Compounds for the treatment of disease and methods of screening same

Abstract

The present invention relates to compounds for use in the prevention or treatment of neurodegenerative diseases associated with stress granule formation. The compounds are selected from lipoic acid, lipoamide, dihydrolipoic acid, and dihydrolipoamide. The present invention also relates to methods for identifying compounds that modulate one or more condensate-associated characteristics, including condensate-associated molecules, comprising (a) contacting a compound with a cellular composition containing one or more condensates or capable of forming one or more condensates, and (b) determining one or more condensate-associated characteristics. Modulation of a characteristic indicates that the compound modulates one or more condensate-associated characteristics compared to a reference.

Description

[Technical field]

[0001] The present invention relates to a compound for use in the prevention or treatment of neurodegenerative diseases associated with the formation of stress granules. The compound is selected from lipoic acid, lipoamide, dihydrolipoic acid, and dihydrolipoamide. The present invention further relates to a method for identifying a compound that modulates one or more condensate-associated characteristics, including condensate-associated molecules, comprising (a) contacting a compound with a cellular composition that includes one or more condensates or that can form one or more condensates, and (b) determining one or more condensate-associated characteristics. Modulation of a characteristic indicates that the compound modulates one or more condensate-associated characteristics compared to a reference. [Background technology]

[0002] Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease with a poor prognosis and few treatment options. Currently, only two FDA-approved drugs are available, riluzole and edaravone, both of which only slow disease progression by a few months. The actions of riluzole and edaravone are not fully understood and most likely do not directly target the pathological mechanisms underlying the disease. Therefore, new approaches are critically needed. ALS is mostly sporadic, although ~10% are monogenic. Familial ALS-associated mutations are frequently found in RNA-binding proteins (RBPs), such as FUS and TDP-43. These RBPs have characteristic low-complexity domains (LCDs).

[0003] The exact mechanism of ALS pathogenesis is unclear, and it is unclear whether aggregates and oligomers are toxic or whether they cause protein loss of function and how this results in downstream effects. However, mutant TDP-43 and FUS tend to mislocalize in the cytoplasm, promoting the formation of disease-associated stress granules and abnormal cytoplasmic aggregation. In particular, it has been hypothesized that the pathological stability of stress granules may be disease-related. This suggests that regardless of whether the pathogenesis is due to a toxic gain of function or protein sequestration and loss of function, it may be possible to ameliorate the disease by dissolving stress granules and / or aggregates.

[0004] Stress granules are liquid, non-membrane-bound compartments, and recent studies have shown that LCD-containing proteins form these compartments by liquid-liquid phase separation. In principle, therefore, it may be possible to target physicochemical properties that promote stress granule formation. Indeed, there are known compounds that disrupt this phase separation, particularly alcohols such as 1,6-hexanediol. However, these compounds have 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, non-membrane-bound compartments as well. There are many other liquid compartments, especially in the nucleus.

[0005] What is needed are methods to identify compounds that more specifically modulate phase separation of specific compartments for effective and specific ALS treatments.

[0006] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. Summary of the Invention [Means for solving the problem]

[0007] In some aspects, the disclosure provides methods for identifying a compound that modulates one or more condensate-associated characteristics, including condensate-associated molecules, the method comprising: (a) contacting the compound with a cellular composition that includes one or more condensates or is capable of forming one or more condensates; and (b) determining the one or more condensate-associated characteristics, wherein modulation of the characteristics indicates that the compound modulates the one or more condensate-associated characteristics relative to a reference.

[0008] In some embodiments, determining the characteristic associated with the one or more condensates is based on any one or more of: (i) the number of condensates with and / or without condensate-associated molecules, (ii) the size of the one or more condensates, (iii) the location of the one or more condensates, (iv) the distribution of the one or more condensates, (v) the surface area of ​​the one or more condensates, (vi) the composition of the one or more condensates, (vii) the fluidity of the one or more condensates, (viii) solidification of the one or more condensates, (ix) dissolution of the one or more condensates, (x) the presence and / or amount of fibril formation, (xi) the location of the condensate-associated molecules, (xii) partitioning of the condensate-associated molecules into one or more condensates, and (xiii) aggregation of the condensate-associated molecules.

[0009] In some embodiments, the one or more condensates are present within one or more cells in the cellular composition.

[0010] In some embodiments, the methods described herein further comprise subjecting the cell composition to condensate-forming conditions prior to determining one or more condensate-associated characteristics.

[0011] In some embodiments, the methods described herein further comprise subjecting the cellular composition to condensate forming conditions prior to contacting the cellular composition with the compound.

[0012] In some embodiments, the condensate forming condition is one or more of: (i) an oxidative stressor, (ii) a mitochondrial electron transport chain inhibitor, (iii) a thermal stressor, (iv) an osmotic stressor, (v) a hyperosmotic stressor, and (vi) glycolysis inhibition.

[0013] 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.

[0014] In some embodiments, the cells in the cell composition express a condensate-associated molecule. In some embodiments, the cell composition comprises HeLA cells, iPSC cells, or iPSC MN cells.

[0015] In some embodiments, the methods described herein further comprise imaging at least a portion of the cellular composition.

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

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

[0018] 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.

[0019] In some embodiments, the reference is a second condensate. In some embodiments, the reference is a second cellular composition.

[0020] In some embodiments, the methods described herein further comprise evaluating the identified compounds using a second cell-based assay.

[0021] In some embodiments, the methods described herein further comprise evaluating the identified compounds using a biochemical assay.

[0022] In some embodiments, the methods described herein further comprise evaluating the identified compounds using an in vivo assay.

[0023] In another aspect, the present disclosure provides a method for identifying a compound useful for treating a disease, comprising identifying a compound according to any one of the methods described in the present invention.In some embodiments, the disease is a neurodegenerative disease.In some embodiments, the neurodegenerative disease is ALS.

[0024] The present invention relates to compounds for the treatment of neurodegenerative diseases associated with the formation of stress granules, in particular for the treatment of amyotrophic lateral sclerosis.

[0025] One aspect of the invention relates to a compound 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(sulfanyl)octanamide; CAS No. 3884-47-7), for use in a method of preventing or treating a neurodegenerative disease associated with the formation of stress granules.

[0026] In particular, neurodegenerative diseases are associated with the formation of stress granules in the cytosol of cells contained within the affected tissue.

[0027] In particular, lipoic acid and / or lipoamide may be used in the R-, S- or racemic form.

[0028] Those skilled in the art will be aware that any of the compounds specifically mentioned can exist as pharmaceutically acceptable salts of the compounds.Pharmaceutically acceptable salts include ionized drugs and oppositely charged counterions.Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, besylate, bitartrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate. Non-limiting examples of pharma- ceutically acceptable cationic salt forms include aluminum, benzathine, calcium, ethylenediamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine, and zinc.

[0029] In some embodiments, the compounds of the invention are administered according to the following dosing schedule: A daily dose of 600-1,600 mg of compound is administered.

[0030] Advantageously, a daily dose of 600 mg may result in a plasma concentration of the compounds of the invention of between 8 and 30 μM.

[0031] In some embodiments, the neurodegenerative disease associated with the formation of stress granules is selected from amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease.

[0032] In some embodiments, the pharmaceutical composition for use in the method of preventing or treating a neurodegenerative disease associated with the formation of stress granules comprises a compound of the invention listed in paragraph

[0025] .

[0033] In some embodiments, the pharmaceutical composition is formulated for oral administration.

[0034] In some embodiments, the neurodegenerative disease associated with stress granule formation is amyotrophic lateral sclerosis.

[0035] Alternatively, there is provided a dosage form for use in a method for preventing or treating a neurodegenerative disease associated with the formation of stress granules, the dosage form comprising a compound of the present invention, specifically a compound listed in paragraph

[0025] .

[0036] The dosage form may be an enteral administration such as nasal, buccal, rectal, transdermal, or oral administration, or an inhalation form, or a suppository. Alternatively, parenteral administration may be used, such as subcutaneous, intravenous, intrahepatic, or intramuscular injection forms. Optionally, a pharmaceutically acceptable carrier and / or excipient may be present.

[0037] In some embodiments, the dosage form is formulated for oral administration.

[0038] In some embodiments, the neurodegenerative disease associated with the formation of stress granules is selected from amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease.

[0039] Alternatively, a therapeutic aspect of the present invention can be formulated as a method for preventing or treating a neurodegenerative disease associated with the formation of stress granules, the method comprising administering to a patient in need thereof a compound of the present invention, in particular the compounds listed in paragraph

[0025] .

[0040] In some embodiments, the compound is administered in a daily dose of 600 to 1,600 mg.

[0041] In some embodiments, the compound is administered orally.

[0042] In some embodiments, the neurodegenerative disease associated with the formation of stress granules is selected from amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease.

[0043] In some embodiments, the neurodegenerative disease associated with stress granule formation is amyotrophic lateral sclerosis. In one aspect, the invention relates to a method of suppressing or inhibiting stress granule formation in a cell, the method comprising: - Lipoic acid, Lipoamide, Dihydrolipoic acid, Dihydrolipoamide, heterotricyclic compounds, in particular anthraquinone or anthraquinone derivatives, such as 1,4-dihydroxyanthraquinone, - acridine or an acridine derivative, such as quinacrine or aminoacridine or mitoxantrone, - tetracyclic compounds, and surfactants, in particular cetylpyridinium chloride, The present invention includes the use of a compound selected from the group consisting of

[0044] In some embodiments, the compound is provided to the culture medium in which the cells are incubated.

[0045] Those skilled in the art will also appreciate that changes in form and detail of the implementations described herein may be made without departing from the scope of the disclosure, and while various advantages, aspects, and objects have been described with reference to various implementations, the scope of the disclosure should not be limited by reference to such advantages, aspects, and objects. [Brief description of the drawings]

[0046] [Figure 1A] Screening of small compounds that affect the formation of FUS-containing stress granules in HeLa cells ex vivo. Diagram showing subcellular localization of FUS-GFP in unstressed HeLa cells, cells stressed with negative control compound solvent (DMSO), and cells stressed with positive controls dimercaprol (an arsenic chelator) and emetine (which stabilizes polysomes and inhibits stress granule formation). Stress leads to nuclear export of FUS and the formation of stress granules (cytoplasmic FUS-containing droplets). [Figure 1B]A workflow to screen small molecules that affect FUS-GFP localization in HeLa cells ex vivo. [Figure 1C] Ranking table of all 1600 compounds screened by Mahalanobis distance (average of 6 fields). Higher values ​​indicate more effective compounds. Several automated measures of FUS localization were combined into one Mahalanobis distance score. Maximum factors were the number and area of ​​cytoplasmic FUS droplets. Using a cutoff value of 130, 47 compounds were selected for further analysis. Dimercaprol and emetine are highlighted. [Figure 1D] Workflow to screen small molecules for their effect on FUS liquid-liquid phase separation of purified FUS-GFP in vitro. [Figure 1E] Table ranking the Z-scores of the change in droplet number and signal partitioning into FUS-GFP droplets (formed under low salt conditions). The higher the positive and negative values, the higher the compound effect. Scores were calculated at 100 μM, the highest concentration at which the negative control compound solvent (DMSO) had no significant effect. Lipoamide, surfactants, and heterotricyclic / tetracyclic compounds are indicated by the color of the data points. See Figure 1F. [Figure 1F] Examples of three hit compound classes: lipoamide, cetylpyridinium chloride (surfactant), and mitoxantrone (heterotricyclic / tetracyclic compound). [Figure 1G] Appearance of droplets with negative control compound solvent (DMSO) or examples of compound classes (cetylpyridinium chloride (surfactant), lipoamide, or mitoxantrone (heterotricyclic)). Note that droplets are larger for cetylpyridinium chloride and lipoamide, and fewer and smaller droplets for mitoxantrone. [Figure 2A] Structure-activity relationships of lipoamide-related compounds that may indicate the mechanism of action. Dose response of FUS-GFP droplet numbers (●, left axis) and nuclear / cytoplasmic signal ratio (○, right axis) in HeLa cells after 1 h pretreatment with lipoamide followed by 1 h of arsenate stress with continued lipoamide treatment. [Figure 2B]Dose response of FUS-GFP droplet number and nucleus / cytoplasm ratio in iPS cells, as in Fig. 2A . [Figure 2C] Dose-response of FUS-GFP droplet numbers and nuclear / cytoplasmic ratios in HeLa cells for a series of compounds related to lipoamide using the same stress / treatment regime as in Figure 2A. [Figure 2D] Summary of compound structures and effects on cytoplasmic FUS droplet numbers and FUS nuclear division. [Figure 3A] Diagram showing the patterns of effect of lipoamide and lipoic acid on various stresses under different regimes that may indicate the mechanism of action. Kinetics of reduction in the number of cytoplasmic FUS-GFP droplets in HeLa cells treated with 10 μM lipoamide (or DMSO solvent control) during continued arsenate stress following 1 hour pretreatment with arsenate. Example images 2 and 100 min after compound addition are shown on the right. Lipoamide can reduce the effect of existing arsenate stress. [Figure 3B] Images of HeLa cells expressing FUS-GFP following 1 hour pretreatment with 10 μM lipoamide (or DMSO control), followed by 3 washes and 1 hour of arsenate stress. Pretreatment with lipoamide has no lasting effect on cells preventing their response to arsenate stress. [Figure 3C] Images of HeLa cells expressing GFP-tagged stress granule markers (EWSR1, TIAL1, PABC1, or G3BP1) following arsenate stress and treatment with 10 μM lipoamide or lipoic acid isomers, arsenate stress and DMSO solvent control, or DMSO without arsenate for 1 hour. Lipoamide and lipoic acid affect many stress granule components. [Figure 3D] Images of HeLa cells expressing FUS-GFP subjected to various stresses [rotenone (mitochondrial), serum deprivation, sorbitol (osmotic), heat, arsenate, or 6-deoxyglucose (glycolysis)] and simultaneously treated with 10 μM lipoamide or lipoic acid isomers. Lipoamide and lipoic acid are effective against multiple stresses, including mitochondrial, osmotic, and oxidative stress. [Figure 4A]Showing that lipoamide does not dissolve the nuclear FUS compartment and other nuclear compartments. Images of HeLa cells expressing protein GFP markers of other non-membrane bound compartments treated with 10 μM compound (or DMSO control) for 1 hour. Where unclear, the location of the nucleus is outlined with a dotted line. Lipoamide does not disrupt P bodies (DCP1A), Cajal bodies (COIL), DNA damage foci (TRP53BP1), or nucleoli (NCB1), whereas mitoxantrone has a specific effect on non-stress granules. [Figure 4B] Recruitment of FUS-GFP to sites of UV laser-induced DNA damage in iPS cells after 1 h of treatment with compounds followed by 1 h of arsenate stress. The top row shows FUS-GFP fluorescence before laser ablation, with arrows pointing to stress granules. The bottom row shows the mean FUS-GFP signal intensity versus DNA damage and one standard deviation above and below the mean. Mitoxantrone blocks nuclear FUS droplet formation at sites of DNA damage below the EC50 for cytoplasmic droplet numbers, whereas lipoamide blocks nuclear FUS droplet formation. [Figure 5A] We show that lipoamide and lipoic acid affect FUS condensate properties and FUS coagulation in ALS-associated mutations in vitro. Schematic illustrating the quantification of fluidity of condensed droplets using optical tweezers. Two droplets are brought into contact to initiate fusion. The time to relax into a single spherical droplet (after adjusting for droplet size) is an indication of the ratio of the droplet's viscosity to surface tension and thus of fluidity. Representative of two independent replicates. [Figure 5B] Droplet size-corrected relaxation times for droplet fusion with either 300 μM lipoamide or an equivalent DMSO solvent control (0.3%). Boxes indicate the 25th, 50th, and 75th percentiles, whiskers indicate the 5th and 95th percentiles. The shorter fusion time with lipoamide indicates lower viscosity and / or higher surface tension. [Figure 5C]Effect of 10 μM Lipoamide on G156E FUS-GFP condensates (formed under dextran clounding) that were "aged" by shaking, compared to an equivalent DMSO solvent control (0.1%). These conditions are the same as those previously published. Representative images after 30 hours of aging, showing fibril formation in the DMSO sample. [Figure 5D] Effect of 10 μM lipoamide on G156E FUS-GFP condensates (formed under dextran clounding) that were "aged" by shaking, compared to an equivalent DMSO solvent control (0.1%). These conditions are the same as those previously published.7 Representative time series images of fluorescence recovery after photobleaching (FRAP) of FUS condensates and fibrils upon aging. [Figure 5E] Comparison of the effect of 10 μM lipoamide on G156E FUS-GFP condensates (formed under dextran clounding) aged with shaking with an equivalent DMSO solvent control (0.1%). These conditions are the same as those previously published7. Quantification of FRAP in Figure 5C. Error bars represent standard deviation. Aged condensates treated with lipoamide maintain a large FUS-GFP mobile fraction and short FRAP half-life, whereas untreated condensates stiffen. Comparison of the effect of lipoamide and lipoic acid on G156E FUS-GFP condensates aged with shaking with an equivalent DMSO solvent control (0.3%). Both compounds delay fibril formation. [Figure 6A] Figure 1. Lipoamide and lipoic acid directly alter FUS phase separation and FUS G156E coagulation in vitro. Droplets formed by 2.8 μM FUS-GFP in vitro with varying concentrations of KCl (which inhibits droplet formation) containing 100 μM lipoamide, lipoic acid, or an equivalent DMSO solvent control (1%). Lipoamide and lipoic acid slightly promote droplet formation. [Figure 6B] The effect of lipoamide and lipoic acid on shaken "aged" G156E FUS-GFP droplets compared to an equivalent DMSO solvent control (0.3%). Both compounds slow fibril formation. [Figure 6C]Representative fluorescence recovery after photobleaching (FRAP) images of FUS droplets and fibrils formed upon aging shown in Figure 6B. Aged droplets treated with lipoamide or lipoic acid maintain rapid FRAP indicative of fluidity. [Figure 6D] Quantification of the fluorescent signal intensity shown in Figure 6C. Error bars represent standard deviation. Treatment with lipoamide and lipoic acid preserves a large FUS-GFP mobile fraction and short FRAP half-life, whereas untreated droplets stiffen. [Figure 7A] Diagram showing that lipoamide accumulates to high concentrations intracellularly without being metabolized. 15N R-(+)- and (±)-lipoamide were synthesized and characterized. Using 15N-edited 1H-detected 1D HSQC NMR experiments, the two amide protons could be selectively detected in biological medium and HeLa cell pellets. The trans-amide proton (resonating at 6.9 ppm) was quantifiable by NMR. At 10 °C and below pH 8.5, the signal intensity was proportional to the lipoamide concentration. See Supplementary Methods for details. [Figure 7B] Lipoamide uptake by HeLa cells was measured by exposing cells to 15N lipoamide, fractionating samples, and measuring 15N lipoamide concentration using the NMR signal intensity of the trans-amide protons. Media containing 100 μM 15N lipoamide was incubated for 1 h in the presence or absence of HeLa cells. After removing the media, cells were washed with media (arsenate-free) and detached with EDTA-trypsin. 15N lipoamide concentration was measured using solution or cell pellet / intracellular NMR. Exemplary spectra of cells stressed with 3 mM arsenate and incubated with R-(+)-lipoamide are shown on the same y-axis scale. [Figure 7C]Cellular uptake was determined by subtracting the signal from the medium incubated with cells (red) from the signal from the medium without cells (blue). This was performed for all four combinations of stressed (3 mM arsenate) or unstressed cells with 15N R-(+) or (±)-lipoamide. For stressed cells treated with 15N R-(+)-lipoamide, the high signal intensity from the washed cell sample (green) is consistent with the magnitude of uptake from the medium calculated from the signal intensities with (red) and without (blue) cells. [Figure 7D] Quantification in Figure 7C showing the uptake rate and calculated intracellular concentration assuming lipoamide is uniformly distributed within the cells (see Supplementary Methods). The uncertainty in the measurements was approximately 30%, and there was no significant difference in uptake between conditions. All measurements showed significant uptake of lipoamide, with cellular concentrations above 1 mM. [Figure 8A] We show that lipoamide and lipoic acid have beneficial effects in in vitro and in vivo ALS models. Lipoic acid inhibits age-induced aggregation of stress granule proteins, but not non-stress granule proteins, in C. elegans. Graph showing toxicity of R-(+)- or S-(-)-lipoic acid and its effect on protein aggregation in worms overexpressing aggregation-prone fluorescently labeled proteins. Incidence of PAB-1 aggregation in pharyngeal muscles was scored as the percentage of cells with >10 aggregates. Incidence of RHO-1 and KIN-19 was scored on a 3-point scale: low, moderate, or high (see Methods). Toxicity was assessed as the percentage of abnormally small or dead worms. Both isomers of lipoic acid significantly inhibited the aggregation of PAB-1, but not RHO-1 or KIN-19, in a dose-dependent manner. Significant changes from DMSO controls are shown by Fisher's exact test. ***p<0.0001, **p<0.001, *p<0.01. Error bars indicate standard error of percentage, n>100 for each sample. [Figure 8B]Lipoic acid inhibits aging-induced aggregation of stress granule proteins, but not non-stress granule proteins, in C. elegans. Z-stack images of the pharynx of worms expressing fluorescently tagged PAB-1, with or without lipoic acid treatment, show a reduced number of aggregates. [Figure 8C] Lipoic acid and lipoamide on axonal dieback in neurons derived from iPS cells expressing FUS P525L associated with familial ALS. A schematic of axonal growth from the cell body through the conduit in neuronal culture is shown on the right. The area indicated in the micrograph in Figure 8D,E is shown. [Figure 8D] Lipoic acid and lipoamide on axonal degeneration and retraction of iPS cell-derived neurons expressing FUS P525L, which is associated with familial ALS. iPS-derived neurons after 60 days in medium containing 0.02% DMSO. Axons of neurons expressing wild-type FUS are stable, whereas axons of neurons expressing FUS P525L are unstable and retract, leaving material around the axonal duct exit. [Figure 8E] Lipoic acid and lipoamide on axonal degeneration and retraction of iPS cell-derived neurons expressing FUS P525L associated with familial ALS. 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 of blinded experiments that also included DMSO (solvent control)-treated neurons shown in Figure 8C. [Figure 8F] Lipoic acid rescues motor deficits in D. melanogaster overexpressing human wild-type FUS or ALS-associated FUS mutations. Overexpression of FUS results in motor deficits and renders flies unable to climb. Treatment with lipoic acid dose-dependently improved the climbing ability of flies expressing wild-type FUS, FUS P525L, or FUS R512C. **p<0.005, *p<0.05, one-way ANOVA. [Figure 8G]Lipoamide also rescues motor function defects in D. melanogaster. Figure 8F shows the same conditions, but with lipoamide instead of lipoic acid. *p<0.05, **p<0.005, Student's t-test. [Figure 8H] Lipoic acid rescues motor deficits in D. melanogaster overexpressing human wild-type FUS or ALS-associated FUS mutations. Overexpression of FUS leads to motor deficits and the flies are unable to climb. **p<0.005, *p<0.05, one-way ANOVA. [Figure 9] iPS-derived neurons after 5 and 60 days in medium containing 0.02% DMSO. Axons of neurons expressing wild-type FUS are stable, whereas axons of neurons expressing FUS P525L are unstable and retract, leaving material around the axonal duct exit. [Figure 10A] Structure-activity relationships of heterotricyclic compounds showing that the tricyclic core is responsible for activity. Dose response of FUS-GFP droplet numbers (●, left axis) and nuclear / cytoplasmic signal ratio (○, right axis) in HeLa cells after 1 h pretreatment with mitoxantrone followed by 1 h of arsenate stress with continued mitoxantrone treatment. [Figure 10B] Dose-response of FUS-GFP droplet numbers and nuclear / cytoplasmic ratios in HeLa cells using the same stress / treatment regimes as in Figure 10A for a series of heterotricyclic hits such as mitoxantrone, quinacrine, and compounds related to the tetracycline antibiotic family. [Figure 10C] Summary of compound structures and effects on cytoplasmic FUS droplet numbers and FUS nuclear division. [Figure 11A]We show that lipoamide reduces intracellular aggregates of wild-type and P525L FUS in iPS cells. We show FUS-GFP localization in isogenic iPS cells expressing either wild-type or P525L FUS-GFP following 1h arsenate stress combined with 1h treatment with 30 μM lipoamide or DMSO negative control. FUS P525L forms large FUS droplets in the cytoplasm but is still sensitive to lipoamide. [Figure 11B] Relative optical density of aggregated FUS assessed by filter retardation assay in iPS cells from FIG. 11A that were either unstressed or stressed for 1 hour following 100 μM lipoamide or DMSO solvent control pretreatment for 1 hour. Significant changes (Student's t-test) are indicated. n=5. [Figure 11C] Western blots of anti-FUS and anti-GAPDH after cell treatment in Figure 11B. No significant changes were observed in the expression levels of FUS relative to GAPDH (Student's t-test, n=3). ***Analysis of variance*** [Figure 12A] Lipoamide rescues the axonal transport deficit caused by expression of FUS P525L in motor neurons. Example kymographs of lysosome trafficking in the distal part of FUS P525L GFP motor neuron axons following treatment with compound vehicle (DMSO) or 2 μM lipoamide for 3 days. LysoTracker fluorescence. [Figure 12B] Percentage of LysoTracker-labeled lysosomes migrating at an average velocity >2 μm / sec after 3 days of treatment of motor neurons expressing either P525L or wild-type FUS with 2 μM lipoamide or solvent control at equivalent DMSO concentrations. n=5 (P525L) or n=3 (wild-type) biological replicates were performed, with 5 axons analyzed per replicate. Lipoamide significantly increased lysosomal trafficking (Student's t-test). ***ANOVA*** [Figure 13A] Characterization of 15N lipoamide by 1H NMR. Chemical structure of 15N lipoamide. [Figure 13B]The resonances in the 1H NMR spectrum can be unambiguously assigned to individual protons of 15N lipoamide in CDCl3. [Figure 13C] 15N filtered NMR experiments showed cis-amide and trans-amide protons in lipoamide (environments 13 and 14, respectively). The relative signal intensities were sensitive to local solution conditions, indicating chemical exchange at 37 °C. [Figure 13D] At pH 8.3, the intensity of both resonances decreased with increasing temperature, indicating local molecular dynamics on the millisecond to microsecond time scale and / or chemical exchange where the signal is diminished by interactions with HO. Below 15 °C, the intensity of the trans-amide resonance (14) approached a plateau, indicating a slow exchange regime where the signal intensity is a clear indicator of concentration. [Figure 13E] At 10 °C, the intensities of the cis- and trans-amide proton resonances increased with decreasing pH, indicating the presence of a second dynamics in the millisecond to microsecond range. Below pH 8.6, the intensities of the trans-amide protons were constant, indicating a slow exchange regime. Figures S3D and S3F show that at 10 °C and below pH 8.6, the integrated signal intensities of the trans-amide protons of lipoamide in 15N-edited 1H NMR experiments are reliable as a proxy for concentration. [Figure 13F] The signal intensity of the trans-amide protons of lipoamide, when dissolved in growth medium, decreased over time at 37° C. but not at 10° C. At 10° C., the signal intensity remained stable for >10 h of the experiment. [Figure 13G] Signal intensity of cis- and trans-amide protons under various experimental conditions. This is a zoomed-in version of Fig. 13B showing only trans-amide protons and adding one additional condition: v) cells disrupted with Triton X-100 and DNase I (iii). Taken together i) to iv) suggest that while lipoamide is taken up by HeLa cells in a mobile form, most of the molecule is unmodified (uptake quantified in Fig. 13C-D). [Figure 13H] Zoomed-in spectra of i) and v) in Figure 13G. The spectra change dramatically upon cell disruption with Triton X-100 and DNase I, indicating that lipoamide is chemically modified upon disruption of the cellular compartment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] The invention, in some aspects, includes methods for identifying condensate-modulating compounds and the utility of such compounds in these applications, such as the treatment of disease.

