m, 2H, CH2CONH2), 1.94 (m, 1H, SSCH2CH 顺 ), 1.81–1.59 (m, 4H), 1.59–1.41 (m, 2H, CH2CH2CH2CONH2). 1313C NMR (101 MHz, chloroform-d) δ 174.90 (d), 56.40, 40.26, 38.50, 35.60, 35.52, 34.64, 28.85, 25.14. ESI-MS: m / z = 207.06 (M+H+). Electrospray (M+H+) ion detection. The data indicate that 15 N is labeled at ~99%. IR: 3340 cm -1 , 3174 cm -1 (CONH2), 2936 cm -1 , 2921 cm -1 , 2865 cm -1 , 2850 cm -1 (C-H), 2788 cm -1 , 2550 cm -1 , 2360 cm -1 , 2341 cm -1 , 2161 cm -1 , 2036 cm -1 , 1751 cm -1 , 1650 cm -1 , 1627 cm -1 (C=O), 1461 cm -1 , 1411 cm -1 , 1369 cm -1 , 1343 cm -1 , 1317 cm -1 , 1294 cm -1 , 1260 cm -1 , 1213 cm -1 , 1197 cm -1 , 1135 cm -1 , 1036 cm -1 , 1004 cm -1 , 914 cm -1 , 878 cm -1 , 808 cm -1 , 794 cm -1 . Melting point: 121.5 °C, Rf = 0.60 (DCM:MeOH = 20:1), [α] 25 +105.6 (c = 2.0, CHCl3).

[0426] Density functional theory (DFT) calculations were performed to confirm the assignment of the lipoamide 1 1H NMR spectrum. The optimized structure of lipoamide was generated using Gaussian098, and its shielding tensor was calculated to enable the determination of the isotropic and anisotropic components. The DFT calculations used the 6-31G(d) basis set 72The B3LYP density functional. DFT calculations confirm that the cis amide proton 13 should have a greater chemical shift than the trans amide proton 14 ( Figure 13A ), and that the proton bonded to carbon 3 closest to the proton bonded to carbon 2 should have a greater chemical shift than the other protons on carbon 3 ( Figure 13A ).

[0427] 15 N-lipoamide's ( 15 N) 1 H NMR

[0428] Obtained on a narrow-bore Varian solution-state spectrometer 1 H-detected 15 N-edited 1 H sensitivity-enhanced HSQCNMR (hereafter referred to as ( 15 N) 1 H) spectra, the spectrometer operating at a fixed field strength of 14.1 T and equipped with a room-temperature probe.

[0429] The free induction decay was recorded with an acquisition time of 0.0624, and 8 kHz scans and a 1 s recovery delay were recorded over 1000 s. Typically, 10,000 transients were collected, with a total experimental time of 3 hours 1 minute. The J-coupling between the amide proton and 15 N in the H2O sample was determined to be 96 Hz, so the transfer time of 1 / 4J in the INEPT part of the pulse sequence was set to 2.6 ms. Under these settings, 15 N ammonia or ammonium ions would be undetectable. 15 Chemical modification of 15 N lipoamide, including covalent attachment to apoenzyme, can produce substantial changes in the ( 1 N)

[0430] If the conditions (including ionic strength, buffer composition, temperature, and pH) are the same, the integrated NMR signal intensity is proportional to the concentration 73 . Since the amide protons in lipoamide protons are expected to be unstable in water, chemical exchange must also be considered 74 . To ensure appropriate conditions were selected, ( 15 N) 1 H NMR spectra were obtained in different solvents (chloroform-d, H2O, medium, medium with 3 mM KAsO2, and a 50:50 mixture of medium with 3 mM KAsO2 and EDTA-trypsin) ( Figure 13B , C). Only in pure water and chloroform-d were the signal intensities of the amide proton resonances comparable. In the other solvents, the cis amide proton signal decreased, indicating chemical exchange (​Figure 13B ), and thus the trans - amide protons were used for concentration quantification. To determine the temperature and pH sensitivity of the trans - amide proton signal, 1 mM lipoamide in culture medium was obtained at different temperatures ( Figure 13D ) and pH ( Figure 13E ) and its ( 15 N) 1 H spectrum was recorded. Both amide protons showed chemical exchange under high - temperature and high - pH conditions, while the trans - amide proton was less affected ( Figure 13D , E). To determine whether lipoamide degrades over time, the signal from the trans - amide proton was monitored for 10 h at 37 °C and 10 °C. At 37 °C but not at 10 °C, the signal intensity decayed slowly ( Figure 13F ), indicating slow hydrolysis to form ammonia. Thus, at 10 °C and below pH 8.6, the integrated signal from the trans - amide proton resonance is a good measure of the 15 N lipoamide concentration.

