Compounds for treating diseases and methods for screening same

Abstract

The present invention relates to a compound for use in the prevention or treatment of neurodegenerative diseases associated with the formation of stress particles. The compound is selected from the group consisting of lipoic acid, lipoic acid amide, dihydrolipoic acid and dihydrolipoic acid amide. The invention further relates to a method of identifying a compound that modulates a property associated with one or more agglomerates comprising an agglomerate-related molecule, comprising: (a) contacting the compound with a cellular composition comprising one or more agglomerates or a cellular composition capable of forming one or more agglomerates, and (b) determining a property associated with the one or more agglomerates. The modulation of the property as compared to a reference indicates that the compound modulates a property associated with the one or more agglomerates.

Description

[0001] This application is a divisional application of the invention patent application with application number 201980082295.2, application date October 14, 2019, and invention name “Compounds for treating diseases and screening methods thereof”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority from 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

[0004] Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a poor prognosis and limited treatment options. Currently, only two FDA-approved drugs are available: riluzole and edaravone, but both can only slow disease progression for a few months. The effects of riluzole and edaravone are not fully understood, and they probably do not directly target the underlying pathogenesis of the disease. Therefore, new approaches are urgently needed. Most types of ALS are sporadic, but about 10% are monogenic disorders. Familial ALS-associated mutations often occur in RNA-binding proteins (RBPs) such as FUS and TDP-43. These RBPs have characteristic low complexity domains (LCDs).

[0005] The precise mechanisms of ALS pathogenesis remain unclear; whether aggregates or oligomers are toxic or whether they lead to loss of protein function, and how this leads to downstream effects, is unclear. However, mutant TDP-43 and FUS frequently translocate to the cytoplasm, promoting the formation of disease-associated stress granules and abnormal cytoplasmic aggregates. One hypothesis, in particular, suggests that pathological stability of stress granules may be involved in the disease. This suggests that dissolving stress granules and / or aggregates could alleviate the disease, regardless of whether the pathogenesis is through toxic gain of function or loss of function due to protein sequestration.

[0006] Stress granules are liquid-like, membrane-less compartments, and recent studies have shown that proteins containing LCDs form these compartments through liquid-liquid phase separation. Therefore, in principle, it is possible to target this physicochemically driven mechanism of stress granule formation. Compounds known to disrupt this phase separation exist, notably 1,6-hexanediol and similar alcohols. However, these compounds present two problems: first, they require extremely high concentrations (1–10%) and are toxic. Second, their effects are not specific to stress granules and affect other liquid-like, membrane-less compartments as well. Many other compartments are also liquid-like, particularly in the cell nucleus.

[0007] There exists a need for a method to identify compounds that more specifically modulate phase separation of certain compartments to effectively and specifically treat ALS.

[0008] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety. Summary of the Invention

[0009] In some aspects, the present disclosure provides a method for identifying a compound that modulates a property associated with one or more condensates comprising a condensate-associated molecule, the method comprising: (a) contacting the compound with a cellular composition comprising one or more condensates or a cellular composition capable of forming one or more condensates, and (b) measuring a property associated with the one or more condensates, wherein modulation of the property compared to a reference indicates that the compound modulates the property associated with the one or more condensates.

[0010] In some embodiments, determining a property associated with the one or more coagulants is based on any one or more of the following: (i) the number of coagulants that contain and / or do not contain coagulant-associated molecules; (ii) the size of the one or more coagulants; (iii) the location of the one or more coagulants; (iv) the distribution of the one or more coagulants; (v) the surface area of ​​the one or more coagulants; (vi) the composition of the one or more coagulants; (vii) the fluidity of the one or more coagulants; (viii) the coagulation of the one or more coagulants; (ix) the dissolution of the one or more coagulants; (x) the presence and / or amount of fiber formation; (xi) the location of the coagulant-associated molecules; (xii) the partitioning of the coagulant-associated molecules into the one or more coagulants; and (xiii) the aggregation of the coagulant-associated molecules.

[0011] In some embodiments, the one or more aggregates are within one or more cells in the cell composition.

[0012] In some embodiments, the methods described herein further comprise subjecting the cell composition to aggregate-forming conditions prior to determining one or more aggregate-related properties.

[0013] In some embodiments, the methods described herein further comprise subjecting the cell composition to condensate-forming conditions prior to contacting the compound with the cell composition.

[0014] In some embodiments, the complex-forming conditions are any one or more of: (i) an oxidative stressor; (ii) a mitochondrial electron transport chain inhibitor; (iii) a heat stressor; (iv) an osmotic stressor; (v) a hypertonic stressor; and (vi) glycolysis inhibition.

[0015] 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 the group consisting of FUS, EWSR1, TIAL1, PABPC1, and G3BP1.

[0016] 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.

[0017] In some embodiments, the methods described herein further comprise imaging at least a portion of the cellular composition.

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

[0019] In some embodiments, the methods described herein further comprise contacting at least a portion of the cell composition with a stain.

[0020] In some embodiments, the methods described herein further comprise contacting at least a portion of the cell composition with a DNA damaging condition. In some embodiments, the DNA damaging condition is laser irradiation.

[0021] In some embodiments, the reference is a second aggregate. In some embodiments, the reference is a second cell composition.

[0022] In some embodiments, the methods described herein further comprise evaluating the identified compound using a second cellular assay.

[0023] In some embodiments, the methods described herein further comprise evaluating the identified compounds using a biochemical assay.

[0024] In some embodiments, the methods described herein further comprise evaluating the identified compounds using an in vivo assay.

[0025] 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.

[0026] The present invention relates to compounds useful in the treatment of neurodegenerative diseases associated with stress granule formation, in particular for the treatment of amyotrophic lateral sclerosis.

[0027] 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(mercapto)octylamide; CAS No. 3884-47-7).

[0028] In particular, such neurodegenerative diseases are associated with the formation of stress granules in the cytosol of cells contained within the tissue affected by the disease.

[0029] In particular, lipoic acid and / or lipoamide can be used in the form of the R-form, the S-form or the racemate.

[0030] 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 ionized medicine and oppositely charged counterions. Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, benzenesulfonate, bitartrate, bromide, carbonate, chloride, citrate, edetate, edisylate, pamoate, etopoate, fumarate, glucoheptonate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, methanesulfonate, methylbromide, methylsulfate, mucate, naphthylsulfonate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, toluenesulfonate, triethyl iodide, 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.

[0031] In some embodiments, the compounds of the present invention are administered according to a dosage regimen where a daily dose of 600 mg to 1,600 mg of the compound is administered.

[0032] Advantageously, a daily dose of 600 mg can produce plasma concentrations of 8 μM to 30 μM of the compound of the invention.

[0033] In some embodiments, the neurodegenerative disease associated with stress granule formation is selected from the group consisting of amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease.

[0034] In some embodiments, a pharmaceutical composition for use in a method for preventing or treating a neurodegenerative disease associated with stress granule formation comprises a compound of the present invention as described in paragraph 0025.

[0035] In some embodiments, the pharmaceutical composition is formulated for oral administration.

[0036] In some embodiments, the neurodegenerative disease associated with stress granule formation is amyotrophic lateral sclerosis.

[0037] Alternatively, a dosage form for use in a method for preventing or treating a neurodegenerative disease associated with stress granule formation is provided, wherein the dosage form comprises a compound of the present invention, particularly a compound as described in paragraph 0025.

[0038] The dosage form can be for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalation form or suppository. Alternatively, parenteral administration, such as subcutaneous, intravenous, intrahepatic or intramuscular injection, can be used. Optionally, a pharmaceutically acceptable carrier and / or excipient may be present.

[0039] In some embodiments, the dosage form is formulated for oral administration.

[0040] 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.

[0041] Alternatively, the therapeutic aspect according to the present invention may be formulated as a method for preventing or treating a neurodegenerative disease associated with stress granule formation, wherein the method comprises administering to a patient in need thereof a compound of the present invention, in particular a compound as described in paragraph 0025.

[0042] In some embodiments, the compound is administered at a daily dose of 600 mg to 1,600 mg.

[0043] In some embodiments, the compound is administered orally.

[0044] 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.

[0045] In some embodiments, the neurodegenerative disease associated with stress granule formation is amyotrophic lateral sclerosis.

[0046] In certain aspects, the present invention relates to a method for reducing or inhibiting stress granule formation in a cell, wherein the method comprises administering a compound selected from the group consisting of:

[0047] -Lipoic acid, lipoamide, dihydrolipoic acid, dihydrolipoamide,

[0048] - heterotricyclic compounds, in particular anthraquinone or anthraquinone derivatives, such as 1,4-dihydroxyanthraquinone,

[0049] - acridine or an acridine derivative, for example quinacrine or aminoacridine or mitoxantrone;

[0050] - a tetracyclic compound, and

[0051] - Surfactants, especially cetylpyridinium chloride

[0052] In some embodiments, the compound is provided in the culture medium in which the cells are cultured.

[0053] Those skilled in the art will also understand that the form and details of the implementations described herein may be changed without departing from the scope of the present disclosure. Additionally, although various advantages, aspects, and objects have been described with reference to various implementations, the scope of the present disclosure should not be limited by reference to such advantages, aspects, and objects. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1Screening of small molecule compounds affecting FUS stress granule formation in HeLa cells in vitro is shown. a) Subcellular localization of FUS GFP in un-stressed HeLa cells, cells stressed with compound solvent (DMSO) negative control, and cells stressed with dimercaprol (an arsenic-chelating drug) and ipecac (which prevents stress granule formation by stabilizing polysomes) positive controls. Stress causes nuclear export of FUS and formation of stress granules (FUS-containing cytoplasmic liquid droplets). b) Screening workflow of small molecules affecting FUS GFP localization in HeLa cells in vitro. c) Mahalanobis distance ranking for all 1600 screened compounds (average from six fields), where high values mean greater compound effect. 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. A cutoff of 130 was used to select 47 compounds for further analysis. Dimercaprol and ipecac are highlighted. d) Screening workflow of small molecules affecting FUS liquid-liquid phase separation of purified FUS GFP in vitro. e) Z-score ranking of droplet number and signal partitioning into FUS GFP droplets (formed under low salt conditions) changes, where larger positive or negative values mean greater compound effect. Scores were calculated at the maximum concentration where the compound solvent (DMSO) negative control had no significant effect; 100 mM. Sulfϊnalamide, surfactants, and heterotricyclics are indicated by data point color, see f). f) Examples of the three hit classes; sulfϊnalamide, cetylpyridinium chloride (surfactant), mitoxantrone (heterotricyclic). g) Appearance of droplets for the compound solvent (DMSO) negative control or examples of the following compound classes: cetylpyridinium chloride (surfactant), sulfϊnalamide, or mitoxantrone (heterotricyclic). Note that droplets for cetylpyridinium chloride and sulfϊnalamide are larger, while the liquid for mitoxantrone is smaller.

[0055] Figure 2 Structure-activity relationship of sulfϊnalamide-related compounds is shown, which reveals possible mechanisms of action. a) Dose response of HeLa cell FUS GFP droplet number (·, left axis) and nuclear / cytoplasmic signal ratio (o, right axis) after 1 h pre-treatment with sulfϊnalamide, followed by 1 h arsenate stress, and continued treatment with sulfϊnalamide. b) Dose response of iPS cell FUS GFP droplet number and nuclear / cytoplasmic ratio as in a). c) Dose response of HeLa cell FUS GFP droplet number and nuclear / cytoplasmic ratio for a series of sulfϊnalamide-related compounds using the same stress / treatment protocol as in a). d) Summary of compound structures and effects on cytoplasmic FUS droplet number and FUS nuclear partitioning.​​

[0056] Figure 3 The mode of action of thioctic amide and lipoic acid under different protocols on different stresses is shown, and possible mechanisms of action are reported. a) Kinetics of loss of cytoplasmic FUS GFP droplets in HeLa cells pre-stressed with arsenate for 1 hour, then treated with 10 μΜ thioctic amide (or DMSO solvent control), then continued arsenate stress. Example images at 2 minutes and 100 minutes after addition of compound are shown on the right. Thioctic amide can reverse the effects of existing arsenate stress. b) Images of HeLa cells expressing FUS GFP that have been pre-treated with 10 μΜ thioctic amide (or DMSO control) for 1 hour, then washed 3 times, then stressed with arsenate for 1 hour. Thioctic amide pre-treatment has no lasting effect on the cells to prevent their response to arsenate stress. c) Images of HeLa cells expressing GFP-tagged stress granule markers (EWSR1, TIAL1, PABC1, or G3BP1) after 1 hour of arsenate stress and 10 μΜ thioctic amide or isomer of lipoic acid, arsenate stress with DMSO solvent control, or DMSO without arsenate. Thioctic amide 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 deprivation, sorbitol (osmotic), heat, arsenate, or 6-deoxy glucose (glycolysis) - while being treated with 10 μΜ thioctic amide or isomer of lipoic acid. Thioctic amide and lipoic acid are active against multiple stresses, including mitochondrial, osmotic, and oxidative stresses.

[0057] Figure 4 Thioctic amide does not solubilize the nuclear FUS compartment and other nuclear compartments is shown. a) Images of HeLa cells expressing GFP-tagged proteins of other membrane-free bounded compartments after 1 hour of treatment with 10 μΜ compound (or DMSO control). Where unclear, the location of the nucleus is indicated with a dashed outline. Thioctic amide does not disrupt P-bodies (DCP1A), Cajal bodies (COIL), DNA damage foci (TRP53BP1), or nucleoli (NCB1), while mitoxantrone does have a non-stress granule-specific effect. b) FUS GFP recruitment to sites of UV laser-induced DNA damage in iPS cells after 1 hour of treatment with compound followed by 1 hour of arsenate stress. Top row shows FUS GFP fluorescence before laser cutting, with stress granules indicated with arrows. Bottom row shows the average FUS GFP signal intensity response to DNA damage, as well as one standard deviation above and below the mean. Mitoxantrone prevents the formation of nuclear FUS droplets at DNA damage sites below the EC50 for cytoplasmic droplets, while thioctic amide does prevent the formation of nuclear FUS droplets.

[0058] Figure 5Figure 3. Lipoic acid and lipoic acid are shown to affect the properties of FUS condensates and the coagulation of ALS-associated mutant FUS in vitro. a) Schematic diagram showing the quantification of condensate droplet fluidity using optical tweezers. The time it takes for two droplets to contact and begin to fuse, relaxing into a single spherical droplet (once adjusted for droplet size), is a measure of the ratio of the droplet's viscosity to its surface tension - a measure of fluidity. Representative of two independent replicates. b) Droplet size-corrected relaxation times for droplet fusion with 300 μM lipoamide or an equivalent DMSO solvent control (0.3%). Boxes represent the 25th, 50th, and 75th percentiles, and whiskers represent the 5th and 95th percentiles. Lipoic acid reduces fusion time, indicating lower viscosity and / or greater surface tension. c–e) Effects of 10 μM lipoamide on G156E FUS GFP aggregates (formed under dextran crowding) during aging with shaking, relative to an equivalent DMSO solvent control (0.1%). These conditions match those previously published.7 c) Representative images after 30 hours of aging, showing fibril formation in the DMSO sample. d) Representative fluorescence recovery after photobleaching (FRAP) time series of FUS aggregates and fibers during aging. e) Quantification of FRAP in c). Error bars represent standard deviation. Aged aggregates treated with lipoamide maintain a large FUS GFP mobile fraction and a short FRAP half-life relative to an equivalent DMSO solvent control (0.3%), while untreated aggregates reverse the effects of lipoamide and lipoic acid on the aging of G156E FUS GFP aggregates during aging. Both compounds delayed fibril formation.

[0059] Figure 6 Figure 3. Lipoamide and lipoic acid directly alter FUS phase separation and FUS G156E coagulation in vitro. a) Droplets formed in vitro from 2.8 μM FUS GFP in the presence of varying concentrations of KCl (which inhibits droplet formation) and 100 μM lipoamide, lipoic acid, or an equivalent DMSO solvent control (1%). Lipoamide and lipoic acid subtly promoted droplet formation. b) Effects of lipoamide and lipoic acid on "aging" of G156E FUS GFP droplets upon oscillation, relative to an equivalent DMSO solvent control (0.3%). Both compounds delayed fibril formation. c) Representative fluorescence recovery after photobleaching (FRAP) of formed FUS droplets and fibers during aging shown in b). Aged droplets treated with lipoamide or lipoic acid retained fast FRAP, indicating mobility. d) Quantification of fluorescence signal intensity shown in c). Error bars represent standard deviation. While untreated droplets hardened, lipoamide and lipoic acid treatment allowed the droplets to retain a large FUS GFP mobile fraction and a short FRAP half-life.

[0060] Figure 7 It was shown that lipoamide accumulates to high concentrations in cells without being metabolized. a) Synthesized and characterized 15 NR-(+) and (±)-lipoamide. Use 15 Edited by N 1 H-detected 1D HSQC NMR experiments can selectively detect both amide protons in biological media and HeLa cell pellets. The trans amide proton (resonating at 6.9 ppm) can be quantified by NMR. Below pH 8.5 and 10°C, the signal intensity is proportional to the lipoamide concentration. For details, see Supplementary Methods. 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 removing the culture medium, 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) is consistent with substantial uptake from the culture medium calculated from the signal intensity with (red) and without cells (cyan). d) Quantification of c) shows the percentage of uptake and the calculated intracellular concentration, assuming that lipoamide is evenly 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, with cellular concentrations >1 mM.

[0061] Figure 8Lipoic acid and lipoic acid demonstrate beneficial effects in ALS models in vitro and in vivo. a, b) In Caenorhabditis elegans, lipoic acid reduces aging-induced aggregation of stress granules, but not stress granule proteins. a) Toxicity and effects of R-(+)- or S-(-)-lipoic acid on protein aggregation in worms overexpressing aggregation-prone fluorescently labeled proteins. The incidence of PAB-1 aggregation in pharyngeal muscle was scored based on the proportion of cells with >10 aggregates. The incidence of RHO-1 and KIN-19 was scored as low, moderate, or high—see Methods. Toxicity was assessed based on the proportion of abnormally small or dead animals. Both lipoic acid isomers caused a strong, dose-dependent reduction in PAB-1 aggregation, but not RHO-1 or KIN-19 aggregation. Significant changes compared to DMSO control are indicated by 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) Confocal microscopy Z-stacks through the pharynx of worms expressing fluorescently labeled PAB-1, treated or not with lipoic acid, show reduced aggregate numbers. c–e) Lipoic acid and lipoamide are associated with familial ALS in neuronal dieback derived from iPS cells expressing FUS P525L. c) Schematic diagram of neuronal culture showing the channel where axons grow from the cell body on the right. The region shown in the micrographs in d and e) is indicated. d) iPS-derived neurons after 60 days of culture with 0.02% DMSO. Neurons expressing wild-type FUS have stable axons, while neurons expressing FUS P525L have unstable axons that die, leaving material around the axon's exit point from the channel. e) iPS-derived neurons expressing FUS P525L after 60 days in culture in the presence of 2 μM lipoamide or racemic R-(+)- or S-(-)-lipoic acid. Representative images from a blinded experiment, which also includes DMSO (solvent control)-treated neurons shown in c). f) Lipoic acid restores motor function deficits in Drosophila melanogaster overexpressing human wild-type FUS or ALS-associated FUS mutations. FUS overexpression leads to motor deficits, and the animals are unable to climb. Lipoic acid treatment showed a dose-dependent increase in climbing ability in animals expressing wild-type FUS, FUS P525L, or FUS R512C. **p<0.005, p<0.05, one-way ANOVA. g) Lipoic acid also restores motor function deficits in Drosophila melanogaster. Comparable conditions to f) are shown using lipoamide treatment instead of lipoic acid. *p<0.05, **p<0.005, Student's t-test. h) Lipoic acid rescues motor deficits in Drosophila melanogaster overexpressing wild-type human FUS or an ALS-associated FUS mutation. Overexpression of FUS leads to motor deficits and the inability of animals to climb.**p<0.005, p<0.05, one-way ANOVA.

[0062] Figure 9 iPS-derived neurons after 5 and 60 days of culture with 0.02% DMSO are shown. Neurons expressing wild-type FUS have stable axons, while neurons expressing FUS P525L have unstable axons that die back, leaving material around the axon's exit point from the channel.

[0063] Figure 10 The structure-activity relationship of heterotricyclic compounds suggests that the tricyclic core is responsible for their activity. a) Dose response of FUS GFP droplet number (●, left axis) and nuclear / cytoplasmic signal ratio (○, right axis) in HeLa cells following 1-hour pretreatment with mitoxantrone, followed by 1-hour arsenate stress and subsequent mitoxantrone treatment. b) Dose response of FUS GFP droplet number and nuclear / cytoplasmic ratio in HeLa cells using the same stress / treatment protocol as in a) for a series of compounds related to mitoxantrone and other heterotricyclics, quinacrine, and tetracycline antibiotic families. c) Summary of compound structure and effects on cytoplasmic FUS droplet number and FUS nuclear compartmentalization.

[0064] Figure 11 Lipoic acid amide 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 lipoic acid amide or DMSO negative control. FUS P525L leads to the formation of larger cytoplasmic FUS droplets, which remain sensitive to lipoic acid amide. b) The relative optical density of aggregated FUS was assessed by filter blocking of the iPS cells in a) after pretreatment with 100 μM thioamide or DMSO solvent control for 1 hour, followed by 1 hour arsenate stress or no stress. Significant changes are shown (Student's t-test), n=5. c) Following cell treatment in b), immunoblotting with anti-FUS and anti-GAPDH was performed. No significant changes in FUS expression levels were detected relative to GAPDH (Student's t-test, n=3).

[0065] ***anova***

[0066] Figure 12Figure 3: Lipoic acid amide restores axonal transport defects caused by FUS P525L expression in motor neurons. a) Example kymograph of lysosomal movement in the distal part of the axon of FUS P525L GFP motor neurons after 3 days of treatment with compound solvent (DMSO) or 2 μM lipoamide. 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 lipoamide or equivalent DMSO concentration solvent control. n=5 (P525L) or n=3 (wild-type) biological replicates, with 5 axonal bundles analyzed per replicate. Lipoic acid amide significantly increases lysosomal trafficking (Student's T test). ***anova***

[0067] 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 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. 15 Edited by N 1 In H NMR experiments, 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 >10 h of experiment. g) Signal intensities of cis and trans amide protons under different experimental conditions. This is Figure 13BAn expanded version of , which shows only trans amide protons and includes an additional condition: v) cells disrupted with TritonX-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 of the molecule is unmodified ( Figure 13C Uptake is quantified in D). h) Expanded views of the spectra in g), i), and v). After cell disruption with Triton X-100 and DNase I, the spectra change significantly, indicating chemical modifications of lipoamide upon disruption of cellular compartments. DETAILED DESCRIPTION

[0068] In some aspects, the invention includes methods for identifying compounds that modulate condensates and uses thereof, such as the usefulness of such compounds in treating disease.