[0048] Traditionally, the various membrane-bound compartments (organelles) of the cell were thought to be a promiscuous mess of macromolecules and metabolites, all of which mixed freely and diffused randomly within the regions of the membrane that demarcate specific compartments (nucleus, cytosol, mitochondria, endoplasmic reticulum, etc.). Cellular processes were thought to be controlled by the interactions of these molecules, leading to biochemical reactions between molecules (enzymatic activities such as transcription, RNA processing, metabolic reactions, signaling molecule reactions such as phosphorylation, etc.) through the induction of specific binding sites. For example, kinases diffuse until they happen to bind to a protein that they recognize as a target, and by binding to that protein, they phosphorylate it.

[0049] Recent studies have revealed that macromolecules are separated into condensates within membrane-bound organelles based on the intrinsic physical properties of proteins, RNA, and / or DNA. Macromolecules come together in a phase-separation reaction, i.e., condense into droplets, concentrating certain protein and / or nucleic acid molecules within the condensate while excluding other specific proteins and / or nucleic acids.

[0050] The process of condensate formation is not driven by a specific, simple stoichiometric binding event, as previously thought, but by phase separation reactions - structural layers within cells that pack macromolecules throughout the cell into small compartments, enhancing reaction specificity and enhancing reactions by increasing their local concentrations.

[0051] Remarkably, because condensates are liquid and reversible, different condensates within a cell can change and dissolve completely or change slightly in molecular composition in response to changes in cell physiology (e.g., signaling events, changes in the concentration of a single macromolecule, or changes in the local environment). This is therefore a major mechanism controlling almost all reactions within a cell, synergizing with previously understood mechanisms of specific binding events.

[0052] Some diseases, including neurodegenerative diseases such as ALS, are believed to result from abnormal aggregation of proteins that accumulate inside cells. Previous attempts to find drugs that inhibit or destroy these aggregates have not been based on a new understanding of condensates. The disclosure of this application is based, at least in part, on the inventors' unique insight that in this case, phase separation of macromolecules into condensates is an intermediate step that occurs before the proteins subsequently form abnormal aggregates, and thus new compounds useful for treating such diseases can be discovered using assays that directly observe the effect of compounds on the formation and properties of condensates.

[0053] In some embodiments, the methods disclosed in the present invention allow for the identification of compounds that modulate certain aspects of condensate behavior. For example, some embodiments of the methods disclosed in the present invention allow for the identification of compounds that modulate fluidity but do not form condensates. Furthermore, using the methods described in the present invention, the inventors have identified compounds that appear to be specific to the condensate of interest, rather than simply inhibiting the formation of all condensates. Such specificity may increase efficacy and reduce side effects such as toxicity.

[0054] The present application provides novel methods for identifying compounds that modulate one or more characteristics of intracellular and extracellular condensates and are therefore useful in the treatment of condensate-associated diseases. The methods focus on changes in the behavior or characteristics of one or more condensates, allowing for the identification of compounds without necessarily having knowledge of the specific molecular target of each compound. This allows for the rapid screening of potentially useful compounds with simple and elegant behavioral assays. Exemplary characteristics of condensates include, but are not limited to, (i) the number of condensates with and / or without condensate-associated molecules, (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 fluidity of one or more condensates, (viii) the solidification of one or more condensates, (ix) the dissolution of one or more condensates, (x) the presence and / or amount of fibril formation, (xi) the location of condensate-associated molecules, (xii) the partitioning of condensate-associated molecules into one or more condensates, and (xiii) the aggregation of condensate-associated molecules. Compounds with desired properties can be identified by assessing their 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 characteristics of one or more condensates. Compounds with desired properties can also be identified by assessing their ability to modulate some characteristics of condensates but not others, or to modulate some condensates but not others. The methods described in this invention have been used to identify compounds that target the physicochemical properties of condensates formed by liquid-liquid phase separation.

[0055] Thus, some aspects of the invention provide methods for screening for compounds that modulate one or more condensate-associated characteristics and / or are useful in methods of treating disease. Some aspects of the invention provide methods for high-throughput screening for compounds that modulate one or more condensate-associated characteristics and / or are useful in methods of treating disease.

[0056] In some embodiments, the method includes (a) contacting a cellular composition that includes one or more condensates or that can form one or more condensates with a compound, and (b) determining a characteristic associated with the one or more condensates. In some embodiments, the modulation of the characteristic indicates that the compound modulates a characteristic associated with the one or more condensates. In some embodiments, the modulation of the characteristic indicates that the compound modulates a characteristic associated with the one or more condensates relative to a reference. In some embodiments, the method includes determining a plurality of characteristics associated with the one or more condensates.

[0057] In some embodiments, the method includes (a) contacting a cellular composition comprising one or more condensates or a cellular composition capable of forming one or more condensates with a plurality of candidate compounds, and (b) determining a characteristic associated with the one or more condensates, where modulation of the characteristic indicates that the candidate compound modulates the characteristic associated with the one or more condensates compared to a reference, thereby obtaining a compound that modulates the characteristic of the one or more condensates.

[0058] In some embodiments of the invention, methods are provided for identifying a compound that modulates a characteristic associated with a first set of one or more condensates comprising a first condensate-associated molecule, the methods including (a) contacting a compound with a cellular composition comprising the first and second sets of one or more condensates or a cellular composition capable of forming the first and second sets of one or more condensates, and (b) determining a characteristic associated with the first set of one or more condensates, and (c) determining a characteristic associated with the second set of one or more condensates.

[0059] In some embodiments of the invention, methods are provided for identifying a compound that modulates a characteristic 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 cellular composition comprising or capable of forming the first and second sets of one or more condensates, and (b) determining a characteristic associated with the first set of one or more condensates, and (c) determining a characteristic associated with the second set of one or more condensates.

[0060] In some embodiments of the invention, methods of screening (e.g., high throughput screening) for compounds that modulate a characteristic associated with a first set of one or more condensates comprising a first condensate-associated molecule are provided, the methods including: (a) contacting a plurality of candidate compounds with a cellular composition comprising the first set of one or more condensates or a cellular composition capable of forming the first set of one or more condensates; (b) determining a characteristic associated with the first set of one or more condensates; (c) contacting the plurality of candidate compounds with a cellular composition comprising a second set of one or more condensates comprising a second condensate-associated molecule or a cellular composition capable of forming the second set of one or more condensates; and (d) determining a characteristic associated with the second set of one or more condensates.

[0061] In some embodiments, modulation of the feature associated with the first set that differs from modulation of the feature associated with the second set indicates that the candidate compound modulates the feature associated with the first set of one or more condensates, thereby obtaining a compound that modulates the feature associated with the first set of one or more condensates. In some embodiments, modulation of the feature associated with the second set that differs from modulation of the feature associated with the first set indicates that the compound modulates the feature associated with the second set of one or more condensates relative to the reference, thereby obtaining a compound that modulates the feature associated with the second set of one or more condensates.

[0062] <Definition> For purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the set forth definition shall control.

[0063] 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, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. These terms also include post-translational modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like.

[0064] The term "polynucleotide" or "nucleic acid" as used herein 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 derivatized nucleotide bases. The backbone of a polynucleotide can include sugar and phosphate groups (usually found in RNA or DNA), or modified or substituted sugar or phosphate groups. The backbone of a polynucleotide can include repeating units such as N-(2-aminoethyl)-glycine linked by peptide bonds (e.g., peptide nucleic acid). Alternatively, the backbone of a polynucleotide can include polymers of synthetic subunits such as phosphoramidates, and thus can be phosphoramidate oligodeoxynucleosides (P-NH2) or mixed phosphoramidate-phosphodiester oligomers.

[0065] As used herein, the terms "comprise," "have," "contain," and "include," and other similar forms and their grammatical equivalents are intended to be equivalent and to be variable in that the item or items following any one of these words are not meant to be an exhaustive list of such items or to be limited to only the items listed. For example, an article "comprising" components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can include not only components A, B, and C, but also one or more other components. Thus, "comprise" and similar forms and their grammatical equivalents are intended and understood to include disclosure of embodiments "consisting essentially of" or "consisting of."

[0066] Where a range of values ​​is presented, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed in the disclosure, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both limits, either or both of those included limits are also included in the disclosure.

[0067] As used herein, reference to a value or parameter with "about" includes (and describes) the variation that is directed to the value or parameter itself. For example, a statement "about X" includes the statement "X."

[0068] As used in this specification, including the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0069] As used herein, "condensates" refers to non-membrane-encapsulated compartments formed by one or more phase separations (including all stages of phase separation) of proteins and / or other macromolecules.

[0070] As used herein, "condensate-associated molecule" refers to a molecule that is found within or on condensates under physiological or pathological conditions.

[0071] The term "stress granules" in the present context relates to liquid non-membrane-bound compartments that are located within the nucleus or cytosol of cells and contain proteins and RNA, which appear when cells are under stress.

[0072] The term "neurodegenerative disease" in the context of this specification relates to a medical condition characterized by the progressive loss of neuronal structure or function. Non-limiting examples of neurodegenerative diseases include amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease.

[0073] As used herein, the term "treating" any disease or disorder (e.g., ALS) or "treatment" thereof refers, in one embodiment, to improving the disease or disorder (slowing or stopping or inhibiting the progression of the disease or at least one clinical symptom thereof). In another embodiment, "treating" or "treatment" refers to reducing or improving at least one physical parameter, including one that is not discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. As used herein, the term "preventing" any disease or disorder (e.g., ALS) or "preventing" thereof refers to inhibiting (including complete inhibition) the progression or onset of the disease or disorder (e.g., delaying the progression or onset of the disease or disorder). Methods for assessing the treatment and / or prevention of a disease are generally known in the art, unless otherwise stated below.

[0074] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0075] <Methods for screening and identifying compounds> In some embodiments of the present application, methods are provided for identifying compounds that modulate one or more condensate-associated characteristics and / or compounds that are useful in methods of treating disease. In some embodiments of the present invention, methods are provided for screening compounds that modulate one or more condensate-associated characteristics and / or compounds that are useful in methods of treating disease. In some embodiments of the present invention, methods are provided for high-throughput screening compounds that modulate one or more condensate-associated characteristics and / or compounds that are useful in methods of treating disease.

[0076] The techniques for identifying compounds are described in more detail below. Those skilled in the art will recognize from the description provided that several embodiments are possible within the scope and spirit of the disclosure of this application.

[0077] In some aspects of the invention, methods are provided for identifying compounds that modulate one or more condensate-associated characteristics, including condensate-associated molecules, the methods comprising: (a) contacting the compound with a cellular composition that includes one or more condensates or is capable of forming one or more condensates; and (b) determining the one or more condensate-associated characteristics, where the modulation of the characteristics indicates that the compound modulates the one or more condensate-associated characteristics relative to a reference. In some embodiments, the methods comprise determining a single characteristic associated with one or more condensates. In some embodiments, the methods comprise determining multiple characteristics associated with one or more condensates.

[0078] In some aspects of the present invention, a method for screening (e.g., high-throughput screening) a compound that modulates one or more condensate-associated characteristics is provided, the method comprising evaluating a plurality of compounds in a screen using any of the methods described in the present invention. In some embodiments, the method for screening (e.g., high-throughput screening) a compound that modulates one or more condensate-associated characteristics, including condensate-associated molecules, comprises (a) contacting a cellular composition that includes one or more condensates or a cellular composition that can form one or more condensates with any of a plurality of candidate compounds, and (b) determining one or more condensate-associated characteristics, the modulation of the characteristics indicating that the candidate compound modulates one or more condensate-associated characteristics compared to a reference, thereby obtaining a compound that modulates one or more condensate characteristics. In some embodiments, the method comprises determining a plurality of characteristics associated with one or more condensates. In some embodiments, the plurality of compounds comprises at least any of 100, 200, 300, 500, 800, 1000, 1500, 3,000, 5,000, 10,000, or more candidate compounds. In some embodiments, multiple candidate compounds can be contacted with the cellular composition in separate reactions (e.g., separate wells or vessels). In some embodiments, multiple candidate compounds can be contacted with the cellular composition simultaneously in separate reactions.

[0079] In some embodiments of the invention, a method is provided for identifying a compound that modulates a characteristic 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 cellular composition comprising or capable of forming the first and second sets of one or more condensates; (b) determining a characteristic associated with the first set of one or more condensates; and (c) determining a characteristic associated with the second set of one or more condensates, wherein modulation of the characteristic associated with the first set that differs from modulation of the characteristic associated with the second set indicates that the compound modulates the characteristic associated with the first set of one or more condensates, compared to a reference.

[0080] In some embodiments of the invention, methods are provided for identifying a compound that modulates a characteristic 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 cellular composition comprising the first set of one or more condensates or a cellular composition capable of forming the first set of one or more condensates; (b) determining a characteristic associated with the first set of one or more condensates; (c) contacting the compound with a cellular composition comprising a second set of one or more condensates comprising a second condensate-associated molecule or a cellular composition capable of forming the second set of one or more condensates; and (d) determining a characteristic associated with the second set of one or more condensates, wherein modulation of the characteristic associated with the first set that differs from modulation of the characteristic associated with the second set indicates that the compound modulates the characteristic associated with the first set of one or more condensates compared to a reference.

[0081] In some embodiments of the invention, a method of screening (e.g., high throughput screening) for a compound that modulates a characteristic associated with a first set of one or more condensates comprising a first condensate-associated molecule is provided, the method comprising: (a) contacting a plurality of candidate compounds with a cellular composition comprising or capable of forming a first and second set of one or more condensates; (b) determining a characteristic associated with the first set of one or more condensates; and (c) determining a characteristic associated with the second set of one or more condensates, wherein modulation of the characteristic associated with the first set that differs from modulation of the characteristic associated with the second set indicates that the compound modulates the characteristic associated with the first set of one or more condensates compared to a reference, thereby obtaining a compound that modulates the characteristic associated with the first set of one or more condensates.

[0082] In some aspects of the invention, a method for screening (e.g., high-throughput screening) for a compound that modulates a feature associated with a first set of one or more condensates comprising a first condensate-associated molecule is provided, the method comprising: (a) contacting a plurality of candidate compounds with a cellular composition comprising the first set of one or more condensates or a cellular composition capable of forming the first set of one or more condensates; (b) determining a feature associated with the first set of one or more condensates; (c) contacting a plurality of candidate compounds with a cellular composition comprising a second set of one or more condensates comprising a second condensate-associated molecule or a cellular composition capable of forming the second set of one or more condensates; and (d) determining a feature associated with the second set of one or more condensates, where modulation of the feature associated with the first set that is different from modulation of the feature associated with the second set indicates that the compound modulates a feature associated with the first set of one or more condensates compared to a reference, thereby obtaining a compound that modulates a feature 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 the one or more condensates are paraspeckles.

[0083] In some aspects of the invention, methods are provided for identifying compounds that modulate one or more stress granule-associated characteristics, the methods comprising: (a) contacting a compound with a cellular composition comprising one or more stress granules or capable of forming one or more stress granules; and (b) determining the one or more stress granule-associated characteristics, where modulation of the characteristics indicates that the compound modulates the one or more stress granule-associated characteristics relative to a reference. In some embodiments, the methods comprise determining a single characteristic associated with one or more stress granules. In some embodiments, the methods comprise determining multiple characteristics associated with one or more stress granules.

[0084] In some aspects of the invention, a method for screening (e.g., high throughput screening) for compounds that modulate one or more stress granule-associated characteristics is provided, the method comprising: (a) contacting a plurality of candidate compounds with a cellular composition that includes one or more stress granules or is capable of forming one or more stress granules; and (b) determining one or more stress granule-associated characteristics, where modulation of the characteristics indicates that the candidate compound modulates the one or more stress granule-associated characteristics relative to a reference, thereby obtaining a compound that modulates the one or more stress granule characteristics. In some embodiments, the method comprises determining a plurality of characteristics associated with one or more stress granules. In some embodiments, the plurality of compounds comprises at least any of 100, 200, 300, 500, 800, 1000, 1500, 3,000, 5,000, 10,000, or more candidate compounds. In some embodiments, the plurality of candidate compounds can be contacted with the cellular composition in separate reactions (e.g., separate wells or vessels). In some embodiments, the plurality of candidate compounds can be contacted with the cellular composition simultaneously in separate reactions.

[0085] In some embodiments of the invention, methods are provided for identifying compounds that modulate one or more stress granule-associated features, the methods including (a) contacting a candidate compound with a cellular composition comprising, or capable of forming, one or more stress granules and one or more paraspeckles, condensates formed around sites of DNA damage, P bodies, Cajal bodies, and / or PML bodies, (b) determining features associated with the one or more stress granules, and (c) determining features associated with the one or more paraspeckles, condensates formed around sites of DNA damage, P bodies, Cajal bodies, and / or PML bodies, wherein modulation of the one or more stress granule-associated features that differs from modulation of features associated with the one or more paraspeckles, condensates formed around sites of DNA damage, P bodies, Cajal bodies, and / or PML bodies, relative to a reference, indicates that the compound modulates the one or more stress granule-associated features.

[0086] In some embodiments of the invention, methods are provided for identifying a compound that modulates one or more stress granule-associated characteristics, the methods including (a) contacting a compound with a cellular composition comprising one or more stress granules or a cellular composition capable of forming one or more stress granules, (b) determining one or more stress granule-associated characteristics, (c) determining one or more stress granule-associated characteristics, and (d) determining one or more stress granule-associated characteristics, and (e) determining one or more stress granule-associated characteristics, and (f) determining one or more stress granule-associated characteristics, and (g) determining one or more stress granule-associated characteristics, and (h) determining one or more stress granule-associated characteristics, and (i) determining one or more stress granule-associated characteristics, and (ii) determining one or more stress granule-associated characteristics, and (iii) determining one or more stress granule-associated characteristics, and (iv) determining one or more stress granule-associated characteristics, and (v) determining one or more stress granule-associated characteristics, and and (d) determining a characteristic associated with one or more paraspeckles, condensates formed around sites of DNA damage, P bodies, Cajal bodies, and / or PML bodies, wherein modulation of the characteristic associated with one or more stress granules that is different from modulation of the characteristic associated with one or more paraspeckles, condensates formed around sites of DNA damage, P bodies, Cajal bodies, and / or PML bodies, relative to the reference, indicates that the compound modulates one or more stress granule-associated characteristics.

[0087] In some embodiments of the invention, methods are provided for screening (e.g., high throughput screening) for compounds that modulate one or more paraspeckle-associated features, the methods including (a) contacting a cellular composition that contains, or is capable of forming, one or more stress granules and one or more paraspeckles, condensates that form around sites of DNA damage, P bodies, Cajal bodies, and / or PML bodies with a plurality of candidate compounds; (b) contacting one or more candidate compounds with a cellular composition that contains, or is capable of forming, one or more stress granules, and one or more paraspeckles, condensates that form around sites of DNA damage, P bodies, Cajal bodies, and / or PML bodies with a plurality of candidate compounds; (c) determining a feature associated with one or more paraspeckles, condensates formed around sites of DNA damage, P bodies, Cajal bodies, and / or PML bodies, wherein modulation of the one or more stress granule associated features that is different from modulation of the feature associated with the one or more paraspeckles, condensates formed around sites of DNA damage, P bodies, Cajal bodies, and / or PML bodies compared to the reference indicates that the compound modulates the one or more stress granule associated features, thereby obtaining a compound that modulates the one or more stress granule associated features.

[0088] In some embodiments of the invention, methods are provided for screening (e.g., high throughput screening) for compounds that modulate one or more stress granule-associated characteristics, the methods including (a) contacting a plurality of candidate compounds with a cellular composition comprising one or more stress granules or a cellular composition capable of forming one or more stress granules, (b) determining one or more stress granule-associated characteristics, (c) determining one or more stress granule-associated characteristics of the cellular composition comprising one or more paraspeckles, condensates formed around sites of DNA damage, P bodies, Cajal bodies, and / or PML bodies, or determining one or more paraspeckles, condensates formed around sites of DNA damage, P bodies, Cajal bodies, and / or PML bodies. and (d) determining a characteristic associated with one or more paraspeckles, condensates formed around sites of DNA damage, P bodies, Cajal bodies, and / or PML bodies, wherein modulation of the one or more stress granule associated characteristics that is different from modulation of the characteristic associated with the one or more paraspeckles, condensates formed around sites of DNA damage, P bodies, Cajal bodies, and / or PML bodies relative to the reference indicates that the compound modulates the one or more stress granule associated characteristics, thereby obtaining a compound that modulates the one or more stress granule associated characteristics.

[0089] In some embodiments, the method further comprises repeating the method steps with a plurality of compounds. For example, in some embodiments, the method comprises repeating the method steps with at least any of 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 multiple concentrations of the compounds.

[0090] In some embodiments, the methods described in the present invention include contacting a compound with a cellular composition that includes one or more condensates or a cellular composition that can form one or more condensates. Those skilled in the art will readily recognize that cellular processes, including the state of condensates and their components, are dynamic. Thus, the methods described in the present invention include contacting a composition, such as a cellular composition, with a compound at any time during the life cycle of one or more condensates. For example, the methods include contacting a cellular composition with a compound when a condensate-associated molecule is present at any location in the cell, when present in any amount, or when it has any post-translational modification state, such as the presence, absence, or amount of phosphorylated residues. In some aspects, the methods also include contacting a cell with a compound when, for example, one or more condensates are present at any location in the cell, when present in any amount (including absence), when undergoing morphological changes, such as changes in size or fluidity, or when undergoing a change in composition.