[0431] Cell uptake was measured by comparing the signal intensity S of the trans - amide proton of lipoamide obtained in the absence (- cells, sample i, Figure 2 A) and presence (+ cells, sample ii, Figure 2 A) of HeLa cells. The measured uptake fraction U is given by:

[0432]

[0433] After uptake, the amount (in moles) of added lipoamide (add) becomes distributed between the intra - cellular (cell) and extra - cellular (out) environments. This can be expressed in terms of concentration c and volume V:

[0434] c add V add = c cell V cell + c out V out

[0435] The total volume of the cells is given by Vcell = V1Ncell, where y1 is the total volume of a single cell and Ncell is the number of cells. Vcell << Vadd, so we assume Vadd = Vo. The uptake fraction can also be expressed in terms of these concentrations and volumes:

[0436]

[0437] Rearranging based on the amount of added lipoamide and the measured uptake fraction U gives expressions for the intra - cellular and extra - cellular concentrations.

[0438]

[0439]

[0440] We approximate HeLa cells as spheres with a radius of 10 -5 mV1 = 4.19x10 -15 m 3 . In our experiment, N cell = 10 6 , C add = 100 μM and V add = 600 μl.

[0441] We have no evidence that peak broadening is related to dissolution in the phospholipid membrane. However, in principle, the signal intensity lost during uptake can be attributed to incorporation into the membrane rather than the cytoplasm.

[0442] Calculations show that this is implausible. The number of phospholipid molecules in the plasma membrane can be estimated based on the footprint of each lipid molecule A L = 0.5 nm 2 75 . Assuming a spherical cell, the surface area of a single cell is A1 = 1.3×10 -9 m 2 . Thus, the total number of phospholipids on the molecules taken up by the cell is N uptake = c cell V cell N A , where N A is Avogadro's number. The ratio of lipoamide to lipid molecules is given by:

[0443]

[0444] For the average value U = 0.35 (i.e., 35% uptake) observed in the experiment, we would expect R = 4.9, i.e., 4.9 lipoamide molecules per plasma membrane lipid molecule. The plasma membrane is not the only membrane in the cell, but even if it accounts for 10% of the total phospholipids, approximately 1 lipoamide molecule is required for every 2 phospholipid molecules.

Claims

1. A method for identifying a compound that modulates a property associated with one or more condensates comprising condensate-associated molecules, the method comprising: (a) contacting the compound with a cell composition comprising one or more condensates or a cell composition capable of forming one or more condensates, and (b) determining a property associated with the one or more condensates based on the number of the one or more condensates comprising condensate-associated molecules, the cellular location of the one or more condensates, and the cellular distribution of the one or more condensates, to identify the compound that modulates the property associated with the one or more condensates, Among them, wherein modulation of the property, compared to a reference, indicates that the compound modulates a property associated with the one or more condensates.

2. The method according to claim 1, wherein the property associated with the one or more condensates is determined based on any one or more of the following: (i) the size of the one or more condensates; (ii) the surface area of the one or more condensates and / or the condensate-associated molecules; (iii) the composition of the one or more condensates; (iv) the mobility of the one or more condensates; (v) the solidification of the one or more condensates; (vi) the dissolution of the one or more condensates; (vii) the presence and / or amount of fiber formation; (viii) the location of the condensate-associated molecules; (ix) the partitioning of the condensate-associated molecules into the one or more condensates; and (x) the aggregation of the condensate-associated molecules.

3. The method according to claim 1, wherein the one or more condensates are within one or more cells of the cell composition.

4. The method according to claim 1, further comprising subjecting the cell composition to condensate formation conditions prior to step (b).

5. The method according to claim 1, further comprising subjecting the cell composition to condensate formation conditions prior to step (a).

6. The method according to claim 4 or 5, wherein the condensate formation conditions are any one or more of the following: (i) an oxidative stressor; (ii) a mitochondrial electron transport chain inhibitor; (iii) a heat stressor; (iv) an osmotic stressor; (v) a hyperosmotic stressor; and (vi) glycolysis inhibition.

7. The method according to claim 1, wherein the condensate-associated molecule is a polypeptide.

8. The method according to claim 1, wherein the condensate-associated molecule is a wild-type polypeptide.

9. The method according to claim 1, wherein the condensate-associated molecule is a mutant polypeptide.

10. The method according to any one of claims 7–9, wherein the condensate-associated molecule is selected from the group consisting of FUS, TDP–43, EWSR1, TIAL1, PABPC1, and G3BP1.

11. The method according to claim 1, wherein the cells in the cell composition express the condensate-associated molecule.

12. The method according to claim 1, wherein the cell composition comprises HeLa, iPSC or iPSC MN cells.

13. The method according to claim 1, further comprising imaging at least a portion of the cell composition.

14. The method according to claim 1, further comprising contacting at least a portion of the cell composition with a fixative.

15. The method according to claim 1, further comprising contacting at least a portion of the cell composition with a stain.