[0069] Traditionally, the various membrane-bound compartments of a cell (organelles) were thought of as a crowded jumble of macromolecules and metabolites, all freely intermingling with one another and randomly diffusing within the boundaries of the membrane that delineated that particular compartment (nucleus, cytosol, mitochondria, endoplasmic reticulum, etc.). Cellular processes were thought to be regulated by the interactions of these molecules, driven by specific binding sites that led to biochemical reactions between the molecules (enzymatic activity, such as transcription, RNA processing, metabolic reactions, signaling 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.

[0070] From recent work, it is now clear that macromolecules are sorted into condensates within membrane-bounded organelles based on the intrinsic physical properties of proteins, RNA, and / or DNA. The macromolecules assemble or condense into liquid droplets in a phase separation reaction, resulting in the enrichment of specific protein and / or nucleic acid molecules within the condensate while excluding other specific proteins and / or nucleic acids.

[0071] The process of forming condensates is driven not by specific, simple stoichiometric binding events, as previously thought, but by phase-separation reactions – layered structures within cells that organize macromolecules throughout the cell into subcompartments to increase reaction specificity and drive reactions through higher local concentrations.

[0072] Remarkably, condensates are fluid and reversible, so that upon changes in cellular physiology (e.g., signaling events, changes in the concentration of one of the macromolecules, or other changes in the local environment), different condensates within the cell will change, sometimes completely dissolving, sometimes more subtly altering their molecular composition. This is therefore a major mechanism for regulating almost all reactions within the cell, synergizing with previously understood mechanisms of specific binding events.

[0073] Several 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 this application is based, at least in part, on the inventors' unique insight that, in this context, macromolecular phase separation into condensates is an intermediate step that occurs before proteins subsequently form abnormal aggregates, and thus new useful compounds for treating such diseases can be discovered by using assays that directly monitor the effects of compounds on condensate formation and properties.

[0074] 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 flowability but not condensate formation. Furthermore, using the methods described herein, the inventors have identified compounds that, rather than simply disrupting all condensate formation, exhibit specificity for the target condensate. This specificity can potentially improve efficacy and reduce side effects such as toxicity.

[0075] The present application provides novel methods for identifying compounds that modulate one or more properties of cellular and extracellular condensates and are therefore useful for treating condensate-associated diseases. The methods focus on changes in the behavior or properties of one or more condensates, allowing the identification of compounds without having to know the specific molecular target of each compound. This allows one to quickly 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-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 coagulation 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 one or more condensates; and (xiii) the aggregation of condensate-associated molecules. Compounds with desired properties can be identified by evaluating the ability to modulate some (e.g., any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) or all of the properties of one or more coacervates. Additionally, compounds with desired properties can be identified by evaluating the ability to modulate some but not other characteristics of coacervates, or the ability to modulate some but not other coacervates. Using the methods described herein, compounds have been identified that target the physicochemistry of coacervates formed by liquid-liquid phase separation.

[0076] Thus, in some aspects, provided herein are methods for screening compounds that modulate properties associated with one or more condensates and / or are useful in methods for treating disease. In some aspects, provided herein are methods for high-throughput screening of compounds that modulate properties associated with one or more condensates and / or are useful in methods for treating disease.

[0077] 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 a property indicates that the compound modulates a property associated with one or more condensates. In some embodiments, modulation of a property indicates that the compound modulates a property associated with one or more condensates compared to a reference. In some embodiments, the method comprises determining multiple properties associated with one or more condensates.

[0078] 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) measuring 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.

[0079] In some aspects, provided herein are methods for identifying a compound that modulates a property associated with a first group 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 groups of one or more condensates, or with a cell composition capable of forming the first and second groups of one or more condensates, and (b) determining a property associated with the first group of one or more condensates, and (c) determining a property associated with the second group of one or more condensates.

[0080] In some aspects, provided herein are methods for identifying a compound that modulates a property associated with a first group 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 groups of one or more condensates, or with a cell composition capable of forming the first and second groups of one or more condensates, and (b) determining a property associated with the first group of one or more condensates, and (c) determining a property associated with the second group of one or more condensates.

[0081] In some aspects, provided herein are methods for screening (e.g., high-throughput screening) for compounds that modulate 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 the first set of one or more condensates or with a cell composition capable of forming the first set of one or more condensates, and (b) determining the property associated with 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 the property associated with the second set of one or more condensates.

[0082] In some embodiments, modulation of a property associated with the first group is different from modulation of a property associated with the second group, indicating that the candidate compound modulates a property associated with the first group of one or more coacervates, thereby obtaining a compound that modulates a property associated with the first group of one or more coacervates. In some embodiments, modulation of a property associated with the second group is different from modulation of a property associated with the first group, as compared to a reference, indicating that the compound modulates a property associated with the second group of one or more coacervates, thereby obtaining a compound that modulates a property associated with the second group of one or more coacervates.

[0083] definition

[0084] For purposes of interpreting this specification, the following definitions will apply, and whenever appropriate, terms used in the singular will also include the plural, and vice versa. In the event of a conflict between any definition set forth below and any document incorporated herein by reference, the set forth definition will control.

[0085] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may 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 multimers of amino acid residues. Full-length proteins and fragments thereof are included within this definition. The term also includes post-translational modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like.

[0086] As used herein, the term "polynucleotide" or "nucleic acid" refers to a polymeric form of nucleotides 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 derived nucleotide bases. The backbone of a polynucleotide may contain sugar and phosphate groups (as commonly found in RNA or DNA), or modified or substituted sugar or phosphate groups. The backbone of a polynucleotide may contain repeating units linked by peptide bonds (e.g., peptide nucleic acids), such as N-(2-aminoethyl)glycine. Alternatively, the backbone of a polynucleotide may contain a polymer of synthetic subunits, such as phosphoramidates, and thus may be an oligodeoxynucleoside phosphoramidate (P–NH2) or a mixed phosphoramidate-phosphodiester oligomer.

[0087] As used herein, the terms "comprising," "having," "containing," and "including," and other similar forms and grammatical equivalents thereof, are intended to be equivalent in meaning and to be open-ended, in that one or more items following any of these words is not meant to be an exhaustive list of the one or more items, or to be limited to only the listed one or more items. For example, an article that "comprising" components A, B, and C may consist of components A, B, and C (i.e., contain only components A, B, and C), or may contain not only components A, B, and C, but also one or more other components. Thus, it is intended and understood that "comprising" and its similar forms and its grammatical equivalents include embodiments that disclose "consisting essentially of" or "consisting of."

[0088] Where a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range and any other stated or intervening value in that range is encompassed within the disclosure, subject to any specifically excluded limits in that range, unless the context clearly dictates otherwise. 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 disclosure.

[0089] Reference herein to "about" a value or parameter includes (and describes) variations involving the value or parameter itself. For example, a description referring to "about X" includes a description of "X."

[0090] As used herein, including in the appended claims, the singular forms "a," "an," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0091] As used herein, "coacervate" refers to a membrane-less compartment formed by phase separation (including all phases of phase separation) of one or more proteins and / or other macromolecules.

[0092] As used herein, "condensate-associated molecule" refers to a molecule that can be found in or on condensates under physiological or pathological conditions.

[0093] In the context of the present specification, the term "stress granules" relates to liquid, membrane-free compartments located within the nucleus or cytosol of a cell and containing proteins and RNA, which appear when the cell is under stress.

[0094] In the context of this specification, the term "neurodegenerative disease" relates to a medical condition characterized by a progressive loss of 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.

[0095] As used herein, the term "treating" any disease or condition (e.g., ALS) refers, in one embodiment, to ameliorating the disease or condition (e.g., slowing or arresting or reducing the development of the disease or at least one clinical symptom thereof). In another embodiment, "treating" refers to alleviating or improving at least one physical parameter, including those that may not be discernible by the patient. In yet another embodiment, "treating" refers to modulating the disease or condition physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. As used herein, the term "preventing" any disease or condition (e.g., ALS) refers to inhibiting (including completely inhibiting) the development or onset of the disease or condition (e.g., delaying the development or onset of the disease or condition). Unless specifically described below, methods for evaluating the treatment and / or prevention of a disease are generally known in the art.

[0096] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0097] Methods for screening and identifying compounds

[0098] In some aspects of the present application, methods are provided for identifying compounds that modulate properties associated with one or more condensates and / or are useful in methods for treating a disease. In some aspects, methods are provided for screening compounds that modulate properties associated with one or more condensates and / or are useful in methods for treating a disease. In some aspects, methods are provided for high-throughput screening of compounds that modulate properties associated with one or more condensates and / or are useful in methods for treating a disease.

[0099] Techniques for identifying compounds are described in more detail below.One skilled in the art will recognize that, given the description provided, many embodiments are possible within the scope and spirit of the present disclosure.

[0100] In some aspects, provided herein are methods for identifying a compound that modulates a property associated with one or more condensates comprising a condensate-associated molecule, 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) measuring 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. In some embodiments, the method comprises measuring a single property associated with the one or more condensates. In some embodiments, the method comprises measuring multiple properties associated with the one or more condensates.

[0101] In some aspects, provided herein are methods for screening (e.g., high-throughput screening) compounds that modulate properties associated with one or more condensates, the method comprising evaluating each of a plurality of compounds in a screen using any of the methods described herein. In some embodiments, a method for screening (e.g., high-throughput screening) compounds that modulates properties 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 properties associated with the one or more condensates, wherein modulation of the property compared to a reference indicates that the candidate compound modulates properties associated with the one or more condensates, thereby obtaining a compound that modulates properties of one or more condensates. In some embodiments, the method comprises determining multiple 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, multiple candidate compounds are contacted with the cell composition in separate reactions (e.g., separate wells or containers). In some embodiments, multiple candidate compounds are contacted with the cell composition simultaneously in separate reactions.

[0102] In some aspects, provided herein are methods for identifying a compound that modulates a property associated with a first group of one or more condensates comprising a first condensate-associated molecule, the method comprising: (a) contacting the compound with a cellular composition comprising the first and second groups of one or more condensates, or with a cellular composition capable of forming the first and second groups of one or more condensates, (b) determining a property associated with the first group of one or more condensates, and (c) determining a property associated with the second group of one or more condensates, wherein the modulation of the property associated with the first group is different from the modulation of the property associated with the second group compared to a reference, indicating that the compound modulates a property associated with the first group of one or more condensates.

[0103] In some aspects, provided herein are methods for 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 first set of properties associated with the compound is different from modulation of the second set of properties associated with the compound as compared to a reference, indicating that the compound modulates a property associated with the first set of one or more condensates.

[0104] In some aspects, provided herein are methods for screening (e.g., high-throughput screening) compounds that modulate properties associated with a first group 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 group of one or more condensates, or with a cell composition capable of forming the first and second groups of one or more condensates, (b) determining properties associated with the first group of one or more condensates, and (c) determining properties associated with the second group of one or more condensates, wherein the modulation of the properties associated with the first group is different from the modulation of the properties associated with the second group compared to a reference, indicating that the compound modulates properties associated with the first group of one or more condensates, thereby obtaining a compound that modulates properties associated with the first group of one or more condensates.

[0105] In some aspects, provided herein are methods for screening (e.g., high-throughput screening) compounds that modulate 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 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 a 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 associated with the second set of one or more condensates, wherein the modulation of the property associated with the first set is different from the modulation of the property associated with the second set 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. 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 are stress granules and the second set of one or more condensates are paraspeckles.

[0106] In some aspects, provided herein is a method for identifying a compound that modulates a property associated with one or more stress granules, the method 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) measuring a property associated with the one or more stress granules, wherein the modulation of the property compared to a reference indicates that the compound modulates a property associated with the one or more stress granules. In some embodiments, the method comprises measuring a single property associated with the one or more stress granules. In some embodiments, the method comprises measuring multiple properties associated with the one or more stress granules.

[0107] In some aspects, provided herein is a method for screening (e.g., high-throughput screening) a compound that modulates a property associated with one or more stress granules, the method 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 the property associated with the one or more stress granules, wherein the modulation of the property indicates that the candidate compound modulates the property associated with the one or more stress granules compared to a reference, thereby obtaining a compound that modulates the property of one or more stress granules. In some embodiments, the method includes determining a plurality of properties associated with the one or more stress granules. In some embodiments, the plurality of compounds include 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, a plurality of candidate compounds are contacted with the cell composition in a separate reaction (e.g., a separate well or container). In some embodiments, a plurality of candidate compounds are contacted with the cell composition simultaneously in a separate reaction.

[0108] In some aspects, provided herein are methods for identifying a compound that modulates a property associated with one or more stress granules, the method comprising: (a) contacting the compound with a cell composition comprising one or more stress granules and one or more paraspikes, condensates, P bodies, Cajal bodies, and / or PML bodies formed around sites of DNA damage, or with a cell composition capable of forming one or more stress granules and one or more paraspikes, condensates, P bodies, Cajal bodies, and / or PML bodies formed around sites of DNA damage, (b) determining a property associated with the one or more stress granules, and (c) determining a property associated with the one or more paraspikes, condensates, P bodies, Cajal bodies, and / or PML bodies formed around sites of DNA damage, wherein the modulation of the property associated with the one or more stress granules is different from the modulation of the property associated with the one or more paraspikes, condensates, P bodies, Cajal bodies, and / or PML bodies formed around sites of DNA damage as compared to a reference, indicating that the compound modulates a property associated with the one or more stress granules.

[0109] In some aspects, provided herein are methods for identifying a compound that modulates a characteristic associated with one or more stress granules, the method comprising: (a) contacting the compound with a cellular composition comprising the one or more stress granules or with a cellular composition capable of forming the one or more stress granules, (b) determining the characteristic associated with the one or more stress granules, (c) contacting the compound with a cellular composition comprising one or more paraspeckles, condensates formed around sites of DNA damage, P bodies, Cajal bodies, and / or PML bodies, or with a cellular composition capable of forming one or more paraspeckles, condensates, P bodies, Cajal bodies, and / or PML bodies, and (d) determining a characteristic associated with the one or more paraspeckles, condensates, P bodies, Cajal bodies, and / or PML bodies, wherein modulation of the characteristic associated with the one or more stress granules is different from modulation of the characteristic associated with the one or more paraspeckles, condensates, P bodies, Cajal bodies, and / or PML bodies formed around sites of DNA damage as compared to a reference, indicating that the compound modulates a characteristic associated with the one or more stress granules.

[0110] In some aspects, provided herein are methods for screening (e.g., high-throughput screening) for compounds that modulate properties associated with one or more paraspikes, condensates formed around sites of DNA damage, 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 the one or more paraspikes, condensates formed around sites of DNA damage, P bodies, Cajal bodies, and / or PML bodies, or with a cell capable of forming the one or more stress granules and the one or more paraspikes, condensates formed around sites of DNA damage, P bodies, Cajal bodies, and / or PML bodies; The invention further comprises contacting a cell composition with a cell, (b) determining a property associated with the one or more stress granules, and (c) determining a property associated with the one or more paraspikes, condensates formed around sites of DNA damage, P bodies, Cajal bodies and / or PML bodies, wherein the modulation of the property associated with the one or more stress granules is different from the modulation of the property associated with the one or more paraspikes, condensates formed around sites of DNA damage, P bodies, Cajal bodies and / or PML bodies compared to a reference, indicating that the compound modulates the property associated with the one or more stress granules, thereby obtaining a compound that modulates the property associated with the one or more stress granules.

[0111] In some aspects, provided herein are methods for screening (e.g., high throughput screening) for 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 the property associated with the one or more stress granules, (c) contacting a plurality of candidate compounds with a cell composition comprising one or more paraspeckles, condensates, P bodies, Cajal bodies, and / or PML bodies formed around sites of DNA damage, or with a cell composition capable of forming the one or more paraspeckles, condensates, P bodies, Cajal bodies, and / or PML bodies formed around sites of DNA damage, The invention further comprises contacting a cell composition with a paraplaque, condensates, P bodies, Cajal bodies and / or PML bodies formed around DNA damage sites, and (d) measuring properties associated with the one or more paraplaques, condensates, P bodies, Cajal bodies and / or PML bodies formed around DNA damage sites, wherein the modulation of the properties associated with the one or more stress granules is different from the modulation of the properties associated with the one or more paraplaques, condensates, P bodies, Cajal bodies and / or PML bodies formed around DNA damage sites compared to a reference, indicating that the compound modulates the properties associated with the one or more stress granules, thereby obtaining a compound that modulates the properties associated with the one or more stress granules.

[0112] In some embodiments, the method further comprises repeating the method steps for a plurality of compounds. For example, in some embodiments, the method comprises repeating the method steps for any compound 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 method steps with a plurality of concentrations of the compound.

[0113] In some embodiments, the methods described herein include contacting the compound with a cell composition comprising one or more condensates or a cell composition capable of forming one or more condensates. It will be readily appreciated by those skilled in the art that cellular processes, including the state of condensates and their components, are dynamic. Therefore, 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 method includes contacting the cell composition with a compound when the condensate-related molecule is at any position in the cell, in any amount, or with any post-translational modification state (such as the presence, absence, or level of phosphorylated residues). In some aspects, the method may also include contacting the cell with a compound, for example, when one or more condensates are at any position in the cell, in any amount (including absence), undergoing morphological changes (such as size or mobility changes, or composition changes).

[0114] In some embodiments, the cell composition comprises one or more coacervates prior to contact with the compound. In some embodiments, the method further comprises subjecting the cell composition to coacervate-forming conditions prior to contacting the compound with a cell composition comprising one or more coacervates or with a cell composition capable of forming one or more coacervates. In some embodiments, the cell composition does not comprise one or more coacervates prior to contact with the compound, and the method comprises subjecting the cell composition to coacervate-forming conditions to form the one or more coacervates. In some embodiments, the cell composition does not comprise one or more coacervates prior to contact with the compound, and the method comprises subjecting the cell composition to coacervate-forming conditions after contacting the cell composition with the compound to form the one or more coacervates. In some embodiments, the cell composition does not comprise one or more coacervates prior to contact with the compound, and one or more coacervates are formed while in contact with the compound. In some embodiments, the coacervates are formed both simultaneously and after addition of the compound. In some embodiments, the cell composition is subjected to coacervate-forming conditions prior to determining properties associated with the one or more coacervates. In some embodiments, the cell composition comprises one or more coacervates, and additional coacervates of the one or more coacervates are formed while contacting the cell composition with the compound. In some embodiments, the cell composition comprises one or more coacervates, and additional coacervates of the one or more coacervates are formed after contacting the cell composition with the compound. In some embodiments, the cell composition comprises one or more coacervates, and additional coacervates of the one or more coacervates are formed while and after contacting the cell composition with the compound.

[0115] Thus, the methods described herein include contacting a cell composition with a compound, wherein (i) the cell composition comprises one or more target coacervates; and / or (ii) the one or more target coacervates are formed while and / or after the cell composition is contacted 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 coacervates; and / or (ii) is capable of forming one or more coacervates, wherein the one or more coacervates are formed while and / or after the cell composition is contacted with the compound. In some embodiments, the methods described herein include contacting a compound with a cell composition comprising one or more coacervates or a cell composition capable of forming one or more coacervates.

[0116] The condensate-forming conditions may comprise adding a condensate-inducing agent. 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 hypertonic 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.

[0117] 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-associated molecules. In some embodiments, the second condensate is a condensate that contains condensate-associated molecules. In some embodiments, the first condensate and the second condensate are located in different parts of the cell composition. In some embodiments, the first condensate and the second condensate are located in different parts of the cell. In some embodiments, the first condensate and the second condensate are located 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 has not been contacted with the compound. In some embodiments, the reference is a cell composition that has not been treated with condensate-forming conditions. In some embodiments, the reference is a cell composition treated with a reference compound.

[0118] In some embodiments, a reference is prepared in a manner that allows for the evaluation of meaningful results for a compound. For example, in some embodiments, the reference is a cell composition, wherein the cell composition is prepared in a similar manner to the cell composition contacted with the compound, except that the reference cell composition is not subjected to the test compound or is not subjected to the same steps as the contact with the compound. In some embodiments, the reference is a cell composition contacted with a reference compound (e.g., a positive or negative control compound).

[0119] Properties related to condensates

[0120] In some embodiments, a property associated with one or more coacervates is determined based on any one or more of the following: (i) the number of coacervates that include and / or do not include coacervate-associated molecules; (ii) the size of the one or more coacervates; (iii) the location of the one or more coacervates; (iv) the distribution of the one or more coacervates; (v) the surface area of ​​the one or more coacervates; (vi) the composition of the one or more coacervates, (vii) the fluidity of the one or more coacervates; (viii) the coagulation of the one or more coacervates; (ix) the dissolution of the one or more coacervates; (x) the presence and / or amount of fiber formation; (xi) the location of the coacervate-associated molecules; (xii) the partitioning of the coacervate-associated molecules into the coacervates; and (xiii) the aggregation of the coacervate-associated molecules. In some embodiments, the method comprises determining a first property associated with one or more coacervates and a second property associated with one or more coacervates. In some embodiments, the method comprises determining a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth property associated with the one or more coacervates. Exemplary techniques that can be used to determine properties are disclosed in the Examples.

[0121] In some embodiments, the method comprises 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 comprising and / or not comprising a condensate-related molecule, 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 flowability of one or more condensates, (viii) the coagulation 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 a condensate-related molecule, (xii) the partitioning of the condensate-related molecule into the condensate, and (xiii) the aggregation of the condensate-related molecule. In some embodiments, the first property is (i) the number of condensates comprising and / or not comprising a condensate-related molecule, 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 comprising and / or not comprising a condensate-related molecule, 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 comprising and / or not comprising a condensate-related molecule, 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 comprising and / or not comprising a condensate-related molecule, 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 comprising and / or not comprising a condensate-related molecule, 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 comprising and / or not comprising a condensate-related molecule, and the second property is (vii) the flowability of the one or more condensates. In some embodiments, the first property is (i) the number of condensates comprising and / or not comprising a condensate-related molecule, and the second property is (viii) the coagulation of the one or more condensates. In some embodiments, the first property is (i) the number of condensates comprising and / or not comprising a condensate-related molecule, 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 comprising and / or not comprising a condensate-related molecule, 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 include and / or do not include 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 include and / or do not include 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 include and / or do not include condensate-associated molecules, and the second property is (xiii) the aggregation of the condensate-associated molecules.