[0091] In some embodiments, the cellular composition comprises one or more condensates prior to contacting with the compound. In some embodiments, the method further comprises providing condensate-forming conditions to the cellular composition prior to contacting the compound with the cellular composition comprising one or more condensates or the cellular composition capable of forming one or more condensates. In some embodiments, the cellular composition does not comprise one or more condensates prior to contacting with the compound, and the method comprises providing condensate-forming conditions to the cellular composition to form one or more condensates. In some embodiments, the cellular composition does not comprise one or more condensates prior to contacting with the compound, and the method comprises providing condensate-forming conditions to the cellular composition to form one or more condensates after the cellular composition is contacted with the compound. In some embodiments, the cellular composition does not comprise one or more condensates prior to contacting with the compound, and forms one or more condensates upon contacting with the compound. In some embodiments, the condensates occur simultaneously with and after the addition of the compound. In some embodiments, the condensate-forming conditions are provided to the cellular composition prior to determining a characteristic associated with the one or more condensates. In some embodiments, the cellular composition comprises one or more condensates, and additional condensates of the one or more condensates occur simultaneously with contacting the cellular composition with the compound. In some embodiments, the cellular composition comprises one or more condensates, and additional condensates of the one or more condensates occur after contacting the cellular composition with the compound. In some embodiments, the cellular composition comprises one or more condensates, and additional condensates of the one or more condensates occur simultaneously and after contacting the cellular composition with the compound.

[0092] Thus, the methods described herein include contacting a cellular composition with a compound, where (i) the cellular composition comprises one or more target condensates, and / or (ii) the one or more target condensates are generated simultaneously and / or after contacting the cellular composition with the compound. In some embodiments, the methods described herein include contacting a cellular composition with a compound, where (i) the cellular composition comprises one or more target condensates, and / or (ii) the one or more condensates are capable of forming, where the one or more condensates are generated simultaneously and / or after contacting the cellular composition with the compound. In some embodiments, the methods described herein include contacting a cellular composition that comprises one or more condensates or a cellular composition that is capable of forming one or more condensates with a compound.

[0093] The condensate forming conditions can include the addition of a condensation-inducing agent. In some embodiments, the method includes subjecting the cell composition to any one or more of: (i) an oxidative stressor, (ii) a mitochondrial electron transport chain inhibitor, (iii) a thermal stressor, (iv) an osmotic stressor, (v) a hyperosmotic stressor, and (vi) a glycolysis inhibitor. 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 inhibitor is 6-deoxyglucose in the absence of glucose. In some embodiments, the thermal stressor is exposing the cell composition to a temperature of about 40-45°C, for example, about 42°C.

[0094] 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 include a condensate-associated molecule. In some embodiments, the second condensate is a condensate that includes a condensate-associated molecule. 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 the first composition and the reference is the second cell composition. In some embodiments, the reference is a cell composition that is not contacted with the compound. In some embodiments, the reference is a cell composition that was not treated with condensate-forming conditions. In some embodiments, the reference is a cell composition that was treated with a reference compound.

[0095] In some embodiments, the reference is prepared so that the compound can be evaluated for meaningful results.For example, in some embodiments, the reference is a cell composition, which is prepared in the same manner as the cell composition contacted with the compound, except that the reference cell composition is not exposed to the compound being tested, or the reference cell composition is not subjected to the same step of contacting with the compound.In some embodiments, the reference is a cell composition that is contacted with a reference compound, such as a positive or negative control compound.

[0096] Condensate-related features In some embodiments, determining the one or more condensate-associated features is based on any one or more of: (i) the number of condensates with and / or without condensate-associated molecules, (ii) the size of the one or more condensates, (iii) the location of the one or more condensates, (iv) the distribution of the one or more condensates, (v) the surface area of ​​the one or more condensates, (vi) the composition of the one or more condensates, (vii) the fluidity of the one or more condensates, (viii) the solidification of the one or more condensates, (ix) the dissolution of the one or more condensates, (x) the presence and / or amount of fibril formation, (xi) the location of the condensate-associated molecules, (xii) the partitioning of the condensate-associated molecules into condensates, and (xiii) the aggregation of the condensate-associated molecules. In some embodiments, the method includes determining a first feature associated with the one or more condensates and a second feature associated with the one or more condensates. In some embodiments, the method includes determining a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth feature associated with the one or more condensates. Exemplary techniques that can be used to determine the characteristics are disclosed in the Examples.

[0097] In some embodiments, the method includes determining a first characteristic associated with one or more condensates and a second characteristic associated with one or more condensates, In some embodiments, the first characteristic is (i) a number of condensates with and / or without condensate-associated molecules, and the second characteristic is any one (or a plurality, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) of (ii) a size of one or more condensates, (iii) a location of one or more condensates, (iv) a distribution of one or more condensates, (v) a surface area of ​​one or more condensates, (vi) a composition of one or more condensates, (vii) a fluidity of one or more condensates, (viii) a solidification of one or more condensates, (ix) a dissolution of one or more condensates, (x) a presence and / or amount of fibril formation, (xi) a location of a condensate-associated molecule, (xii) a partitioning of a condensate-associated molecule into condensates, and (xiii) an aggregation of a condensate-associated molecule. In some embodiments, the first characteristic is (i) the number of condensates with and / or without condensate-associated molecules, and the second characteristic is (ii) the size of the one or more condensates. In some embodiments, the first characteristic is (i) the number of condensates with and / or without condensate-associated molecules, and the second characteristic is (iii) the location of the one or more condensates. In some embodiments, the first characteristic is (i) the number of condensates with and / or without condensate-associated molecules, and the second characteristic is (iv) the distribution of the one or more condensates. In some embodiments, the first characteristic is (i) the number of condensates with and / or without condensate-associated molecules, and the second characteristic is (v) the surface area of ​​the one or more condensates. In some embodiments, the first characteristic is (i) the number of condensates with and / or without condensate-associated molecules, and the second characteristic is (vi) the composition of the one or more condensates. In some embodiments, the first characteristic is (i) the number of condensates with and / or without condensate-associated molecules, and the second characteristic is (vii) the fluidity of the one or more condensates. In some embodiments, the first characteristic is (i) the number of condensates with and / or without condensate-associated molecules, and the second characteristic is (viii) solidification of one or more condensates.In some embodiments, the first characteristic is (i) the number of condensates with and / or without condensate-associated molecules, and the second characteristic is (ix) dissolution of one or more condensates. In some embodiments, the first characteristic is (i) the number of condensates with and / or without condensate-associated molecules, and the second characteristic is (x) the presence and / or amount of fibril formation. In some embodiments, the first characteristic is (i) the number of condensates with and / or without condensate-associated molecules, and the second characteristic is (xi) the location of condensate-associated molecules. In some embodiments, the first characteristic is (i) the number of condensates with and / or without condensate-associated molecules, and the second characteristic is (xii) the partitioning of condensate-associated molecules into condensates. In some embodiments, the first characteristic is (i) the number of condensates with and / or without condensate-associated molecules, and the second characteristic is (xiii) aggregation of condensate-associated molecules.

[0098] In some embodiments, the method includes determining a first feature associated with one or more condensates and a second feature associated with one or more condensates. In some embodiments, the first feature is (ii) a size of one or more condensates and the second feature is (iii) a location of one or more condensates, (iv) a distribution of one or more condensates, (v) a surface area of ​​one or more condensates, (vi) a composition of one or more condensates, (vii) a flowability of one or more condensates, (viii) a solidification of one or more condensates, (ix) a dissolution of one or more condensates, (x) a presence and / or amount of fibril formation, (xi) a location of condensate-associated molecules, (xii) a partitioning of condensate-associated molecules into condensates, and (xiii) an aggregation of condensate-associated molecules. In some embodiments, the first feature is (ii) a size of one or more condensates and the second feature is (iii) a location of one or more condensates. In some embodiments, the first characteristic is (ii) the size of the one or more condensates and the second characteristic is (iv) the distribution of the one or more condensates. In some embodiments, the first characteristic is (ii) the size of the one or more condensates and the second characteristic is (v) the surface area of ​​the one or more condensates. In some embodiments, the first characteristic is (ii) the size of the one or more condensates and the second characteristic is (vi) the composition of the one or more condensates. In some embodiments, the first characteristic is (ii) the size of the one or more condensates and the second characteristic is (vii) the flowability of the one or more condensates. In some embodiments, the first characteristic is (ii) the size of the one or more condensates and the second characteristic is (viii) the solidification of the one or more condensates. In some embodiments, the first characteristic is (ii) the size of the one or more condensates and the second characteristic is (ix) the dissolution of the one or more condensates. In some embodiments, the first characteristic is (ii) the size of the one or more condensates and the second characteristic is (x) the presence and / or amount of fibril formation.In some embodiments, the first characteristic is (ii) a size of the one or more condensates and the second characteristic is (xi) a location of the condensate-associated molecules. In some embodiments, the first characteristic is (ii) a size of the one or more condensates and the second characteristic is (xii) a partitioning of the condensate-associated molecules into condensates. In some embodiments, the first characteristic is (ii) a size of the one or more condensates and the second characteristic is (xiii) an aggregation of the condensate-associated molecules.

[0099] In some embodiments, the method includes determining a first feature associated with one or more condensates and a second feature associated with one or more condensates. In some embodiments, the first feature is (iii) the location of one or more condensates and the second feature is (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 fluidity of one or more condensates, (viii) the solidification of one or more condensates, (ix) the dissolution of one or more condensates, (x) the presence and / or amount of fibril formation, (xi) the location of condensate-associated molecules, (xii) the partitioning of condensate-associated molecules into condensates, and (xiii) the aggregation of condensate-associated molecules. In some embodiments, the first feature is (iii) the location of one or more condensates and the second feature is (iv) the distribution of one or more condensates. In some embodiments, the first feature is (iii) the location of one or more condensates and the second feature is (v) the surface area of ​​one or more condensates. In some embodiments, the first characteristic is (iii) the location of one or more condensates and the second characteristic is (vi) the composition of one or more condensates. In some embodiments, the first characteristic is (iii) the location of one or more condensates and the second characteristic is (vii) the fluidity of one or more condensates. In some embodiments, the first characteristic is (iii) the location of one or more condensates and the second characteristic is (viii) the solidification of one or more condensates. In some embodiments, the first characteristic is (iii) the location of one or more condensates and the second characteristic is (ix) the dissolution of one or more condensates. In some embodiments, the first characteristic is (iii) the location of one or more condensates and the second characteristic is (x) the presence and / or amount of fibril formation. In some embodiments, the first characteristic is (iii) the location of one or more condensates and the second characteristic is (xi) the location of a condensate-associated molecule. In some embodiments, the first characteristic is (iii) the location of one or more condensates and the second characteristic is (xii) the partitioning of condensate-associated molecules into condensates.In some embodiments, the first characteristic is (iii) the location of one or more condensates and the second characteristic is (xiii) an aggregation of condensate-associated molecules.

[0100] In some embodiments, the method includes determining a first feature associated with one or more condensates and a second feature associated with one or more condensates. In some embodiments, the first feature is (iv) a distribution of one or more condensates and the second feature is any one (or more than one, such as 2, 3, 4, 5, 6, 7, 8, or 9) of (v) a surface area of ​​one or more condensates, (vi) a composition of one or more condensates, (vii) a fluidity of one or more condensates, (viii) solidification of one or more condensates, (ix) dissolution of one or more condensates, (x) presence and / or amount of fibril formation, (xi) location of condensate-associated molecules, (xii) partitioning of condensate-associated molecules into condensates, and (xiii) aggregation of condensate-associated molecules. In some embodiments, the first feature is (iv) a distribution of one or more condensates and the second feature is (v) a surface area of ​​one or more condensates. In some embodiments, the first feature is (iv) a distribution of one or more condensates and the second feature is (vi) a composition of one or more condensates. In some embodiments, the first characteristic is (iv) a distribution of one or more condensates and the second characteristic is (vii) a fluidity of one or more condensates. In some embodiments, the first characteristic is (iv) a distribution of one or more condensates and the second characteristic is (viii) a solidification of one or more condensates. In some embodiments, the first characteristic is (iv) a distribution of one or more condensates and the second characteristic is (ix) dissolution of one or more condensates. In some embodiments, the first characteristic is (iv) a distribution of one or more condensates and the second characteristic is (x) the presence and / or amount of fibril formation. In some embodiments, the first characteristic is (iv) a distribution of one or more condensates and the second characteristic is (xi) the location of a condensate-associated molecule. In some embodiments, the first characteristic is (iv) a distribution of one or more condensates and the second characteristic is (xii) partitioning of condensate-associated molecules into condensates. In some embodiments, the first characteristic is (iv) a distribution of one or more condensates and the second characteristic is (xiii) aggregation of condensate-associated molecules.

[0101] In some embodiments, the method includes determining a first characteristic associated with one or more condensates and a second characteristic associated with one or more condensates. In some embodiments, the first characteristic is (v) a surface area of ​​one or more condensates and the second characteristic is any one (or more than one, such as 2, 3, 4, 5, 6, 7, or 8) of (vi) a composition of one or more condensates, (vii) a fluidity of one or more condensates, (viii) solidification of one or more condensates, (ix) dissolution of one or more condensates, (x) presence and / or amount of fibril formation, (xi) location of condensate-associated molecules, (xii) partitioning of condensate-associated molecules into condensates, and (xiii) aggregation of condensate-associated molecules. In some embodiments, the first characteristic is (v) a surface area of ​​one or more condensates and the second characteristic is (vi) a composition of one or more condensates. In some embodiments, the first characteristic is (v) a surface area of ​​one or more condensates and the second characteristic is (vii) a fluidity of one or more condensates. In some embodiments, the first characteristic is (v) a surface area of ​​one or more condensates and the second characteristic is (viii) solidification of one or more condensates. In some embodiments, the first characteristic is (v) a surface area of ​​one or more condensates and the second characteristic is (ix) dissolution of one or more condensates. In some embodiments, the first characteristic is (v) a surface area of ​​one or more condensates and the second characteristic is (x) the presence and / or amount of fibril formation. In some embodiments, the first characteristic is (v) a surface area of ​​one or more condensates and the second characteristic is (xi) the location of condensate-associated molecules. In some embodiments, the first characteristic is (v) a surface area of ​​one or more condensates and the second characteristic is (xii) partitioning of condensate-associated molecules into condensates. In some embodiments, the first characteristic is (v) a surface area of ​​one or more condensates and the second characteristic is (xiii) aggregation of condensate-associated molecules.

[0102] In some embodiments, the method includes determining a first characteristic associated with one or more condensates and a second characteristic associated with one or more condensates. In some embodiments, the first characteristic is (vi) a composition of one or more condensates and the second characteristic is any one (or more than one, such as 2, 3, 4, 5, 6, or 7) of (vii) a fluidity of one or more condensates, (viii) a solidification of one or more condensates, (ix) a dissolution of one or more condensates, (x) a presence and / or amount of fibril formation, (xi) a location of condensate-associated molecules, (xii) a partitioning of condensate-associated molecules into condensates, and (xiii) an aggregation of condensate-associated molecules. In some embodiments, the first characteristic is (vi) a composition of one or more condensates and the second characteristic is (vii) a fluidity of one or more condensates. In some embodiments, the first characteristic is (vi) a composition of one or more condensates and the second characteristic is (viii) a solidification of one or more condensates. In some embodiments, the first characteristic is (vi) a composition of one or more condensates and the second characteristic is (ix) a dissolution of one or more condensates. In some embodiments, the first characteristic is (vi) the composition of one or more condensates and the second characteristic is (x) the presence and / or amount of fibril formation. In some embodiments, the first characteristic is (vi) the composition of one or more condensates and the second characteristic is (xi) the location of condensate-associated molecules. In some embodiments, the first characteristic is (vi) the composition of one or more condensates and the second characteristic is (xii) the partitioning of condensate-associated molecules into condensates. In some embodiments, the first characteristic is (vi) the composition of one or more condensates and the second characteristic is (xiii) the aggregation of condensate-associated molecules.

[0103] In some embodiments, the method includes determining a first characteristic associated with one or more condensates and a second characteristic associated with one or more condensates. In some embodiments, the first characteristic is (vii) a fluidity of one or more condensates and the second characteristic is (viii) solidification of one or more condensates, (ix) dissolution of one or more condensates, (x) presence and / or amount of fibril formation, (xi) location of condensate-associated molecules, (xii) partitioning of condensate-associated molecules into condensates, and (xiii) aggregation of condensate-associated molecules. In some embodiments, the first characteristic is (vii) a fluidity of one or more condensates and the second characteristic is (viii) solidification of one or more condensates. In some embodiments, the first characteristic is (vii) a fluidity of one or more condensates and the second characteristic is (ix) dissolution of one or more condensates. In some embodiments, the first characteristic is (vii) a fluidity of one or more condensates and the second characteristic is (x) presence and / or amount of fibril formation. In some embodiments, the first characteristic is (vii) the fluidity of one or more condensates and the second characteristic is (xi) the location of condensate-associated molecules. In some embodiments, the first characteristic is (vii) the fluidity of one or more condensates and the second characteristic is (xii) the partitioning of condensate-associated molecules into condensates. In some embodiments, the first characteristic is (vii) the fluidity of one or more condensates and the second characteristic is (xiii) the aggregation of condensate-associated molecules.

[0104] In some embodiments, the method includes determining a first feature associated with one or more condensates and a second feature associated with one or more condensates. In some embodiments, the first feature is (viii) solidification of one or more condensates and the second feature is any one (or more than one, such as two, three, four, or five) of (ix) dissolution of one or more condensates, (x) presence and / or amount of fibril formation, (xi) location of condensate-associated molecules, (xii) partitioning of condensate-associated molecules into condensates, and (xiii) aggregation of condensate-associated molecules. In some embodiments, the first feature is (viii) solidification of one or more condensates and the second feature is (ix) dissolution of one or more condensates. In some embodiments, the first feature is (viii) solidification of one or more condensates and the second feature is (x) presence and / or amount of fibril formation. In some embodiments, the first feature is (viii) solidification of one or more condensates and the second feature is (xi) location of condensate-associated molecules. In some embodiments, the first characteristic is (viii) solidification of one or more condensates and the second characteristic is (xii) partitioning of condensate-associated molecules into condensates. In some embodiments, the first characteristic is (viii) solidification of one or more condensates and the second characteristic is (xiii) aggregation of condensate-associated molecules.

[0105] In some embodiments, the method includes determining a first feature associated with one or more condensates and a second feature associated with one or more condensates. In some embodiments, the first feature is (ix) dissolution of one or more condensates and the second feature is any one (or more, such as two, three, or four) of (x) presence and / or amount of fibril formation, (xi) location of condensate-associated molecules, (xii) partitioning of condensate-associated molecules into condensates, and (xiii) aggregation of condensate-associated molecules. In some embodiments, the first feature is (ix) dissolution of one or more condensates and the second feature is (x) presence and / or amount of fibril formation. In some embodiments, the first feature is (ix) dissolution of one or more condensates and the second feature is (xi) location of condensate-associated molecules. In some embodiments, the first feature is (ix) dissolution of one or more condensates and the second feature is (xii) partitioning of condensate-associated molecules into condensates. In some embodiments, the first feature is (ix) dissolution of one or more condensates and the second feature is (xiii) aggregation of condensate-associated molecules.

[0106] In some embodiments, the method includes determining a first feature associated with one or more condensates and a second feature associated with one or more condensates. In some embodiments, the first feature is (x) the presence and / or amount of fibril formation and the second feature is any one (or more, such as two or three) of (xi) the location of condensate-associated molecules, (xii) partitioning of condensate-associated molecules into condensates, and (xiii) aggregation of condensate-associated molecules. In some embodiments, the first feature is (x) the presence and / or amount of fibril formation and the second feature is (xi) the location of condensate-associated molecules. In some embodiments, the first feature is (x) the presence and / or amount of fibril formation and the second feature is (xii) partitioning of condensate-associated molecules into condensates. In some embodiments, the first feature is (x) the presence and / or amount of fibril formation and the second feature is (xiii) aggregation of condensate-associated molecules.

[0107] In some embodiments, the method includes determining a first feature associated with one or more condensates and a second feature associated with one or more condensates. In some embodiments, the first feature is (xi) a location of a condensate-associated molecule and the second feature is (xii) partitioning of the condensate-associated molecule into condensates and / or (xiii) aggregation of the condensate-associated molecule. In some embodiments, the first feature is (xi) a location of a condensate-associated molecule and the second feature is (xii) partitioning of the condensate-associated molecule into condensates.

[0108] In some embodiments, the method includes determining a first characteristic associated with one or more condensates and a second characteristic associated with one or more condensates, in some embodiments, the first characteristic is (xii) partitioning of the condensate-associated molecules into condensates and the second characteristic is (xiii) aggregation of the condensate-associated molecules.

[0109] In some embodiments, the compound modulates one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all) features selected from: (i) the number of condensates with and / or without condensate-associated molecules, (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 fluidity of one or more condensates, (viii) the solidification of one or more condensates, (ix) the dissolution of one or more condensates, (x) the presence and / or amount of fibril formation, (xi) the location of condensate-associated molecules, (xii) the partitioning of condensate-associated molecules into condensates, and (xiii) the aggregation of condensate-associated molecules.

[0110] In some embodiments, the compound (i) modulates the number of condensates with and / or without condensate-associated molecules. In some embodiments, the compound (ii) modulates the size of one or more condensates. In some embodiments, the compound (iii) modulates the location of one or more condensates. In some embodiments, the compound (iv) modulates the distribution of one or more condensates. In some embodiments, the compound (v) modulates the surface area of ​​one or more condensates. In some embodiments, the compound (vi) modulates the composition of one or more condensates. In some embodiments, the compound (vii) modulates the fluidity of one or more condensates. In some embodiments, the compound (viii) modulates the solidification of one or more condensates. In some embodiments, the compound (ix) modulates the dissolution of one or more condensates. In some embodiments, the compound (x) modulates the presence and / or amount of fibril formation. In some embodiments, the compound (xi) modulates the location of condensate-associated molecules. In some embodiments, the compound (xii) modulates the partitioning of condensate-associated molecules into condensates. In some embodiments, the compound (xiii) modulates the aggregation of condensate-associated molecules.

[0111] In some embodiments, the compound does not modulate one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all) features selected from: (i) the number of condensates with and / or without condensate-associated molecules, (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 fluidity of one or more condensates, (viii) the solidification of one or more condensates, (ix) the dissolution of one or more condensates, (x) the presence and / or amount of fibril formation, (xi) the location of condensate-associated molecules, (xii) the partitioning of condensate-associated molecules into condensates, and (xiii) the aggregation of condensate-associated molecules.

[0112] In some embodiments, the compound does not (i) modulate the number of condensates with and / or without condensate-associated molecules. In some embodiments, the compound does not (ii) modulate the size of one or more condensates. In some embodiments, the compound does not (iii) modulate the location of one or more condensates. In some embodiments, the compound does not (iv) modulate the distribution of one or more condensates. In some embodiments, the compound does not (v) modulate the surface area of ​​one or more condensates. In some embodiments, the compound does not (vi) modulate the composition of one or more condensates. In some embodiments, the compound does not (vii) modulate the fluidity of one or more condensates. In some embodiments, the compound does not (viii) modulate the solidification of one or more condensates. In some embodiments, the compound does not (ix) modulate the dissolution of one or more condensates. In some embodiments, the compound does not (x) modulate the presence and / or amount of fibril formation. In some embodiments, the compound does not (xi) modulate the location of condensate-associated molecules. In some embodiments, the compound does not (xii) modulate the partitioning of condensate-associated molecules into condensates. In some embodiments, the compound does not (xiii) modulate the aggregation of condensate-associated molecules.

[0113] In some embodiments, the compound modulates one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) features selected from: (i) the number of condensates with and / or without condensate-associated molecules, (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 fluidity of one or more condensates, (viii) the solidification of one or more condensates, (ix) the dissolution of one or more condensates, (x) the presence and / or amount of fibril formation, (xi) the location of condensate-associated molecules, (xii) the partitioning of condensate-associated molecules into condensates, and (xiii) the aggregation of condensate-associated molecules, and does not modulate other features of these 13.

[0114] In some embodiments, the compound modulates a first characteristic associated with one or more condensates and does not modulate a second characteristic associated with one or more condensates, where the first characteristic is different from the second characteristic and the characteristic is selected from the group consisting of (i) the number of condensates with and / or without condensate-associated molecules, (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 fluidity of one or more condensates, (viii) the solidification of one or more condensates, (ix) the dissolution of one or more condensates, (x) the presence and / or amount of fibril formation, (xi) the location of condensate-associated molecules, (xii) the partitioning of condensate-associated molecules into condensates, and (xiii) the aggregation of condensate-associated molecules, and does not modulate any other characteristic among these thirteen.