16. The method according to claim 1, further comprising contacting at least a portion of the cell composition with DNA damage conditions.

17. The method according to claim 16, wherein the DNA damage condition is laser irradiation.

18. The method according to claim 1, wherein the reference is a second condensate.

19. The method according to claim 1, wherein the reference is a second cell composition.

20. The method according to claim 1, further comprising evaluating the identified compound using a second cell level assay.

21. The method according to claim 1, further comprising evaluating the identified compound using a biochemical assay.

22. The method according to claim 1, further comprising evaluating the identified compound using an in vivo assay.

23. A method of identifying a compound for treating a disease, the method comprising identifying a compound according to the method of any one of claims 1–22.

24. The method according to claim 23, wherein the disease is a neurodegenerative disease.

25. The method according to claim 24, wherein the neurodegenerative disease is amyotrophic lateral sclerosis ALS.

26. The method according to claim 1, wherein the one or more condensates are membrane-less encapsulated compartments formed by phase separation of one or more of proteins, DNA, and RNA.

27. The method according to claim 1, wherein the one or more condensates are selected from the group consisting of stress granules, P-bodies, Cajal bodies, promyelocytic leukemia protein PML bodies, paraspeckles, DNA damage focus condensates, divisomes, P-granules, histone locus bodies, multivesicular bodies, neuronal RNA granules, nuclear gems, nuclear pores, nuclear stress bodies, nucleoli, Oct1 / PTF / transcription OPT domains, perinuclear compartments, PML oncogenic domains, polycomb bodies, processing bodies, Sam68 nuclear bodies, and splicing speckles.

28. Use of a compound identified by the method of any one of claims 1–27 in the preparation of a medicament for treating an RNA-binding protein RBP-related disease in an individual, wherein the RBP-related disease is characterized in that the RBP is mislocalized from a nuclear location to a cytoplasmic location, wherein the treatment comprises administering to the individual an effective amount of the medicament, and wherein the medicament restores the nuclear location of the RBP.

29. The use according to claim 28, wherein in the case of the RBP-related disease, the RBP forms or segregates into condensates at the cytoplasmic location.

30. The use according to claim 29, wherein the condensate is a stress granule.

31. The use according to claim 28, wherein the RBP is selected from the group consisting of FUS, TDP–43, EWSR1, TIAL1, PABPC1, and G3BP1.

32. The use according to claim 28, wherein the RBP is FUS.

33. The use according to claim 28, wherein the RBP is TDP–43.

34. The use according to claim 29, wherein the RBP is compartmentalized into the condensate at the cytoplasmic location.

35. The use according to claim 29, wherein the drug i) reduces the number of the condensates containing the RBP at the cytoplasmic location; ii) increases the mobility of the condensates containing the RBP at the cytoplasmic location; and / or iii) reduces and / or prevents the coagulation of the condensates containing the RBP at the cytoplasmic location.

36. The use according to claim 29, wherein the drug does not affect other condensates and / or condensate-related molecules in the individual.

37. The use according to claim 36, wherein the other condensates are selected from the group consisting of stress granules not containing the RBP, P bodies, PML bodies, Cajal bodies, paraspeckles, and DNA damage focus condensates.

38. The use according to claim 28, wherein the nuclear location of the RBP contains DNA damage sites.

39. The use according to claim 28, wherein the RBP-related disease is a neurodegenerative disease.

40. The use according to claim 39, wherein the neurodegenerative disease is ALS.

41. The use according to claim 39, wherein the drug restores the neuronal defects caused by the RBP.

42. The use according to claim 41, wherein the neuronal defects include axonal transport defects and / or axonal death.

43. The use according to claim 28, wherein the compound is selected from the group consisting of lipoic acid and its salts, lipoamide and its salts, dihydrolipoic acid and its salts, and dihydrolipoamide and its salts.

44. A compound identified by the method according to any one of claims 1–27.

45. The compound according to claim 44, selected from the group consisting of lipoic acid and its salts, lipoamide and its salts, dihydrolipoic acid and its salts, and dihydrolipoamide and its salts.

46. A pharmaceutical composition comprising the compound according to claim 44 or 45.

47. The pharmaceutical composition according to claim 46, further comprising a pharmaceutically acceptable excipient.

48. The pharmaceutical composition according to claim 46, formulated for oral administration.

49. Use of the compound according to claim 44 or 45 or the pharmaceutical composition according to any one of claims 46–48 in the preparation of a drug for the treatment or prevention of a neurodegenerative disease associated with the formation of stress granules in an individual.

50. The use according to claim 49, wherein the drug is for oral administration.

51. The use according to claim 49, wherein the neurodegenerative disease associated with the formation of stress granules is ALS.

52. The method according to claim 1, wherein the cell distribution is based on the ratio of one or more condensates containing condensate-related molecules in the nucleus and the cytoplasm.

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