[0122] In some embodiments, the method comprises determining a first property associated with the one or more coacervates and a second property associated with the one or more coacervates. In some embodiments, the first property is (ii) the size of the one or more coacervates, and the second property is any one (or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) of (iii) the location of the one or more coacervates, (iv) the distribution of the one or more coacervates, (v) the surface area of ​​the one or more coacervates, (vi) the composition of the one or more coacervates, (vii) the fluidity of the one or more coacervates, (viii) the coagulation of the one or more coacervates, (ix) the dissolution of the one or more coacervates, (x) the presence and / or amount of fiber formation, (xi) the location of the coacervate-associated molecules, (xii) the partitioning of the coacervate-associated molecules into the coacervate, and (xiii) the aggregation of the coacervate-associated molecules. In some embodiments, the first property is (ii) the size of the one or more coacervates, and the second property is (iii) the location of the one or more coacervates. In some embodiments, the first characteristic is (ii) the size of the one or more coacervates, and the second characteristic is (iv) the distribution of the one or more coacervates. In some embodiments, the first characteristic is (ii) the size of the one or more coacervates, and the second characteristic is (v) the surface area of ​​the one or more coacervates. In some embodiments, the first characteristic is (ii) the size of the one or more coacervates, and the second characteristic is (vi) the composition of the one or more coacervates. In some embodiments, the first characteristic is (ii) the size of the one or more coacervates, and the second characteristic is (vii) the fluidity of the one or more coacervates. In some embodiments, the first characteristic is (ii) the size of the one or more coacervates, and the second characteristic is (viii) the coagulation of the one or more coacervates. In some embodiments, the first characteristic is (ii) the size of the one or more coacervates, and the second characteristic is (ix) the dissolution of the one or more coacervates. In some embodiments, the first characteristic is (ii) the size of the one or more coacervates, and the second characteristic is (x) the presence and / or amount of fiber formation. In some embodiments, the first characteristic is (ii) the size of the one or more coacervates, and the second characteristic is (xi) the location of the molecules associated with the coacervates. 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.

[0123] 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 (iii) the location of the one or more condensates, and the second property is (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 flowability of the one or more condensates, (viii) the coagulation 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 condensate, and (xiii) the aggregation of the condensate-associated molecules (or more than one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10). 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 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 flowability 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 coagulation 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 condensate. 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.

[0124] In some embodiments, the method comprises determining a first property associated with the one or more coacervates and a second property associated with the one or more coacervates. In some embodiments, the first property is (iv) the distribution of the one or more coacervates, 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 coacervates, (vi) the composition of the one or more coacervates, (vii) the fluidity of the one or more coacervates, (viii) the coagulation of the one or more coacervates, (ix) the dissolution of the one or more coacervates, (x) the presence and / or amount of fiber formation, (xi) the location of the coacervate-associated molecules, (xii) the partitioning of the coacervate-associated molecules into the coacervate, and (xiii) the aggregation of the coacervate-associated molecules. In some embodiments, the first property is (iv) the distribution of the one or more coacervates, and the second property is (v) the surface area of ​​the one or more coacervates. In some embodiments, the first property is (iv) the distribution of the one or more coacervates, and the second property is (vi) the composition of the one or more coacervates. In some embodiments, the first characteristic is (iv) the distribution of one or more coagulants, and the second characteristic is (vii) the fluidity of the one or more coagulants. In some embodiments, the first characteristic is (iv) the distribution of one or more coagulants, and the second characteristic is (viii) the solidification of the one or more coagulants. In some embodiments, the first characteristic is (iv) the distribution of one or more coagulants, and the second characteristic is (ix) the dissolution of the one or more coagulants. In some embodiments, the first characteristic is (iv) the distribution of one or more coagulants, and the second characteristic is (x) the presence and / or amount of fiber formation. In some embodiments, the first characteristic is (iv) the distribution of one or more coagulants, and the second characteristic is (xi) the location of the coagulant-associated molecules. In some embodiments, the first characteristic is (iv) the distribution of one or more coagulants, and the second characteristic is (xii) the partitioning of the coagulant-associated molecules into the coagulants. In some embodiments, the first characteristic is (iv) the distribution of one or more coagulants, and the second characteristic is (xiii) the aggregation of the coagulant-associated molecules.

[0125] In some embodiments, the method comprises determining a first property associated with the one or more coacervates and a second property associated with the one or more coacervates. In some embodiments, the first property is (v) the surface area of ​​the one or more coacervates, 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 coacervates, (vii) the fluidity of the one or more coacervates, (viii) the coagulation of the one or more coacervates, (ix) the dissolution of the one or more coacervates, (x) the presence and / or amount of fiber formation, (xi) the location of the coacervate-associated molecules, (xii) the partitioning of the coacervate-associated molecules into the coacervate, and (xiii) the aggregation of the coacervate-associated molecules. In some embodiments, the first property is (v) the surface area of ​​the one or more coacervates, and the second property is (vi) the composition of the one or more coacervates. In some embodiments, the first property is (v) the surface area of ​​the one or more coacervates, and the second property is (vii) the fluidity of the one or more coacervates. In some embodiments, the first characteristic is (v) the surface area of ​​the one or more coagulants, and the second characteristic is (viii) the coagulation of the one or more coagulants. In some embodiments, the first characteristic is (v) the surface area of ​​the one or more coagulants, and the second characteristic is (ix) the dissolution of the one or more coagulants. In some embodiments, the first characteristic is (v) the surface area of ​​the one or more coagulants, and the second characteristic is (x) the presence and / or amount of fiber formation. In some embodiments, the first characteristic is (v) the surface area of ​​the one or more coagulants, and the second characteristic is (xi) the location of the coagulant-associated molecules. In some embodiments, the first characteristic is (v) the surface area of ​​the one or more coagulants, and the second characteristic is (xii) the partitioning of the coagulant-associated molecules into the coagulant. In some embodiments, the first characteristic is (v) the surface area of ​​the one or more coagulants, and the second characteristic is (xiii) the aggregation of the coagulant-associated molecules.

[0126] In some embodiments, the method comprises determining a first property associated with the one or more coacervates and a second property associated with the one or more coacervates. In some embodiments, the first property is (vi) the composition of the one or more coacervates, and the second property is any one (or more, e.g., 2, 3, 4, 5, 6, or 7) of (vii) the fluidity of the one or more coacervates, (viii) the coagulation of the one or more coacervates, (ix) the dissolution of the one or more coacervates, (x) the presence and / or amount of fiber formation, (xi) the location of the coacervate-associated molecules, (xii) the partitioning of the coacervate-associated molecules into the coacervate, and (xiii) the aggregation of the coacervate-associated molecules. In some embodiments, the first property is (vi) the composition of the one or more coacervates, and the second property is (vii) the fluidity of the one or more coacervates. In some embodiments, the first property is (vi) the composition of the one or more coacervates, and the second property is (viii) the coagulation of the one or more coacervates. In some embodiments, the first property is (vi) the composition of the one or more coacervates, and the second property is (ix) the dissolution of the one or more coacervates. In some embodiments, the first characteristic is (vi) the composition of the one or more coacervates, and the second characteristic is (x) the presence and / or amount of fiber formation. In some embodiments, the first characteristic is (vi) the composition of the one or more coacervates, and the second characteristic is (xi) the location of the coacervate-associated molecules. In some embodiments, the first characteristic is (vi) the composition of the one or more coacervates, and the second characteristic is (xii) the partitioning of the coacervate-associated molecules into the coacervates. In some embodiments, the first characteristic is (vi) the composition of the one or more coacervates, and the second characteristic is (xiii) the aggregation of the coacervate-associated molecules.

[0127] In some embodiments, the method comprises determining a first property associated with the one or more coacervates and a second property associated with the one or more coacervates. In some embodiments, the first property is (vii) fluidity of the one or more coacervates, and the second property is any one (or more, such as 2, 3, 4, 5, or 6) of (viii) coagulation of the one or more coacervates, (ix) dissolution of the one or more coacervates, (x) the presence and / or amount of fiber formation, (xi) the location of the coacervate-associated molecules, (xii) the partitioning of the coacervate-associated molecules into the coacervate, and (xiii) the aggregation of the coacervate-associated molecules. In some embodiments, the first property is (vii) fluidity of the one or more coacervates, and the second property is (viii) coagulation of the one or more coacervates. In some embodiments, the first property is (vii) fluidity of the one or more coacervates, and the second property is (ix) dissolution of the one or more coacervates. In some embodiments, the first property is (vii) fluidity of the one or more coacervates, and the second property is (x) the presence and / or amount of fiber formation. In some embodiments, the first property is (vii) the mobility 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 (vii) the mobility of the one or more condensates, and the second property is (xii) the partitioning of the condensate-associated molecules into the condensate. In some embodiments, the first property is (vii) the mobility of the one or more condensates, and the second property is (xiii) the aggregation of the condensate-associated molecules.

[0128] In some embodiments, the method comprises determining a first property associated with the one or more coacervates and a second property associated with the one or more coacervates. In some embodiments, the first property is (viii) coagulation of the one or more coacervates, and the second property is any one (or more, e.g., 2, 3, 4, or 5) of (ix) dissolution of the one or more coacervates, (x) the presence and / or amount of fiber formation, (xi) the location of the coacervate-associated molecules, (xii) the partitioning of the coacervate-associated molecules into the coacervate, and (xiii) the aggregation of the coacervate-associated molecules. In some embodiments, the first property is (viii) coagulation of the one or more coacervates, and the second property is (ix) dissolution of the one or more coacervates. In some embodiments, the first property is (viii) coagulation of the one or more coacervates, and the second property is (x) the presence and / or amount of fiber formation. In some embodiments, the first property is (viii) coagulation of the one or more coacervates, and the second property is (xi) the location of the coacervate-associated molecules. In some embodiments, the first property is (viii) solidification of the one or more coacervates, and the second property is (xii) partitioning of the coacervate-associated molecules into the coacervates. In some embodiments, the first property is (viii) solidification of the one or more coacervates, and the second property is (xiii) aggregation of the coacervate-associated molecules.

[0129] In some embodiments, the method comprises determining a first property associated with the one or more coacervates and a second property associated with the one or more coacervates. In some embodiments, the first property is (ix) dissolution of the one or more coacervates, and the second property is any one (or more, e.g., 2, 3, or 4) of (x) the presence and / or amount of fiber formation, (xi) the location of the coacervate-associated molecules, (xii) the partitioning of the coacervate-associated molecules into the coacervate, and (xiii) the aggregation of the coacervate-associated molecules. In some embodiments, the first property is (ix) dissolution of the one or more coacervates, and the second property is (x) the presence and / or amount of fiber formation. In some embodiments, the first property is (ix) dissolution of the one or more coacervates, and the second property is (xi) the location of the coacervate-associated molecules. In some embodiments, the first property is (ix) dissolution of the one or more coacervates, and the second property is (xii) the partitioning of the coacervate-associated molecules into the coacervate. In some embodiments, the first property is (ix) dissolution of the one or more coacervates, and the second property is (xiii) the aggregation of the coacervate-associated molecules.

[0130] In some embodiments, the method comprises determining a first property associated with the one or more coacervates and a second property associated with the one or more coacervates. 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, e.g., 2 or 3) of (xi) the location of the coacervate-associated molecules, (xii) the partitioning of the coacervate-associated molecules into the coacervate, and (xiii) the aggregation of the coacervate-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 coacervate-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 coacervate-associated molecules into the coacervate. 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 coacervate-associated molecules.

[0131] 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 (xii) the partitioning of the condensate-associated molecules into the condensate 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 condensate.

[0132] 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) partitioning of the condensate-associated molecules into the condensate, and the second property is (xiii) aggregation of the condensate-associated molecules.

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

[0134] In some embodiments, the compound modulates (i) the number of coacervates that include and / or do not include the coacervate-associated molecule. In some embodiments, the compound modulates (ii) the size of the one or more coacervates. In some embodiments, the compound modulates (iii) the location of the one or more coacervates. In some embodiments, the compound modulates (iv) the distribution of the one or more coacervates. In some embodiments, the compound modulates (v) the surface area of ​​the one or more coacervates. In some embodiments, the compound modulates (vi) the composition of the one or more coacervates. In some embodiments, the compound modulates (vii) the fluidity of the one or more coacervates. In some embodiments, the compound modulates (viii) the coagulation of the one or more coacervates. In some embodiments, the compound modulates (ix) the dissolution of the one or more coacervates. 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 coacervate-associated molecules. In some embodiments, the compound modulates (xii) the partitioning of the coacervate-associated molecules into the coacervates. In some embodiments, the compound modulates (xiii) the aggregation of the coacervate-associated molecules.

[0135] 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) properties selected from: (i) the number of coacervates that contain and / or do not contain the coacervate-associated molecule; (ii) the size of the one or more coacervates; (iii) the location of the one or more coacervates; (iv) the distribution of one or more coacervates; (v) the surface area of ​​the one or more coacervates; (vi) the composition of the one or more coacervates; (vii) the fluidity of the one or more coacervates; (viii) the coagulation of the one or more coacervates; (ix) the dissolution of the one or more coacervates; (x) the presence and / or amount of fiber formation; (xi) the location of the coacervate-associated molecules; (xii) the partitioning of the coacervate-associated molecules into the coacervates; and (xiii) the aggregation of the coacervate-associated molecules.

[0136] In some embodiments, the compound does not modulate (i) the number of coacervates that include and / or do not include the coacervate-associated molecule. In some embodiments, the compound does not modulate (ii) the size of the one or more coacervates. In some embodiments, the compound does not modulate (iii) the location of the one or more coacervates. In some embodiments, the compound does not modulate (iv) the distribution of one or more coacervates. In some embodiments, the compound does not modulate (v) the surface area of ​​the one or more coacervates. In some embodiments, the compound does not modulate (vi) the composition of the one or more coacervates. In some embodiments, the compound does not modulate (vii) the fluidity of the one or more coacervates. In some embodiments, the compound does not modulate (viii) the coagulation of the one or more coacervates. In some embodiments, the compound does not modulate (ix) the dissolution of the one or more coacervates. 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 coacervate-associated molecules. In some embodiments, the compound does not modulate (xii) the partitioning of the coacervate-associated molecules into the coacervates. In some embodiments, the compound does not modulate (xiii) aggregation of the condensate-associated molecules.

[0137] In some embodiments, the compound modulates one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) properties selected from the group consisting of: (i) the number of condensates comprising and / or not comprising 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 fluidity of the one or more condensates; (viii) the coagulation 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.

[0138] In some embodiments, the compound modulates a first property related to one or more condensates and does not modulate a second property related to the one or more condensates, wherein the first property is different from the second property, and wherein the properties are selected from the group consisting of (i) the number of condensates comprising and / or not comprising 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 fluidity of the one or more condensates; (viii) the coagulation 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.

[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: (i) the number of condensates comprising and / or not comprising 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 fluidity of the one or more condensates; (viii) the coagulation 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 segregation of the condensate-associated molecule into condensates in a first portion of the cellular composition (e.g., a first portion of the cells), and does not modulate one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) of the following: (i) the number of condensates comprising and / or not comprising 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 fluidity of the one or more condensates; (viii) the coagulation 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 segregation of the condensate-associated molecule into condensates in a second portion of the cellular composition (e.g., a second portion of the cells).

[0140] In some embodiments, the compound modulates one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) properties selected from the group consisting of: (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 fluidity of the one or more condensates; (viii) the coagulation 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 condensation of the condensate-associated molecule into 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 the group consisting of: (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 fluidity of the one or more condensates; (viii) the coagulation 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; and (xii) the partitioning of the condensate-associated molecule into the condensate in the cell nucleus.

[0141] In some embodiments, the compound modulates one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) properties selected from the group consisting of: (i) the number of coacervates that contain and / or do not contain the coacervate-associated molecule; (ii) the size of the one or more coacervates; (iii) the location of the one or more coacervates; (iv) the distribution of the one or more coacervates; (v) the surface area of ​​the one or more coacervates; (vi) the composition of the one or more coacervates; (vii) the fluidity of the one or more coacervates; (viii) the coagulation of the one or more coacervates; (ix) the dissolution of the one or more coacervates; (x) the presence and / or amount of fiber formation; (xi) the location of the coacervate-associated molecule; (xii) the orientation of the coacervate-associated molecule toward the coacervate of the first group of one or more coacervates. and does not regulate one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) properties selected from the group consisting of: (i) the number of coacervates that contain and / or do not contain the coacervate-associated molecules; (ii) the size of the one or more coacervates; (iii) the location of the one or more coacervates; (iv) the distribution of the one or more coacervates; (v) the surface area of ​​the one or more coacervates; (vi) the composition of the one or more coacervates; (vii) the fluidity of the one or more coacervates; (viii) the coagulation of the one or more coacervates; (ix) the dissolution of the one or more coacervates; (x) the presence and / or amount of fiber formation; (xi) the location of the coacervate-associated molecules; and (xii) the partitioning of the coacervate-associated molecules into coacervates for a second group of one or more coacervates.

[0142] In some embodiments, the compound modulates one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) properties selected from the group consisting of: (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 fluidity of the one or more condensates; (viii) the coagulation 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 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) characteristics of the present invention include: (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 fluidity of the one or more condensates; (viii) the coagulation 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; and (xii) the partitioning of the condensate-associated molecule into one or more of the following condensates: paranuclear spots, condensates formed around sites of DNA damage, P bodies, Cajal bodies, and PML bodies.

[0143] In some embodiments, the method is a method for identifying a compound that modulates one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or all) properties selected from the group consisting of: (i) the number of coacervates that contain and / or do not contain the coacervate-associated molecule; (ii) the size of the one or more coacervates; (iii) the location of the one or more coacervates; (iv) the distribution of the one or more coacervates; (v) the surface area of ​​the one or more coacervates; (vi) the composition of the one or more coacervates; (vii) the fluidity of the one or more coacervates; (viii) the coagulation of the one or more coacervates; (ix) the dissolution of the one or more coacervates; (x) the presence and / or amount of fiber formation; (xi) the location of the coacervate-associated molecules; (xii) the partitioning of the coacervate-associated molecules into the coacervates; and (xiii) the aggregation of the coacervate-associated molecules.

[0144] In some embodiments, the method is a method for identifying a compound that modulates (i) the number of coacervates that include and / or do not include the coacervate-associated molecule. In some embodiments, the method is a method for identifying a compound that modulates (ii) the size of one or more coacervates. In some embodiments, the method is a method for identifying a compound that modulates (iii) the location of one or more coacervates. In some embodiments, the method is a method for identifying a compound that modulates (iv) the distribution of one or more coacervates. In some embodiments, the method is a method for identifying a compound that modulates (v) the surface area of ​​one or more coacervates. In some embodiments, the method is a method for identifying a compound that modulates (vi) the composition of one or more coacervates. In some embodiments, the method is a method for identifying a compound that modulates (vii) the fluidity of one or more coacervates. In some embodiments, the method is a method for identifying a compound that modulates (viii) the coagulation of one or more coacervates. In some embodiments, the method is a method for identifying a compound that modulates (ix) the solubility of one or more coacervates. In some embodiments, the method is a method for identifying a compound that modulates (x) the presence and / or amount of fiber formation. In some embodiments, the method is a method for identifying a compound that modulates (xi) the location of the condensate-associated molecule. In some embodiments, the method is a method for identifying a compound that modulates (xii) the partitioning of the condensate-associated molecule into the condensate. In some embodiments, the method is a method for identifying a compound that modulates (xiii) the aggregation of the condensate-associated molecule.

[0145] 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) properties selected from the group consisting of: (i) the number of coacervates that contain and / or do not contain the coacervate-associated molecule; (ii) the size of the one or more coacervates; (iii) the location of the one or more coacervates; (iv) the distribution of the one or more coacervates; (v) the surface area of ​​the one or more coacervates; (vi) the composition of the one or more coacervates; (vii) the fluidity of the one or more coacervates; (viii) the coagulation of the one or more coacervates; (ix) the dissolution of the one or more coacervates; (x) the presence and / or amount of fiber formation; (xi) the location of the coacervate-associated molecules; (xii) the partitioning of the coacervate-associated molecules into the coacervates; and (xiii) the aggregation of the coacervate-associated molecules.

[0146] In some embodiments, the compound does not modulate (i) the number of coacervates that include and / or do not include the coacervate-associated molecule. In some embodiments, the compound does not modulate (ii) the size of the one or more coacervates. In some embodiments, the compound does not modulate (iii) the location of the one or more coacervates. In some embodiments, the compound does not modulate (iv) the distribution of one or more coacervates. In some embodiments, the compound does not modulate (v) the surface area of ​​the one or more coacervates. In some embodiments, the compound does not modulate (vi) the composition of the one or more coacervates. In some embodiments, the compound does not modulate (vii) the fluidity of the one or more coacervates. In some embodiments, the compound does not modulate (viii) the coagulation of the one or more coacervates. In some embodiments, the compound does not modulate (ix) the dissolution of the one or more coacervates. 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 coacervate-associated molecules. In some embodiments, the compound does not modulate (xii) the partitioning of the coacervate-associated molecules into the coacervates. In some embodiments, the compound does not modulate (xiii) aggregation of the condensate-associated molecules.

[0147] In some embodiments, the method is a method for 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 coacervates that contain and / or do not contain the coacervate-associated molecule; (ii) the size of the one or more coacervates; (iii) the location of the one or more coacervates; (iv) the distribution of the one or more coacervates; (v) the surface area of ​​the one or more coacervates; (vi) the composition of the one or more coacervates; (vii) the fluidity of the one or more coacervates; (viii) the coagulation of the one or more coacervates; (ix) the dissolution of the one or more coacervates; (x) the presence and / or amount of fiber formation; (xi) the location of the coacervate-associated molecules; (xii) the partitioning of the coacervate-associated molecules into the coacervates; and (xiii) the aggregation of the coacervate-associated molecules, and does not modulate another of the thirteen properties.

[0148] 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 group consisting of: (i) the number of coacervates that contain and / or do not contain the coacervate-associated molecule; (ii) the size of the one or more coacervates; (iii) the location of the one or more coacervates; (iv) the distribution of the one or more coacervates; (v) the surface area of ​​the one or more coacervates; (vi) the composition of the one or more coacervates; (vii) the fluidity of the one or more coacervates; (viii) the coagulation of the one or more coacervates; (ix) the dissolution of the one or more coacervates; (x) the presence and / or amount of fibril formation; (xi) the location of the coacervate-associated molecule; (xii) the delivery of the coacervate-associated molecule to the first portion of the cell composition (e.g., The invention relates to a method for regulating the partitioning of aggregates into aggregates in a first portion of the cell composition (e.g., a first portion of the cell composition) without regulating 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 aggregates that contain and / or do not contain the aggregate-associated molecule; (ii) the size of the one or more aggregates; (iii) the location of the one or more aggregates; (iv) the distribution of the one or more aggregates; (v) the surface area of ​​the one or more aggregates; (vi) the composition of the one or more aggregates; (vii) the fluidity of the one or more aggregates; (viii) the coagulation of the one or more aggregates; (ix) the dissolution of the one or more aggregates; (x) the presence and / or amount of fiber formation; (xi) the location of the aggregate-associated molecule; and (xii) the partitioning of the aggregate-associated molecule into aggregates in a second portion of the cell composition (e.g., a second portion of the cell composition).