[0115] In some embodiments, the compound is capable of detecting one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) changes selected from: (i) the number of condensates with and / or without condensate-associated molecules; (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 fluidity of one or more condensates; (viii) the solidification of one or more condensates; (ix) the dissolution of one or more condensates; (x) the presence and / or amount of fibril formation; (xi) the location of condensate-associated molecules; (xii) partitioning of condensate-associated molecules into condensates in a first portion of a cell composition, such as a first portion of cells. (v) the surface area of ​​one or more condensates; (vi) the composition of one or more condensates; (vii) the fluidity of one or more condensates; (viii) the solidification of one or more condensates; (ix) the dissolution of one or more condensates; (x) the presence and / or amount of fibril formation; (xi) the location of condensate-associated molecules; (xii) partitioning of condensate-associated molecules into condensates in a second portion of the cell composition, such as a second portion of the cells.

[0116] In some embodiments, the compound is capable of detecting one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) events selected from: (i) the number of condensates with and / or without condensate-associated molecules, (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 fluidity of one or more condensates, (viii) the solidification of one or more condensates, (ix) the dissolution of one or more condensates, (x) the presence and / or amount of fibril formation, (xi) the location of condensate-associated molecules, (xii) the partitioning of condensate-associated molecules into condensates in the cytoplasm. and does not modulate one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) features selected from: (i) the number of condensates with and / or without condensate-associated molecules, (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 fluidity of one or more condensates, (viii) the solidification of one or more condensates, (ix) the dissolution of one or more condensates, (x) the presence and / or amount of fibril formation, (xi) the location of condensate-associated molecules, (xii) the partitioning of condensate-associated molecules into condensates in the nucleus.

[0117] In some embodiments, the compound is capable of detecting one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) characteristics selected from: (i) the number of condensates with and / or without condensate-associated molecules; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of ​​the one or more condensates; (vi) the composition of the one or more condensates; (vii) the fluidity of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fibril formation; (xi) the location of the condensate-associated molecules; (xii) the partitioning of the condensate-associated molecules into condensates for the first set of one or more condensates. (viii) solidification of one or more condensates; (ix) dissolution of one or more condensates; (x) presence and / or amount of fibril formation; (xi) location of condensate-associated molecules; (xii) partitioning of condensate-associated molecules into condensates;

[0118] In some embodiments, the compound modulates one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) features selected from (i) the number of condensates with and / or without condensate-associated molecules, (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 fluidity of one or more condensates, (viii) the solidification of one or more condensates, (ix) the dissolution of one or more condensates, (x) the presence and / or amount of fibril formation, (xi) the location of condensate-associated molecules, (xii) the partitioning of condensate-associated molecules into condensates in one or more stress granules, and modulates one or more nuclear partitioning. (viii) solidification of one or more condensates; (ix) dissolution of one or more condensates; (x) presence and / or amount of fibril formation; (xi) location of condensate-associated molecules; (xii) partitioning of condensate-associated molecules into condensates; (xiii) the presence and / or amount of fibril formation; (xiv) the amount of condensate-associated molecules; (xv) the amount of condensate-associated molecules; (xvi) the amount of condensate-associated molecules; (xvi) the amount of condensate-associated molecules; (xv ...

[0119] In some embodiments, the method is for identifying a compound that modulates one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all) features selected from: (i) the number of condensates with and / or without condensate-associated molecules; (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 fluidity of one or more condensates; (viii) the solidification of one or more condensates; (ix) the dissolution of one or more condensates; (x) the presence and / or amount of fibril formation; (xi) the location of condensate-associated molecules; (xii) the partitioning of condensate-associated molecules into condensates; and (xiii) the aggregation of condensate-associated molecules.

[0120] In some embodiments, the method is (i) a method for identifying a compound that modulates the number of condensates with and / or without condensate associated molecules. In some embodiments, the method is (ii) a method for identifying a compound that modulates the size of one or more condensates. In some embodiments, the method is (iii) a method for identifying a compound that modulates the location of one or more condensates. In some embodiments, the method is (iv) a method for identifying a compound that modulates the distribution of one or more condensates. In some embodiments, the method is (v) a method for identifying a compound that modulates the surface area of ​​one or more condensates. In some embodiments, the method is (vi) a method for identifying a compound that modulates the composition of one or more condensates. In some embodiments, the method is (vii) a method for identifying a compound that modulates the fluidity of one or more condensates. In some embodiments, the method is (viii) a method for identifying a compound that modulates the solidification of one or more condensates. In some embodiments, the method is (ix) a method for identifying a compound that modulates the dissolution of one or more condensates. In some embodiments, the method is (x) a method for identifying a compound that modulates the presence and / or amount of fibril formation. In some embodiments, the method is (xi) a method for identifying a compound that modulates the location of a condensate associated molecule. In some embodiments, the method is (xii) a method for identifying a compound that modulates the partitioning of a condensate associated molecule into condensates. In some embodiments, the method is (xiii) a method for identifying a compound that modulates aggregation of condensate-associated molecules.

[0121] In some embodiments, the compound does not modulate one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all) features selected from: (i) the number of condensates with and / or without condensate-associated molecules, (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 fluidity of one or more condensates, (viii) the solidification of one or more condensates, (ix) the dissolution of one or more condensates, (x) the presence and / or amount of fibril formation, (xi) the location of condensate-associated molecules, (xii) the partitioning of condensate-associated molecules into condensates, and (xiii) the aggregation of condensate-associated molecules.

[0122] In some embodiments, the compound does not (i) modulate the number of condensates with and / or without condensate-associated molecules. In some embodiments, the compound does not (ii) modulate the size of one or more condensates. In some embodiments, the compound does not (iii) modulate the location of one or more condensates. In some embodiments, the compound does not (iv) modulate the distribution of one or more condensates. In some embodiments, the compound does not (v) modulate the surface area of ​​one or more condensates. In some embodiments, the compound does not (vi) modulate the composition of one or more condensates. In some embodiments, the compound does not (vii) modulate the fluidity of one or more condensates. In some embodiments, the compound does not (viii) modulate the solidification of one or more condensates. In some embodiments, the compound does not (ix) modulate the dissolution of one or more condensates. In some embodiments, the compound does not (x) modulate the presence and / or amount of fibril formation. In some embodiments, the compound does not (xi) modulate the location of condensate-associated molecules. In some embodiments, the compound does not (xii) modulate the partitioning of condensate-associated molecules into condensates. In some embodiments, the compound does not (xiii) modulate the aggregation of condensate-associated molecules.

[0123] In some embodiments, a method is for identifying a compound that modulates one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) features selected from: (i) the number of condensates with and / or without condensate-associated molecules; (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 fluidity of one or more condensates; (viii) the solidification of one or more condensates; (ix) the dissolution of one or more condensates; (x) the presence and / or amount of fibril formation; (xi) the location of condensate-associated molecules; (xii) the partitioning of condensate-associated molecules into condensates; and (xiii) the aggregation of condensate-associated molecules, and does not modulate any other feature of these 13.

[0124] In some embodiments, the method modulates one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) features selected from: (i) the number of condensates with and / or without condensate-associated molecules; (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 fluidity of one or more condensates; (viii) the solidification of one or more condensates; (ix) the dissolution of one or more condensates; (x) the presence and / or amount of fibril formation; (xi) the location of condensate-associated molecules; (xii) partitioning of condensate-associated molecules into condensates in a first portion of a cell composition, such as a first portion of cells. (viii) solidification of one or more condensates; (ix) dissolution of one or more condensates; (x) presence and / or amount of fibril formation; (xi) location of condensate-associated molecules; (xii) partitioning of condensate-associated molecules into condensates in a second portion of the cellular composition, such as a second portion of the cells, and which does not modulate one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) features selected from: (i) the number of condensates with and / or without condensate-associated molecules; (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 fluidity of one or more condensates; (viii) solidification of one or more condensates; (ix) dissolution of one or more condensates; (x) presence and / or amount of fibril formation; (xi) location of condensate-associated molecules; (xii) partitioning of condensate-associated molecules into condensates in a second portion of the cellular composition, such as a second portion of the cells.

[0125] In some embodiments, the method modulates one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) features selected from: (i) the number of condensates with and / or without condensate-associated molecules, (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 fluidity of one or more condensates, (viii) the solidification of one or more condensates, (ix) the dissolution of one or more condensates, (x) the presence and / or amount of fibril formation, (xi) the location of condensate-associated molecules, (xii) the partitioning of condensate-associated molecules into condensates in the cytoplasm. (ix) dissolution of one or more condensates; (x) the presence and / or amount of fibril formation; (xi) the location of condensate-associated molecules; (xii) partitioning of condensate-associated molecules into condensates; (xiii) the presence and / or amount of fibril formation; (xiv) the distribution of one or more condensates in the nucleus; and (xv) the presence and / or amount of fibril formation; (xvi) the location of condensate-associated molecules; (xvi) the distribution of one or more condensates in the nucleus;

[0126] In some embodiments, the method modulates one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) characteristics of a first set of one or more condensates selected from: (i) the number of condensates with and / or without condensate-associated molecules; (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 fluidity of one or more condensates; (viii) the solidification of one or more condensates; (ix) the dissolution of one or more condensates; (x) the presence and / or amount of fibril formation; (xi) the location of condensate-associated molecules; (xii) the partitioning of condensate-associated molecules into condensates. and for a second set of one or more condensates, the compound does not modulate one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) features selected from: (i) the number of condensates with and / or without condensate-associated molecules; (ii) the size of the one or more condensates; (iii) the location of the one or more condensates; (iv) the distribution of the one or more condensates; (v) the surface area of ​​the one or more condensates; (vi) the composition of the one or more condensates; (vii) the fluidity of the one or more condensates; (viii) the solidification of the one or more condensates; (ix) the dissolution of the one or more condensates; (x) the presence and / or amount of fibril formation; (xi) the location of the condensate-associated molecules; and (xii) the partitioning of condensate-associated molecules into condensates.

[0127] In some embodiments, the method modulates one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) features selected from (i) the number of condensates with and / or without condensate-associated molecules, (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 fluidity of one or more condensates, (viii) the solidification of one or more condensates, (ix) the dissolution of one or more condensates, (x) the presence and / or amount of fibril formation, (xi) the location of condensate-associated molecules, (xii) the partitioning of condensate-associated molecules into condensates in one or more stress granules, and one or more nuclear paraspeckle features. (viii) solidification of one or more condensates; (ix) dissolution of one or more condensates; (x) presence and / or amount of fibril formation; (xi) location of condensate-associated molecules; (xii) partitioning of condensate-associated molecules into condensates; (xiii) the presence and / or amount of fibril formation; (xiv) the presence and / or amount of fibril formation; (xv) the presence and / or amount of fibril formation; (xvi) the presence and / or amount of fibril formation; (xv ...

[0128] In some embodiments, the determination is performed within about 60 days, e.g., 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 after contact with the compound. In some embodiments, the determination is performed about 5 seconds after contact with the compound, e.g., 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.

[0129] In some embodiments, the method includes determining the characteristic before and after contacting the compound. In some embodiments, the method further includes comparing the characteristic before and after contacting the compound.

[0130] In some embodiments, the method further comprises repeating the determining step of the method. For example, in some embodiments, the method comprises repeating the determining step of the method at least any of 2, 3, 4, 5, 10, or more times. In some embodiments, the method comprises performing the determining step on a first portion of the cellular composition and a second portion of the cellular composition, such as a first cell and a second cell in the cellular composition. In some embodiments, the method comprises performing the determining step on a third, fourth, fifth, sixth, or more portion of the cellular composition, such as a third, fourth, fifth, sixth, or more cell in the cellular composition. In some embodiments, the method comprises performing the determining step on a first portion of the cells in the cellular composition and a second portion of the cells in the cellular composition, such as a cytoplasm and a nucleus, or a first organelle and a second organelle. In some embodiments, the method comprises performing the determining step on a third, fourth, fifth, sixth, or more portion of the cells in the cellular composition.

[0131] In some embodiments, the determining step of the method is repeated after a time interval, e.g., 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 more. In some embodiments, the determining step is based on the same characteristic when repeated. In some embodiments, the method further includes comparing the characteristic over time, such as comparing the number of condensates with a day between determinations.

[0132] Visualization of the condensates can aid in determining one or more condensate-associated features. Condensates can be visualized by a variety of methods, including microscopy, such as stereomicroscopy, bright-field microscopy, polarized light microscopy, phase contrast microscopy, differential interference contrast microscopy, fluorescence microscopy, total internal reflection fluorescence microscopy, confocal microscopy, or multiphoton excitation microscopy. Analysis, such as counting the number of condensates and measuring the size of the condensates, can be determined by a variety of methods, either manually or in an automated manner, for example, by imaging or directly by microscopy. To facilitate this, in some embodiments, the condensates and / or condensate-associated molecules can be labeled, such as labeled with a fluorescent dye. In some embodiments, the condensate-associated molecules include fluorescent labels, such as fluorescent proteins (e.g., GFP, RFP, YFP, etc.). In some embodiments, the method includes contacting at least a portion of the cell composition with a label. In some embodiments, the label is a labeled binding molecule, such as an antibody (e.g., a labeled secondary antibody) or a biotin-binding protein. In some embodiments, the label is a stain, such as a stain specific for an organelle.

[0133] In some embodiments, the method further comprises imaging at least a portion of the cellular composition, such as a field of view. In some embodiments, the method further comprises contacting at least a portion of the cellular composition with a fixative. In some embodiments, the method further comprises contacting at least a portion of the cellular composition with a stain. In some embodiments, the method further comprises contacting at least a portion of the cellular composition with a DNA damaging condition. In some embodiments, the DNA damaging condition is laser irradiation.

[0134] The characteristic can be determined for a portion or all of the cellular composition. Thus, in some embodiments, the method includes determining the characteristic in a portion of the cellular composition. In some embodiments, the method includes determining the characteristic in the entire cellular composition. In some embodiments, the method includes determining the characteristic in one or more cells in the cellular composition. In some embodiments, the method includes determining the characteristic in a single cell in the cellular composition.

[0135] The characteristics can also be determined for some or all of the cells in the cellular composition. Thus, in some embodiments, the method includes determining the characteristics in a portion of one or more cells in the cellular composition. In some embodiments, the method includes determining the characteristics in a portion of a single cell in the cellular composition. In some embodiments, the method includes determining the characteristics in the cytoplasm of the cellular composition. In some embodiments, the method includes determining the characteristics in the nucleus of the cellular composition. In some embodiments, the method includes determining the characteristics in an organelle.

[0136] <Number of condensates> In some embodiments, the one or more condensate-associated characteristics are based on the number of condensates that contain and / or do not contain a condensate-associated molecule. In some embodiments, the one or more condensate-associated characteristics are based on the number of condensates that ... do not contain a condensate-associated molecule. Thus, in some embodiments of the present invention, a method for identifying a compound that modulates one or more condensate-associated characteristics including a condensate-associated molecule is provided, the method comprising: (a) contacting a cellular composition that contains one or more condensates or a cellular composition that can form one or more condensates with the compound; and (b) determining the one or more condensate-associated characteristics, the modulation of the characteristics being indicative of the compound modulating the one or more condensate-associated characteristics compared to a reference, the characteristic being modulation of the number of condensates that contain and / or do not contain a condensate-associated molecule. In some embodiments of the invention, a method is provided for identifying a compound that modulates the number of condensates with and / or without condensate-associated molecules, the method comprising (a) contacting the compound with a cellular composition that contains one or more condensates or is capable of forming one or more condensates, and (b) determining the number of condensates with and / or without condensate-associated molecules, where modulation of the number of condensates indicates that the compound modulates the number of condensates with and / or without condensate-associated molecules compared to a reference.

[0137] The number of condensates can be determined for a portion or all of the cellular composition. Thus, in some embodiments, the method includes determining the number of condensates with and / or without condensate-associated molecules in a portion of the cellular composition. In some embodiments, the method includes determining the number of condensates with and / or without condensate-associated molecules in the entire cellular composition. In some embodiments, the method includes determining the number of condensates with and / or without condensate-associated molecules in one or more cells in the cellular composition. In some embodiments, the method includes determining the number of condensates with and / or without condensate-associated molecules in a single cell in the cellular composition.

[0138] The number of condensates can also be determined for some or all of the cells in the cell composition. Thus, in some embodiments, the method includes determining the number of condensates with and / or without condensate-associated molecules in a portion of one or more cells in the cell composition. In some embodiments, the method includes determining the number of condensates with and / or without condensate-associated molecules in a portion of a single cell in the cell composition. In some embodiments, the method includes determining the number of condensates with and / or without condensate-associated molecules in the cytoplasm. In some embodiments, the method includes determining the number of condensates with and / or without condensate-associated molecules in the nucleus. In some embodiments, the method includes determining the number of condensates with and / or without condensate-associated molecules in an organelle.

[0139] In some embodiments, the method includes determining the number of condensates that contain condensate-associated molecules, determining the number of condensates that do not contain condensate-associated molecules, and comparing the number of condensates that contain condensate-associated molecules to the number of condensate-associated molecules. In some embodiments, the method includes determining the number of condensates that contain and / or do not contain condensate-associated molecules in a first portion of the cell composition, such as any portion of the cell composition disclosed herein, determining the number of condensates that contain and / or do not contain condensate-associated molecules in a second portion of the cell composition, such as any portion of the cell composition disclosed herein, and comparing the number of condensates that contain and / or do not contain condensate-associated molecules in the first portion to the number of condensates that contain and / or do not contain condensate-associated molecules in the second portion. In some embodiments, the method includes determining the number of condensates that contain condensate-associated molecules in the cytoplasm of the cell composition, determining the number of condensates that contain condensate-associated molecules in the nucleus of the cell composition, and comparing the number of condensates that contain condensate-associated molecules in the cytoplasm and the nucleus.

[0140] In some embodiments, the number of condensates that contain condensate-associated molecules is increased relative to the reference. In some embodiments, the number of condensates that contain condensate-associated molecules is decreased relative to the reference. In some embodiments, the number of condensates that do not contain condensate-associated molecules is increased relative to the reference. In some embodiments, the number of condensates that do not contain condensate-associated molecules is decreased relative to the reference.

[0141] In some embodiments, the number of condensates with and / or without condensate-associated molecules in the first portion of the cell composition is decreased compared to the reference, and the number of condensates with and / or without condensate-associated molecules in the second portion of the cell composition is not decreased compared to the reference. In some embodiments, the number of condensates with and / or without condensate-associated molecules in the first portion of the cell composition is increased compared to the reference, and the number of condensates with and / or without condensate-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 condensates with condensate-associated molecules in the cytoplasm is decreased compared to the reference, and the number of condensates with condensate-associated molecules in the nucleus is not decreased compared to the reference. In some embodiments, the number of condensates with condensate-associated molecules in the cytoplasm is decreased compared to the reference, and the number of condensates with condensate-associated molecules in the nucleus is increased compared to the reference.

[0142] In some embodiments, the number of condensates with and / or without condensate-associated molecules in the first portion of the cellular composition is increased relative to the second portion of the cellular composition, or the number of condensates with and / or without condensate-associated molecules in the second portion of the cellular composition is increased relative to the first portion of the cellular composition, In some embodiments, the first portion of the cellular composition is cytoplasmic and the second portion of the cellular composition is nuclear, or the first portion of the cellular composition is nuclear and the second portion of the cellular composition is cytoplasmic.

[0143] <Size of the condensate> In some embodiments, the characteristic associated with one or more condensates is based on the size of one or more condensates. Thus, in some embodiments of the present invention, a method for identifying a compound that modulates a characteristic associated with one or more condensates, including condensate-associated molecules, is provided, the method comprising: (a) contacting a 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 characteristic associated with one or more condensates, the modulation of the characteristic indicating that the compound modulates the characteristic associated with one or more condensates compared to a reference, the characteristic being modulation of the size of one or more condensates. In some embodiments of the present invention, a method for identifying a compound that modulates the size of one or more condensates is provided, the method comprising: (a) contacting a 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 size of one or more condensates, the modulation of the size indicating that the compound modulates the size of one or more condensates compared to a reference. In some embodiments, the size is an average of two or more condensates.

[0144] The size of the condensate can be determined for a portion or all of the cellular composition. Thus, in some embodiments, the method includes determining the size of one or more condensates in a portion of the cellular composition. In some embodiments, the method includes determining the size of one or more condensates in the entire cellular composition. In some embodiments, the method includes determining the size of one or more condensates in one or more cells in the cellular composition. In some embodiments, the method includes determining the size of one or more condensates in a single cell in the cellular composition.

[0145] The size of the condensate can also be determined for some or all of the cells in the cell composition. Thus, in some embodiments, the method includes determining the size of one or more condensates in a portion of one or more cells in the cell composition. In some embodiments, the method includes determining the size of one or more condensates in a portion of a single cell in the cell composition. In some embodiments, the method includes determining the size of one or more condensates in the cytoplasm. In some embodiments, the method includes determining the size of one or more condensates in the nucleus. In some embodiments, the method includes determining the size of one or more condensates in an organelle.

[0146] In some embodiments, the size of the one or more condensates increases relative to the reference, hi some embodiments, the size of the one or more condensates decreases relative to the reference.

[0147] In some embodiments, the size of one or more condensates in the first portion of the cell composition is decreased compared to the reference, and the size of one or more condensates in the second portion of the cell composition is not decreased compared to the reference. In some embodiments, the size of one or more condensates in the first portion of the cell composition is increased compared to the reference, and the size of one or more condensates in the second portion of the cell composition is not increased compared to the reference. In some embodiments, the first portion of the cell composition is the cytoplasm and the second portion of the cell composition is the nucleus, or the first portion of the cell composition is the nucleus and the second portion of the cell composition is the cytoplasm. In some embodiments, the size of one or more condensates in the cytoplasm is decreased compared to the reference, and the size of one or more condensates in the nucleus is not decreased compared to the reference. In some embodiments, the size of one or more condensates in the cytoplasm is decreased compared to the reference, and the size of one or more condensates in the nucleus is increased compared to the reference.

[0148] In some embodiments, the size of one or more condensates in the first portion of the cellular composition is increased relative to the second portion of the cellular composition, or the size of one or more condensates in the second portion of the cellular composition is increased relative to the first portion of the cellular composition, In some embodiments, the first portion of the cellular composition is cytoplasm and the second portion of the cellular composition is nucleus, or the first portion of the cellular composition is nucleus and the second portion of the cellular composition is cytoplasm.

[0149] <Position of condensate> In some embodiments, the one or more condensate-associated features are based on the location of one or more condensates. Thus, in some embodiments of the present invention, a method for identifying a compound that modulates one or more condensate-associated features, including condensate-associated molecules, is provided, the method comprising: (a) contacting a compound with a cellular composition that includes one or more condensates or a cellular composition that can form one or more condensates; and (b) determining one or more condensate-associated features, the modulation of the features indicating that the compound modulates one or more condensate-associated features compared to a reference, the feature being modulation of the location of one or more condensates. In some embodiments of the present invention, a method for identifying a compound that modulates one or more condensate locations is provided, the method comprising: (a) contacting a compound with a cellular composition that includes one or more condensates or a cellular composition that can form one or more condensates; and (b) determining the location of one or more condensates, the modulation of the locations indicating that the compound modulates one or more condensate locations compared to a reference.

[0150] The location of the condensate can be determined for a portion or all of the cellular composition. Thus, in some embodiments, the method includes determining the location of one or more condensates in a portion of the cellular composition. In some embodiments, the method includes determining the location of one or more condensates in the entire cellular composition. In some embodiments, the method includes determining the location of one or more condensates in one or more cells in the cellular composition. In some embodiments, the method includes determining the location of one or more condensates in a single cell in the cellular composition.

[0151] The location of the condensate can also be determined for some or all of the cells in the cellular composition. Thus, in some embodiments, the method includes determining the location of one or more condensates in a portion of one or more cells in the cellular composition. In some embodiments, the method includes determining the location of one or more condensates in a portion of a single cell in the cellular composition. In some embodiments, the method includes determining the location of one or more condensates in the cytoplasm. In some embodiments, the method includes determining the location of one or more condensates in the nucleus. In some embodiments, the method includes determining the location of one or more condensates in an organelle.