[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 the group consisting of: (i) the number of coacervates that contain and / or do not contain the coacervate-associated molecule; (ii) the size of the one or more coacervates; (iii) the location of the one or more coacervates; (iv) the distribution of the one or more coacervates; (v) the surface area of ​​the one or more coacervates; (vi) the composition of the one or more coacervates; (vii) the fluidity of the one or more coacervates; (viii) the coagulation of the one or more coacervates; (ix) the dissolution of the one or more coacervates; (x) the presence and / or amount of fiber formation; (xi) the location of the coacervate-associated molecule; (xii) the orientation of the coacervate-associated molecule toward Compartmentalization within condensates in the cytoplasm, and without regulating 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 fluidity of the one or more condensates; (viii) the coagulation 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; and (xii) the partitioning of the condensate-associated molecules into condensates in the cell nucleus.

[0150] 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 the group consisting of: (i) the number of coacervates that contain and / or do not contain the coacervate-associated molecule; (ii) the size of the one or more coacervates; (iii) the location of the one or more coacervates; (iv) the distribution of the one or more coacervates; (v) the surface area of ​​the one or more coacervates; (vi) the composition of the one or more coacervates; (vii) the fluidity of the one or more coacervates; (viii) the coagulation of the one or more coacervates; (ix) the dissolution of the one or more coacervates; (x) the presence and / or amount of fiber formation; (xi) the location of the coacervate-associated molecule; (xii) the direction of the coacervate-associated molecule to the first group of one or more and / or does not adjust one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) properties selected from the group consisting of: (i) the number of coacervates that contain and / or do not contain the coacervate-associated molecules; (ii) the size of the one or more coacervates; (iii) the location of the one or more coacervates; (iv) the distribution of the one or more coacervates; (v) the surface area of ​​the one or more coacervates; (vi) the composition of the one or more coacervates; (vii) the fluidity of the one or more coacervates; (viii) the coagulation of the one or more coacervates; (ix) the dissolution of the one or more coacervates; (x) the presence and / or amount of fiber formation; (xi) the location of the coacervate-associated molecules; and (xii) the partitioning of the coacervate-associated molecules into coacervates for a second group of one or more coacervates.

[0151] 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 the group consisting of: (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 fluidity of the one or more condensates; (viii) the coagulation 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 in one or more stress granules, and does not Modulating 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 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 coagulation 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; and (xii) the partitioning of the condensate-associated molecule into condensates for one or more of paranuclear spots, condensates formed around sites of DNA damage, P bodies, Cajal bodies, and PML bodies.

[0152] In some embodiments, the assay is performed within about 60 days of contacting 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 after about 5 seconds of contacting 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.

[0153] In some embodiments, the method comprises measuring the property before and after exposure to the compound. In some embodiments, the method further comprises comparing the property before and after exposure to the compound.

[0154] In some embodiments, described method further comprises the determination step of repeating described method.For example, in some embodiments, described method comprises the determination step of repeating described method at least about 2,3,4,5,10 or more times.In some embodiments, described method comprises the second part (such as the first cell and the second cell in the cell composition) of the first part of described cell composition and described cell composition is carried out described determination step.In some embodiments, described method comprises the third, fourth, fifth, sixth or more parts (such as the third, fourth, fifth, sixth or more cells in the cell composition) of described cell composition is carried out described determination step.In some embodiments, described method comprises the first part of the cell in described cell composition and the second part of the cell in described cell composition is carried out described determination step, for example, in cytoplasm and in nucleus, or in the first organelle and the second organelle.In some embodiments, described method comprises the third, fourth, fifth, sixth or more parts of the cell in described cell composition is carried out described determination step.

[0155] In some embodiments, the determining 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 determining step is based on the same characteristic when repeated. In some embodiments, the method further comprises comparing the characteristic over time, for example, comparing the number of aggregates between two measurements taken one day apart.

[0156] 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, brightfield microscopy, polarizing 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 manual or automatic methods, and can be performed, for example, from an image or directly from a 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 molecule comprises a fluorescent marker, 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 marker. In some embodiments, the marker is a labeled binding molecule, such as an antibody (e.g., a labeled secondary antibody) or a biotin-binding protein. In some embodiments, the marker is a stain, such as a stain specific for an organelle.

[0157] 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 a DNA damaging condition. In some embodiments, the DNA damaging condition is laser irradiation.

[0158] Can measure part or all of the characteristic of cell composition.Therefore, in some embodiments, described method comprises the characteristic of measuring a part of cell composition.In some embodiments, described method comprises the characteristic of measuring whole cell composition.In some embodiments, described method comprises the characteristic of measuring one or more cells in cell composition.In some embodiments, described method comprises the characteristic of measuring single cell in cell composition.

[0159] The properties of some or all cells in a 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 a cell composition. In some embodiments, the method comprises determining the properties of a portion of a single cell in a cell composition. In some embodiments, the method comprises determining the properties of the cytoplasm in a cell composition. In some embodiments, the method comprises determining the properties of the nucleus in a cell composition. In some embodiments, the method comprises determining the properties of an organelle.

[0160] The number of condensates

[0161] In some embodiments, the property associated with the one or more condensates is based on the number of condensates that include and / or do not include condensate-associated molecules. In some embodiments, the property associated with the one or more condensates is based on the number of condensates that include condensate-associated molecules. In some embodiments, the property associated with the one or more condensates is based on the number of condensates that do not include condensate-associated molecules. Thus, in some embodiments, provided herein is a method for identifying a compound that modulates a property associated with one or more condensates that include condensate-associated molecules, the method comprising: (a) contacting the compound with a cell composition that includes one or more condensates or with a cell composition that is 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 include and / or do not include the condensate-associated molecules. In some embodiments, provided herein is a method for identifying a compound that modulates the number of aggregates that include and / or do not include the aggregate-associated molecule, the method comprising: (a) contacting the compound with a cell composition that includes one or more aggregates or with a cell composition that is capable of forming one or more aggregates, and (b) measuring the number of aggregates that include and / or do not include the aggregate-associated molecule, wherein a modulation of the number of aggregates compared to a reference indicates that the compound modulates the number of aggregates that include and / or do not include the aggregate-associated molecule.

[0162] The number of aggregates can be determined for a portion or all of a cell composition. Thus, in some embodiments, the method comprises determining the number of aggregates that include and / or do not include aggregate-associated molecules in a portion of a cell composition. In some embodiments, the method comprises determining the number of aggregates that include and / or do not include aggregate-associated molecules in the entire cell composition. In some embodiments, the method comprises determining the number of aggregates that include and / or do not include aggregate-associated molecules in one or more cells in the cell composition. In some embodiments, the method comprises determining the number of aggregates that include and / or do not include aggregate-associated molecules in a single cell in the cell composition.

[0163] The number of aggregates of a portion or all of the cells in the cell composition can also be determined. Thus, in some embodiments, the method comprises determining the number of aggregates that contain and / or do not contain aggregate-associated molecules in a portion of one or more cells in the cell composition. In some embodiments, the method comprises determining the number of aggregates that contain and / or do not contain aggregate-associated molecules in a portion of a single cell in the cell composition. In some embodiments, the method comprises determining the number of aggregates that contain and / or do not contain aggregate-associated molecules in the cytoplasm. In some embodiments, the method comprises determining the number of aggregates that contain and / or do not contain aggregate-associated molecules in the nucleus. In some embodiments, the method comprises determining the number of aggregates that contain and / or do not contain aggregate-associated molecules in an organelle.

[0164] In some embodiments, the method comprises determining the number of aggregates comprising aggregate-associated molecules; determining the number of aggregates not comprising aggregate-associated molecules; and comparing the number of aggregates comprising aggregate-associated molecules to the number of aggregate-associated molecules. In some embodiments, the method comprises determining the number of aggregates comprising and / or not comprising aggregate-associated molecules in a first portion of a cell composition (e.g., any portion of a cell composition disclosed herein); determining the number of aggregates comprising and / or not comprising aggregate-associated molecules in a second portion of a cell composition (e.g., any portion of a cell composition disclosed herein); and comparing the number of aggregates comprising and / or not comprising aggregate-associated molecules in the first portion to the number of aggregates comprising and / or not comprising aggregate-associated molecules in the second portion. In some embodiments, the method comprises determining the number of aggregates comprising aggregate-associated molecules in the cytoplasm of a cell composition; determining the number of aggregates comprising aggregate-associated molecules in the nucleus of a cell composition; and comparing the number of aggregates comprising aggregate-associated molecules in the cytoplasm to the number of aggregates in the nucleus.

[0165] In some embodiments, the number of coacervates comprising coacervate-associated molecules is increased compared to a reference. In some embodiments, the number of coacervates comprising coacervate-associated molecules is decreased compared to a reference. In some embodiments, the number of coacervates not comprising coacervate-associated molecules is increased compared to a reference. In some embodiments, the number of coacervates not comprising coacervate-associated molecules is decreased compared to a reference.

[0166] In some embodiments, the number of aggregates that include and / or do not include aggregate-associated molecules in the first portion of the cell composition is reduced compared to a reference, and the number of aggregates that include and / or do not include aggregate-associated molecules in the second portion of the cell composition is not reduced compared to the reference. In some embodiments, the number of aggregates that include and / or do not include aggregate-associated molecules in the first portion of the cell composition is increased compared to the reference, and the number of aggregates that include and / or do not include aggregate-associated 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 number of aggregates that include aggregate-associated molecules in the cytoplasm is reduced compared to the reference, and the number of aggregates that include aggregate-associated molecules in the nucleus is not reduced compared to the reference. In some embodiments, the number of aggregates that include aggregate-associated molecules in the cytoplasm is reduced compared to the reference, and the number of aggregates that include aggregate-associated molecules in the nucleus is increased compared to the reference.

[0167] In some embodiments, the number of aggregates that include and / or do not include aggregate-associated molecules is increased in the first portion of the cell composition compared to the second portion of the cell composition, or the number of aggregates that include and / or do not include aggregate-associated molecules is increased in the second portion of the cell composition 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.

[0168] Size of aggregates

[0169] 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 for identifying a compound that modulates a property associated with one or more condensates comprising a condensate-associated molecule, 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 size of the one or more condensates. In some embodiments, provided herein are methods for 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 compared to a reference indicates that the compound modulates the size of the one or more condensates. In some embodiments, the size is an average of two or more condensates.

[0170] The size of aggregates can be determined for a portion or all of a cell composition. Thus, in some embodiments, the method comprises determining the size of one or more aggregates in a portion of the cell composition. In some embodiments, the method comprises determining the size of one or more aggregates in the entire cell composition. In some embodiments, the method comprises determining the size of one or more aggregates in one or more cells in the cell composition. In some embodiments, the method comprises determining the size of one or more aggregates in a single cell in the cell composition.

[0171] The size of aggregates of some or all cells in a cell composition can also be determined. Thus, in some embodiments, the method comprises determining the size of one or more aggregates in a portion of one or more cells in a cell composition. In some embodiments, the method comprises determining the size of one or more aggregates in a portion of a single cell in a cell composition. In some embodiments, the method comprises determining the size of one or more aggregates in the cytoplasm. In some embodiments, the method comprises determining the size of one or more aggregates in the nucleus. In some embodiments, the method comprises determining the size of one or more aggregates in an organelle.

[0172] In some embodiments, the one or more coacervates are increased in size compared to a reference. In some embodiments, the one or more coacervates are decreased in size compared to a reference.

[0173] In some embodiments, the size of one or more aggregates in the first portion of the cell composition is reduced compared to a reference, and the size of one or more aggregates in the second portion of the cell composition is not reduced compared to the reference. In some embodiments, the size of one or more aggregates in the first portion of the cell composition is increased compared to the reference, and the size of one or more aggregates 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 aggregates in the cytoplasm is reduced compared to the reference, and the size of one or more aggregates in the nucleus is not reduced compared to the reference. In some embodiments, the size of one or more aggregates in the cytoplasm is reduced compared to the reference, and the size of one or more aggregates in the nucleus is increased compared to the reference.

[0174] In some embodiments, the size of one or more aggregates 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 aggregates 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.

[0175] Location of condensate

[0176] In some embodiments, the property associated with the one or more condensates is based on the location of the one or more condensates. Thus, in some embodiments, provided herein are methods for identifying a compound that modulates a property associated with one or more condensates comprising a condensate-associated molecule, 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 location of the one or more condensates. In some embodiments, provided herein are methods for 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 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 compared to a reference indicates that the compound modulates the location of the one or more condensates.

[0177] The location of the aggregates 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 aggregates in a portion of the cell composition. In some embodiments, the method comprises determining the location of one or more aggregates in the entire cell composition. In some embodiments, the method comprises determining the location of one or more aggregates in one or more cells in the cell composition. In some embodiments, the method comprises determining the location of one or more aggregates in a single cell in the cell composition.

[0178] The location of condensates can also be determined for a portion or all of the cells in a 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 in the cell composition. In some embodiments, the method comprises determining the location of one or more condensates in a portion of a single cell in 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.

[0179] Distribution of condensates

[0180] In some embodiments, the property associated with the one or more condensates is 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 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 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 the property associated with the one or more condensates, wherein the property is modulation of the distribution of one or more condensates. In some embodiments, provided herein are methods for 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 one or more condensates, wherein modulation of the distribution compared to a reference indicates that the compound modulates the distribution of one or more condensates.

[0181] In some embodiments, the distribution of one or more condensates is based on the number of condensates containing condensate-related molecules in a cellular location (such as any one or more of the cytosol, nucleus, organelles, or any part thereof). In some embodiments, when the number of condensates containing condensate-related 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) is compared, the number of condensates containing condensate-related molecules in the cellular location (such as the cytosol or nucleus) is reduced, the distribution of one or more condensates is determined to be regulated by the compound. In some embodiments, when the number of condensates containing condensate-related 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) is compared, the number of condensates containing condensate-related molecules in the cellular location (such as the cytosol or nucleus) is increased, the distribution of one or more condensates is determined to be regulated by the compound.

[0182] In some embodiments, the distribution of one or more condensates is based on the number of condensates that contain condensate-associated molecules in one or more portions of a cell composition (e.g., one or more fields of view or one or more portions of an image of a cell composition). In some embodiments, when the number of condensates that contain 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) decreases, the distribution of one or more condensates is determined to be modulated by the compound. In some embodiments, when the number of condensates that contain condensate-associated molecules in one or more portions of a 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) increases, the distribution of one or more condensates is determined to be modulated by the compound.

[0183] In some embodiments, the distribution of condensates is determined by calculating the ratio (e.g., percentage, thousandth, etc.) of the condensates in the first part of the cell composition to the condensates in the second part of the cell composition. In some embodiments, the method comprises measuring the number of condensates comprising condensate-related molecules in the first part of the cell composition, measuring the number of condensates comprising condensate-related molecules in the second part of the cell composition, and calculating the ratio of the number of condensates comprising condensate-related molecules in the first and second parts of the cell composition. In some embodiments, the first part of the cell composition is the first part of the cell and the second part of the cell, such as the cytoplasm, the nucleus, or the organelle. In some embodiments, the first part of the cell composition is the cytoplasm and the second part is the nucleus. In some embodiments, compared to a reference (e.g., a cell composition not in contact with the compound or a cell composition in contact with a control compound), the ratio increases. In some embodiments, compared to a reference, the ratio decreases. In some embodiments, compared to a reference, the ratio of nuclei to cytoplasmic condensates increases. In some embodiments, compared to a reference, the ratio of nuclei to cytoplasmic condensates decreases. In some embodiments, the distribution of one or more aggregates is determined to be modulated by the compound when the ratio of aggregates in a first portion of the cell composition (e.g., nuclei) to a second portion of the cell composition (e.g., cytosol) increases compared to a reference. In some embodiments, the distribution of one or more aggregates is determined to be modulated by the compound when the ratio of aggregates in a first portion of the cell composition (e.g., nuclei) to a second portion of the cell composition (e.g., cytosol) decreases compared to a reference.

[0184] In some embodiments, imaging techniques are used to determine the distribution of one or more aggregates.

[0185] Surface area of ​​condensate

[0186] 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 for identifying a compound that modulates a property associated with one or more condensates comprising a condensate-associated molecule, 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 surface area of ​​the one or more condensates. In some embodiments, provided herein are methods for 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 with 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 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.

[0187] The surface area of ​​a portion or all of the aggregates in a cell composition can be measured. Thus, in some embodiments, the method comprises measuring the surface area of ​​one or more aggregates in a portion of the cell composition. In some embodiments, the method comprises measuring the surface area of ​​one or more aggregates in the entire cell composition. In some embodiments, the method comprises measuring the surface area of ​​one or more aggregates in one or more cells in the cell composition. In some embodiments, the method comprises measuring the surface area of ​​one or more aggregates in a single cell in the cell composition.

[0188] The surface area of ​​aggregates of a portion or all of the cells in a cell composition can also be determined. Thus, in some embodiments, the method comprises determining the surface area of ​​one or more aggregates in a portion of one or more cells in a cell composition. In some embodiments, the method comprises determining the surface area of ​​one or more aggregates in a portion of a single cell in a cell composition. In some embodiments, the method comprises determining the surface area of ​​one or more aggregates in the cytoplasm. In some embodiments, the method comprises determining the surface area of ​​one or more aggregates in the nucleus. In some embodiments, the method comprises determining the surface area of ​​one or more aggregates in an organelle.

[0189] In some embodiments, the surface area of ​​the one or more coacervates is increased compared to a reference. In some embodiments, the surface area of ​​the one or more coacervates is decreased compared to a reference.

[0190] In some embodiments, the surface area of ​​the one or more aggregates in the first portion of the cell composition is reduced compared to a reference, and the surface area of ​​the one or more aggregates in the second portion of the cell composition is not reduced compared to the reference. In some embodiments, the surface area of ​​the one or more aggregates in the first portion of the cell composition is increased compared to the reference, and the surface area of ​​the one or more aggregates 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 aggregates in the cytoplasm is reduced compared to the reference, and the surface area of ​​the one or more aggregates in the nucleus is not reduced compared to the reference. In some embodiments, the surface area of ​​the one or more aggregates in the cytoplasm is reduced compared to the reference, and the surface area of ​​the one or more aggregates in the nucleus is increased compared to the reference.

[0191] In some embodiments, the surface area of ​​the one or more aggregates is increased in the first portion of the cell composition compared to the second portion of the cell composition, or the surface area of ​​the one or more aggregates is increased in the second portion of the cell composition 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.

[0192] Composition of condensates

[0193] In some embodiments, the property associated with the one or more condensates is based on the composition of the one or more condensates. Thus, in some embodiments, provided herein are methods for identifying a compound that modulates a property associated with one or more condensates comprising a condensate-associated molecule, 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 composition of the one or more condensates. In some embodiments, provided herein are methods for 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 with 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 compared to a reference indicates that the compound modulates the composition of the one or more condensates.

[0194] In some embodiments, the composition of the coacervate is determined by detecting the presence of one or more macromolecules contained in the coacervate, measuring the amount of one or more macromolecules contained in the coacervate, calculating the ratio of one macromolecule to a second macromolecule contained in the coacervate, and / or comparing the amount of one or more macromolecules contained in the coacervate with the size and / or surface area of ​​the coacervate. In some embodiments, the composition of the coacervate is determined by detecting the presence of one or more macromolecules (e.g., 1, 2, 3, 4, 5, 6, 7 or more macromolecules) contained in the coacervate. In some embodiments, the composition of the coacervate is determined by measuring the amount of one or more macromolecules (e.g., 1, 2, 3, 4, 5, 6, 7 or more macromolecules) contained in the coacervate. In some embodiments, the composition of the coacervate is determined by calculating the ratio of one macromolecule to a second macromolecule contained in the coacervate. In some embodiments, the composition of the coacervate is determined by comparing the amount of one or more macromolecules contained in the coacervate with the size and / or surface area of ​​the coacervate, e.g., 1, 2, 3, 4, 5, 6, 7 or more macromolecules. In some embodiments, the macromolecule is a coacervate-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.

[0195] The composition of the condensate of a portion or all of the cell composition can be determined. Thus, in some embodiments, the method comprises determining the composition of one or more condensates in a portion of the cell composition. In some embodiments, the method comprises determining the composition of one or more condensates in the entire cell composition. In some embodiments, the method comprises determining the composition of one or more condensates in one or more cells in the cell composition. In some embodiments, the method comprises determining the composition of one or more condensates in a single cell in the cell composition.

[0196] The composition of the condensate of a portion or all of the cell composition can be determined. Thus, in some embodiments, the method comprises determining the composition of one or more condensates in a portion of the cell composition. In some embodiments, the method comprises determining the composition of one or more condensates in the entire cell composition. In some embodiments, the method comprises determining the composition of one or more condensates in one or more cells in the cell composition. In some embodiments, the method comprises determining the composition of one or more condensates in a single cell in the cell composition.

[0197] Fluidity of condensates

[0198] In some embodiments, the property related to the one or more condensates is based on the fluidity 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 molecules associated with 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 property related to the one or more condensates, wherein a modulation of the property compared to a reference indicates that the compound modulates the property related to the one or more condensates, wherein the property is a modulation of the fluidity of the one or more condensates. In some embodiments, provided herein is a method of identifying a compound that modulates the fluidity 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 fluidity of the one or more condensates, wherein a modulation of the fluidity compared to a reference indicates that the compound modulates the fluidity of the one or more condensates. In some embodiments, the fluidity is based on a measurement that takes into account two or more condensates, e.g., an average or a distribution of the fluidity of two or more condensates.

[0199] The fluidity of one or more condensates in a portion or all of a cell composition can be determined. Thus, in some embodiments, the method comprises determining the fluidity of one or more condensates in a portion of a 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 determining the fluidity of one or more condensates in the entire cell composition. In some embodiments, the method comprises determining the fluidity of one or more condensates in one or more cells in the cell composition. In some embodiments, the method comprises determining the fluidity of one or more condensates in a single cell in the cell composition. In some embodiments, the method comprises determining the fluidity of one or more condensates in a portion of one or more cells in the cell composition. In some embodiments, the method comprises determining the fluidity of one or more condensates in a portion of a single cell in the cell composition. In some embodiments, the method comprises determining the fluidity of one or more condensates in the cytoplasm. In some embodiments, the method comprises determining the fluidity of one or more condensates in the nucleus. In some embodiments, the method comprises determining the fluidity of one or more condensates in an organelle.

[0200] In some embodiments, when the mobility of one or more condensates is increased compared to a reference (including a reference condensate), the mobility of the one or more condensates is determined to be regulated by the compound. In some embodiments, when the mobility of one or more condensates is reduced compared to a reference (including a reference condensate), the mobility of the one or more condensates is determined to be regulated by the compound. In some embodiments, the mobility of the one or more condensates in the first part of the cell composition is reduced compared to the reference, and the mobility of the one or more condensates in the second part of the cell composition is not reduced compared to the reference. In some embodiments, the mobility of the one or more condensates in the first part of the cell composition is increased compared to the reference, and the mobility of the one or more condensates in the second part of the cell composition is not increased compared to the reference. 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, the mobility of the one or more condensates in the cytoplasm is reduced compared to the reference, and the mobility of the one or more condensates in the nucleus is not reduced compared to the reference. In some embodiments, the fluidity of one or more condensates in the cytoplasm is reduced compared to a reference, and the fluidity of one or more condensates in the nucleus is increased compared to a reference.

[0201] In some embodiments, the fluidity of one or more aggregates in the first portion of the cell composition is increased compared to the second portion of the cell composition, or the fluidity of one or more aggregates 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.

[0202] In some embodiments, the fluidity of one or more coacervates is assessed by one or more of the following: droplet size, surface tension, phase diagram, equilibrium state, droplet coarsening, and hardening.

[0203] In some embodiments, imaging techniques are used to determine the fluidity of one or more coacervates.

[0204] Solidification of condensates

[0205] In some embodiments, the property associated with the one or more condensates is based on the coagulation of the one or more condensates. Thus, in some embodiments, provided herein are methods for identifying a compound that modulates a property associated with one or more condensates comprising a condensate-associated molecule, 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) measuring 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 coagulation of the one or more condensates. In some embodiments, provided herein are methods for identifying a compound that modulates coagulation 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) measuring coagulation of the one or more condensates, wherein modulation of coagulation compared to a reference indicates that the compound modulates coagulation of the one or more condensates. In some embodiments, the coagulation is based on a measurement that considers two or more condensates, such as an average or distribution of coagulation of two or more condensates.

[0206] Coagulation of one or more condensates or their products can be measured in part or all of a cell composition. Thus, in some embodiments, the method comprises measuring coagulation of one or more condensates in a portion of a 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 coagulation of one or more condensates in the entire cell composition. In some embodiments, the method comprises measuring coagulation of one or more condensates in one or more cells in the cell composition. In some embodiments, the method comprises measuring coagulation of one or more condensates in a single cell in the cell composition. In some embodiments, the method comprises measuring coagulation of one or more condensates in a portion of one or more cells in the cell composition. In some embodiments, the method comprises measuring coagulation of one or more condensates in a portion of a single cell in the cell composition. In some embodiments, the method comprises measuring coagulation of one or more condensates in the cytoplasm. In some embodiments, the method comprises measuring coagulation of one or more condensates in the nucleus. In some embodiments, the method comprises measuring coagulation of one or more condensates in an organelle.

[0207] In some embodiments, when the coagulation of one or more condensates is increased compared to a reference (including a reference condensate or its product), the coagulation of the one or more condensates or its product is determined to be regulated by the compound. In some embodiments, when the coagulation of one or more condensates is reduced compared to a reference (including a reference condensate), the coagulation of the one or more condensates is determined to be regulated by the compound. In some embodiments, the coagulation of the one or more condensates in the first part of the cell composition is reduced compared to the reference, and the coagulation of the one or more condensates in the second part of the cell composition is not reduced compared to the reference. In some embodiments, the coagulation of the one or more condensates in the first part of the cell composition is increased compared to the reference, and the coagulation of the one or more condensates in the second part of the cell composition is not increased compared to the reference. 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, the coagulation of the one or more condensates in the cytoplasm is reduced compared to the reference, and the coagulation of the one or more condensates in the nucleus is not reduced compared to the reference. In some embodiments, coagulation of one or more condensates in the cytoplasm is reduced compared to a reference, and coagulation of one or more condensates in the nucleus is increased compared to a reference.

[0208] In some embodiments, coagulation of one or more aggregates is increased in a first portion of the cell composition compared to a second portion of the cell composition, or coagulation of one or more aggregates is increased in a second portion of the cell composition 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.

[0209] In some embodiments, the compound modulates the liquid-to-gel transition of the one or more coacervates compared to a reference. In some embodiments, the compound modulates the liquid-to-gel transition of the one or more coacervates compared to a reference, but does not modulate the gel-to-solid transition of the one or more coacervates compared to a reference. In some embodiments, the compound modulates the gel-to-solid transition of the one or more coacervates compared to a reference. In some embodiments, the compound modulates the gel-to-solid transition of the one or more coacervates compared to a reference, but does not modulate the liquid-to-gel transition of the one or more coacervates compared to a reference.

[0210] In some embodiments, solidification of the one or more coagulants is assessed by one or more of droplet size, surface tension, phase diagram, equilibrium state, droplet coarsening, hardening, and fiber and / or aggregate formation.

[0211] In some embodiments, the solidification of one or more coagulants is determined using imaging techniques. In some embodiments, the solidification of one or more coagulants is determined by measuring the formation of aggregates or fibers. In some embodiments, the solidification of one or more coagulants is determined using filtration-based techniques.

[0212] Dissolution of aggregates

[0213] In some embodiments, the property associated with the one or more condensates is based on the dissolution of the one or more condensates. Thus, in some embodiments, provided herein are methods for identifying a compound that modulates a property associated with one or more condensates comprising a condensate-associated molecule, 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) measuring 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 dissolution of the one or more condensates. In some embodiments, provided herein are methods for identifying a compound that modulates dissolution 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) measuring dissolution of the one or more condensates, wherein modulation of dissolution compared to a reference indicates that the compound modulates dissolution of the one or more condensates. In some embodiments, the dissolution is based on a measurement that considers two or more condensates, such as an average or distribution of dissolution of two or more condensates.

[0214] The dissolution of one or more aggregates in a portion or all of a cell composition can be determined. Thus, in some embodiments, the method comprises determining the dissolution of one or more aggregates in a portion of a cell composition. In some embodiments, the method comprises determining the dissolution of one or more aggregates in the entire cell composition. In some embodiments, the method comprises determining the dissolution of one or more aggregates in one or more cells in the cell composition. In some embodiments, the method comprises determining the dissolution of one or more aggregates in a single cell in the cell composition. In some embodiments, the method comprises determining the dissolution of one or more aggregates in a portion of one or more cells in the cell composition. In some embodiments, the method comprises determining the dissolution of one or more aggregates in a portion of a single cell in the cell composition. In some embodiments, the method comprises determining the dissolution of one or more aggregates in the cytoplasm. In some embodiments, the method comprises determining the dissolution of the one or more aggregates in the nucleus. In some embodiments, the method comprises determining the dissolution of the one or more aggregates in an organelle.

[0215] In some embodiments, the dissolution of one or more aggregates is determined to be modulated by the compound when the dissolution of one or more aggregates is increased compared to a reference. In some embodiments, the dissolution of one or more aggregates is determined to be modulated by the compound when the dissolution of one or more aggregates is decreased compared to a reference. In some embodiments, the dissolution of one or more aggregates in the first portion of the cell composition is decreased compared to a reference, and the dissolution of one or more aggregates in the second portion of the cell composition is not decreased compared to a reference. In some embodiments, the dissolution of one or more aggregates in the first portion of the cell composition is increased compared to a reference, and the dissolution of one or more aggregates in the second portion of the cell composition is not increased compared to a 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 dissolution of one or more aggregates in the cytoplasm is decreased compared to a reference, and the dissolution of one or more aggregates in the nucleus is not decreased compared to a reference. In some embodiments, the dissolution of one or more aggregates in the cytoplasm is decreased compared to a reference, and the dissolution of one or more aggregates in the nucleus is increased compared to a reference.

[0216] In some embodiments, the solubilization of one or more aggregates in the first portion of the cell composition is increased compared to the second portion of the cell composition, or the solubilization of one or more aggregates 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.

[0217] In some embodiments, the method comprises subjecting the cell composition to aggregate-forming conditions prior to contacting the compound with the cell composition. In some embodiments, lysis is assessed by one or more of the number of aggregates, the size of the aggregates (e.g., a decrease in size), or the disappearance of the aggregates.

[0218] In some embodiments, imaging techniques are used to determine the dissolution of one or more coacervates.

[0219] Presence and / or amount of fibrillation

[0220] In some embodiments, the property associated with the one or more condensates is based on the presence and / or amount of fiber formation. In some embodiments, the property associated with the one or more condensates is based on the presence of fiber formation. In some embodiments, the property associated with the one or more condensates is based on the amount of fiber formation. Thus, in some embodiments, provided herein is a method for identifying a compound that modulates a property associated with one or more condensates comprising a condensate-associated molecule, 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 presence and / or amount of fiber formation. In some embodiments, provided herein is a method for 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 compared to a reference indicates that the compound modulates the presence and / or amount of fiber formation.

[0221] Fiber formation can be detected and measured by a variety of methods, such as by visualizing the fibers using microscopy and / or fiber binding assays, such as those described in the Examples.

[0222] The presence and / or amount of fibrogenesis in a portion or all of the cell composition can be measured. Therefore, in some embodiments, the method comprises measuring the presence and / or amount of fibrogenesis in a portion of the cell composition. In some embodiments, the method comprises measuring the presence and / or amount of fibrogenesis in the entire cell composition. In some embodiments, the method comprises measuring the presence and / or amount of fibrogenesis in one or more cells in the cell composition. In some embodiments, the method comprises measuring the presence and / or amount of fibrogenesis in a single cell in the cell composition.

[0223] The presence and / or amount of fibrogenesis in a portion or all of the cells in a cell composition can also be determined. Therefore, in some embodiments, the method comprises determining the presence and / or amount of fibrogenesis in a portion of one or more cells in a cell composition. In some embodiments, the method comprises determining the presence and / or amount of fibrogenesis in a portion of a single cell in a cell composition. In some embodiments, the method comprises determining the presence and / or amount of fibrogenesis in the cytoplasm. In some embodiments, the method comprises determining the presence and / or amount of fibrogenesis in the nucleus. In some embodiments, the method comprises determining the presence and / or amount of fibrogenesis in an organelle.

[0224] 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.

[0225] In some embodiments, the amount of fibril formation in the first portion of the cell composition is reduced compared to a reference, and the amount of fibril formation in the second portion of the cell composition is not reduced compared to the reference. In some embodiments, the amount of fibril formation in the first portion of the cell composition is increased compared to a reference, and the amount of fibril formation 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 amount of fibril formation in the cytoplasm is reduced compared to a reference, and the amount of fibril formation in the nucleus is not reduced compared to the reference. In some embodiments, the amount of fibril formation in the cytoplasm is reduced compared to a reference, and the amount of fibril formation in the nucleus is increased compared to the reference.

[0226] In some embodiments, the amount of fibril formation is increased in the first portion of the cell composition compared to the second portion of the cell composition, or the amount of fibril formation is increased in the second portion of the cell composition 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.

[0227] Location of condensate-associated molecules

[0228] In some embodiments, the property related to the one or more condensates is based on the location of the one or more condensates. Accordingly, in some embodiments, provided herein are methods of identifying a compound that modulates a property related to one or more condensates of a molecule comprising a condensate, 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 property related to the one or more condensates, wherein a modulation of the property compared to a reference indicates that the compound modulates the property related to the one or more condensates, wherein the property is a 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 cellular composition comprising one or more condensates or with a cellular composition capable of forming one or more condensates, and (b) determining the location of the one or more condensates, wherein a modulation of the location compared to a reference indicates that the compound modulates the location of the one or more condensates.

[0229] The location of the condensate can be determined for a portion or the entire cellular composition. Accordingly, in some embodiments, the method comprises determining the location of the one or more condensates in a portion of the cellular composition. In some embodiments, the method comprises determining the location of the one or more condensates in the entire cellular composition. In some embodiments, the method comprises determining the location of the one or more condensates in one or more cells of the cellular composition. In some embodiments, the method comprises determining the location of the one or more condensates in a single cell of the cellular composition.

[0230] The location of the condensate can also be determined for a portion or the entire cell in the cellular composition. Accordingly, in some embodiments, the method comprises determining the location of the one or more condensates in a portion of one or more cells of the cellular composition. In some embodiments, the method comprises determining the location of the one or more condensates in a portion of a single cell of the cellular 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.

[0231] In some embodiments, the location of the one or more condensates is determined using an imaging technique.

[0232] Segregation of condensate-associated molecules into condensates

[0233] In some embodiments, the property associated with the one or more condensates is based on the partitioning of condensate-associated molecules into the one or more condensates. Thus, in some embodiments, provided herein are methods 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 with a cell composition capable of forming one or more condensates, and (b) measuring 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 for identifying a compound that modulates the partitioning of the condensate-associated molecules into 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) measuring 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.

[0234] Partitioning of condensate-associated molecules into condensates can be measured for a portion or all of a cell composition. Thus, in some embodiments, the method comprises measuring the partitioning of condensate-associated molecules into one or more condensates in a portion of a cell composition. In some embodiments, the method comprises measuring the partitioning of condensate-associated molecules into one or more condensates in the entire cell composition. In some embodiments, the method comprises measuring the partitioning of condensate-associated molecules into one or more condensates in one or more cells in a cell composition. In some embodiments, the method comprises measuring the partitioning of condensate-associated molecules into one or more condensates in a single cell in a cell composition.

[0235] Partitioning of condensate-associated molecules into condensates can also be measured for a portion or all of the cells in a cell composition. Thus, in some embodiments, the method comprises measuring the partitioning of condensate-associated molecules into one or more condensates in a portion of one or more cells in a cell composition. In some embodiments, the method comprises measuring the partitioning of the condensate-associated molecules into the one or more condensates in a portion of a single cell in a cell composition. In some embodiments, the method comprises measuring the partitioning of the condensate-associated molecules into the one or more condensates in the cytoplasm. In some embodiments, the method comprises measuring the partitioning of the condensate-associated molecules into the one or more condensates in the nucleus. In some embodiments, the method comprises measuring the partitioning of the condensate-associated molecules into the one or more condensates in an organelle.

[0236] In some embodiments, partitioning of condensate-associated molecules into one or more condensates is increased compared to a reference. In some embodiments, partitioning of condensate-associated molecules into one or more condensates is decreased compared to a reference.

[0237] In some embodiments, the partitioning of condensate-associated molecules into one or more condensates is reduced in the first portion of the cell composition compared to a reference, and the partitioning of condensate-associated molecules into one or more condensates is not reduced in the second portion of the cell composition compared to the reference. In some embodiments, the partitioning of condensate-associated molecules into one or more condensates is increased in the first portion of the cell composition compared to a reference, and the partitioning of condensate-associated molecules into one or more condensates is not increased in the second portion of the cell composition 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-associated molecules into one or more condensates is reduced in the cytoplasm compared to a reference, and the partitioning of condensate-associated molecules into one or more condensates is not reduced in the nucleus compared to the reference. In some embodiments, the partitioning of condensate-associated molecules into one or more condensates is reduced in the cytoplasm compared to a reference, and the partitioning of condensate-associated molecules into one or more condensates is increased in the nucleus compared to the reference.

[0238] In some embodiments, the partitioning of condensate-associated molecules into one or more condensates is increased in the first portion of the cell composition compared to the second portion of the cell composition, or the partitioning of condensate-associated molecules into one or more condensates is increased in the second portion of the cell composition 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.

[0239] Aggregation of condensate-associated molecules

[0240] In some embodiments, the property associated with the one or more condensates is based on aggregation of condensate-associated molecules. Thus, in some embodiments, provided herein are methods 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 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 aggregation of the condensate-associated molecules. In some embodiments, provided herein are methods for identifying a compound that modulates 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 aggregation of the condensate-associated molecules, wherein modulation of the aggregation compared to a reference indicates that the compound modulates aggregation of the condensate-associated molecules.

[0241] Aggregation of condensate-associated molecules can be measured for a portion or all of a cell composition. Thus, in some embodiments, the method comprises measuring aggregation of condensate-associated molecules in a portion of a cell composition. In some embodiments, the method comprises measuring aggregation of condensate-associated molecules in the entire cell composition. In some embodiments, the method comprises measuring aggregation of condensate-associated molecules in one or more cells in a cell composition. In some embodiments, the method comprises measuring aggregation of condensate-associated molecules in a single cell in a cell composition.

[0242] Aggregation of condensate-associated molecules in some or all cells of a cell composition can also be measured. Thus, in some embodiments, the method comprises measuring aggregation of condensate-associated molecules in a portion of one or more cells in a cell composition. In some embodiments, the method comprises measuring aggregation of condensate-associated molecules in a portion of a single cell in a cell composition. In some embodiments, the method comprises measuring aggregation of condensate-associated molecules in the cytoplasm. In some embodiments, the method comprises measuring aggregation of condensate-associated molecules in the nucleus. In some embodiments, the method comprises measuring aggregation of condensate-associated molecules in an organelle.

[0243] In some embodiments, the aggregation of the condensate-associated molecules is increased compared to a reference. In some embodiments, the aggregation of the condensate-associated molecules is decreased compared to a reference.

[0244] In some embodiments, the aggregation of condensate-associated molecules in the first part of the cell composition is reduced compared to the reference, and the aggregation of condensate-associated molecules in the second part of the cell composition is not reduced compared to the reference. In some embodiments, the aggregation of condensate-associated molecules in the first part of the cell composition is increased compared to the reference, and the aggregation of condensate-associated molecules in the second part of the cell composition is not increased compared to the reference. 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, the aggregation of condensate-associated molecules in the cytoplasm is reduced compared to the reference, and the aggregation of condensate-associated molecules in the nucleus is not reduced compared to the reference. In some embodiments, the aggregation of condensate-associated molecules in the cytoplasm is reduced compared to the reference, and the aggregation of condensate-associated molecules in the nucleus is increased compared to the reference.

[0245] In some embodiments, aggregation of condensate-associated molecules is increased in the first portion of the cell composition compared to the second portion of the cell composition, or aggregation of condensate-associated molecules is increased in the second portion of the cell composition 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.

[0246] Compound

[0247] As used herein, "compound" refers to any agent. 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 U.S. 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.

[0248] In some embodiments, the compound is capable of selectively and non-covalently interacting with a biomolecule (particularly a protein or nucleic acid) under conditions prevalent in living cells, wherein the compound and the biomolecule form a 10 -4 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 comprises up to five hydrogen bond donors, up to ten hydrogen bond acceptors, and is characterized by an octanol-water partition coefficient logP of less than 5.6. These are the so-called "Lipinski" rule 5 for drug-like compounds (originally referring to molecules between 160 and 500 Da). It should be understood that any of these embodiments are applicable and can be combined with any compound described herein (including the preceding paragraphs).

[0249] Cells and aggregates

[0250] In some embodiments, the cell composition comprises a microorganism or an animal cell. In some embodiments, the cell composition comprises a human cell. In some embodiments, the cell composition comprises a neuron. In some embodiments, the cell composition includes a cancer cell. In some embodiments, the cell composition comprises a cell that is or is derived from an induced pluripotent stem cell (iPS cell), a HeLa cell, or a HEK293 cell. In some embodiments, the cell composition comprises an unregulated condensate. In some embodiments, the cell composition comprises a cell containing a mutation associated with a disease. In some embodiments, the cell composition comprises a cell having one or more characteristics of a neurodegenerative or proliferative disease. In some embodiments, the cell composition comprises a cell expressing 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 being linked to or fused with a fluorescent protein.

[0251] In some embodiments, cells in the cell composition express the condensate-associated molecule. In some embodiments, expression can include any 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 in 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 in the cell composition. In some embodiments, the condensate-associated molecule is heterologous to the cells.

[0252] Many condensates are well known in the art. Examples of known condensates include schizosomes, P particles, histone locus bodies, multivesicular bodies, neuronal RNA particles, nuclear gems, nuclear pores, nuclear speckles, nuclear stress bodies, nucleoli, Oct1 / PTF / transcription (OPT) domains, paraspots, 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 have formed, but have not yet been described. Many condensates can be identified using a microscope. In some embodiments, the method further comprises identifying one or more condensates. In some embodiments, the one or more condensates are cell condensates. In some embodiments, the one or more condensates are intracellular condensates. In some embodiments, the one or more condensates are intracellular condensates in one or more cells in the cell composition. In some embodiments, the one or more condensates are one or more stress granules. In some embodiments, the first group of one or more condensates are one or more stress granules. In some embodiments, the second group of one or more condensates are one or more paranuclear speckles, condensates formed around DNA damage sites, P bodies, Cajal bodies and PML bodies.

[0253] In some embodiments, the condensate is selected from the group consisting of stress granules, P bodies, Cajal bodies, PML bodies, paraplaques (e.g., paranuclear plaques), DNA damage focus condensates, mitotic bodies, 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 splice speckles. For example, Banani et al., Nat Rev Mol CellBiol, 18, 2017, "Biomolecular condensates: organizers of cellular biochemistry"; Brangwynne et al., Science, 324, 2009, "Germline Pgranules are liquid droplets that localize by controlled dissolution / condensation"; Patel et al., Cell, 162, 2015, "A Exemplary condensates are discussed in "Liquid-to-Solid Phase Transition of the ALS Protein Accelerated by Disease Mutation"; Alberti, S., Current Biology, 27, R1089–R1107, 2017, "PhaseSeparation in Biology."

[0254] condensate-associated molecules

[0255] In some embodiments, the coagulant-associated molecule is a polynucleotide or a polypeptide. In some embodiments, the coagulant-associated molecule is a polypeptide. In some embodiments, the coagulant-associated molecule is a wild-type polypeptide. In some embodiments, the coagulant-associated molecule is a mutant polypeptide. In some embodiments, the coagulant-associated molecule is FUS, EWSR1, TIAL1, PABPC1, or G3BP1, or a mutant thereof. In some embodiments, the coagulant-associated molecule is FUS.

[0256] Additional assays

[0257] In some embodiments, the method further comprises evaluating the compound by one or more additional assays, such as one or more of a second cellular assay, a biochemical assay, and an in vivo assay.