[0152] <Condensate distribution> In some embodiments, the one or more condensate-related features are based on the distribution of one or more condensates, such as the spatial distribution of one or more condensates in a cell. Thus, in some embodiments of the present invention, a method for identifying a compound that modulates one or more condensate-related features, including condensate-associated molecules, is provided, the method comprising: (a) contacting a compound with a cellular composition that includes one or more condensates or a cellular composition that can form one or more condensates; and (b) determining one or more condensate-related features, the modulation of the features indicating that the compound modulates the one or more condensate-related features compared to a reference, the feature being modulation of the distribution of one or more condensates. In some embodiments of the present invention, a method for identifying a compound that modulates the distribution of one or more condensates is provided, the method comprising: (a) contacting a compound with a cellular composition that includes one or more condensates or a cellular composition that can form one or more condensates; and (b) determining the distribution of one or more condensates, the modulation of the distribution indicating that the compound modulates the distribution of one or more condensates compared to a reference.

[0153] In some embodiments, the distribution of one or more condensates is based on the number of condensates comprising a condensate-associated molecule in a cellular location, such as the cytosol, nucleus, organelle, or any one or more of these. In some embodiments, the distribution of one or more condensates is determined to be modulated by the compound if the number of condensates comprising a condensate-associated molecule in a cellular location, such as the cytosol or nucleus, is decreased compared to the number of condensates comprising a condensate-associated molecule in a cellular location of a reference cellular composition, such as a cellular composition not contacted with the compound or a cellular composition contacted with a reference compound. In some embodiments, the distribution of one or more condensates is determined to be modulated by the compound if the number of condensates comprising a condensate-associated molecule in a cellular location, such as the cytosol or nucleus, is increased compared to the number of condensates comprising a condensate-associated molecule in a cellular location of a reference cellular composition, such as a cellular composition not contacted with the compound or a cellular composition contacted with a reference compound.

[0154] In some embodiments, the distribution of one or more condensates is based on the number of condensates containing condensate-associated molecules in one or more portions of the cellular composition, such as one or more fields of view or one or more portions of the image of the cellular composition. In some embodiments, the distribution of one or more condensates is determined to be modulated by the compound if the number of condensates containing condensate-associated molecules in one or more portions of the cellular composition, such as a portion of the cytosol or nucleus, is decreased compared to the number of condensates containing condensate-associated molecules in one or more portions of the reference cellular composition, such as one or more portions of the cellular composition not contacted with the compound or one or more portions of the cellular composition contacted with the reference compound. In some embodiments, the distribution of one or more condensates is determined to be modulated by the compound if the number of condensates containing condensate-associated molecules in one or more portions of the cellular composition, such as a portion of the cytosol or nucleus, is increased compared to the number of condensates containing condensate-associated molecules in one or more portions of the reference cellular composition, such as one or more portions of the cellular composition not contacted with the compound or one or more portions of the cellular composition contacted with the reference compound.

[0155] In some embodiments, the distribution of condensates is determined by calculating a ratio (e.g., percentage, parts per million, etc.) of condensates in a first portion of the cellular composition and a second portion of the cellular composition. In some embodiments, the method includes determining the number of condensates comprising condensate-associated molecules in the first portion of the cellular composition, determining the number of condensates comprising condensate-associated molecules in the second portion of the cellular composition, and calculating the ratio of the number of condensates comprising condensate-associated molecules in the first portion of the cellular composition and the second portion of the cellular composition. In some embodiments, the first portion of the cellular composition is a first portion of the cell and a second portion of the cell, such as the cytoplasm, the nucleus, or an organelle. In some embodiments, the first portion of the cellular composition is the cytoplasm and the second portion is the nucleus. In some embodiments, the ratio is increased relative to a reference, such as a cellular composition not contacted with the compound or a cellular composition contacted with a reference compound. In some embodiments, the ratio is decreased relative to the reference. In some embodiments, the ratio of nuclear condensates to cytoplasmic condensates is increased relative to the reference. In some embodiments, the ratio of nuclear condensates to cytoplasmic condensates is decreased relative to the reference. In some embodiments, the distribution of one or more condensates is determined to be modulated by the compound if the proportion of condensates in a first portion of the cellular composition, such as the nucleus, and in a second portion of the cellular composition, such as the cytosol, is increased relative to a reference. In some embodiments, the distribution of one or more condensates is determined to be modulated by the compound if the proportion of condensates in a first portion of the cellular composition, such as the nucleus, and in a second portion of the cellular composition, such as the cytosol, is decreased relative to a reference.

[0156] In some embodiments, the distribution of one or more condensates is determined using an imaging technique.

[0157] <Surface area of ​​condensate> In some embodiments, the characteristic associated with one or more condensates is based on the surface area of ​​one or more condensates. Thus, in some embodiments of the present invention, a method for identifying a compound that modulates a characteristic associated with one or more condensates, including condensate-associated molecules, is provided, the method comprising: (a) contacting a 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 characteristic associated with one or more condensates, the characteristic being indicative of the compound modulating the characteristic associated with one or more condensates compared to a reference, the characteristic being a modulation of the surface area of ​​one or more condensates. In some embodiments of the present invention, a method for identifying a compound that modulates the surface area of ​​one or more condensates is provided, the method comprising: (a) contacting a 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 surface area of ​​one or more condensates, the characteristic being indicative of the compound modulating the surface area of ​​one or more condensates compared to a reference. In some embodiments, the surface area is an average of two or more condensates.

[0158] The surface area of ​​the condensate can be determined for a portion or all of the cellular composition. Thus, in some embodiments, the method includes determining the surface area of ​​one or more condensates in a portion of the cellular composition. In some embodiments, the method includes determining the surface area of ​​one or more condensates in the entire cellular composition. In some embodiments, the method includes determining the surface area of ​​one or more condensates in one or more cells in the cellular composition. In some embodiments, the method includes determining the surface area of ​​one or more condensates in a single cell in the cellular composition.

[0159] The surface area of ​​the condensate can also be determined for some or all of the cells in the cell composition. Thus, in some embodiments, the method includes determining the surface area of ​​one or more condensates in a portion of one or more cells in the cell composition. In some embodiments, the method includes determining the surface area of ​​one or more condensates in a portion of a single cell in the cell composition. In some embodiments, the method includes determining the surface area of ​​one or more condensates in the cytoplasm. In some embodiments, the method includes determining the surface area of ​​one or more condensates in the nucleus. In some embodiments, the method includes determining the surface area of ​​one or more condensates in an organelle.

[0160] In some embodiments, the surface area of ​​the one or more condensates is increased relative to the reference, hi some embodiments, the surface area of ​​the one or more condensates is decreased relative to the reference.

[0161] In some embodiments, the surface area of ​​one or more condensates in the first portion of the cellular composition is decreased compared to the reference, and the size of one or more condensates in the second portion of the cellular composition is not decreased compared to the reference. In some embodiments, the surface area of ​​one or more condensates in the first portion of the cellular composition is increased compared to the reference, and the surface area of ​​one or more condensates in the second portion of the cellular composition is not increased compared to the reference. In some embodiments, the first portion of the cellular composition is the cytoplasm and the second portion of the cellular composition is the nucleus, or the first portion of the cellular composition is the nucleus and the second portion of the cellular composition is the cytoplasm. In some embodiments, the surface area of ​​one or more condensates in the cytoplasm is decreased compared to the reference, and the surface area of ​​one or more condensates in the nucleus is not decreased compared to the reference. In some embodiments, the surface area of ​​one or more condensates in the cytoplasm is decreased compared to the reference, and the surface area of ​​one or more condensates in the nucleus is increased compared to the reference.

[0162] In some embodiments, the surface area of ​​one or more condensates in the first portion of the cellular composition is increased relative to the second portion of the cellular composition, or the surface area of ​​one or more condensates in the second portion of the cellular composition is increased relative to the first portion of the cellular composition, In some embodiments, the first portion of the cellular composition is cytoplasm and the second portion of the cellular composition is nucleus, or the first portion of the cellular composition is nucleus and the second portion of the cellular composition is cytoplasm.

[0163] <Composition of the condensate> In some embodiments, the characteristic associated with one or more condensates is based on the composition of one or more condensates. Thus, in some embodiments of the present invention, a method for identifying a compound that modulates a characteristic associated with one or more condensates, including a condensate-associated molecule, is provided, the method comprising: (a) contacting a compound with a cellular composition that includes one or more condensates or a cellular composition that can form one or more condensates; and (b) determining a characteristic associated with one or more condensates, the modulation of the characteristic indicating that the compound modulates the characteristic associated with one or more condensates compared to a reference, the characteristic being modulation of the composition of one or more condensates. In some embodiments of the present invention, a method for identifying a compound that modulates the composition of one or more condensates is provided, the method comprising: (a) contacting a compound with a cellular composition that includes one or more condensates or a cellular composition that can form one or more condensates; and (b) determining a composition of one or more condensates, the modulation of the composition indicating that the compound modulates the composition of one or more condensates compared to a reference.

[0164] In some embodiments, the composition of the condensate is determined by detecting the presence of one or more macromolecules in the condensate, detecting the amount of one or more macromolecules in the condensate, calculating the ratio of one macromolecule to a second macromolecule in the condensate, and / or comparing the amount of one or more macromolecules in the condensate to the size and / or surface area of ​​the condensate. In some embodiments, the composition of the condensate is determined by detecting the presence of one or more macromolecules in the condensate, e.g., 1, 2, 3, 4, 5, 6, 7, or more macromolecules. In some embodiments, the composition of the condensate is determined by detecting the amount of one or more macromolecules in the condensate, e.g., 1, 2, 3, 4, 5, 6, 7, or more macromolecules. In some embodiments, the composition of the condensate is determined by calculating the ratio of one macromolecule to a second macromolecule in the condensate. In some embodiments, the composition of the condensate is determined by comparing the amount of one or more macromolecules in the condensate, e.g., 1, 2, 3, 4, 5, 6, 7, or more macromolecules, in the condensate to the size and / or surface area of ​​the condensate. In some embodiments, the macromolecules are condensate-associated molecules. In some embodiments, the polymer is a polynucleotide or a polypeptide. In some embodiments, the polymer is a polypeptide. In some embodiments, the polymer is a wild-type polypeptide. In some embodiments, the polymer is a mutant polypeptide. In some embodiments, the polymer is FUS, EWSR1, TIAL1, PABPC1, or G3BP1, or a mutant thereof.

[0165] The composition of the condensate can be determined for a portion or all of the cellular composition. Thus, in some embodiments, the method includes determining the composition of one or more condensates in a portion of the cellular composition. In some embodiments, the method includes determining the composition of one or more condensates in the entire cellular composition. In some embodiments, the method includes determining the composition of one or more condensates in one or more cells in the cellular composition. In some embodiments, the method includes determining the composition of one or more condensates in a single cell in the cellular composition.

[0166] The composition of the condensate can also be determined for some or all of the cells in the cellular composition. Thus, in some embodiments, the method comprises determining the composition of one or more condensates in a portion of one or more cells in the cellular composition. In some embodiments, the method comprises determining the composition of one or more condensates in a portion of a single cell in the cellular composition. In some embodiments, the method comprises determining the composition of one or more condensates in the cytoplasm. In some embodiments, the method comprises determining the composition of one or more condensates in the nucleus. In some embodiments, the method comprises determining the composition of one or more condensates in an organelle.

[0167] <Fluidity of condensed matter> In some embodiments, the characteristic associated with one or more condensates is based on the fluidity of one or more condensates. Thus, in some embodiments of the present invention, a method for identifying a compound that modulates a characteristic associated with one or more condensates, including condensate-associated molecules, is provided, the method comprising: (a) contacting a 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 characteristic associated with one or more condensates, the modulation of the characteristic indicating that the compound modulates the characteristic associated with one or more condensates compared to a reference, the characteristic being modulation of the fluidity of one or more condensates. In some embodiments of the present invention, a method for identifying a compound that modulates the fluidity of one or more condensates is provided, the method comprising: (a) contacting a compound with a cellular composition comprising one or more condensates or a cellular composition capable of forming one or more condensates; and (b) determining the fluidity of one or more condensates, the modulation of the fluidity indicating that the compound modulates the fluidity of one or more condensates compared to a reference. In some embodiments, the fluidity is based on a measurement of two or more condensates, for example, an average or distribution of the fluidities of two or more condensates.

[0168] The fluidity of a condensate can be determined for one or more condensates in some or all of a cellular composition. Thus, in some embodiments, the method includes determining the fluidity of one or more condensates in a portion of a cellular composition, such as one or more fields of view or one or more portions of an image of a cellular composition. In some embodiments, the method includes determining the fluidity of one or more condensates in the entire cellular composition. In some embodiments, the method includes determining the fluidity of one or more condensates in one or more cells in the cellular composition. In some embodiments, the method includes determining the fluidity of one or more condensates in a single cell in the cellular composition. In some embodiments, the method includes determining the fluidity of one or more condensates in a portion of one or more cells in the cellular composition. In some embodiments, the method includes determining the fluidity of one or more condensates in a portion of a single cell in the cellular composition. In some embodiments, the method includes determining the fluidity of one or more condensates in the cytoplasm. In some embodiments, the method includes determining the fluidity of one or more condensates in the nucleus. In some embodiments, the method includes determining the fluidity of one or more condensates in an organelle.

[0169] In some embodiments, the fluidity of the one or more condensates is determined to be modulated by the compound if the fluidity of the one or more condensates is increased compared to a reference comprising a reference condensate. In some embodiments, the fluidity of the one or more condensates is determined to be modulated by the compound if the fluidity of the one or more condensates is decreased compared to a reference comprising a reference condensate. In some embodiments, the fluidity of the one or more condensates in a first portion of the cellular composition is decreased compared to the reference and the fluidity of the one or more condensates in a second portion of the cellular composition is not decreased compared to the reference. In some embodiments, the fluidity of the one or more condensates in a first portion of the cellular composition is increased compared to the reference and the fluidity of the one or more condensates in a second portion of the cellular composition is not increased compared to the reference. In some embodiments, the first portion of the cellular composition is the cytoplasm and the second portion of the cellular composition is the nucleus, or the first portion of the cellular composition is the nucleus and the second portion of the cellular composition is the cytoplasm. In some embodiments, the fluidity of the one or more condensates in the cytoplasm is decreased compared to the reference and the fluidity of the one or more condensates in the nucleus is not decreased compared to the reference. In some embodiments, the fluidity of the one or more condensates in the cytoplasm is decreased compared to the reference and the fluidity of the one or more condensates in the nucleus is increased compared to the reference.

[0170] In some embodiments, the fluidity of one or more condensates in a first portion of the cellular composition is increased relative to a second portion of the cellular composition, or the fluidity of one or more condensates in the second portion of the cellular composition is increased relative to the first portion of the cellular composition, In some embodiments, the first portion of the cellular composition is cytoplasm and the second portion of the cellular composition is nucleus, or the first portion of the cellular composition is nucleus and the second portion of the cellular composition is cytoplasm.

[0171] In some embodiments, the fluidity of one or more condensates is assessed by one or more of droplet size, surface tension, phase diagrams, equilibrium states, droplet coarsening, and hardening.

[0172] In some embodiments, the fluidity of one or more condensates is determined using an imaging technique.

[0173] <Solidification of condensate> In some embodiments, the characteristic associated with one or more condensates is based on the coagulation of one or more condensates. Thus, in some embodiments of the present invention, a method is provided for identifying a compound that modulates a characteristic associated with one or more condensates, including a condensate-associated molecule, the method comprising: (a) contacting a 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 characteristic associated with one or more condensates, the modulation of the characteristic being indicative of the compound modulating the characteristic associated with one or more condensates compared to a reference, the characteristic being modulation of coagulation of one or more condensates. In some embodiments of the present invention, a method is provided for identifying a compound that modulates coagulation of one or more condensates, the method comprising: (a) contacting a compound with a cellular composition comprising one or more condensates or a cellular composition capable of forming one or more condensates, and (b) determining the coagulation of one or more condensates, the modulation of coagulation being indicative of the compound modulating coagulation of one or more condensates compared to a reference. In some embodiments, the coagulation is based on a measurement of two or more condensates, for example, an average or distribution of coagulation of two or more condensates.

[0174] The solidification of condensates can be determined for one or more condensates or products thereof in some or all of the cellular composition. Thus, in some embodiments, the method includes determining the solidification of one or more condensates in a portion of the cellular composition, such as one or more fields of view or one or more portions of an image of the cellular composition. In some embodiments, the method includes determining the solidification of one or more condensates in the entire cellular composition. In some embodiments, the method includes determining the solidification of one or more condensates in one or more cells in the cellular composition. In some embodiments, the method includes determining the solidification of one or more condensates in a single cell in the cellular composition. In some embodiments, the method includes determining the solidification of one or more condensates in a portion of one or more cells in the cellular composition. In some embodiments, the method includes determining the solidification of one or more condensates in a portion of a single cell in the cellular composition. In some embodiments, the method includes determining the solidification of one or more condensates in the cytoplasm. In some embodiments, the method includes determining the solidification of one or more condensates in the nucleus. In some embodiments, the method includes determining the solidification of one or more condensates in an organelle.

[0175] In some embodiments, the solidification of one or more condensates or products thereof is determined to be modulated by the compound if the solidification of one or more condensates is increased compared to a reference comprising a reference condensate or products thereof. In some embodiments, the solidification of one or more condensates is determined to be modulated by the compound if the solidification of one or more condensates is decreased compared to a reference comprising a reference condensate. In some embodiments, the solidification of one or more condensates in a first portion of the cell composition is decreased compared to the reference and the solidification of one or more condensates in a second portion of the cell composition is not decreased compared to the reference. In some embodiments, the solidification of one or more condensates in a first portion of the cell composition is increased compared to the reference and the solidification of one or more condensates in a second portion of the cell composition is not increased compared to the reference. In some embodiments, the first portion of the cell composition is the cytoplasm and the second portion of the cell composition is the 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 solidification of one or more condensates in the cytoplasm is decreased compared to the reference and the solidification of one or more condensates in the nucleus is not decreased compared to the reference. In some embodiments, solidification of one or more condensates in the cytoplasm is decreased compared to a reference and solidification of one or more condensates in the nucleus is increased compared to a reference.

[0176] In some embodiments, solidification of one or more condensates in a first portion of the cellular composition is increased relative to a second portion of the cellular composition, or solidification of one or more condensates in the second portion of the cellular composition is increased relative to the first portion of the cellular composition, In some embodiments, the first portion of the cellular composition is cytoplasm and the second portion of the cellular composition is nuclei, or the first portion of the cellular composition is nuclei and the second portion of the cellular composition is cytoplasm.

[0177] In some embodiments, the compound modulates the liquid-gel transition of one or more condensates relative to a reference. In some embodiments, the compound modulates the liquid-gel transition of one or more condensates and does not modulate the gel-solid transition of one or more condensates relative to a reference. In some embodiments, the compound modulates the gel-solid transition of one or more condensates relative to a reference. In some embodiments, the compound modulates the gel-solid transition of one or more condensates and does not modulate the liquid-gel transition of one or more condensates relative to a reference.

[0178] In some embodiments, solidification of one or more condensates is assessed by one or more of droplet size, surface tension, phase diagrams, equilibrium states, droplet coarsening, hardening, and fiber and / or aggregate formation.

[0179] In some embodiments, the clotting of one or more condensates is determined using an imaging technique. In some embodiments, the clotting of one or more condensates is determined by measuring the formation of aggregates or fibrils. In some embodiments, the clotting of one or more condensates is determined using a filtration-based technique.

[0180] <Dissolution of condensate> In some embodiments, the characteristic associated with one or more condensates is based on the dissolution of one or more condensates. Thus, in some embodiments of the present invention, a method for identifying a compound that modulates a characteristic associated with one or more condensates, including a condensate-associated molecule, is provided, the method comprising: (a) contacting a 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 characteristic associated with one or more condensates, the modulation of the characteristic indicating that the compound modulates the characteristic associated with one or more condensates compared to a reference, the characteristic being modulation of dissolution of one or more condensates. In some embodiments of the present invention, a method for identifying a compound that modulates dissolution of one or more condensates is provided, the method comprising: (a) contacting a compound with a cellular composition comprising one or more condensates or a cellular composition capable of forming one or more condensates; and (b) determining the dissolution of one or more condensates, the modulation of dissolution indicating that the compound modulates dissolution of one or more condensates compared to a reference. In some embodiments, the dissolution is based on a measurement consisting of two or more condensates, for example, an average or distribution of dissolution of two or more condensates.

[0181] Dissolution of condensates can be determined for one or more condensates in some or all of the cellular composition. Thus, in some embodiments, the method includes determining dissolution of one or more condensates in a portion of the cellular composition. In some embodiments, the method includes determining dissolution of one or more condensates in the entire cellular composition. In some embodiments, the method includes determining dissolution of one or more condensates in one or more cells in the cellular composition. In some embodiments, the method includes determining dissolution of one or more condensates in a single cell in the cellular composition. In some embodiments, the method includes determining dissolution of one or more condensates in a portion of one or more cells in the cellular composition. In some embodiments, the method includes determining dissolution of one or more condensates in a portion of a single cell in the cellular composition. In some embodiments, the method includes determining dissolution of one or more condensates in the cytoplasm. In some embodiments, the method includes determining dissolution of one or more condensates in the nucleus. In some embodiments, the method includes determining dissolution of one or more condensates in an organelle.

[0182] In some embodiments, the dissolution of one or more condensates is determined to be modulated by the compound if the dissolution of one or more condensates is increased compared to the reference. In some embodiments, the dissolution of one or more condensates is determined to be modulated by the compound if the dissolution of one or more condensates is decreased compared to the reference. In some embodiments, the dissolution of one or more condensates in a first portion of the cellular composition is decreased compared to the reference and the dissolution of one or more condensates in a second portion of the cellular composition is not decreased compared to the reference. In some embodiments, the dissolution of one or more condensates in a first portion of the cellular composition is increased compared to the reference and the dissolution of one or more condensates in a second portion of the cellular composition is not increased compared to the reference. In some embodiments, the first portion of the cellular composition is the cytoplasm and the second portion of the cellular composition is the nucleus, or the first portion of the cellular composition is the nucleus and the second portion of the cellular composition is the cytoplasm. In some embodiments, the dissolution of one or more condensates in the cytoplasm is decreased compared to the reference and the dissolution of one or more condensates in the nucleus is not decreased compared to the reference. In some embodiments, the dissolution of one or more condensates in the cytoplasm is decreased compared to the reference and the dissolution of one or more condensates in the nucleus is increased compared to the reference.

[0183] In some embodiments, lysis of one or more condensates in a first portion of the cellular composition is increased relative to a second portion of the cellular composition, or lysis of one or more condensates in the second portion of the cellular composition is increased relative to the first portion of the cellular composition, In some embodiments, the first portion of the cellular composition is cytoplasmic and the second portion of the cellular composition is nuclear, or the first portion of the cellular composition is nuclear and the second portion of the cellular composition is cytoplasmic.

[0184] In some embodiments, the method includes subjecting the cellular composition to condensate forming conditions prior to contacting the cellular composition with the compound, in some embodiments, lysis is assessed by one or more of the number of condensates, the size of the condensates, e.g., a reduction in size, or the disappearance of condensates.

[0185] In some embodiments, the dissolution of one or more condensates is determined using an imaging technique.

[0186] Presence and / or amount of fibrosis In some embodiments, the one or more condensate-associated features are based on the presence and / or amount of fibril formation. In some embodiments, the one or more condensate-associated features are based on the presence of fibril formation. In some embodiments, the one or more condensate-associated features are based on the amount of fibril formation. Thus, some embodiments of the present invention provide a method for identifying a compound that modulates one or more condensate-associated features, including condensate-associated molecules, the method comprising: (a) contacting a 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 feature associated with one or more condensates, the modulation of the feature being indicative of the compound modulating the one or more condensate-associated features relative to a reference, the feature being modulation of the presence and / or amount of fibril formation. Some embodiments of the present invention provide a method for identifying a compound that modulates the presence and / or amount of fibril formation, the method comprising: (a) contacting a compound with a cellular composition comprising one or more condensates or a cellular composition capable of forming one or more condensates, and (b) determining the presence and / or amount of fibril formation, the modulation of the presence and / or amount of fibril formation being indicative of the compound modulating the presence and / or amount of fibril formation relative to a reference.

[0187] Fibril formation can be detected and measured in a variety of ways, such as by using a microscope to visualize fibrils, and / or a fibril binding assay, such as the fibril binding assay described in the Examples.

[0188] The presence and / or amount of fibrosis can be determined for a portion or all of the cell composition. Thus, in some embodiments, the method includes determining the presence and / or amount of fibrosis in a portion of the cell composition. In some embodiments, the method includes determining the presence and / or amount of fibrosis in the entire cell composition. In some embodiments, the method includes determining the presence and / or amount of fibrosis in one or more cells in the cell composition. In some embodiments, the method includes determining the presence and / or amount of fibrosis in a single cell in the cell composition.