[0258] In some embodiments, the biochemical assay is a method for identifying compounds that modulate properties associated with aggregates.

[0259] 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-forming conditions, wherein (if formed) the condensate comprises the precursor molecule; and (c) measuring a property associated with the condensate, wherein modulation of the property compared to a reference identifies the compound as a compound that modulates the property. In some embodiments, the condensate-forming conditions are reduced salt concentration. In some embodiments, the method is repeated using different condensate-forming conditions.

[0260] 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 associated with the condensate, wherein modulation of the property compared to a reference identifies the compound as a compound that modulates the property. In some embodiments, the biochemical assay method further comprises, after mixing the compound and the condensate to form the reaction composition, subjecting the reaction composition to an aging condition. In some embodiments, the aging condition is incubation, shaking, and / or heating.

[0261] In some embodiments, a property associated with the coacervates is determined based on any one or more of: (i) the number of coacervates; (ii) the size of the coacervates; (iii) the partitioning of precursor molecules into the coacervates; (iv) the surface area of ​​the coacervates; (v) the composition of the coacervates; (vii) the fluidity of the coacervates; (viii) the solidification of the coacervates; (ix) the aggregation of precursor molecules; (x) the dissolution of the coacervates; and (xi) the presence and / or amount of fiber formation.

[0262] 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 the group consisting of FUS, EWSR1, TIAL1, PABPC1, and G3BP1.

[0263] In some embodiments, the biochemical assay method further comprises imaging the reaction composition after subjecting the reaction composition to condensate-forming conditions. In some embodiments, the reaction composition comprises a fixed ratio of compound and precursor molecule. In some embodiments, the biochemical assay method is repeated using two or more different ratios of compound and precursor molecule.

[0264] In some embodiments, the reference is a reaction composition that does not contain the compound.

[0265] Exemplary embodiments

[0266] The implementation methods provided are:

[0267] Embodiment 1. A compound for use in a method for preventing or treating a neurodegenerative disease associated with stress granule formation, wherein the compound is selected from lipoic acid

[0268] -lipoamide,

[0269] - dihydrolipoic acid, and

[0270] -Dihydrolipoamide.

[0271] Embodiment 2. The compound for use in a method for preventing or treating a neurodegenerative disease associated with stress granule formation according to embodiment 1, comprising a dosing regimen of:

[0272] - Administering a daily dose of 600 mg to 1,600 mg of the compound.

[0273] Embodiment 3. The compound for use in a method for 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.

[0274] Embodiment 4. A pharmaceutical composition for use in a method for preventing or treating neurodegeneration associated with stress granule formation, comprising a compound according to any one of Embodiments 1 to 3.

[0275] Embodiment 5. The pharmaceutical composition for use in a method for preventing or treating amyotrophic lateral sclerosis according to embodiment 4, wherein the pharmaceutical composition is formulated for oral administration.

[0276] Embodiment 6. The pharmaceutical composition according to embodiment 4 or 5, wherein the neurodegenerative disease associated with stress granule formation is amyotrophic lateral sclerosis.

[0277] Embodiment 7. A dosage form for use in a method of preventing or treating amyotrophic lateral sclerosis, comprising a compound according to any one of Embodiment 1, in particular administered at the dose specified in Embodiment 2.

[0278] Embodiment 8. The dosage form of embodiment 7, wherein the dosage form is formulated for oral administration.

[0279] Embodiment 9. The dosage form of embodiment 7 or 8, wherein the neurodegenerative disease associated with stress granule formation is amyotrophic lateral sclerosis.

[0280] Embodiment 10. A method for treating or preventing a neurodegenerative disease associated with stress granule formation, comprising administering the compound according to embodiment 1 to a patient in need thereof.

[0281] 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.

[0282] Embodiment 12. The method for treating or preventing a neurodegenerative disease according to embodiment 10 or 11, wherein the compound is administered orally.

[0283] 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.

[0284] Embodiment 14. A method for reducing or inhibiting the formation of stress granules in a cell, comprising administering a compound selected from the group consisting of:

[0285] -Lipoic acid

[0286] -lipoamide,

[0287] - dihydrolipoic acid, and

[0288] -dihydrolipoamide,

[0289] - heterotricyclic compounds, in particular anthraquinone or anthraquinone derivatives (such as 1,4-dihydroxyanthraquinone), acridine or acridine derivatives (such as quinacrine or aminoacridine or mitoxantrone);

[0290] - a tetracyclic compound, and

[0291] - Surfactants, in particular cetylpyridinium chloride.

[0292] Further exemplary embodiments

[0293] The embodiments provided are also:

[0294] E1. A method of identifying a compound that modulates a property related to one or more condensates of a condensate-associated molecule, the method comprising: (a) contacting the compound with a cellular composition comprising one or more condensates or a cellular composition capable of forming one or more condensates, and (b) determining a property related to the one or more condensates, wherein a modulation of the property compared to a reference indicates that the compound modulates a property related to the one or more condensates.

[0295] E2. The method of embodiment E1, wherein the property related to the one or more condensates is determined based on any one or more of: (i) the number of condensates comprising and / or not comprising 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 fluidity of the one or more condensates; (viii) the coagulation 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 segregation of the condensate-associated molecule into the one or more condensates; and (xiii) the aggregation of the condensate-associated molecule.

[0296] E3. The method of embodiment E1 or E2, wherein the one or more condensates are within one or more cells of the cellular composition.

[0297] E4. The method of any one of embodiments E1-E3, further comprising subjecting the cellular composition to condensate-forming conditions prior to step (b).

[0298] E5. The method of any one of embodiments E1-E3, further comprising subjecting the cellular composition to condensate-forming conditions prior to step (a).

[0299] E6. The method of embodiment E4 or E5, wherein the condensate-forming conditions are any one or more of: (i) an oxidative stressor; (ii) a mitochondrial electron transport chain inhibitor; (iii) a heat stressor; (iv) an osmotic stressor; (v) a hypertonic stressor; and (vi) a glycolysis inhibition.

[0300] E7. The method of any one of embodiments E1-E6, wherein the condensate-associated molecule is a polypeptide.

[0301] E8. The method of any one of embodiments E1-E7, wherein the aggregate-associated molecule is a wild-type polypeptide.

[0302] E9. The method of any one of embodiments E1-E7, wherein the aggregate-associated molecule is a mutant polypeptide.

[0303] E10. The method of any one of embodiments E1-E9, wherein the aggregate-associated molecule is selected from the group consisting of FUS, EWSR1, TIAL1, PABPC1, and G3BP1.

[0304] E11. The method of any one of embodiments E1-E10, wherein the cells in the cellular composition express the aggregate-associated molecule.

[0305] E12. The method of any one of embodiments E1-E11, wherein the cellular composition comprises HeLA, iPSC, or iPSC MN cells.

[0306] E13. The method of any one of embodiments E1-E12, further comprising imaging at least a portion of the cellular composition.

[0307] E14. The method of any one of embodiments E1-E13, further comprising contacting at least a portion of the cellular composition with a fixative.

[0308] E15. The method of any one of embodiments E1-E14, further comprising contacting at least a portion of the cellular composition with a stain.

[0309] E16. The method of any one of embodiments E1-E15, further comprising contacting at least a portion of the cellular composition with a DNA damaging condition.

[0310] E17. The method of embodiment E16, wherein the DNA damaging condition is laser irradiation.

[0311] E18. The method of any one of embodiments E1-E17, wherein the reference is a second aggregate.

[0312] E19. The method of any one of embodiments E1-E17, wherein the reference is a second cellular composition.

[0313] E20. The method of any one of embodiments E1-E19, further comprising evaluating the identified compound using a second cellular level assay.

[0314] E21. The method of any one of embodiments E1-E20, further comprising evaluating the identified compound using a biochemical assay.

[0315] E22. The method of any one of embodiments E1-E21, further comprising evaluating the identified compound using an in vivo assay.

[0316] 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 to E22.

[0317] E24. The method according to embodiment E23, wherein the disease is a neurodegenerative disease.

[0318] E25. The method of embodiment E24, wherein the neurodegenerative disease is ALS.

[0319] E26. The method according to any one of the preceding embodiments E1 to E25, wherein the condensate is a membrane-less compartment formed by phase separation of protein and one or more other macromolecules, in particular DNA and / or RNA.

[0320] E27. A method according to E27, wherein the condensates are selected from the group consisting of stress granules, P bodies, Cajal bodies, promyelocytic leukemia protein (PML) bodies, paraspeckles (e.g., paranuclear spots), DNA damage focus condensates, mitotic bodies, 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.

[0321] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present disclosure. The present invention is further illustrated by the following examples and drawings, from which further embodiments and advantages can be derived. These examples are intended to illustrate the present invention and are not intended to limit its scope.

[0322] Example

[0323] Example 1

[0324] Using FUS as a model LCD / RBP stress granule protein to screen compounds

[0325] The present inventors developed a cell-based screen 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 primarily localized to the nucleus, where it is partially excluded from the nucleolus and localized to small spots called parapuncta ( 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 the compound library for 1 hour and then stressed with 1 mM potassium arsenate (still in the presence of the compound). Figure 1 B) and monitored the localization of FUS to stress granules. Arsenate disrupts antioxidant responses by reacting with thiol groups, blocks the tricarboxylic acid (TCA) cycle by reacting with thiols in the essential lipoyl moiety, and causes general oxidative damage. In untreated stressed cells, FUS is partially exported to the cytoplasm, where it associates with other proteins and mRNAs and phase separates to form liquid stress granules surrounded by FUS-depleted cytoplasm ( Figure 1 A).

[0326] Many compounds affect stress granule formation. Multiparameter image analysis (including the number of cytoplasmic puncta, the number of nuclear puncta, and nuclear / cytoplasmic compartmentalization) was used to rank compounds by the strength 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 polysome-stabilizing compound emetine, which prevents mRNA release, and the heavy metal chelating compound dimercaprol, which should chelate stressors. Edaravone, which is considered an antioxidant and used as an ALS treatment in Japan and the United States, had no effect on FUS localization in arsenate-stressed cells. New classes of compounds that tend to have a strong effect on FUS localization (generally reducing the number of stress granules) include cardiac glycosides, heterotricyclic and tetracyclic compounds (anthraquinones and acridines), surfactants, and benzimidazoles.

[0327] Compounds that can directly interact with FUS are the main target, so the present inventors conducted follow-up screening of compounds for their effects on FUS droplet formation in vitro ( Figure 1 D). The inventors analyzed the top 47 strongest hits from the cell-based screen. The hits were identified by their effect on the number of FUS droplets formed and the partitioning of FUS into the droplets formed in vitro under low salt conditions in the presence of 1 mM DTT to simulate a reducing intracellular environment ( Figure 1 E). Among 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, heterotri- and tetracyclic compounds, and lipoamide, the latter two of which are plausible therapeutic agents ( Figure 1 F).

[0328] In this non-equilibrium snapshot, heterotricyclic and tetracyclic compounds tended to reduce coacervate formation in a dose-dependent manner, and the resulting droplets were smaller. In contrast, surfactants and lipoamide tended to increase the number of coacervate droplets, increase partitioning into droplets, and produce larger droplets ( Figure 1 E, G). Surfactants are not plausible therapeutic agents because they permeabilize cell membranes, and they are present in the library due to their use as topical antimicrobial agents.

[0329] Stress granule formation is associated with the export of FUS from the nucleus, typically by stress granule proteins containing LCD / RBP. Persistent stress granules or stress granule proteins may cause deleterious nuclear dysfunction, including a reduction in FUS DNA damage response. Restoring the nuclear localization of this protein may be beneficial.

[0330] Therefore, the dose-dependent activity of lipoamide, lipoic acid, and related compounds was analyzed. In HeLa cells expressing FUS GFP, dose-response analysis showed that lipoamide reduced the number of stress granules and increased nuclear / cytoplasmic FUS compartmentalization with similar EC50 values ​​( Figure 2 A). Not all selected compounds exhibit this behavior; for example, mitoxantrone (a heterotricyclic anthraquinone) reduces nuclear / cytoplasmic FUS compartmentalization and also reduces stress granule numbers ( Figure 10 The present inventors confirmed these key activities of lipoamide using an induced pluripotent stem cell (iPSC) line expressing FUS GFP under arsenate stress. This also showed that lipoamide reduced the number of stress granules and restored the nuclear FUS location ( Figure 2 B). Thus, lipoamide appears to reverse both the export of FUS from the nucleus and its incorporation into stress granules in response to stress. Lipoic acid was later shown to have the same effect (see below). FUS returning to the nucleus may be beneficial, mimicking the state of unstressed cells, suggesting that lipoamide is a more promising candidate.

[0331] Lipoic acid is related to lipoic acid, a naturally occurring compound. Lipoic acid has two stereoisomers, with R-(+)-lipoic acid naturally occurring in cells and synthesized in mitochondria, while S-(-)-lipoic acid is not. The mixture of the two isomers is racemic lipoic acid or (±)-lipoic acid. It exists in cells at very low free concentrations and is typically covalently bound to proteins as a lipoyl moiety via a secondary amide. This binding form is similar to lipoamide. These compounds are dithiols, and the thiol of the lipoyl moiety is used as a hydrogen carrier by various enzymes, including one that enters the tricarboxylic acid (TCA) cycle and one that is not. The R-(+) isomer can be interconverted between the oxidized (dithiol) and reduced (cyclic disulfide) states in cells by dihydrolipoamide dehydrogenase, and although the evidence for a direct antioxidant effect is controversial, these compounds are considered antioxidants. Importantly, both lipoic acid and lipoamide are nontoxic, and lipoic acid has well-characterized pharmacokinetics: it is nontoxic, with an oral dose of 1,600 mg giving humans plasma concentrations of 8 to 30 μM. This is highly promising, as the concentration of approximately 10 μM used for in vitro characterization can be achieved in humans—lipoic acid has a long history of use in the treatment of diabetic neuropathy at doses of approximately 600 mg / day. Overall, lipoamide and lipoic acid dissolve stress granules, return FUS to the nucleus of stressed cells, interact directly with FUS, and have low toxicity. Therefore, they appear plausible candidates for use as therapeutic agents.

[0332] Lipoamide / lipoic acid effects appear to be independent of enzyme or antioxidant effects

[0333] The activity of lipoamide in subsequent in vitro screening ( Figure 1 D, E) showed that it has a direct effect on the phase separation of stress granule proteins. Therefore, the inventors tried to exclude more common mechanisms of activity in the cell-level screen, such as stressor sequestration, enzymatic action or antioxidant activity. The inventors tested this using lipoamide and lipoic acid-related compounds ( Figure 2 C, D).

[0334] Screening was performed using arsenate stress, which may induce general oxidative damage, impair antioxidant responses by reacting with thiol groups, and block the TCA cycle by reacting with thiol groups in the critical lipoyl moiety. To determine the contribution of the extent of direct arsenate reaction with lipoic acid, antioxidant effects, and enzymatic / glycolytic effects 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 of them had comparable EC50 in reducing the number of stress granules after 1 h of 1 mM arsenate stress.

[0335] This suggests that lipoic acid is inactive by supporting glycolysis, as free lipoamide is not a normal component of cellular metabolism and should not be able to function as a coenzyme / be added to the apoenzyme by lipoate-protein ligase. It also suggests that lipoic acid does not act directly as an antioxidant, as the reduced (dihydrolipoic acid) form should be more active than the oxidized (lipoic acid) form, although a redox cycle involving dihydrolipoamide dehydrogenase (in which only the naturally occurring R-(+) isomer is likely active) could be recruited. We also note that menadione (a pro-vitamin K and a classic antioxidant) did not reduce stress granule numbers in the initial HeLa cell screen. Arsenate reacts with thiols, so 1,3-propanedithiol and dihydrolipoic acid should be equally capable of directly chemically inactivating arsenate stress, but 1,3-propanedithiol had no effect at concentrations up to 100 μM. Furthermore, the EC50 of lipoic acid indicates that arsenate must be inactivated at a stoichiometry of ~1:50. Finally, any enzymatic role in lipoic acid activity, either as an antioxidant and recycling of the lipoyl moiety in support of glycolysis or via dihydrolipoamide dehydrogenase, should be specific for naturally occurring R-(+)-lipoic acid. However, S-(-)-lipoic acid has very similar activity.

[0336] Overall, this suggests some alternative mechanism of action; perhaps a stress signalling mechanism or a direct effect on the physicochemistry of stress granule protein phase separation.

[0337] Lipoic acid / lipoic acid reverses stress granule formation induced by multiple types of cellular stress

[0338] A cell-based screen was performed to analyze only FUS by pre-treating cells with the compounds prior to arsenate stress.To gain insight into the breadth of effects of lipoamide and lipoic acid and to gain further insight into the possible mechanisms, their effects on HeLa cells were fully characterized.

[0339] To determine whether lipoic acid could cause the dissolution of existing stress granules, the present inventors pre-stressed HeLa cells expressing FUS-GFP cells and then treated them with lipoamide. Time-lapse microscopy showed that the addition of 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 is likely too rapid to represent a transcriptional / translational response.

[0340] To test whether treatment of cells with thioctic amide can produce a lasting resistance to stress granule formation in the absence of continuous thioctic amide treatment, HeLa cells were pre-treated with 10 mM thioctic amide for 1 hour before testing arsenate stress for 1 hour in the absence of thioctic amide. This did not prevent stress granule formation, indicating that there is no lasting thioctic amide-induced cellular adaptation to resist stress Figure 3 B).

[0341] It is expected that useful therapeutic agents can prevent the localization of all PLD-containing stress granule proteins to stress granules. Using 1 mM arsenate on a panel of HeLa cell lines expressing GFP fusions of stress granule proteins (EWSR1, TIAL1, PABPC1, G3BP1) the inventors imposed stress in the absence of treatment or using 10 mM thioctic acid or thioctic amide. In the absence of treatment, and in the absence of racemic, S-(-)- or R-(+)-thioctic acid or thioctic amide, all proteins localized to stress granules Figure 3 C) As FUS is not required for stress granule formation, this means that FUS is neither the only, nor necessarily the main target of thioctic amide or thioctic acid in the cell. Rather, all LCD / RBP proteins are affected, and one protein that is essential for stress granule formation can be the target. This is a promising property for neurodegenerative disease therapy, as many stress granule-associated proteins are relevant to different diseases, and suggests that there can be a therapeutic effect on pathologies resulting from mutations in other stress granule proteins.

[0342] The stressor (arsenate) would react with the thiol group of thioctic amide. Therefore, to exclude the possibility that thioctic amide only works by removing the stressor, it was further tested whether thioctic acid can prevent stress granule formation triggered by other stresses than arsenate (oxidative) stress: mitochondrial electron transport chain inhibition (methyl viologen), heat stress (42°C), hypertonic stress (sorbitol, a non-metabolic sugar), glycolysis inhibition (6-deoxy glucose in the absence of glucose) or serum starvation. 10 mM racemic thioctic amide or racemic, R-(+) or S-(-)-thioctic acid all reduced stress granule formation in HeLa cells using mitochondrial, hypertonic or arsenate stress Figure 3 D). Thus, these compounds do not affect stress granules formed after all stresses, but are not specific to arsenate stress.

[0343] Overall, these data show that thioctic amide and thioctic acid completely inhibit the formation of stress granules under multiple stresses. Their mode of action on different proteins and different stresses is consistent with a complex effect on phase separation of important LCD stress granule proteins like TIA1 or a step in stress granule formation signaling shared by specific stresses.

[0344] Lipoic acid amide does not affect other cytoplasmic or nuclear fluid compartments

[0345] Stress granules are one of several similar cytoplasmic, liquid-like compartments containing RNA. Notably, these also include processing bodies (P bodies). Various stress granule proteins also form nuclear bodies: paranuclear spots and foci at sites of DNA damage. There are many other nuclear liquid-like compartments. We then used a panel of cell lines expressing GFP fusions to ask whether lipoamide also affects these other bodies. For the analysis, mitoxantrone was included as a representative of the heterotricyclic / tetracyclic class of compounds ( Figure 1 F) Excessive action on the fluid-like compartment may lead to side effects of the treatment.

[0346] Under conditions that allow the solubilization of stress granules, lipoamide did 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 effects of lipoamide are specific to the signaling and / or physicochemistry that drive stress granule formation, stress granule phase separation, and / or specific signaling pathways. Compared to many other compartments, stress granules are typically formed lytically and dissolve rapidly.

[0347] FUS is recruited to sites of DNA damage, and the potential mechanism that contributes to the pathogenesis of ALS is a reduced DNA damage response due to FUS sequestration 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 arsenate-stressed iPSCs expressing FUS GFP to test whether lipoamide affects the recruitment of FUS to sites of DNA damage induced by focused UV laser irradiation ( Figure 4 B). 20 μM lipoamide (which dissolves stress granules) had no significant effect on the recruitment of FUS GFP to sites of DNA damage. 1 μM lipoamide (which does not affect stress granules) increased the recruitment of FUS GFP to sites of DNA damage, which may be beneficial. In contrast, mitoxantrone blocked the recruitment of FUS GFP to sites of DNA damage at concentrations insufficient to dissolve cytoplasmic FUS droplets. Thus, the effects of lipoamide are specific for the cytoplasmic FUS liquid compartment, a therapeutically useful feature.

[0348] Lipoamide / lipoic acid directly interact with FUS to reduce fibril formation and stiffening

[0349] The present inventors selected lipoic acid and lipoamide for further characterization because lipoamide affects FUS droplet formation in vitro ( Figure 1 D, E). This results in the formation of larger droplets, similar to the effect of surfactants in the library. This suggests faster Ostwald ripening / coarsening, likely driven by increased surface tension. Continuing to use FUS as a well-characterized phase-separating stress granule protein, we tested whether lipoic acid or lipoamide has additional effects on phase separation in vitro.

[0350] The effect of lipoamide on the fluidity (surface tension and viscosity) of FUS eGFP aggregates was determined. Here, the aggregate droplets were brought together using optical tweezers and the rates of fusion and relaxation into spheres were measured ( Figure 5 A). Lipoic acid amide increases fluidity threefold ( Figure 5 B). In summary, the combined results of the in vitro analyses suggest that the observed ( Figure 1 E) The increase in droplet size and number was caused by the faster generation of larger droplets due to increased mobility, followed by sedimentation of the larger droplets (see Materials and Methods for more details). Since lipoamide / lipoic acid did not cause dissolution of FUS droplets in vitro, we were able to examine the effects of lipoamide and lipoic acid on fibril formation and stiffening by first analyzing FUS aggregates formed under (dextran-induced) crowding conditions ( Figure 5 C–E).