[0189] The presence and / or amount of fibrogenesis can also be determined for some or all of the cells in the cell composition. Thus, in some embodiments, the method comprises determining the presence and / or amount of fibrogenesis in a portion of one or more cells in the cell composition. In some embodiments, the method comprises determining the presence and / or amount of fibrogenesis in a portion of a single cell in the 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 a cellular organelle.

[0190] In some embodiments, the amount of fibrogenesis is increased relative to the reference, hi some embodiments, the amount of fibrogenesis is decreased relative to the reference.

[0191] In some embodiments, the amount of fibrogenesis in the first portion of the cell composition is decreased compared to the reference, and the amount of fibrogenesis in the second portion of the cell composition is not decreased compared to the reference. In some embodiments, the amount of fibrogenesis in the first portion of the cell composition is increased compared to the reference, and the amount of fibrogenesis 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 cytoplasm, and the second portion of the cell composition is nucleus, or the first portion of the cell composition is nucleus, and the second portion of the cell composition is cytoplasm. In some embodiments, the amount of fibrogenesis in the cytoplasm is decreased compared to the reference, and the amount of fibrogenesis in the nucleus is not decreased compared to the reference. In some embodiments, the amount of fibrogenesis in the cytoplasm is decreased compared to the reference, and the amount of fibrogenesis in the nucleus is increased compared to the reference.

[0192] In some embodiments, the amount of fibrogenesis in a first portion of the cellular composition is increased relative to a second portion of the cellular composition, or the amount of fibrogenesis in the second portion of the cellular composition is increased relative to the first portion of the cellular composition, In some embodiments, the first portion of the cellular composition is cytoplasmic and the second portion of the cellular composition is nuclear, or the first portion of the cellular composition is nuclear and the second portion of the cellular composition is cytoplasmic.

[0193] <Location of condensate-related molecules > In some embodiments, the one or more condensate-associated features are based on the location of one or more condensates. Thus, in some embodiments of the present invention, a method for identifying a compound that modulates one or more condensate-associated features, including condensate-associated molecules, is provided, the method comprising: (a) contacting a compound with a cellular composition that includes one or more condensates or a cellular composition that can form one or more condensates; and (b) determining one or more condensate-associated features, the modulation of the features indicating that the compound modulates one or more condensate-associated features compared to a reference, the feature being modulation of the location of one or more condensates. In some embodiments of the present invention, a method for identifying a compound that modulates one or more condensate locations is provided, the method comprising: (a) contacting a compound with a cellular composition that includes one or more condensates or a cellular composition that can form one or more condensates; and (b) determining the location of one or more condensates, the modulation of the locations indicating that the compound modulates one or more condensate locations compared to a reference.

[0194] The location of the condensate can be determined for a portion or all of the cellular composition. Thus, in some embodiments, the method includes determining the location of one or more condensates in a portion of the cellular composition. In some embodiments, the method includes determining the location of one or more condensates in the entire cellular composition. In some embodiments, the method includes determining the location of one or more condensates in one or more cells in the cellular composition. In some embodiments, the method includes determining the location of one or more condensates in a single cell in the cellular composition.

[0195] The location of the condensate can also be determined for some or all of the cells in the cellular composition. Thus, in some embodiments, the method includes determining the location of one or more condensates in a portion of one or more cells in the cellular composition. In some embodiments, the method includes determining the location of one or more condensates in a portion of a single cell in the cellular composition. In some embodiments, the method includes determining the location of one or more condensates in the cytoplasm. In some embodiments, the method includes determining the location of one or more condensates in the nucleus. In some embodiments, the method includes determining the location of one or more condensates in an organelle.

[0196] In some embodiments, the location of the one or more condensates is determined using an imaging technique.

[0197] <Division of condensate-related molecules into condensates> In some embodiments, the one or more condensate-associated characteristics are based on the division of the condensate-associated molecules into one or more condensates. Thus, in some embodiments of the present invention, a method for identifying a compound that modulates one or more condensate-associated characteristics, including condensate-associated molecules, is provided, the method comprising: (a) contacting a compound with a cellular composition that includes one or more condensates or a cellular composition that can form one or more condensates; and (b) determining one or more condensate-associated characteristics, the modulation of the characteristics indicating that the compound modulates the one or more condensate-associated characteristics compared to a reference, the characteristic being modulation of the division of the condensate-associated molecules into one or more condensates. In some embodiments of the present invention, a method for identifying a compound that modulates the division of the condensate-associated molecules into one or more condensates is provided, the method comprising: (a) contacting a compound with a cellular composition that includes one or more condensates or a cellular composition that can form one or more condensates; and (b) determining the division of the condensate-associated molecules into one or more condensates, the modulation of the division indicating that the compound modulates the division of the condensate-associated molecules into one or more condensates compared to a reference.

[0198] The partitioning of condensate-associated molecules into condensates can be determined for a portion or all of the cellular composition. Thus, in some embodiments, the method comprises determining the partitioning of condensate-associated molecules into one or more condensates in a portion of the cellular composition. In some embodiments, the method comprises determining the partitioning of condensate-associated molecules into one or more condensates in the entire cellular composition. In some embodiments, the method comprises determining the partitioning of condensate-associated molecules into one or more condensates in one or more cells in the cellular composition. In some embodiments, the method comprises determining the partitioning of condensate-associated molecules into one or more condensates in a single cell in the cellular composition.

[0199] The partitioning of condensate-associated molecules into condensates can also be determined for some or all of the cells in the cell composition. Thus, in some embodiments, the method comprises determining the partitioning of condensate-associated molecules into one or more condensates in a portion of one or more cells in the cell composition. In some embodiments, the method comprises determining the partitioning of condensate-associated molecules into one or more condensates in a portion of a single cell in the cell composition. In some embodiments, the method comprises determining the partitioning of condensate-associated molecules into one or more condensates in the cytoplasm. In some embodiments, the method comprises determining the partitioning of condensate-associated molecules into one or more condensates in the nucleus. In some embodiments, the method comprises determining the partitioning of condensate-associated molecules into one or more condensates in an organelle.

[0200] In some embodiments, partitioning of a condensate-associated molecule into one or more condensates is increased relative to the reference, hi some embodiments, partitioning of a condensate-associated molecule into one or more condensates is decreased relative to the reference.

[0201] In some embodiments, the partitioning of the condensate-associated molecules into one or more condensates in the first portion of the cell composition is decreased compared to the reference, and the partitioning of the condensate-associated molecules into one or more condensates in the second portion of the cell composition is not decreased compared to the reference. In some embodiments, the partitioning of the condensate-associated molecules into one or more condensates in the first portion of the cell composition is increased compared to the reference, and the partitioning of the condensate-associated molecules into one or more condensates in the second portion of the cell composition is not increased compared to the reference. In some embodiments, the first portion of the cell composition is the cytoplasm and the second portion of the cell composition is the nucleus, or the first portion of the cell composition is the nucleus and the second portion of the cell composition is the cytoplasm. In some embodiments, the partitioning of the condensate-associated molecules into one or more condensates in the cytoplasm is decreased compared to the reference, and the partitioning of the condensate-associated molecules into one or more condensates in the nucleus is not decreased compared to the reference. In some embodiments, the partitioning of the condensate-associated molecules into one or more condensates in the cytoplasm is decreased compared to the reference, and the partitioning of the condensate-associated molecules into one or more condensates in the nucleus is increased compared to the reference.

[0202] In some embodiments, partitioning of condensate-associated molecules into one or more condensates in a first portion of the cellular composition is increased relative to a second portion of the cellular composition, or partitioning of condensate-associated molecules into one or more condensates in the second portion of the cellular composition is increased relative to the first portion of the cellular composition, In some embodiments, the first portion of the cellular composition is cytoplasmic and the second portion of the cellular composition is nuclear, or the first portion of the cellular composition is nuclear and the second portion of the cellular composition is cytoplasmic.

[0203] <Aggregation of condensate-related molecules> In some embodiments, the one or more condensate-associated characteristics are based on the aggregation of condensate-associated molecules. Thus, in some embodiments of the present invention, a method for identifying a compound that modulates one or more condensate-associated characteristics, including condensate-associated molecules, is provided, the method comprising: (a) contacting a compound with a cellular composition that includes one or more condensates or a cellular composition that can form one or more condensates; and (b) determining one or more condensate-associated characteristics, the modulation of the characteristics indicating that the compound modulates the one or more condensate-associated characteristics compared to a reference, the characteristic being modulation of the aggregation of condensate-associated molecules. In some embodiments of the present invention, a method for identifying a compound that modulates the aggregation of condensate-associated molecules is provided, the method comprising: (a) contacting a compound with a cellular composition that includes one or more condensates or a cellular composition that can form one or more condensates; and (b) determining the aggregation of condensate-associated molecules, the modulation of the aggregation indicating that the compound modulates the aggregation of condensate-associated molecules compared to a reference.

[0204] The aggregation of condensate-associated molecules can be determined for a portion or all of the cellular composition. Thus, in some embodiments, the method includes determining the aggregation of condensate-associated molecules in a portion of the cellular composition. In some embodiments, the method includes determining the aggregation of condensate-associated molecules in the entire cellular composition. In some embodiments, the method includes determining the aggregation of condensate-associated molecules in one or more cells in the cellular composition. In some embodiments, the method includes determining the aggregation of condensate-associated molecules in a single cell in the cellular composition.

[0205] The aggregation of condensate-associated molecules can also be determined for some or all of the cells in the cell composition. Thus, in some embodiments, the method includes determining the aggregation of condensate-associated molecules in a portion of one or more cells in the cell composition. In some embodiments, the method includes determining the aggregation of condensate-associated molecules in a portion of a single cell in the cell composition. In some embodiments, the method includes determining the aggregation of condensate-associated molecules in the cytoplasm. In some embodiments, the method includes determining the aggregation of condensate-associated molecules in the nucleus. In some embodiments, the method includes determining the aggregation of condensate-associated molecules in an organelle.

[0206] In some embodiments, aggregation of condensate-associated molecules is increased relative to the reference, hi some embodiments, aggregation of condensate-associated molecules is decreased relative to the reference.

[0207] In some embodiments, the aggregation of condensate-associated molecules in the first portion of the cell composition is decreased relative to the reference, and the aggregation of condensate-associated molecules in the second portion of the cell composition is not decreased relative to the reference. In some embodiments, the aggregation of condensate-associated molecules in the first portion of the cell composition is increased relative to the reference, and the aggregation of condensate-associated molecules in the second portion of the cell composition is not increased relative to the reference. In some embodiments, the first portion of the cell composition is the cytoplasm and the second portion of the cell composition is the nucleus, or the first portion of the cell composition is the nucleus and the second portion of the cell composition is the cytoplasm. In some embodiments, the aggregation of condensate-associated molecules in the cytoplasm is decreased relative to the reference, and the aggregation of condensate-associated molecules in the nucleus is not decreased relative to the reference. In some embodiments, the aggregation of condensate-associated molecules in the cytoplasm is decreased relative to the reference, and the aggregation of condensate-associated molecules in the nucleus is increased relative to the reference.

[0208] In some embodiments, aggregation of condensate-associated molecules in a first portion of the cellular composition is increased relative to a second portion of the cellular composition, or aggregation of condensate-associated molecules in the second portion of the cellular composition is increased relative to the first portion of the cellular composition, In some embodiments, the first portion of the cellular composition is cytoplasmic and the second portion of the cellular composition is nuclear, or the first portion of the cellular composition is nuclear and the second portion of the cellular composition is cytoplasmic.

[0209] <Compound> As used herein, a "compound" refers to any agent. In some embodiments, the compound is a small molecule, a polypeptide, a lipid, or a nucleic acid. In some embodiments, the compound is an approved compound, e.g., a compound approved for medical use by the U.S. Food and Drug Administration. 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, a glycoside, a phenazine, a phenol, a polyketide, a terpene, or a 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, e.g., siRNA, miRNA, or mRNA. In some embodiments, the compound is a non-natural compound. In some embodiments, the compound is a natural compound. When multiple candidate compounds are used in screening, the multiple candidate compounds can be of the same type or different types.

[0210] In some embodiments, the compound can selectively interact with a biomolecule (particularly a protein or a nucleic acid) non-covalently under conditions prevalent in a living cell, and the compound and the biomolecule can be combined at least once. -4They form complexes with a dissociation constant kd of 1.0 mol / L or less. In some embodiments, the compounds have a molecular weight of more than 160 Da but less than 1000 Da, particularly less than 700 Da, more particularly less than 500 Da, and the compounds are characterized by containing up to 5 hydrogen bond donors, up to 10 hydrogen bond acceptors, and an octanol / water partition coefficient logP of less than 5.6. These are the so-called "Lipinski's rule" of 5 for drug-like compounds (originally referring to molecules between 160 and 500 Da). It is understood that any of these embodiments may be applied to and combined with any compound described herein (including the preceding paragraph).

[0211] <Cells and condensates> In some embodiments, the cellular composition comprises a microbial or animal cell. In some embodiments, the cellular composition comprises a human cell. In some embodiments, the cellular composition comprises a neuron. In some embodiments, the cellular composition comprises a cancer cell. In some embodiments, the cellular 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 cellular composition comprises a dysregulated condensate. In some embodiments, the cellular composition comprises a cell that comprises a disease-associated mutation. In some embodiments, the cellular composition comprises a cell that has one or more characteristics of a neurodegenerative or proliferative disease. In some embodiments, the cellular composition comprises a cell that expresses a protein labeled with a fluorescent protein. In some embodiments, the protein is a protein that is known to condense into condensates. In some embodiments, the condensate-associated molecule is labeled, such as by attachment or fusion to a fluorescent protein.

[0212] In some embodiments, the cells in the cell composition express the condensate-associated molecule. In some embodiments, expression can include any of gene duplication, transcription, and translation. In some embodiments, the condensate-associated molecule is a polynucleotide, such as RNA, and 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, and the condensate-associated molecule is translated in the cells in the cell composition. In some embodiments, the condensate-associated molecule is heterologous to the cell.

[0213] Many condensates are known in the art. Examples of known condensates include cleavage bodies, P granules, histone locus bodies, multivesicular bodies, neuronal RNA granules, nuclear gems, nuclear pores, nuclear speckles, nuclear stress bodies, nucleoli, Oct1 / PTF / transcription (OPT) domains, paraspeckles, juxtanucleolar compartments, PML nuclear structures, PML oncogenic domains, polycomb bodies, processing bodies, Sam68 nuclear structures, stress granules, or splicing speckles. Many more condensates are known to form but have not yet been described. Many condensates can be identified using microscopy. In some embodiments, the method further comprises identifying one or more condensates. In some embodiments, the one or more condensates are cellular condensates. In some embodiments, the one or more condensates are present within one or more cells in the cellular composition. In some embodiments, the one or more condensates are one or more stress granules. In some embodiments, the first set of one or more condensates are one or more stress granules. In some embodiments, the second set of one or more condensates is one or more of nuclear paraspeckles, condensates formed around sites of DNA damage, P bodies, Cajal bodies, and PML bodies.

[0214] In some embodiments, the condensate is selected from the group consisting of stress granules, P bodies, Cajal bodies, PML bodies, paraspeckles, e.g., nuclear paraspeckles, DNA damage focus condensates, schizonts, P granules, histone locus bodies, multivesicular bodies, neuronal RNA granules, nuclear gems, nuclear pores, nuclear stress bodies, nucleoli, Oct1 / PTF / transcription (OPT) domains, paranuclear compartments, PML oncogenic domains, polycomb bodies, processing bodies, Sam68 nuclear structures, and splicing speckles. Exemplary condensates are described, for example, in Banani et al., Nat Rev Mol Cell Biol, 18, 2017, "Biomolecular condensates: organizers of cellular biochemistry"; Brangwynne et al., Science, 324, 2009, "Germline P granules are liquid droplets that localize by controlled dissolution / condensation"; Patel et al., Cell, 162, 2015, "A Liquid-to-Solid Phase Transition of the ALS Protein Accelerated by Disease Mutation"; Alberti, S., Current Biology, 27, R1089-R1107, 2017, "Phase Separation in Biology".

[0215] <Condensate-related molecules> In some embodiments, the condensate associated molecule is a polynucleotide or a polypeptide. 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 FUS, EWSR1, TIAL1, PABPC1, or G3BP1, or a mutant thereof. In some embodiments, the condensate associated molecule is FUS.

[0216] Additional Assays In some embodiments, the method further comprises evaluating the compound by one or more additional assays, such as one or more of a second cell-based assay, a biochemical assay, and an in vivo assay.

[0217] In some embodiments, the biochemical assay is a method for identifying compounds that modulate a condensate-associated characteristic.

[0218] In some embodiments, the biochemical assay method includes (a) mixing a compound and a precursor molecule to form a reaction composition, (b) subjecting the reaction composition to condensate-forming conditions, whereby the condensates upon formation comprise the precursor molecule, and (c) determining a characteristic associated with the condensate, where modulation of the characteristic relative to a reference identifies the compound as a compound that modulates the characteristic. In some embodiments, the condensate-forming condition is a reducing salt concentration. In some embodiments, the method is repeated using different condensate-forming conditions.

[0219] In some embodiments, the biochemical assay method includes (a) mixing a condensate comprising a precursor molecule with a compound to form a reaction composition, and (b) determining a characteristic associated with the condensate, where modulation of the characteristic relative to a reference identifies the compound as a compound that modulates the characteristic. In some embodiments, the biochemical assay method further includes subjecting the reaction composition to aging conditions after mixing the compound with the condensate to form the reaction composition. In some embodiments, the aging conditions are incubation, shaking, and / or heat.

[0220] In some embodiments, determining the condensate-related characteristic is based on any one or more of: (i) number of condensates, (ii) size of the condensates, (iii) partitioning of precursor molecules into condensates, (iv) surface area of ​​the condensates, (v) composition of the condensates, (vii) fluidity of the condensates, (viii) solidification of the condensates, (ix) aggregation of precursor molecules, (x) dissolution of the condensates, and (xi) presence and / or amount of fibril formation.

[0221] 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.

[0222] In some embodiments, the biochemical assay 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 is repeated with two or more different ratios of compound and precursor molecule.

[0223] In some embodiments, the reference is a reaction composition that does not include the compound.

[0224] Exemplary embodiments Some of the embodiments are provided below.

[0225] Embodiment 1. A compound for use in a method for preventing a neurodegenerative disease associated with the formation of stress granules, comprising: - Lipoic acid, - Lipoamide, - Dihydrolipoic acid, and - Dihydrolipoamide A compound selected from:

[0226] Embodiment 2. Dosing regimen - Administration of said compound in a daily dose of 600 to 1,600 mg 2. A compound for use in a method for preventing a neurodegenerative disease associated with the formation of stress granules, as described in embodiment 1, comprising:

[0227] Embodiment 3. A compound for use in a method for preventing a neurodegenerative disease associated with the formation of stress granules according to embodiment 1 or 2, wherein said neurodegenerative disease associated with the formation of stress granules is amyotrophic lateral sclerosis.

[0228] Embodiment 4. A pharmaceutical composition for use in a method for preventing or treating neurodegeneration associated with the formation of stress granules, comprising a compound according to any one of embodiments 1 to 3.

[0229] 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.

[0230] Embodiment 6. The pharmaceutical composition of embodiment 4 or 5, wherein the neurodegenerative disease associated with the formation of stress granules is amyotrophic lateral sclerosis.

[0231] Embodiment 7. A dosage form for use in a method for preventing or treating amyotrophic lateral sclerosis comprising a compound as defined in any one of embodiments 1, in particular administered in a dose as defined in embodiment 2.

[0232] Embodiment 8. The dosage form of embodiment 7, wherein the dosage form is formulated for oral administration.

[0233] Embodiment 9. The dosage form of embodiment 7 or 8, wherein the neurodegenerative disease associated with the formation of stress granules is amyotrophic lateral sclerosis.

[0234] Embodiment 10. A method for treating or preventing a neurodegenerative disease associated with the formation of stress granules, comprising administering to a patient in need thereof a compound according to embodiment 1.

[0235] Embodiment 11. The method for treating or preventing a neurodegenerative disease according to embodiment 10, wherein the compound is administered in a daily dose of 600 to 1,600 mg.

[0236] Embodiment 12. A method for treating or preventing a neurodegenerative disease according to embodiment 10 or 11, wherein the compound is administered orally.

[0237] Embodiment 13. A method for treating or preventing a neurodegenerative disease according to any one of embodiments 10 to 12, wherein the neurodegenerative disease associated with the formation of stress granules is amyotrophic lateral sclerosis.

[0238] Embodiment 14. A method for suppressing or inhibiting the formation of stress granules in a cell, comprising: - Lipoic acid, - Lipoamide, - Dihydrolipoic acid, and - dihydrolipoamide, heterotricyclic compounds, in particular anthraquinones or anthraquinone derivatives, such as 1,4-dihydroxyanthraquinone, acridines or acridine derivatives, such as quinacrine or aminoacridine or mitoxantrone, - tetracyclic compounds, and - surfactants, especially cetylpyridinium chloride The method of claim 1, comprising the use of a compound selected from

[0239] <Other Exemplary Embodiments> Some of the embodiments are also provided below.

[0240] E1. A method for identifying a compound that modulates one or more condensate-associated characteristics, including condensate-associated molecules, the method comprising: (a) contacting the compound with a cellular composition comprising one or more condensates or capable of forming one or more condensates; and (b) determining the one or more condensate-associated characteristics, wherein modulation of the characteristic indicates that the compound modulates the one or more condensate-associated characteristics relative to a reference.

[0241] E2. The method of embodiment E1, wherein determining the characteristic associated with the one or more condensates is based on any one or more of: (i) the number of condensates with and / or without condensate-associated molecules, (ii) the size of the one or more condensates, (iii) the location of the one or more condensates, (iv) the distribution of the one or more condensates, (v) the surface area of ​​the one or more condensates, (vi) the composition of the one or more condensates, (vii) the fluidity of the one or more condensates, (viii) solidification of the one or more condensates, (ix) dissolution of the one or more condensates, (x) the presence and / or amount of fibril formation, (xi) the location of the condensate-associated molecules, (xii) partitioning of the condensate-associated molecules into one or more condensates, and (xiii) aggregation of the condensate-associated molecules.

[0242] E3. The method of embodiment E1 or E2, wherein the one or more condensates are present within one or more cells in the cellular composition.

[0243] E4. The method of any one of embodiments E1-E3, further comprising subjecting the cell composition to condensate-forming conditions prior to step (b).

[0244] E5. The method of any one of embodiments E1-E3, further comprising subjecting the cell composition to condensate-forming conditions prior to step (a).

[0245] 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 thermal stressor, (iv) an osmotic stressor, (v) a hyperosmotic stressor, and (vi) glycolysis inhibition.

[0246] E7. The method of any one of embodiments E1-E6, wherein the condensate-associated molecule is a polypeptide.

[0247] E8. The method of any one of embodiments E1-E7, wherein the condensate-associated molecule is a wild-type polypeptide.

[0248] E9. The method of any one of embodiments E1-E7, wherein the condensate-associated molecule is a mutant polypeptide.

[0249] E10. The method of any one of embodiments E1-E9, wherein the condensate-associated molecule is selected from the group consisting of FUS, EWSR1, TIAL1, PABPC1, and G3BP1.

[0250] E11. The method of any one of embodiments E1-E10, wherein the cells in the cell composition express a condensate-associated molecule.

[0251] E12. The method of any one of embodiments E1-E11, wherein the cell composition comprises HeLA cells, iPSC cells, or iPSC MN cells.

[0252] E13. The method of any one of embodiments E1-E12, further comprising imaging at least a portion of the cellular composition.

[0253] E14. The method of any one of embodiments E1-E13, further comprising contacting at least a portion of the cell composition with a fixative.

[0254] E15. The method of any one of embodiments E1-E14, further comprising contacting at least a portion of the cell composition with a staining agent.

[0255] E16. The method of any one of embodiments E1-E15, further comprising contacting at least a portion of the cell composition with DNA damaging conditions.

[0256] E17. The method of any one of embodiments E16, wherein the DNA damaging condition is laser irradiation.

[0257] E18. The method of any one of embodiments E1-E17, wherein the reference is a second condensate.

[0258] E19. The method of any one of embodiments E1-E17, wherein the reference is a second cell composition.

[0259] E20. The method of any one of embodiments E1-E19, further comprising evaluating the identified compound using a second cell-based assay.

[0260] E21. The method of any one of embodiments E1-E20, further comprising evaluating the identified compounds using a biochemical assay.