[0351] We determined whether lipoic acid or lipoamide affects the conditions under which FUS phase separation occurs in vitro. We saw only a minor effect of 100 μM lipoic acid or lipoamide, slightly increasing the minimum KCl concentration at which phase separation occurs ( Figure 6 A) This suggests that lipoic acid or lipoamide have little or no effect on the system's chemical potential, which can alter the phase diagram and equilibrium state. Instead, these compounds affect coarsening, which is a change in the kinetics of reaching equilibrium.

[0352] Therefore, we analyzed stiffening and fibril formation, another kinetic phenomenon. In vitro FUS droplets "age" over time, first stiffening 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 delayed fibril formation of G156E FUS ( Figure 6 B). In addition, both compounds delayed the hardening of FUS droplets, and the droplets retained a large FUS mobility fraction ( Figure 6 C) This occurs in the presence of 1 mM DTT (approximately 1:30 excess of reducing agent), strongly suggesting a non-antioxidant mechanism for lipoic acid / lipoamide.

[0353] The prevention of hardening and faster coarsening suggests a direct interaction of FUS to increase "mobility" at concentrations reached in cells. This may allow stress granules to dissolve more easily and may reduce the 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 arsenate-stressed cells ( Figure 11 B, C).

[0354] In vitro data indicate that the mechanism of action of lipoamide requires a transient and weak interaction between lipoamide and intracellular FUS. Capturing this interaction is challenging for conventional tools of structural biology. Because this mechanism is relevant inside cells, lipoamide must enter the cell in its unmodified form at sufficiently high concentrations to exert its effects. Lipoic acid and lipoic acid have physical effects on FUS, but large amounts of the compounds relative to the protein are required, for example, 100 μM lipoamide with 2.8 μM FUS ( Figure 6 A) Since cytoplasmic FUS concentrations are low μM, the compound must accumulate in the cell to make this plausible: it must enter the cell, be concentrated, and not be metabolized. The signaling mechanism does not require any of these mechanisms.

[0355] Finally, mechanistic details were analyzed at the submolecular level by using NMR of a FUS prion-like N-terminal LCD to determine the putative site of lipoamide interaction. The LCD can phase separate in vitro to form aggregates, and the individual residues can be resolved and isolated in the presence of 1 H– 15 N heteronuclear single quantum coherence spectrum. 1 H– 15 N analysis revealed environmental changes at 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 was no single clear site of interaction between lipoamide and the FUS LCD, mitoxantrone caused a weak shift in the LCD, consistent with a weak interaction with a tyrosine residue. This suggests that lipoamide does not act through direct, high-affinity protein binding. This leaves open several possibilities: perhaps lipoamide interacts with the FUS LCD only upon phase separation, potentially reducing viscosity, or perhaps interacts with the coacervate phase interface, potentially increasing surface tension. Such an interaction could reduce the formation or swelling of aggregated forms of FUS that participate in coacervate stiffening and reduce fluidity.

[0356] Lipoic acid amide becomes highly enriched in cells

[0357] The concentration of lipoamide required to affect the fluidity of aggregates in vitro relative to proteins was higher than the EC50 for cells, with approximately 100 μM lipoamide and 1 μM FUS ( Figure 1 D, E, Figure 5 In cells, FUS concentrations are low for proteins (low μM) 41 The EC50 of the cells was about 10 μM ( Figure 2 A, Figure 3 D). We also observed a reversal effect of lipoamide on FUS-containing aggregates in vitro (more and larger aggregates) and in cells (fewer stress granules). To reconcile this discrepancy and understand the relevance of the effects outside the cell body, we would like to know the actual concentration of the compound in the cell. In principle, isotope labeling allows direct monitoring of isotope-labeled compounds by appropriate spectroscopy, even in complex environments. To use this approach, we synthesized 15 N-labeled lipoamide ( Figure 7 A) Solution-state NMR experiments were then used to quantitatively detect the covalent attachment of 15 The NH2 protons on the N can be used to determine the lipoamide concentration from the complex mixture inside the cell, while also revealing any chemical modifications of the amide group (manifested as chemical shift changes or spectral changes) (Figure 13).

[0358] After incubation of HeLa cells at 37°C for 1 hour in the absence or presence of lipoamide, the amount of lipoic acid removed from the culture medium by the cells was quantified by NMR. 15 N-lipoamide uptake ( Figure 7 B). For the combination of R-(+) or (±)-lipoamide and unstressed or stressed cells, cellular uptake was determined by calculating the difference between the culture medium with or without cells ( Figure 7 C). For one sample, using cells stressed with R-(+)-lipoamide, we confirmed that the strong signal from the cellular fraction was consistent with uptake of a large proportion of lipoamide ( Figure 7 C). Both R-(+) and ±-lipoamide measurements showed uptake of 35 ± 11% of the lipoamide present in the culture medium (n = 3 and 2, respectively). No significant differences were observed in uptake of R-(+) compared to (±)-lipoamide, nor in stressed cells compared to unstressed cells ( Figure 7 D).

[0359] There was no evidence for metabolism or any other chemical modification of lipoamide: NMR signals from cell samples indicated that lipoamide was present in the cells in its unmodified form ( Figure 7 B).

[0360] The approximate intracellular 15 N-lipoamide concentrations—these indicated an average intracellular concentration of 5.0 ± 1.6 mM ( Figure 7 D), significantly higher than the concentrations obtained in vitro under optimal conditions with 1% v / v DMSO in H2O. Thus, lipoamide is readily taken up by HeLa cells (reaching concentrations comparable to abundant cellular metabolites). It exists in its chemically unmodified form ( Figure 13G , H). These concentrations are an order of magnitude higher than those that affect FUS GFP aggregates in vitro, indicating that the physicochemical effects in cells are plausible.

[0361] Lipoic acid / lipoic acid prevents stress granule aggregation in vivo

[0362] Because lipoamide and lipoic acid have large effects on FUS aggregation in vitro, we used a filter-trap retention assay (in which aggregated proteins from cell lysates tend to be retained on the membrane) to test whether lipoamide has beneficial effects on the spontaneous aggregation of wild-type or P525L FUS GFP in iPSCs ( Figure 11 Both cell lines showed some evidence of FUS aggregation, which was reduced after treatment with lipoamide ( Figure 11 B).

[0363] Lipoic acid prevents persistent stress granules / aggregates in vivo

[0364] In search of evidence for the in vivo effects of lipoic acid on stress granule formation and protein aggregation, the inventors turned to C. 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. Therefore, it was tested whether lipoic acid prevents the coagulation of stress granules. C. elegans grown in liquid culture containing R-(+) or S-(-)-lipoic acid showed a dose-dependent decrease in the proportion of animals with aggregation of PAB-1, stress granule proteins with LCD / RBP structures, and orthologs of PABC1. At the highest concentration tested (2mM), there was some toxicity that resulted in 6 to 8% worm mortality. It was not possible to test lipoamide because it precipitated in the worm culture medium.

[0365] To investigate whether the effect of lipoic acid is specific to RBP with LCD, aggregation of two globular proteins, KIN-19 and RHO-1, which have previously been shown to aggregate with age in C. elegans, was tested. Neither of the two proteins has an RNA binding domain or a LCD. No effect of 1.5 mM lipoic acid on RHO-1 aggregation was found. KIN-19 aggregation was slightly reduced, but 1.5 mM lipoic acid is highly toxic.

[0366] Thus, lipoic acid can affect the coagulation of stress granules over a longer time frame of the organism's life, in relation to its behavior in the short term. The behavior of lipoic acid is consistent with a direct interaction with stress granule proteins to reduce stress granule formation and / or stress granule protein aggregation.

[0367] Lipoic acid and lipoamide restore FUS-related defects in neurons and organisms

[0368] In humans, one cause of familial ALS is a mutation in FUS, particularly in the C-terminal NLS. P525L is one such mutation, and iPSC-derived motor neurons (iPSC MNs) from an ALS patient expressing P525L show defects consistent with motor neuron defects in the patient. iPSC MNs can be grown through a silicone channel, with the cell body positioned on one side and the axon protruding through the channel to the far side. This culture can be maintained for a long time (>60 days). However, when expressing P525L FUS, the axon dies over the time period in the absence of an exogenous cellular stressor, despite the P525L FUS MNs having a greater propensity to form stress granules. This is similar to axonal retraction, which leads to motor dysfunction in patients. Therefore, whether lipoamide or lipoic acid could ameliorate these phenotypes was tested. FUS P525L neurons initially showed normal morphology Figure 8 D, Figure 9 ), but by 60 days, axonal material that had died in culture had accumulated around the exit of the neuron from the silicone channel Figure 8 D, Figure 9 ). Including 2 mM lipoic acid or lipoamide in the culture medium prevented this dying. Defective axonal transport was thought to cause the axonal dying. To test this, the inventors analyzed the transport of lysosomes within the axon of iPSC MNs expressing P525L FUS with or without lipoamide Figure 12 ) compared to iPSC MNs expressing WT FUS. Lipoamide restored axonal transport in P525L FUS iPSC MNs to the same level as in WT neurons. In these assays, the inventors did not use treatments to induce stress granule formation in iPSC MNs, although in iPSCs, P525L FUS has a greater propensity to form them Figure 8A) Since no stressor is required to induce axonal dieback, this suggests that lipoic acid either helps iPSC MNs cope with random stress in culture or restores intrinsic defects caused by P525L.

[0369] A fly model was used to further test whether lipoic acid had similar beneficial effects 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 expressing human FUS in Drosophila melanogaster resulted in motor defects, including reduced climbing ability. Expression of P525L or R521C human FUS (both NLS mutants) resulted in even more severe motor defects. Food supplementation with lipoic acid restored motor defects in a dose-dependent manner, restoring the ability to climb 4 cm from ~50% to >80% within 30 seconds. This significantly restored the flies' climbing ability. Therefore, lipoic acid can restore motor neuron defects caused by FUS.

[0370] discuss

[0371] Stress granules are considered to be a key site in the pathogenesis of ALS. They form by liquid-liquid phase separation of proteins, driven by multivalent weak interactions. This is in contrast to the strong enzyme-substrate or protein-protein interactions that are typically targeted by drugs. Therefore, it was unclear whether it was possible to identify drug-like compounds that interfere with phase separation, although it seemed conceptually plausible. The inventors searched for compounds that affect stress granule formation by directly interacting with stress granule proteins containing LCDs, 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 therapeutics for a variety of conditions, but without a clear mechanism of action. Here, it is 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 phenotype caused by FUS mutants associated with familial ALS in neurons and animals.

[0372] The research underlying this invention focused on lipoamide, rather than lipoic acid, as a more effective candidate in vitro. Evidence suggests that the physicochemical mechanism of action is plausible. Lipoamide accumulates in high concentrations in cells and, at these concentrations (using FUS as an in vitro model), affects the fluidity of phase-separated droplets. In cells, lipoamide completely dissolves stress granules ( Figure 3D), but not other membrane-free fluid-like compartments—this includes compartments formed by the same protein in different regions of the cell (paranuclear plaques and around sites of DNA damage) or by other proteins (P bodies, Cajal bodies, and PML bodies) ( Figure 4 Lipoic acid amide also reduced these phase-separated compartments in vitro ( Figure 6 BD), coagulation in cells and on surfaces throughout the animal ( Figure 8 A). Lipoic acid amide affects the behavior of FUS in vitro, but FUS is not required for stress granule formation in cells. Therefore, lipoic acid amide may also interact with FUS-like proteins (other proteins containing LCD / RBP) that are critical for stress granule formation in cells. However, the precise nature of the interaction with FUS or other stress granule proteins containing LCD remains unclear.

[0373] In cells, lipoamide causes the dissolution of stress granules formed under various cellular stresses ( Figure 3 ), whereas lipoamide did not induce the dissolution of FUS aggregate droplets in vitro ( Figure 5 ), which seems to affect the kinetics rather than the thermodynamics of FUS phase separation. One possible reason is that lipoamide accumulates to a significantly high concentration in cells ( Figure 7 ), which may be degraded but not metabolized in cells (Figure 13). The estimated cellular concentration (approximately 5 mM) is an order of magnitude higher than the concentration achievable in vitro (300 μM) and much higher than the concentration required to increase the fluidity and reduce stiffening of phase-separated FUS in vitro ( Figure 5 It is possible that 5 mM lipoamide can dissolve FUS aggregate droplets in vitro. There are other possible explanations. First, the higher fluidity of stress granules may make them more sensitive to cellular stress granule dissolving factors. Second, cells have stress granules, cytoplasm, and nuclear environment, making it a more complex three-phase system (possibly related to the effect of lipoamide on FUS nuclear / cytoplasmic partitioning, Figure 2 Finally, lipoamide has a strong effect on the coagulation of FUS ( Figure 5 ), and this is thought to be driven in part by LCD-LCD interactions that are not required for extracellular phase separation. If LCD-LCD interactions are more important for intracellular phase separation, this could be reflected in the increased sensitivity of stress granules to lipoamide relative to FUS in vitro.

[0374] The cellular targets of lipoamide or lipoic acid could not be clearly determined. Although these compounds altered the properties of FUS aggregates 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 LCD domains and are affected by treatment in cells, including G3BP1, which is thought to nucleate stress granules ( Figure 3 ).

[0375] Lipoic acid and lipoic acid may affect properties shared by multiple FUS-like stress granule proteins or key life-threatening stress granule proteins. If the physicochemical mechanism is correct, then theoretically, lipoic acid modulates transient and weak interactions between FUS molecules to regulate condensate properties rather than having a single binding site. It is perhaps not surprising that despite the apparent effect on the physical properties of FUS condensates in vitro, no interaction was detected by NMR. Figure 5 , Figure 6 It is important to note that this assay only involves FUSLCD in the non-phase separated phase - lipoamide may interact with other regions of FUS (such as the RNA binding domain, which is not present in this assay).

[0376] The precise mechanisms of ALS pathogenesis remain unclear, but lipoamide and lipoic acid appear to be possible therapeutic agents based on the hypothesis that stress granules are key to ALS pathogenesis. The usefulness of lipoamide / lipoic acid depends on whether the formation of stress granules contributes to 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 characterizing lipoic acid because of its known pharmacokinetics and toxicology in humans, although with slightly lower potency in vitro. Lipoic acid has been shown to reduce the aggregation of stress granule proteins in Caenorhabditis elegans, which is a hallmark of aging in said organism and a phenomenon with general similarities to 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 found that lipoic acid rescued defects in axonal stability in neurons in vitro and defects in motor control caused by expression of FUS mutants associated with ALS pathogenesis in Drosophila melanogaster ( Figure 8 C). Lipoic acid has previously shown some efficacy in SOD1 animal models of ALS, and our work demonstrates its efficacy in a stress granule-driven model of ALS pathogenesis 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, suggesting that lipoic acid has surprising plausibility as a therapeutic agent.

[0377] Lipoic acid is a naturally occurring metabolite, and lipoamide is closely related. Despite being related to naturally occurring metabolites, there is no evidence for lipoamide metabolism. Both are considered antioxidants, and lipoamide is indeed briefly considered an antioxidant in prion-related diseases, but the activity is independent of the redox state of lipoic acid on the cell, 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 changes in phase separation a plausible mechanism; especially considering that lipoamide accumulates to high concentrations in cells. For example, this is different from preventing stress granule formation by inhibiting eIF2α phosphorylation by the ISRIB small molecule. Although the detailed mode of action is not clear, it may be a different mode of action from "traditional" compounds that target strong, specific enzyme-substrate or protein-protein interactions.

[0378] In general, the success of the screens used demonstrates that compounds can be discovered that target the physicochemistry of proteins and proves the feasibility of this overall screening approach. This is important in diseases including ALS and others; many different LCD-containing proteins are involved, and many different mutations are associated with familial ALS. Targeting each target separately is not feasible. Instead, compounds can be discovered that make protein species more liquid or soluble in the cytoplasm. This points to a new class of drugs—those that affect the physicochemistry of non-membrane-bounded compartments, or physicochemical drugs.

[0379] Stress granules are formed by phase separation of proteins within the cytoplasm, and several small molecules that modulate phase separation have been previously identified (particularly 1,6-hexanediol), however, lipoic acid and lipoamide appear far more plausible as therapeutic agents. 1,6-Hexanediol concentrations 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 (heterotricyclic compounds, tetracyclic compounds, as well as lipoamide and related compounds) exhibited activity in vitro ( ) at much lower concentrations (tens to hundreds of μM). Figure 1 、 5 ) and on cells ( Figure 1 、 2 , 10) were active at concentrations three to four orders of magnitude lower than that of 1,6-hexanediol. The effect of lipoamide on stress granules is also specific in that it does not affect other membraneless liquid compartments in the cell, and it does not affect the nuclear compartment formed by FUS ( Figure 4 B) or the nuclear compartment 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.

[0380] Furthermore, the anticancer drug mitoxantrone effectively destroys many non-membrane-bounded compartments. Mitoxantrone induces DNA damage, which has been attributed to its role as a topoisomerase inhibitor through DNA binding; however, it appears to destroy rather than increase the number of membrane-free DNA damage resting compartments—a physicochemical mechanism may also be at work here.

[0381] Lipoic acid is a naturally occurring metabolite of particular interest. Recent data have identified another metabolite, ATP, as a hydrotrope, with properties that keep proteins soluble. Solutions that prevent abnormal protein aggregation in disease compartments may support the cell's ability to maintain a lytic environment. Future screens could identify more such molecules.

[0382] method

[0383] BAC recombineering was used to generate a stable Kyoto HeLaBAC cell line expressing a protein with a C-terminal GFP fluorescent marker. This gave 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 culture was supplemented with 1% penicillin-streptomycin and maintained under geneticin (Gibco, 400 μg / ml) selection at 37°C with 5% CO2.

[0384] 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 pre-generated from a healthy female donor. 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 pre-generated from KOLF-C1 clonal iPS cell line generated as part of the Human Induced Pluripotent Stem Cell Initiative (HipSci). KOLF-C1 cells are 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. JS-SL-C1 and KOLF-C1 iPS cell lines were used as isogenic pairs to analyze the DNA damage response and the effect of thioctic acid on P525L FUS. AH-ALS1-F58 iPS cells expressing P525L FUS with a C-terminal GFP fluorescent marker were pre-generated 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. iPS cells were grown in TeSRE8 medium (Stem Cell Technologies) at 37°C and 5% C02.

[0385] As previously described, MNs were generated from AH-ALS1-F58 iPS cells expressing P525L FUS by induced differentiation in Matrigel-coated plates with silicone channels for axons. This results in clusters of somata on one side of the channel, with axons extending through the channel and protruding from the distal side of the channel. Motor neurons (MNs) were generated using AH-ALS1-F58 because they have been previously characterized in axonal transport assays. iPS MNs were used in assays within 4 weeks of completing differentiation, unless otherwise stated.

[0386] All procedures using human cell samples were in accordance with the Helsinki Convention and approved by the ethics committee of the TU Dresden (EK45022009, EK3931 22012).

[0387] Recombinant proteins

[0388] For in vitro droplet formation screen and clotting assays, exactly as previously described 7Recombinant GFP-FUS and GFP-G156EFUS were purified using a baculovirus / insect cell expression system. Briefly, His-MBP-FUS-GFP was purified from cell lysates by Ni-NTA affinity purification, followed by cleavage of His-MBP, concentration by dialysis, and further purification by size-exclusion chromatography. Purified FUS was stored in a buffer consisting of 1 M KCl, 50 mM Tris·HCl, pH 7.4, 5% glycerol, and 1 mM DTT. Prior to use, the FUS concentration in the storage buffer was adjusted to 30 μM.

[0389] Compound

[0390] For ex vivo and in vitro screening, PHARMAKON 1600 libraries were used, prepared as 10 mM stocks in DMSO. For subsequent analysis, compounds were purchased fresh and prepared as 10 mM stocks 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), 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-tetrahydronaphthoanthracene-5,12-dione (R162892, Sigma Aldrich). 15 N- and R-(+)-lipoamide (see below), and by 1 H NMR, 13 The product was characterized by C NMR, mass spectrometry, infrared spectroscopy and melting point. The yield was 50%. 15 N labeling was -99%.

[0391] Ex vivo HeLa cell screening

[0392] Compounds were screened for their effect on FUS GFP localisation in stressed HeLa cells in 384 well format. 4000 cells were seeded per well and incubated for 24 hours, then the media was replaced with 40μl fresh media and compounds were added by acoustic dispensing (Labcyte Echo 550) to a final concentration of 10μM. The final concentration of DMSO in all samples was 0.1%. After 1 hour, potassium arsenate was added from a 5x stock to a final concentration of 1mM and the cells were incubated for a further 1 hour before fixing the cells with 7.4% formaldehyde, staining with 1mg / ml Hoechst 33342 and 1:10,000 CellMask Blue (ThermoFisher). Six fields per well were captured using a 40x NA 1.3 water immersion objective using a CellVoyager CV7000 automated spinning disk confocal microscope (Hamamatsu). Each plate included 48 wells treated with 0.1% DMSO and stressed with arsenate (compound solvent control), as well as 8 untreated unstressed wells with parental Koyoto HeLa cells and 4 untreated unstressed wells. All images were displayed with gamma 0.7 to show both bright stress granules and faint nuclei.

[0393] To perform the initial screen, FUS GFP signal was analysed using KNIME. The cytoplasm was identified from the weak (CellMask Blue) blue fluorescence signal and the nucleus from the strong (Hoechst 33342) blue fluorescence signal. The number of granules and total area of the nucleus and cytoplasm were measured in the green (FUS GFP) channel at a granularity of 9, 10, 11px (cytoplasm) or 1, 5, 6, 7, 8, 9px (nucleus) scale, a texture of 10px scale and an integrated signal intensity. For each parameter, a Z-score (z = (x - μ) / σ z where x is the observation, μ is the control mean and σ is the control standard deviation) was calculated for each plate relative to the DMSO treated control wells and combined into a Mahalanobis distance.

[0394] In vitro purified FUS GFP screening

[0395] Compounds were evaluated in vitro against FUS GFP droplets in a 384-well plate format. The desired volume of compound (in DMSO) for a final concentration of 1, 3, 10, 30, or 100 μM was added to the wells of a 96-well plate containing 3 μl of FUS GFP diluted in 50 mM Tris·HCl pH 7.4 and 1 mM DTT (for low-salt assays) using acoustic partitioning (Labcyte Echo 550). The final DMSO concentration ranged from 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. Compound / FUS GFP and assay buffer were mixed using a standardized pipetting procedure and then aliquoted into four wells of a clear-bottom 384-well plate before immediate imaging using a CellVoyager CV7000 automated spinning disk confocal microscope (as above). Six fields of view of the droplets in suspension were imaged as maximum intensity projections of six focal planes with a 2 μm step size for each sample. The number of droplets and the FUS GFP segregation into the droplets were analyzed using ImageJ with a fixed intensity threshold. Due to the weak time dependence of droplet settling, the number of droplets and segregation were normalized by assuming linear variation over time with reference to the DMSO controls at the beginning and end of each plate row.