[0261] E22. The method of any one of embodiments E1-E21, further comprising evaluating the identified compounds using an in vivo assay.

[0262] E23. A method for identifying a compound useful in the treatment of a disease, comprising identifying a compound according to any one of the methods of embodiments E1-E22.

[0263] E24. The method of embodiment E23, wherein the disease is a neurodegenerative disease.

[0264] E25. The method of embodiment E24, wherein the neurodegenerative disease is ALS.

[0265] E26. The method of any one of the preceding embodiments E1-E25, wherein the condensates are non-membrane-encapsulated compartments formed by phase separation of one or more of the macromolecules, such as proteins, in particular DNA and / or RNA.

[0266] E27. The method of E27, wherein the condensate is selected from the group consisting of stress granules, P bodies, Cajal bodies, promyelocytic leukemia protein (PML) bodies, paraspeckles, e.g., nuclear paraspeckles, DNA damage focus condensates, schizonts, P granules, histone locus bodies, multivesicular bodies, neuronal RNA granules, nuclear gems, nuclear pores, nuclear stress bodies, nucleoli, Oct1 / PTF / transcription (OPT) domains, juxtanucleolar compartments, PML oncogenic domains, polycomb bodies, processing bodies, Sam68 nuclear structures, and splicing speckles.

[0267] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the disclosure of this application. The present invention will be further described below by way of 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.

Examples

[0268] <Screening of Compounds Using FUS as an LCD / RBP Stress Granule Protein Model> The inventors developed a cell-based screening using a HeLa cell line that stably expresses FUS labeled with GFP at approximately endogenous levels. Although not required for the formation of stress granules, FUS is a well-characterized protein with a domain structure specific to stress granule proteins. In the absence of cellular stress, FUS-GFP is mainly localized in the nucleus, but some is displaced from the nucleolus and localized in small spots called paraspeckles (Figure 1A). These are nuclear droplets involved in retaining RNA in the nucleus for a rapid stress response. Cells were pretreated with 10 μM of a compound from a compound library for 1 hour, followed by stressing the cells with 1 mM potassium arsenite (in the presence of the compound) (Figure 1B), and the localization of FUS to stress granules was observed. Arsenite inhibits the antioxidant response by reacting with thiol groups and blocks the tricarboxylic acid (TCA) cycle by reacting with thiols in the lipoyl moiety of the organism, causing overall oxidative damage. In untreated stressed cells, some of the FUS translocates to the cytoplasm, binds to other proteins and mRNAs, and phase-separates to form liquid stress granules surrounded by cytoplasm depleted of FUS (Figure 1A).

[0269] Many compounds affected stress granule formation. Using multiparameter image analysis including cytoplasmic speckle count, nuclear speckle count, and nuclear / cytoplasmic partitioning, compounds were ranked by the strength of their effect on FUS localization in stressed cells (Fig. 1C). Two compounds in the library predicted to inhibit stress granules did so: the polysome-stabilizing compound emetine (which inhibits mRNA release) and the heavy metal chelating compound dimercaprol (which is believed to sequester stressors). Edaravone, a potential antioxidant and used in Japan and the United States to treat ALS, had no effect on FUS localization in arsenate-stressed cells. Novel classes of hit compounds that tended to have a large effect on FUS localization and generally inhibited the number of stress granules included cardiac glycosides, heterotricyclic and tetracyclic compounds (anthraquinones and acridines), surfactants, and benzimidazoles.

[0270] As compounds that can directly interact with FUS were of primary interest, we performed a follow-up screen of compounds for their effect on FUS droplet formation in vitro (Figure 1D). Based on the cell-based screen, 47 of the top potent hit compounds were analyzed. Hit compounds were identified based on their effect on the number of FUS droplets formed in vitro under low-salt conditions in the presence of 1 mM DTT, mimicking the intracellular reducing environment, and on the partitioning of FUS into droplets formed (Figure 1E). Seven of these 47 compounds significantly affected FUS in vitro (Figure 1E), thereby identifying three classes of compounds that directly affect FUS droplet formation: surfactants, heterotricyclic and tetracyclic compounds, and lipoamides, the latter two of which are promising therapeutic agents (Figure 1F).

[0271] In this non-equilibrium snapshot, heterotricyclic and tetracyclic compounds tended to inhibit condensate formation in a dose-dependent manner, and the resulting droplets were smaller, whereas surfactants and lipoamides tended to increase the number of condensates and their division into droplets, and the droplets were larger (Figure 1E, G). Surfactants are included in the library due to their use as topical preservatives and are not promising therapeutic agents, as they permeabilize cell membranes.

[0272] Stress granule formation is associated with translocation of FUS from the nucleus, which is characteristic of LCD / RBP-containing stress granule proteins. Persistent stress granules or stress granule proteins may confer deleterious loss of nuclear function, including a compromised DNA damage response of FUS. Restoring nuclear localization of such proteins would be beneficial.

[0273] Therefore, we analyzed the dose-dependent activity of lipoamide, lipoic acid, and related compounds. In HeLa cells expressing FUS-GFP, dose-response analysis showed that lipoamide had similar EC 50 It was shown that lipoamide inhibited the number of stress granules and at the same time increased the nuclear / cytoplasmic FUS partitioning (Figure 2A). Not all hit compounds showed this behavior, for example, mitoxantrone (a heterotricyclic anthraquinone) reduced the nuclear / cytoplasmic FUS partitioning but inhibited the number of stress granules (Figure 10). We confirmed these important activities of lipoamide using an induced pluripotent stem cell (iPSC) line expressing FUS-GFP under arsenate stress. This also showed that lipoamide inhibited the number of stress granules and restored the location of FUS in the nucleus (Figure 2B). Thus, lipoamide appears to reverse both the translocation of FUS from the nucleus and its incorporation into stress granules in response to stress. Lipoic acid was subsequently shown to have a similar effect (see below). Returning FUS to the nucleus would be beneficial to mimic the state of unstressed cells, indicating that lipoamide is a more promising candidate.

[0274] Lipoamide is related to the natural compound lipoic acid. Lipoic acid has two stereoisomers, of which R-(+)-lipoic acid occurs naturally in cells and is synthesized in mitochondria, whereas S-(-)-lipoic acid is not. A mixture of both isomers is racemic lipoic acid or (±)-lipoic acid. It exists in cells at very low free concentrations and is usually covalently attached to proteins as a lipoyl moiety via a secondary amide. This form of attachment is similar to lipoamide. These compounds are dithiols, and the thiol of the lipoyl moiety is used as a hydrogen carrier by several enzymes, including those that enter and those that are in the tricarboxylic acid (TCA) cycle. The R-(+) isomer can be interconverted between oxidized (dithiol) and reduced (cyclic disulfide) states in cells by dihydrolipoamide dehydrogenase, and these compounds are thought to be antioxidants, although evidence for direct antioxidant activity is debated. Importantly, both lipoic acid and lipoamide are non-toxic. Lipoic acid is also well-characterized pharmacokinetically, non-toxic, and in humans, when administered orally at 1,600 mg, produces plasma concentrations of 8-30 μM. This is very promising, since concentrations of the order of 10 μM, as characterized ex vivo, can be achieved in humans (and lipoic acid has been used for many years in the treatment of diabetic neuropathy at doses of about 600 mg / day). In summary, lipoamide and lipoic acid dissolve stress granules, transport FUS back into the nucleus in stressed cells, interact directly with FUS, and have low toxicity. This makes them promising for use as therapeutic agents.

[0275] The action of lipoamide / lipoic acid appears independent of any enzyme or antioxidant role. The activity of lipoamide in a follow-up in vitro screen (Fig. 1D,E) indicates that it has a direct effect on phase separation of stress granule proteins. We therefore decided to exclude common mechanisms of activity in the cell-based screen, such as stressor sequestration, enzymatic action, or antioxidant activity. We tested this with lipoamide and lipoic acid-related compounds (Fig. 2C,D).

[0276] Screening was performed using arsenate stress. Arsenate stress may cause global oxidative damage, react with thiol groups to prevent antioxidant responses, and react with thiols in the critical lipoyl moiety to block the TCA cycle. To investigate to what extent the direct reaction of arsenate with lipoic acid, antioxidant effects, and enzymatic / glycolytic effects are involved in the activity of lipoic acid compounds, we analyzed the dose-dependent activities of (±)-lipoic acid, (±)-lipoamide, (±)-dihydrolipoic acid, 1,3-propanedithiol, and R-(+)- and S-(-)-lipoic acid in HeLa cells expressing FUS-GFP (Fig. 2C,D). All except 1,3-propanedithiol showed EC values ​​that suppressed the number of stress granules after 1 h of stress with 1 mM arsenate. 50 were equivalent.

[0277] This indicates that lipoic acid is not active by aiding glycolysis, since free lipoamide is not a normal component of cellular metabolism and is not capable of acting as a coenzyme / apoenzyme adduct by lipoate-protein ligase. Although lipoic acid does not directly act as an antioxidant, since the reduced form (dihydrolipoic acid) is more active than the oxidized form (lipoic acid), it has been shown that it may supplement the redox cycle involving dihydrolipoamide dehydrogenase (only the natural R-(+) isomer may be active). It is also noteworthy that menadione (a provitamin K and representative antioxidant) did not suppress the number of stress granules in a screen of primary HeLa cells. As arsenate reacts with thiols, 1,3-propanedithiol and dihydrolipoic acid should be able to directly chemically inactivate arsenate stress as well, but 1,3-propanedithiol was ineffective up to 100 μM. Furthermore, the EC of lipoic acid was not significantly different from that of 1,3-propanedithiol. 50This indicates that a stoichiometry of approximately 1:50 is required to inactivate arsenate. Also, the enzymatic role in the activity of lipoic acid, either as the lipoyl moiety to aid in glycolysis, or through antioxidant activity and recycling by dihydrolipoamide dehydrogenase, has been unique to native R-(+)-lipoic acid. However, S-(-)-lipoic acid had very similar activity.

[0278] These suggest some other mechanism of action, perhaps a stress signaling mechanism or a direct effect on the physicochemical properties of phase separation of stress granule proteins.

[0279] Lipoamide / Lipoic Acid Reverses the Formation of Stress Granules Caused by Several Types of Cellular Stress A cell-based screen was performed by pretreating cells with compounds prior to arsenate stress and analyzing only FUS.To understand the breadth of action of lipoamide and lipoic acid and to gain deeper insight into their potential mechanisms, we comprehensively characterized their effects on HeLa cells.

[0280] To determine whether lipoic acid could dissolve existing stress granules, we pre-stressed HeLa cells expressing FUS-GFP cells and then treated them with lipoamide. Addition of fresh medium supplemented with 10 μM lipoamide and 1 mM arsenate dissolved 80–90% of the cytoplasmic FUS droplets within 20 min, whereas time-lapse microscopy showed that fresh medium supplemented with 1 mM arsenate only slightly reduced the number of stress granules (Figure 3A). This response appears to be extremely rapid for a transcription / translation reaction.

[0281] To examine whether lipoamide treatment of cells confers sustained resistance to stress granule formation even in the absence of continued lipoamide treatment, we pretreated HeLa cells with 10 μM lipoamide for 1 h, and then subjected them to arsenate stress for 1 h without lipoamide, indicating that lipoamide-induced adaptation of cells to stress resistance was not sustained (Fig. 3B).

[0282] Blocking the localization of all PLD-containing stress granule proteins to stress granules was predicted to be a useful therapeutic agent. We stressed a panel of HeLa cell lines expressing GFP fusions of stress granule proteins (EWSR1, TIAL1, PABPC1, G3BP1) with either no treatment or 10 μM lipoic acid or lipoamide with 1 mM arsenate. All proteins localized to stress granules without treatment and did not localize in the presence of racemic lipoic acid, S-(-)- or R-(+)-lipoic acid, or lipoamide (Figure 3C). FUS is not required for stress granule formation, suggesting that FUS is not the only, or necessarily the primary, target of lipoamide or lipoic acid in cells. Rather, all LCD / RBP proteins were affected, potentially targeting proteins essential for stress granule formation. This is a promising property for the treatment of neurodegenerative diseases, as many stress granule-associated proteins are associated with a variety of diseases, suggesting that it may have therapeutic value against pathologies caused by mutations in other stress granule proteins.

[0283] The stressor arsenate reacts with the thiol group of lipoamide. Therefore, to exclude the possibility that lipoamide does not work unless the stressor is removed, we further tested whether lipoic acid could prevent the formation of stress granules induced by stresses other than arsenate (oxidative) stress, namely mitochondrial electron transport chain inhibition (rotenone), heat stress (42°C), hyperosmotic stress (sorbitol, a non-metabolizable sugar), glycolysis inhibition (6-deoxyglucose in the absence of glucose), or serum starvation. 10 μM racemic lipoamide, racemic lipoic acid, R-(+) or S-(-)-lipoic acid all inhibited the formation of stress granules in mitochondrial, hyperosmotic, or arsenate stressed HeLa cells (Figure 3D). Thus, these compounds are not specific for arsenate stress, although they do not affect the stress granules formed after all stresses.

[0284] Taken together, these data indicate that lipoamide and lipoic acid completely inhibit stress granule formation under several stresses. This pattern of action across different proteins and different stresses is consistent with the compounds affecting the phase separation of key LCD stress granule proteins such as TIA1, or a signaling step in stress granule formation that is common to certain stresses.

[0285] Lipoamide does not affect other cytoplasmic compartments or the nuclear fluid compartment. Stress granules are one of several similar RNA-containing liquid compartments in the cytoplasm, including processing bodies (P-bodies) in particular. Some stress granule proteins also form nuclear structures, namely nuclear paraspeckles and foci at sites of DNA damage. Many other nuclear liquid compartments exist. Using a panel of cell lines expressing GFP fusions, we asked whether lipoamide also affects these other structures. In this analysis, we included mitoxantrone as a representative of the heterotricyclic / tetracyclic class of hit compounds (Figure 1F). Excessive effects on liquid compartments may lead to side effects of treatment.

[0286] Under conditions that dissolve stress granules, lipoamide did not affect the localization of RNA processing bodies (P bodies, cytoplasmic), PML bodies (nuclear), or DNA damage foci (nuclear) proteins (Figure 4A). Mitoxantrone, on the other hand, affected all of these compartments to some degree (Figure 4B). Thus, the action of lipoamide is specific to stress granule formation, stress granule phase separation, and / or signaling and / or physicochemical properties that promote specific signaling pathways. Stress granules typically form and dissolve rapidly compared to many other compartments.

[0287] FUS is recruited to sites of DNA damage. Suppression of DNA damage response by FUS incorporation into stress granules in the cytoplasm may be a mechanism involved in the pathogenesis of ALS. The strong association of FUS nuclear localization signal (NLS) mutations (e.g., P525L) with familial ALS mutations supports this hypothesis. Therefore, we tested whether lipoamide affects FUS recruitment to sites of DNA damage induced by focused UV laser irradiation using arsenic acid stressed iPSCs expressing FUS-GFP (Figure 4B). 20 μM lipoamide (which dissolves stress granules) had no significant effect on FUS-GFP recruitment to sites of DNA damage. 1 μM lipoamide (which had no effect on stress granules) increased FUS-GFP recruitment to sites of DNA damage and appeared to be beneficial. In contrast, mitoxantrone inhibited the recruitment of FUS-GFP to sites of DNA damage at concentrations insufficient to dissolve cytoplasmic FUS droplets, thus demonstrating that the action of lipoamide is specific to the cytoplasmic FUS liquid compartment, a therapeutically useful feature.

[0288] <Lipoamide / lipoic acid directly interacts with FUS to inhibit fibrosis and sclerosis> Given that lipoamide affected FUS droplet formation in vitro, we selected lipoic acid and lipoamide for further characterization (Figure 1D,E). Lipoamide caused the formation of larger droplets, similar to the action of the surfactants in the library. This suggests that more rapid Ostwald ripening / coarsening occurred, possibly due to increased surface tension. We then tested whether lipoic acid or lipoamide had other effects on phase separation in vitro for FUS, a well-characterized phase-separating stress granule protein.

[0289] We confirmed the effect of lipoamide on the fluidity (surface tension and viscosity) of FUS-eGFP condensates. Here, droplets of condensates were brought together with optical tweezers and the time it took for them to fuse and relax into spheres was measured (Figure 5A). Lipoamide increased fluidity by 3-fold (Figure 5B). Taken together, the in vitro analysis suggests that the observed increase in droplet size and number (Figure 1E) results from faster generation of larger droplets due to increased fluidity, followed by sedimentation of the larger droplets (see Materials and Methods for details). As lipoamide / lipoic acid did not cause dissolution of FUS droplets in vitro, we first analyzed FUS condensates formed under (dextran-induced) crowding conditions to investigate the effect of lipoamide and lipoic acid on fibril formation and stiffening (Figure 5C–E).

[0290] We asked whether lipoic acid or lipoamide affected the conditions under which FUS phase separates in vitro. We found that 100 μM lipoic acid or lipoamide only had a subtle effect of slightly increasing the minimum KCl concentration at which phase separation occurs (Figure 6A). This suggests that lipoic acid or lipoamide has little or no effect on the chemical potential of the system, which changes the phase diagram or equilibrium state. Rather, the compounds affect coarsening, which is a change in the kinetics of reaching equilibrium.

[0291] Therefore, we analyzed another kinetic phenomenon: stiffening and fibril formation. In vitro FUS droplets “aged” over time, first stiffening and then tending to form amyloid / prion-like fibrils. This was accelerated in G156E FUS, a mutation associated with familial ALS. Both 30 μM lipoic acid and lipoamide delayed fibril formation of G156E FUS (Fig. 6B). Moreover, both compounds delayed stiffening of FUS droplets, leaving a large mobile fraction of FUS in the droplets (Fig. 6C). This occurred in the presence of 1 mM DTT (approximately 1:30 excess of reducing agent), strongly suggesting a non-antioxidant mechanism of lipoic acid / lipoamide.

[0292] The inhibition of hardening and rapid coarsening indicates that direct interaction of FUS increases its "fluidity" at the concentrations achieved in cells. This may allow stress granules to dissolve more easily and inhibit FUS (and other PLD-containing stress granules) aggregation in vivo. We first tested this in vitro in iPSCs using a filter-binding assay that detects insoluble FUS aggregates. This showed that lipoamide inhibited the amount of insoluble FUS, either wild-type or P525L, in arsenate-stressed cells (Figure 11B,C).

[0293] In vitro data indicate that the mechanism of action of lipoamide requires a weak, transient interaction between lipoamide and FUS in granules. Capturing this interaction would be difficult using traditional structural biology techniques. For this mechanism to be meaningful in cells, lipoamide would need to reach the cell interior in its unmodified form and in high enough concentrations to exert its effect. Lipoamide and lipoic acid exert physical effects on FUS, but large amounts are required for the protein, e.g., 100 μM lipoamide for 2.8 μM FUS (Figure 6A). Given the low μM concentration of FUS in the cytoplasm, for this to be possible, accumulation of the compound would have to occur within the cell; i.e., the compound would need to enter the cell, be concentrated, and not be metabolized. A signaling mechanism would not require either of these.

[0294] We also used NMR spectroscopy of the prion-like N-terminal LCD of FUS to dissect the mechanism at the submolecular level and identify the putative site of lipoamide interaction. The LCD can phase separate in vitro to form condensates, and individual residues can be degraded to form 1 H- 15 N heteronuclear single quantum coherence spectra can be assigned to 1 H- 15Analysis of N reveals that the environment of individual residues is altered, as indicated by the corresponding chemical shifts. The magnitude of the chemical shift changes was compared with the use of lipoamide or mitoxantrone versus both. Although no single clear site of interaction of lipoamide with the LCD of FUS was observed, mitoxantrone induced a weak shift throughout the LCD, consistent with a weak interaction with tyrosine residues. This suggests that lipoamide does not act by direct binding to high affinity proteins. This leaves several possibilities: perhaps lipoamide interacts with the LCD of FUS only during phase separation, potentially lowering the viscosity, or it interacts with the interface of the condensates, perhaps increasing the surface tension. Such interactions could inhibit the formation and expansion of aggregated forms of FUS that are responsible for the stiffening and reduced fluidity of the condensates.

[0295] <Lipoamide is highly concentrated in cells> The concentration of lipoamide relative to protein required to affect condensate fluidity in vitro was determined by the EC 50 The concentration of FUS in cells was high (low μM) for a protein, with lipoamide at 100 μM for 1 μM FUS (Fig. 1D, E, Fig. 5). 41 , cellular EC 50 The concentrations of FUS-containing condensates were on the order of 10 μM (Figures 2A and 3D). We also observed opposite effects of lipoamide on FUS-containing condensates in vitro (more and larger condensates) and in cells (fewer stress granules). To reconcile this difference and understand the relevance of the in vitro action in cells, we wanted to know the actual concentration of the compound in cells. In principle, isotopic labeling allows the direct observation of isotopically labeled compounds even in complex environments with the appropriate spectroscopic techniques. To take advantage of this technique, 15 N-labeled lipoamide (Figure 7A) was synthesized. Solution-state NMR studies were then used to confirm its intracellular 15The NH2 protons covalently bonded to N were quantitatively detected to determine the concentration of lipoamide from a complex mixture within cells and to reveal chemical modifications of the amide group (manifested as chemical shift changes or spectral changes) (Figure 13).

[0296] From the medium by cells 15 N-lipoamide uptake was quantified by NMR after incubating lipoamide in the absence or presence of HeLa cells for 1 hour at 37°C (Figure 7B). Cellular uptake was determined by calculating the difference between media incubated with or without cells for combinations of R-(+)-lipoamide or (±)-lipoamide with unstressed or stressed cells (Figure 7C). In one sample, R-(+)-lipoamide and stressed cells, a strong signal from the cellular fraction was identified consistent with uptake of the majority of lipoamide (Figure 7C). Measurements of both R-(+)-lipoamide and (±)-lipoamide indicated that 35±11% (n=3 and 2, respectively) of the lipoamide present in the media was taken up. No significant differences were observed between R-(+)-lipoamide and (±)-lipoamide uptake or between stressed and unstressed cells (Figure 7D).

[0297] No evidence of metabolic or other chemical modifications of lipoamide was observed, and the NMR signal of the cell samples indicated that lipoamide was present in the cells in its unmodified form (Fig. 7B).

[0298] Approximate intracellular 15N-lipoamide concentration can be calculated from uptake rate, cell number, and volume (Supplementary Methods). This shows that the average intracellular concentration is 5.0 ± 1.6 mM (Figure 7D), significantly higher than that obtained under optimal conditions of 1% v / v DMSO in HO in vitro. Thus, lipoamide is readily taken up into HeLa cells and reaches concentrations comparable to abundant intracellular metabolites, which exist in a chemically unmodified form (Figure S13G,H). This concentration is an order of magnitude higher than that observed to affect FUS-GFP condensates in vitro, indicating that it may exert physicochemical effects in cells.

[0299] Lipoamide / lipoic acid prevents stress granule protein aggregation in vivo Given that lipoamide and lipoic acid had a profound effect on FUS aggregation in vitro, we used a filter trap retention assay (which tends to retain aggregated proteins on the membrane from cell lysates) to test whether lipoamide had a beneficial effect on the spontaneous aggregation of wild-type or P525L FUS-GFP in iPSCs (Figure S11). There was some evidence of FUS aggregation in both cell lines, which was reduced after treatment with lipoamide (Figure S11B).

[0300] Lipoic acid prevents persistent stress granule / aggregation in vivo To look for evidence that lipoic acid acts on stress granule formation and protein aggregation in vivo, we turned to Caenorhabditis elegans (nematode). In C. elegans, aging and chronic stress are associated with the aggregation of stress granule proteins (including orthologues of TIAL1 and PABC1). This may parallel the pathogenesis of ALS. Therefore, we tested whether lipoic acid prevents the coagulation of stress granules. All C. elegans grown in liquid culture with R-(+) or S-(-)-lipoic acid showed a dose-dependent decrease in the percentage of worms with aggregated PAB-1, stress granule proteins with LCD / RBP structures, and orthologues of PABC1. The highest concentration tested (2 mM) was somewhat toxic, killing 6–8% of worms. Lipoamide could not be tested because it precipitated in the worm culture medium.

[0301] To test whether the effect of lipoic acid is specific to RBPs with LCDs, we examined the aggregation of two globular proteins, KIN-19 and RHO-1, previously shown to aggregate with age in C. elegans. Neither protein has an RNA-binding domain or an LCD. No effect of 1.5 mM lipoic acid on the aggregation of RHO-1 was observed. A mild reduction in the aggregation of KIN-19 was observed, but 1.5 mM lipoic acid was highly toxic.