[0396] Compound characterization on HeLa and iPS cells

[0397] By live cell imaging, the effect of compound was evaluated under various conditions in HeLa or iPS cells expressing wild type or P525L FUS GFP. Compounds were pretreated for 1 hour or treated with compounds for 1 hour and / or with different combinations of arsenate stress after arsenate pre-stress. Unless otherwise stated, cells were pretreated for 1 hour using 10 μM compounds (from 10 mM stock solution in DMSO) (or an equal volume of DMSO control) and then stressed for 1 hour with 1 mM potassium arsenate in the presence of the compound. An inverted Olympus IX71 microscope was used, with an Olympus 100 × NA 1.4 Plan Apo oil immersion objective and a CoolSNAP HQCCD camera (Photometrics), using a DeltaVision climate control unit (37 ° C, 5% CO 2 ) (Applied Precision), by wide-field epifluorescence to GFP fluorescence imaging. Unless otherwise stated, for a single time point image captured after 1 hour, the kinetics of the pre-stress treatment was analyzed from an image analysis captured at 2 minute intervals for 100 minutes.

[0398] Various cell stresses were obtained by replacing the 1 h 1 mM potassium arsenate treatment with other conditions: 100 μM rotenone (R8875, Sigma Aldrich) from a 1 M stock 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 in H2O for 1 h (osmotic stress). 42°C in normal growth medium for 30 min (heat stress). 100 mM 6-deoxyglucose (D9761, Sigma Aldrich) from a 1 M stock in H2O in glucose-free DMEM (11966025, ThermoFisher Scientific) supplemented with 10% FCS for 1 h (glycolytic stress). Appropriate solvent controls were used.

[0399] 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 and horseradish peroxidase-conjugated anti-mouse or anti-rabbit (Dianova 1:10,000) secondary antibodies. Filter retardation assays for intracellular FUS aggregates were performed as previously described. 56 Briefly, protein extracts were loaded onto 0.2 μm cellulose acetate membranes and then microfiltered to retain aggregated proteins on the membrane. Aggregated FUS was detected as described above.

[0400] Compound dose response in HeLa cells

[0401] In addition to manually preparing serial compound dilutions in culture from 80 μM to ~0.4 nM in 1.189× dilution steps, as in the in vitro HeLa cell screen, a 1-hour 1 mM potassium arsenate stress was used to assess the dose-dependent effects of compounds on HeLa or iPS cells expressing FUS GFP. Small dilution steps were chosen over concentration replicates because they provide greater statistical power from a defined 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 compartmentalization 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 the green image after a 10px weight 0.9 unsharp filter masked by the thresholded nucleus, and nuclear FUS droplets were obtained by intensity thresholding the green image after a 5px weight 0.9 unsharp filter masked to include only the thresholded nucleus and a 10px rolling ball background subtraction. The ratio of cytoplasmic FUS droplets to nuclei was taken as the cytoplasmic FUS droplets per cell per field of view, and the ratio of FUS partitioning to the nucleus and cytoplasm, p, was derived from a = v n / v t , the ratio of nuclear to total green signal per field of view, where p = a / (1–a). These data were logarithmically transformed and fitted with a Rodbard sigmoidal curve to determine EC 50 Six fields of view were captured and analyzed for each condition.

[0402] In vitro FUS GFP coagulation assay

[0403] For in vitro coagulation assays, FUS GFP in storage buffer was diluted in water to obtain 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). Compounds or an equal volume of DMSO were then added to a final concentration of 30 μM compound and 0.3% DMSO. "Aging" was induced by shaking horizontally at 800 rpm at room temperature to induce fibers. Fiber and droplet formation were analyzed by wide-field epifluorescence using a DeltaVision Elite microscope (GE Healthcare Life Sciences) with a PlanApoN 60×NA 1.4 oil-immersion objective (Olympus) and an sCMOS camera (PCO). For salt sensitivity of droplet formation, FUS GFP in storage buffer was diluted in an appropriate concentration of KCl premixed with compound or DMSO to produce a final KCl concentration series with a 30 μM compound concentration and 0.3% DMSO.

[0404] Fluorescence recovery after photobleaching (FRAP) of FUS-GFP droplets and fibers was performed using a Yokogawa CSU-X1 spinning disk head and an Andor iXon EM+DU-897 EMCCD camera on a Nikon TiE inverted microscope with a Nikon Apo 100× NA 1.49 oil-immersion objective. 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 minutes. Recovery curve half-lives and mobile fractions were calculated in ImageJ.

[0405] In vitro DNA cleavage assay

[0406] UV microirradiation of live cells was performed to induce DNA damage. KOLF iPS cells expressing wild-type GFP were stressed for 1 hour by the addition of 1 mM potassium arsenate and then treated with lipoamide, mitoxantrone, or an equal volume of DMSO for 1 hour. A single point in the nucleus was subjected to three UV pulses as described for FRAP, but at 10% laser power. GFP fluorescence was imaged at 1 Hz, and the response intensity was analyzed in ImageJ.

[0407] NMR

[0408] To analyze the interaction of compounds with FUS, the untagged FUS low complexity domain (residues 1 to 163) was expressed, purified, and used 1 H– 15 N heteronuclear single quantum coherence NMR and previously described sample conditions 14Assays were performed in the presence of 500 [mu]M compound or equivalent DMSO solvent control (1%).

[0409] To analyze the cells 15 To investigate the uptake of N-lipoamide, HeLa cells expressing FUS-GFP were grown in 6-well plates in DMEM supplemented with 10% FCS to 10 6 To stress and treat the cells simultaneously, 0.6 ml of PBS supplemented with potassium arsenate and 100 μM 15 The culture medium containing N-racemic or R-(+)-lipoamide was replaced with the culture medium at 37°C for 1 hour. A high concentration of compound was used to maximize the signal. The culture medium was then removed and retained (culture medium sample), the cells were washed with ~2 ml PBS, and then the cells were removed by trypsinization: 0.3 ml TrypLE Express (12604013, ThermoFisher) was added and incubated at 37°C for 5 minutes, and then 0.3 ml culture medium was added to quench the trypsin. The resuspended cells were retained (cell sample). All samples were frozen at -80°C. For no compound (1% DMSO control), 15 N(±)-lipoamide or 15 Wells were prepared for all combinations of NR-(+)-lipoamide with or without potassium arsenate and with or without cells. 1 H detection 15 Edited by N 1 HSQ with enhanced H sensitivity for quantitative 15 N-lipoamide concentration (see Supplementary Information). Solvent, pH, and temperature sensitivities of primary amide proton chemical shifts were determined using mock samples assembled from appropriate solvents and added compounds.

[0410] Neuronal dieback and axonal transport assays

[0411] To analyze axonal dieback transport, AH-ALS1-F58iPS cells expressing P525L FUS or isogenic controls expressing WT FUS were grown with 2 μM compound or an equal volume of DMSO for 60 days. Over this length of culture, neurons expressing WT FUS developed stable axons, while neurons expressing P525L FUS did not. Axonal dieback is visible as accumulation of cellular debris at the axonal exit site. Experiments and analyses were performed blindly, and axonal dieback was qualitatively scored using phase-contrast images captured every 10 to 20 days.

[0412] 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 culture time was selected to ensure the penetration and action of the compound along the length of the axonal channel. 2 μM was selected as the highest concentration without toxic effects (qualitative assessment). The analysis of axonal transport of liposomes was performed as follows: liposomes were labeled by adding 50 nM lysosomal red probe red (ThermoFisher) and a Leica DMI6000 inverted microscope with a 100× NA 1.46 oil immersion objective and an AndoriXON 897 EMCCD camera was used in an incubator (37° C., 5% CO 2 ) at 3 ℃. 1 The proximal or distal end of a silicone channel containing an axon was imaged for 120 s at 3 Hz. For five video micrographs, particle tracking was used to determine the proportion of particles moving faster than 0.2 μm / s. Each video included a variable population of non-mobile background particles; therefore, for each biological replicate, data were normalized to the average proportion of fast-moving lysosomes in DMSO-treated (solvent control) samples.

[0413] Protein aggregation in Caenorhabditis elegans

[0414] The effects of lipoic acid on the aggregation of stress granule proteins in vivo were analyzed using the Caenorhabditis elegans model for stress granule formation and aggregation. As previously described, fluorescently labeled PAB-1 forms numerous 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.

[0415] Animals were exposed to lipoic acid starting at the L4 larval stage in liquid culture in 96-well plates with a total volume of 50 μΐ^per well in S-complete (100 mM NaCl, 50 mM potassium phosphate pH 6, 10 mM potassium citrate, 3 mM MgS04, 3 mM CaCl2, 5 μg / mL cholesterol, 50 μΜ ethylenediaminetetraacetic acid (EDTA), 25 μΜ FeS04, 10 μΜ MnCl2, 10 μΜ ZnS04, 1 μΜ CuS04) supplemented with heat-killed OP50 and 50 μg / mL carbenicillin. For each experiment, a minimum of nine wells were treated with R-(+)- or S-(-)-lipoic acid or an equal volume of DMSO, with 13 animals per well. Toxicity was assessed from the number of dead or abnormally small animals.

[0416] Forty-eight hours after the L4s were shifted from 20 °C to 25 °C (day 2 of adulthood), extensive aggregation of fluorescently tagged PAB-1 and RHO-1 was observed in the pharyngeal muscles and KIN-19 throughout the animal. After fixation with 2 mM levamisole, scoring was performed using a fluorescent stereo microscope (Leica M165 FC, Plan Apo 2.0x objective). For PAB-1, aggregates were primarily observed in the terminal bulb of the pharynx, and aggregation was scored as high (>10 aggregates / animal) or low (<10). For RHO-1, aggregates were scored in the isthmus of the pharynx, and aggregation was scored as high (>50% of the isthmus), medium (<50%), or low (no aggregation). For KIN-19, aggregates were observed throughout the body wall muscles, and aggregation was scored as high (aggregation in the head, middle of the body, and tail), medium (>15 aggregates in the head and middle of the body), or low (>15 aggregates in the head or middle of the body). High magnification images were obtained using a Leica SP8 confocal microscope using a HC Plan Apo CS 26 3x NA 1.40 oil objective, using 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-projection.

[0417] Motor defects in Drosophila melanogaster

[0418] 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 Bloomington 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).

[0419] Climbing test was performed as described above. Briefly, in the presence or absence of (±)-α-lipoic acid (0.43mM or 2.15mM diluted in ethanol), (R)-(+)-α-lipoic acid (0.43mM or 2.15mM diluted in ethanol), (S)-(-)-α-lipoic acid (0.43mM or 2.15mM diluted in ethanol), (±)-α-lipoamide (0.43mM diluted in DMSO) or PP242 / Torkinib (10 μM or 50 μM diluted in DMSO), expression of FUS, eGFP or w1118 flies was grown, then anesthetized, placed in a vial and allowed to adapt to the new vial for 15 minutes. For each fly genotype, the vial was knocked three times on the substrate on the workbench, and a video camera was used to record the flies climbing up the vial wall. The percentage of flies climbing 4 cm in 30 seconds was recorded. Statistical analyses were performed in GraphPad Prism 6 using Student's T test or one-way ANOVA with Tukey or Dunnet's multiple comparison tests.

[0420] Racemic (±) and 15 Synthesis and Characterization of NR-(+)-Lipoic Acid Amide

[0421] 15 N(±)-Lipoic acid amide

[0422]

[0423] (±)-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) at 25° C. under argon atmosphere for 4 hours. The solution was diluted with EtOAc (100 ml) and washed with H 2 O (100 ml) and saturated NaHCO 3 aqueous solution (100 ml). The organic layer was dried over MgSO 4 , 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) was dissolved in DCM (20 ml) and the mixture was stirred for 20 hours. The solution was diluted with DCM (100 ml), washed with H2O (100 ml), saturated aqueous NaHCO3 (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 crude 15 N-lipoamide was further purified by silica gel column chromatography (DCM: MeOH = 30: 1). The solvent was removed under reduced pressure and a yellow solid was obtained. 15 N-Lipoic acid amide. Yield: 4.2 mg (51%).1 H NMR(400MHz,chloroform-d)δ5.62(d,J=31.3Hz,1H,NH cis ),5.37(d,J=31.0Hz,1H,NH trans ),3.60(ddt,1H,SSCH),3.26–3.06(m,2H,SSCH2),2.58–2.40(m,1H,SSCH2CH trans ),2.26(t,J=7.5Hz,2H,CH2CONH2),2.02–1.84(m,1H,SSCH2CH cis ),1.83–1.60(m,4H),1.60–1.39(m,2H,CH2CH2CH2CONH2). 13 C 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. The data show 15 N labeling is ~99%. IR: 3352cm -1 ,3176cm -1 (CONH2), 2937cm -1 ,2898cm -1 ,2865cm -1 ,2783cm -1 (C–H), 1746 cm -1 ,1650cm -1 ,1629cm -1 ,1464cm -1 ,1413cm -1 ,1367cm -1 ,1342cm -1 ,1321cm -1 ,1292cm -1 ,1281cm -1 ,1252cm -1 ,1226cm -1 ,1203cm -1 ,1144cm -1 ,1125cm -1 ,1078cm -1 ,1034cm -1 ,999cm -1 ,950cm -1 ,911cm -1,868cm -1 ,803cm -1 ,734cm -1 ,675cm -1 ,629cm -1 (C=O). Melting point: 130°C, Rf=0.60 (DCM:MeOH=20:1).

[0424] 15 NR-(+)-Lipoic acid amide

[0425]

[0426] 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) at 25 ° C under argon atmosphere for 4 hours. The obtained solution was diluted with EtOAc (100 ml) and washed with H2O (100 ml) and saturated NaHCO3 aqueous 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) was dissolved in DCM (20 ml) and the mixture was stirred for 20 hours. The solution was diluted with DCM (100 ml), washed with H2O (100 ml), saturated aqueous NaHCO3 (100 ml) and then with H2O (100 ml, 2 times). The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure to give crude 15 N-lipoamide was further purified by silica gel column chromatography (DCM: MeOH = 30: 1). The solvent was removed under reduced pressure to obtain a yellow solid 15 N(R)-Lipoic acid amide. Yield: 554.5 mg (51%). 1 H NMR(400MHz,chloroform-d)δ5.56(d,J=4.1Hz,1H,NH cis ),5.34(d,J=4.7Hz,1H,NH trans ),3.60(ddt,1H,SSCH),3.30–3.03(m,2H,SSCH2),2.61–2.35(m,1H,SSCH2CH),2.32–2.18(m,2H,CH2CONH2),1.94(m,1H,SSCH2CH cis ),1.81–1.59(m,4H),1.59–1.41(m,2H,CH2CH2CH2CONH2).13 C 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 show 15 N labeling is ~99%. IR: 3340cm -1 ,3174cm -1 (CONH2), 2936cm -1 ,2921cm -1 ,2865cm -1 ,2850cm -1 (CH),2788cm -1 ,2550cm -1 ,2360cm -1 ,2341cm -1 ,2161cm -1 ,2036cm -1 ,1751cm -1 ,1650cm -1 ,1627cm -1 (C=O),1461cm -1 ,1411cm -1 ,1369cm -1 ,1343cm -1 ,1317cm -1 ,1294cm -1 ,1260cm -1 ,1213cm -1 ,1197cm -1 ,1135cm -1 ,1036cm -1 ,1004cm -1 ,914cm -1 ,878cm -1 ,808cm -1 ,794cm -1 Melting point: 121.5°C, Rf=0.60 (DCM:MeOH=20:1), [α] 25 +105.6 (c=2.0, CHCl3).

[0427] Density functional theory (DFT) calculations were performed to confirm that lipoamide 1Assignment of the H NMR spectrum. Gaussian098 was used to generate the optimized structure of lipoamide, from which the shielding tensor was calculated, enabling the determination of the isotropic and anisotropic components. DFT calculations were performed using the B3LYP density functional with a 6–31G(d) basis set. 7 2. DFT calculations confirmed that the cis-amide proton 13 should have a larger chemical shift than the trans-amide proton 14 ( Figure 13A ), and the carbon 3-bonded proton closest to the carbon 2-bonded proton should have a larger chemical shift than the other protons on carbon 3 ( Figure 13A ).

[0428] 15 N-lipoamide ( 15 N) 1 HNMR

[0429] Obtained on a narrow aperture Varian solution state spectrometer 1 H detection 15 Edited by N 1 H-sensitivity-enhanced HSQCNMR (herein referred to as ( 15 N) 1 H) Spectra, the spectrometer was operated at a fixed field strength of 14.1 T and equipped with a room temperature probe.

[0430] The free induction decay was recorded with an acquisition time of 0.0624 s and an 8 kHz scan with a 1 s recovery delay over 1000 s. Typically, 10,000 transients were collected, with a total experimental time of 3 h 1 min. 15 The J coupling between N was determined at 96 Hz, so the 1 / 4 J transfer time in the INEPT portion of the pulse sequence was set to 2.6 ms. Under these settings, 15 N ammonia or ammonium ions will not be detectable. 15 Chemical modification of N-lipoamide (including covalent attachment to apoenzymes) can be performed in ( 15 N) 1 The HNMR spectrum shows substantial changes. Similarly, dissolution of lipoamide in phospholipid membranes should produce significant peak broadening in cell samples. We observed neither, consistent with freely diffusing lipoamide.

[0431] If the conditions (including ionic strength, buffer composition, temperature and pH) are the same, the integrated NMR signal intensity is proportional to the concentration 7 3. It is expected that since the amide protons in lipoamide should be unstable in water, chemical exchange must also be considered. 74 To ensure the selection of appropriate conditions, ( 15 N)1 H NMR spectrum ( Figure 13B , C). Only in pure water and chloroform-d, the signal intensity of the amide proton resonance is comparable. In other solvents, the cis-amide proton signal is reduced, indicating chemical exchange ( Figure 13B ), so the trans amide proton was used for concentration quantification. In order to determine the temperature and pH sensitivity of the trans amide proton signal, the trans amide proton was detected at different temperatures ( Figure 13D ) and pH( Figure 13E ) to obtain 1 mM lipoamide in the culture medium ( 15 N) 1 H spectrum. Both amide protons exhibit chemical exchange under high temperature and high pH conditions, while the trans amide proton is less affected ( Figure 13D , E). To determine whether lipoamide degrades over time, the signal from the trans amide protons was monitored at 37°C and 10°C for 10 h. The signal intensity decayed slowly at 37°C but not at 10°C ( Figure 13F ), indicating slow hydrolysis to form ammonia. Therefore, at 10 °C and below pH 8.6, the integrated signal from the trans amide proton resonance is 15 A good measure of N-lipoamide concentration.

[0432] By comparing the absence of (-cells, sample i, Figure 2 A) and presence (+ cells, sample ii, Figure 2 A) The signal intensity S of the trans-amide proton of lipoamide obtained under the conditions of HeLa cells was used to measure the cellular uptake. The measured uptake fraction U is given by the following formula:

[0433]

[0434] After uptake, the amount (in moles) of added lipoamide (add) becomes distributed between the intracellular (cell) and extracellular (out) environments. This can be expressed in terms of concentration c and volume V:

[0435] c add V add =c cell V cell +c out V out

[0436] By V cell =V1N cell gives the total volume of the cell, where V1 is the total volume of a single cell and N cell is the number of cells. cell < <V add , so we assume that V add =V oThe fractional uptake can also be expressed in terms of these concentrations and volumes:

[0437]

[0438] Rearrangement gives expressions for the internal and external concentrations of lipoamide based on the amount of lipoamide added and the measured fractional uptake, U.

[0439]

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

[0441] We have no evidence that the peak broadening is related to solubilization in phospholipid membranes; however, in principle, the loss of signal intensity upon uptake could be attributed to uptake into membranes rather than the cytosol.

[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. L =0.5nm 2 . 75 Assuming that the cells are spherical, the surface area of ​​a single cell is A1 = 1.3 × 10 -9 m 2 Therefore, the total number of phospholipids on the molecule taken up by the cell is N uptake =c cell V cell N A , where N A is Avogadro's constant. The ratio of lipoamide to lipid molecules is given by:

[0443]

[0444] For the experimentally observed mean value of U = 0.35 (i.e., 35% uptake), 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 a condensate-associated molecule, the method comprising: (a) contacting the compound with a cell composition comprising one or more coacervates or a cell composition capable of forming one or more coacervates, and (b) determining a property associated with the one or more coagulants, in, Modulation of the property compared to a reference indicates that the compound modulates a property associated with the one or more aggregates.

2. The method of claim 1 , wherein the property associated with the one or more coagulants is determined based on any one or more of the following: (i) the number of aggregates that include and / or do not include the aggregate-associated molecules; (ii) the size of the one or more agglomerates; (iii) the location of the one or more condensates; (iv) distribution of the one or more agglomerates; (v) the surface area of ​​the one or more coagulants; (vi) the composition of the one or more coagulants; (vii) flowability of the one or more coacervates; (viii) solidification of the one or more coagulants; (ix) dissolution of the one or more coacervates; (x) the presence and / or amount of fiber formation; (xi) the location of the condensate-associated molecules; (xii) partitioning of the condensate-associated molecules into the one or more condensates; and (xiii) Aggregation of molecules associated with the condensate.

3. The method of claim 1 or 2, wherein the one or more aggregates are within one or more cells in the cell composition.

4. The method of any one of claims 1-3, further comprising subjecting the cell composition to aggregate-forming conditions prior to step (b).

5. The method of any one of claims 1-3, further comprising subjecting the cell composition to aggregate-forming conditions prior to step (a).

6. The method according to claim 4 or 5, wherein the coagulant forming conditions are any one or more of the following: (i) Oxidative stressors; (ii) mitochondrial electron transport chain inhibitors; (iii) heat stressors; (iv) osmotic stressor; (v) hypertonic stressors; and (vi) Glycolysis inhibition.

7. The method of any one of claims 1-6, wherein the condensate-associated molecule is a polypeptide.

8. The method of any one of claims 1-7, wherein the condensate-associated molecule is a wild-type polypeptide.

9. The method of any one of claims 1-7, wherein the condensate-associated molecule is a mutant polypeptide.

10. The method according to any one of claims 1 to 9, wherein the condensate-associated molecule is selected from the group consisting of FUS, EWSR1, TIAL1, PABPC1, and G3BP1.

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