[0302] Lipoic acid therefore correlates with its short-term in vitro behavior and may affect stress granule coagulation over the long term of an organism's lifespan. The behavior of lipoic acid is consistent with a direct interaction with stress granule proteins that inhibits stress granule formation and / or aggregation of stress granule proteins.

[0303] Lipoic acid and lipoamide reverse FUS-related defects in neurons and organisms In humans, one of the causes of familial ALS is FUS mutations, particularly mutations in the C-terminal NLS. P525L is one such mutation, and iPSC-derived motor neurons (iPSC MNs) expressing P525L in ALS patients show defects consistent with those of the motor neurons in patients. iPSC MNs can be grown using silicone conduits with cell bodies positioned on one side and axons projecting through the conduit to the other side. Such cultures can be maintained for extended periods (>60 days). However, while expression of P525L FUS results in axonal degeneration and retraction within this time period even in the absence of exogenous cellular stressors, P525L FUS MNs have a strong tendency to form stress granules. This is similar to the axonal retraction that leads to motor dysfunction in patients. Therefore, we tested whether lipoamide or lipoic acid could ameliorate these phenotypes. Although FUS P525L neurons initially appeared morphologically normal (Figs. 8D, 9), by day 60 in culture, degenerated and retracted axonal material had accumulated near the neuronal exit of the silicone conduit (Figs. 8D, 9). Addition of 2 μM lipoic acid or lipoamide to the culture medium prevented this degenerative retraction. It is believed that defects in axonal transport lead to axonal degeneration and retraction. To test this, we analyzed the transport of lysosomes in axons of iPS MNs expressing P525L FUS with or without lipoamide compared to iPS MNs expressing WT FUS (Fig. 12). Lipoamide restored axonal transport in P525L FUS iPS MNs to the same level as WT neurons. These assays did not use treatments to induce stress granule formation in iPSC MNs, but P525L FUS is more prone to form stress granules in iPSCs (Fig. 8A). No stressor was required to induce axonal degeneration and retraction, suggesting that lipoic acid either helps iPSC-MNs cope with stochastic stress in culture or restores the intrinsic defects caused by P525L.

[0304] We further tested whether lipoic acid could exert a similar beneficial effect on motor neurons in vivo using a fly model. Drosophila melanogaster has a FUS orthologue, cabeza, that is required for normal neuronal development. cabeza has a shorter N-terminal PLD than FUS, and expression of human FUS in D. melanogaster results in motor defects, including reduced climbing ability. Expression of P525L or R521C human FUS (both NLS mutants) results in even more severe motor defects. Addition of lipoic acid to the diet dose-dependently rescued the motor defects, restoring the ability to climb 4 cm in 30 s from approximately 50% to more than 80%. Thus, the climbing ability of the flies was significantly restored. Thus, lipoic acid can rescue motor neuron defects caused by FUS.

[0305] <Consideration> Stress granules have been implicated as a key site in the pathogenesis of ALS. They form by liquid-liquid phase separation of proteins driven by weak multivalent interactions. This is distinct from the strong enzyme-substrate or protein-protein interactions that drugs typically target. Thus, although conceptually plausible, it was unclear whether drug-like compounds could be identified that inhibit phase separation. We focused on FUS as a well-characterized model protein and searched for compounds that affect stress granule formation by direct interactions with LCD-containing stress granule proteins. 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 several conditions, but their mechanisms of action remain unclear. We now show that lipoamide directly alters the properties of phase-separated FUS droplets, inhibits stress granule formation in cells, inhibits FUS aggregation in vitro and in cells, inhibits stress granule protein aggregation in animals, and rescues phenotypes in neurons and animals resulting from FUS mutants associated with familial ALS.

[0306] The research underlying the present invention focused on lipoamide, rather than lipoic acid, as a more potent hit compound in vitro. Evidence indicates a promising physicochemical mechanism of action. Lipoamide accumulates in cells at high concentrations, and at this concentration (using FUS as a model in vitro) it affects the fluidity of phase-separated droplets. In cells, lipoamide completely dissolves stress granules that form under any stress (Figure 3D), but not other non-membrane-bound liquid compartments. These include compartments formed by the same protein in different regions of the cell (nuclear paraspeckles and around sites of DNA damage) or by other proteins (P-bodies, Cajal bodies, PML bodies) (Figure 4). Lipoamide also inhibits the coagulation of these phase-separated compartments in vitro (Figure 6B-D), in cells, and seemingly in whole animals (Figure 8A). Lipoamide affects FUS behavior in vitro, but is not required for stress granule formation in cells. Thus, lipoamide may also interact with FUS-like proteins (or other LCD / RBP-containing proteins) that are essential for the formation of stress granules in cells, although the exact nature of the interaction of FUS or other LCD-containing stress granule proteins remained unclear.

[0307] In cells, lipoamide dissolved stress granules formed under several cellular stresses (Fig. 3), but did not result in dissolution of FUS condensed droplets in vitro (Fig. 5), appearing to affect the kinetics but not the thermodynamics of FUS phase separation. One possible reason is that lipoamide accumulates in cells to significant high concentrations (Fig. 7) and, although it can be reduced, is not otherwise metabolized (Fig. 13). The estimated cellular concentration (~5 mM) is an order of magnitude higher than the concentration that could be reached in vitro (300 μM) and much higher than the concentration required to increase the fluidity and inhibit stiffening of phase-separated FUS in vitro (Fig. 5). It is possible that 5 mM lipoamide dissolves FUS condensed droplets in vitro. Other explanations are also possible. First, high fluidity of stress granules may increase the sensitivity of cells to stress granule dissolving factors. Second, cells have stress granule, cytoplasmic, and nuclear environments, making the triphasic system more complex (probably related to the effect of lipoamide on the nuclear / cytoplasmic partitioning of FUS, Fig. 2). Finally, lipoamide exerts a strong effect on FUS aggregation (Fig. 5), which may be due in part to LCD-LCD interactions that are not required for phase separation in vitro. If LCD-LCD interactions are more important for phase separation in cells, this could be manifested in the increased sensitivity of stress granules to lipoamide compared to FUS in vitro.

[0308] The cellular targets of lipoamide or lipoic acid could not be clearly identified. Although these compounds alter the properties of FUS condensates in vitro, FUS is not essential for stress granule formation. Thus, FUS cannot be the only cellular target. Many stress granule proteins, such as G3BP1, which is thought to aggregate stress granules, are FUS-like with LCD domains and affected cells upon treatment (Figure 3).

[0309] Lipoamide and lipoic acid may affect common properties of some FUS-like or pivotal stress granule proteins. If the physicochemical mechanism is correct, lipoamide does not have a single binding site, but rather modulates the weak transient interactions between FUS molecules to modulate condensate properties. It may be surprising that we could not detect the interaction by NMR, despite the clear effect on the physical properties of FUS condensates in vitro (Figs. 5, 6). Of note, this assay only targets FUS LCDs in the non-phase separated stage, so lipoamide may interact with other regions of FUS, such as the RNA-binding domain, that are not present in this assay.

[0310] Although the exact mechanism of ALS pathogenesis remains unclear, lipoamide and lipoic acid are promising therapeutic candidates based on the hypothesis that stress granules are involved in ALS pathogenesis. The utility of lipoamide / lipoic acid depends on whether stress granule formation is driving ALS pathogenesis or is part of an essential cellular response to stress. Similarly, it depends on whether aggregation of stress granule proteins is a means to sequester harmful proteins or is intrinsically harmful. Therefore, we characterized the potential of lipoamide and lipoic acid for the treatment of ALS-like diseases. We focused primarily on lipoic acid because of its known pharmacokinetics and toxicity in humans, even if its in vitro potency is somewhat lower. Lipoic acid was shown to suppress the aggregation of stress granule proteins in C. elegans, a hallmark of aging in this organism and a phenomenon with general similarity to the aggregation of LCD-containing proteins in ALS pathology (Figure 8A). We also tested two models of ALS caused by mutations in FUS and confirmed that lipoic acid rescued the deficits in neuronal axon stability in vitro and motor control in D. melanogaster caused by expression of FUS mutants linked to ALS pathology (Figure 8C). Lipoic acid has previously shown some efficacy in the SOD1 animal model of ALS, but our study shows efficacy in a stress granule protein-driven model of ALS pathogenesis through a novel mechanism of action. In humans, daily administration of 600 mg lipoic acid produces plasma concentrations of 8-30 μM, similar to the concentrations used in our cell-based assays, demonstrating the surprising promise of lipoic acid as a therapeutic agent.

[0311] Lipoic acid is a natural metabolite and is closely related to lipoamide. Despite being related to natural metabolites, there was no evidence of metabolism of lipoamide. Both are considered antioxidants, and indeed lipoamide was briefly considered as an antioxidant in prion-related diseases, but activity was independent of the redox state of lipoic acid on cells, and lipoic acid and lipoamide showed activity in the presence of excess reducing agents in vitro. This suggests a nonenzymatic / nonmetabolic and nonredox action, and alterations in phase separation remain a likely mechanism, especially considering that lipoamide accumulates to high concentrations in cells. This is different from, for example, blocking stress granule formation by small molecule inhibition of eIF2α phosphorylation by ISRIB. The detailed mechanism of action is unknown, but it may be a different mechanism of action from "conventional" compounds that target potent and specific enzyme-substrate or protein-protein interactions.

[0312] In general terms, the success of the screen used demonstrates that it is possible to find compounds that target the physicochemical properties of proteins, and demonstrates the practicality of the whole screening approach. This is important in diseases such as ALS. Different LCD-containing proteins are involved and different mutations are associated with familial ALS. It is not possible to target each one individually. Instead, compounds can be found that keep certain proteins in a more mobile state or more soluble in the cytoplasm. This points to a new class of drugs, drugs that affect the physicochemical properties of non-membrane-bound compartments, i.e. physicochemical drugs.

[0313] Stress granules are formed by protein phase separation in the cytoplasm, and although several small molecules have been identified that modulate phase separation (notably 1,6-hexanediol), lipoic acid and lipoamide are by far the most promising therapeutic agents. 1,6-hexanediol requires concentrations of 1-10% (hundreds of mM) for activity in vitro or in cells, a concentration that rapidly becomes toxic. The molecules we identified in our screen (both heterotricyclic and tetracyclic compounds and lipoamide and related compounds) were active in vitro (Figures 1, 5) and in cells (Figures 1, 2, 10) at extremely low concentrations (tens to hundreds of μM), 3-4 orders of magnitude lower than 1,6-hexanediol. The effect of lipoamide on stress granules is also specific in the sense that lipoamide did not affect other membrane-free liquid compartments in cells. Lipoamide did not affect the nuclear compartment formed by FUS (Figure 4B) or by other proteins in either the cytoplasm or nucleus (Figure 4A) and was well tolerated in culture by HeLa cells, iPS cells, and motor neuron cells.

[0314] Incidentally, the anticancer drug mitoxantrone was effective in disrupting many non-membrane-bound compartments. Mitoxantrone induces DNA damage by its role as a topoisomerase inhibitor through DNA binding, but it appears to disrupt rather than increase the number of non-membrane-bound DNA damage repose compartments, possibly through physicochemical mechanisms.

[0315] Interestingly, lipoic acid is a natural metabolite. Recent data have identified another metabolite, ATP, as a hydrotrope with properties that maintain protein solubility. A solution to prevent abnormal protein aggregation at disease sites may lie in aiding the cell's ability to maintain a soluble environment. Future screens may uncover more such molecules.

[0316] <Method> Stable Koyoto HeLa BAC cell lines expressing proteins with a GFP fluorescent marker at the C-terminus were generated using BAC recombineering, which results in near-endogenous expression levels of the fusion protein. In these cell lines, GFP is part of a modified localization and affinity purification (LAP) tag, via a short linker. HeLa cells were cultured in high-glucose DMEM supplemented with 10% FCS. Cultures were supplemented with 1% penicillin-streptomycin and maintained at 37°C, 5% CO2 under geneticin (Gibco, 400 μg / mL) selection.

[0317] Human iPS cell lines expressing FUS with a C-terminal GFP fluorescent marker from three donors were used. All were generated using CRISPR / Cas9 tagging and mutagenesis and have been described previously. In summary, JS-SL-C1 iPS cells expressing either wild-type or P525L FUS-GFP were previously 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 previously generated from the KOLF-C1 clonal iPS cell line generated as part of the Human Induced Pluripotent Stem Cell Initiative (HipSci). KOLF-C1 cells were obtained from a healthy male donor. In these cell lines, GFP is part of a modified localization and affinity purification (LAP) tag, which, via a short linker, delivers identical fusion protein sequences to the Koyoto HeLa BAC cell line. The JS-SL-C1 and KOLF-C1 iPS cell lines were used as an isogenic pair to analyze DNA damage response and lipoamide action against P525L FUS. AH-ALS1-F58 iPS cells expressing P525L FUS with a GFP fluorescent marker at the C-terminus were previously generated from a clonal iPS cell line of 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 cultured in TeSR E8 medium (Stem Cell Technologies) at 37 °C and 5% CO2.

[0318] AH-ALS1-F58 iPS cells expressing P525L FUS were placed on Matrigel-coated plates containing silicone conduits for the axons and induced to differentiate as described above to generate MNs, resulting in a cluster of cell bodies on one side of the conduit and axons extending through the conduit and projecting from the other side of the conduit. AH-ALS1-F58 were used to generate motor neurons (MNs) because they had been previously characterized in axonal transport assays. Unless otherwise stated, iPS MNs were used for assays within 4 weeks of completing differentiation.

[0319] All procedures using human cell samples were in accordance with the Declaration of Helsinki and were approved by the ethical committee of TU Dresden (EK45022009, EK393122012).

[0320] <Recombinant Protein> For in vitro droplet formation screening and clotting assays, recombinant GFP-FUS and GFP-G156E-FUS were purified using a baculovirus / insect cell expression system as described above. 7 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. The composition of the storage buffer for purified FUS was 1 M KCl, 50 mM Tris HCl (pH 7.4), 5% glycerol, and 1 mM DTT, and the FUS concentration was adjusted to 30 μM in the storage buffer before use.

[0321] <Compound> For ex vivo and in vitro screening, the PHARMAKON 1600 library was used and prepared as a 10 mM stock solution in DMSO. Compounds were purchased again for the follow-up assay and prepared as 10 mM stocks in DMSO. Lipamide (T5875, Sigma Aldrich or sc-239160, Santa Cruz Biotechnology), lipoic acid (62320, Sigma Aldrich), R-(+)-lipoic acid (07039, Sigma Aldrich), S-(-)-lipoic acid (08561, Sigma Aldrich), dihydrolipoic acid (T8260, Sigma Aldrich), valeric acid (240370, Sigma Aldrich), 1,3-propanedithiol (P50609, Sigma Aldrich), mitoxantrone (M6545, Sigma Aldrich), N-(2-hydroxyethyl)ethylenediamine (127582, Sigma Aldrich), 1,4-dihydroxyanthraquinone (Q906, Sigma Aldrich), cetylpyridinium chloride (C9002, Sigma Aldrich), quinacrine (Q3251, Sigma Aldrich), 9-aminoacridine (A38401, Sigma Aldrich), 2-amino-5-diethylaminopentane (A48806, Sigma Aldrich), daunorubicin (30450, Sigma Aldrich), 8-acetyl-6,11-dihydroxy-7,8,9,10-tetrahydro-naphthalene-5,12-dione (R162892, Sigma Aldrich). 15 The N racemate and R-(+)-lipamide were synthesized (see below), 1 characterized by 1H NMR, 13 13C NMR, mass spectrometry, infrared spectroscopy, and melting point. The yields were approximately 50% and approximately 99% 15 N-labeled.

[0322] <Ex vivo Screening of HeLa Cells> The effect of compounds on the localization of FUS-GFP in stressed HeLa cells was screened in a 384-well format. 4000 cells were seeded per well and incubated for 24 hours. After that, the medium was replaced with 40 μL of fresh medium, and compounds were added by acoustic dispensing (Labcyte Echo550) to a final concentration of 10 μM. The final concentration of DMSO in all samples was 0.1%. After 1 hour, potassium arsenite was added from a 5-fold stock solution to a final concentration of 1 mM, and the cells were incubated for an additional 1 hour. Then the cells were fixed with 7.4% formaldehyde and stained with 1 mg / mL Hoechst 33342 and 1:10,000 CellMask Blue (ThermoFisher). Six fields of view per well were imaged using a CellVoyager CV7000 automated spinning disk confocal microscope (Yokogawa Electric) equipped with a 40× NA1.3 water immersion objective lens. Each plate included 48 wells (compound solvent control) treated with 0.1% DMSO and stressed with arsenite, 8 wells untreated and unstressed, and 4 wells untreated and unstressed using parental Kyoto HeLa cells. All images were displayed with a gamma of 0.7 so that bright stress granules and faint nuclei could be seen simultaneously.

[0323] For the primary screening, the FUS-GFP signal was analyzed using KNIME. The cytoplasm was identified from the weak (CellMask blue) blue fluorescence signal, and the nucleus was identified from the strong (Hoechst 33342) blue fluorescence signal. For the nucleus and cytoplasm, the number of particles, total area, granularity at scales of 9, 10, and 11 px (cytoplasm) or 1, 5, 6, 7, 8, and 9 px (nucleus), organization at a scale of 10 px, and integrated signal intensity were measured in the green (FUS-GFP) channel. The Z-score (z = (χ - μ) / σ, where χ is the observed value, μ is the mean of the control, and σ is the standard deviation of the control) for each parameter was calculated for the DMSO-treated control of each plate and summarized into the Mahalanobis distance.

[0324] <in vitro purified FUS-GFP screening> The effect of the compound on FUS-GFP droplets in vitro was evaluated in a 384-well plate format. The amount of compound (in DMSO) required for final concentrations of 1, 3, 10, 30, or 100 μM was added by acoustic dispensing (Labcyte Echo550) to wells of a 96-well plate containing 3 μL of FUS-GFP diluted with 50 mM Tris·HCl (pH 7.4), 1 mM DTT (for low-salt assay). The final concentration of DMSO was 0.01 - 1%. Using a Freedom Evo200 liquid handling workstation (TECAN), the FUS-GFP / compound mixture was diluted with 7 μL of assay buffer containing 50 mM Tris·HCl (pH 7.4), 1 mM DTT, 50 mM KCl, and 0.7 μM FUS-GFP. The compound / FUS-GFP and assay buffer were mixed by standard pipetting procedures and subsequently dispensed into 4 wells of a clear-bottom 384-well plate, and then immediately imaged using a CellVoyager CV7000 automated spinning disk confocal microscope (same as above). Droplets in the suspension were imaged as maximum intensity projections of 6 focal planes at 2-μm steps for 6 fields of view per sample. The number of droplets and the partitioning of FUS-GFP into droplets were analyzed using a fixed intensity threshold with ImageJ. The number of droplets and partitioning were normalized assuming a linear change over time with reference to the first and last DMSO controls in each plate column, as the time dependence was weak due to droplet sedimentation.

[0325] <Characterization of the Compound in HeLa Cells and iPS Cells> Compound effects were assessed by live cell imaging in HeLa or iPS cells expressing wild-type or P525L FUS-GFP under various conditions. Various combinations of 1-h compound pretreatment or arsenate prestress followed by 1-h compound treatment and / or arsenate stress were used. Unless otherwise stated, cells were pretreated with 10 μM compound from a 10 mM stock solution in DMSO (or an equivalent amount of DMSO control) for 1 h and then stressed with 1 mM potassium arsenate for 1 h in the still presence of compound. GFP fluorescence was imaged by wide-field epifluorescence using an Olympus IX71 inverted microscope equipped with an Olympus 100x (NA1.4) Plan Apo oil immersion objective and a CoolSNAP HQ CCD camera (Photometrics) using a DeltaVision environmental control unit (37 °C, 5% CO2) (Applied Precision). The dynamics of samples treated after pre-stress was analyzed with images taken at 2 min intervals for 100 min and, for single time points, after 1 h unless otherwise stated.

[0326] Treatment with 1 mM potassium arsenate for 1 h was replaced with other conditions to impose different cell stresses: 100 μM rotenone (R8875, Sigma Aldrich) from a 1 M stock in DMSO for 2 h (mitochondrial stress); 2.5 h in serum-free DMEM (serum starvation stress); 1 h in 4 M stock sorbitol (S1876, Sigma Aldrich) in H2O (osmotic stress); 30 min in normal growth medium at 42 °C (heat stress); 1 h in glucose-free DMEM (11966025, ThermoFisher Scientific) supplemented with 10% FCS with 100 mM 6-deoxyglucose (D9761, Sigma Aldrich) from a 1 M stock in H2O (glycolytic stress). Appropriate solvent controls were used.

[0327] For Western blot and analysis of intracellular FUS aggregates, iPS cells were lysed with RIPA buffer. Western blot was performed in a standard manner using the following antibodies. Mouse anti-FUS (AMAB90549 Sigma Aldrich, diluted 1:500), 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. The filter retardation assay for intracellular FUS aggregates was performed as described above. 56 Briefly, after loading the protein extract onto a 0.2 μm cellulose acetate membrane, microfiltration was performed, leaving the proteins aggregated on the membrane. Aggregated FUS was detected by the method described above.

[0328] <HeLa cells and compound dose response> The dose-dependent effects of compounds on HeLa cells or iPS cells expressing FUS-GFP were evaluated ex vivo using 1 hour of 1 mM potassium arsenite stress, similar to HeLa cell screening in vitro. However, serial compound dilutions in the medium were manually prepared from 80 μM to approximately 0.4 nM in 1.189-fold dilution steps. Smaller dilution steps were chosen instead of concentration replication to obtain greater statistical power from a fixed number of samples. The final DMSO concentration was 0.08% in all samples, and each plate included at least 12 control wells with 0.08% DMSO. The number of cytoplasmic FUS droplets and the nuclear / cytoplasmic partitioning of FUS were analyzed using a custom macro in ImageJ. Nuclei were identified by binarizing the luminance of the blue fluorescence image after applying a 5 px Gaussian blur. Cytoplasmic FUS droplets were masked by the binarized nuclei, and after applying a 10 px weight, 0.9 unsharp mask filter, were identified by binarizing the luminance of the green image. Nuclear FUS droplets were masked to include only the binarized nuclei, and after applying a 5 px weight, 0.9 unsharp mask filter, 10 px rolling ball background subtraction, were identified by binarizing the luminance of the green image. The ratio of cytoplasmic FUS droplets to nuclei was taken as the number of cytoplasmic FUS droplets per cell per field of view, and the ratio p of nuclear to cytoplasmic partitioning of FUS was derived from the ratio of nuclei to the total green signal per field of view (where p = a / (1 - a)). These data were log-transformed and fit to a Rodbard sigmoid curve to determine EC n / v t , i.e., derived from the ratio of nuclei to the total green signal per field of view. These data were log-transformed and fit to a Rodbard sigmoid curve to determine EC 50 . Six fields of view were imaged and analyzed for each condition.

[0329] <in vitro FUS-GFP aggregation assay> For the in vitro aggregation assay, FUS-GFP in storage buffer was diluted with water, and 10 μM FUS, 50 mM Tris·HCl (pH 7.4), and 1 mM DTT were placed in a 20 μL volume in a non-binding clear-bottom 384-well plate (Greiner Bio-One, 781906). Then, the compound or an equal volume of DMSO was added to give final compound concentrations of 30 μM and 0.3% DMSO. Horizontal shaking at 800 rpm was performed at room temperature to induce "aging" to form fibers. The formation of fibers and droplets was analyzed by wide-field epifluorescence using a DeltaVision Elite microscope (GE Healthcare Life Sciences) equipped with a Plan ApoN 60x (NA 1.4) oil immersion objective lens (Olympus) and an sCMOS camera (PCO). For the salt sensitivity of droplet formation, FUS-GFP in storage buffer was diluted with appropriate concentrations of KCl pre-mixed with the compound or DMSO, and a final KCl concentration series was made at a 30 μM compound concentration and 0.3% DMSO.

[0330] 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-897EMCCD camera on a Nikon TiE inverted microscope equipped with a Nikon Apo 100x (NA 1.49) oil immersion objective lens. A 10×10 px region was bleached for 50 nanoseconds with a 6 mW 405 nm laser using an Andor FRAPPA beam delivery unit and then imaged at 5 Hz for 5 minutes. The half-life and mobile fraction of the recovery curve were calculated using ImageJ.

[0331] <ex vivo DNA cleavage assay> Live cells were subjected to UV micro-irradiation to induce DNA damage. KOLF iPS MNs expressing wild-type GFP were stressed with 1 mM potassium arsenate for 1 h and then treated with lipoamide, mitoxantrone, or an equal volume of DMSO for 1 h. A single nuclear spot was exposed to three UV pulses as described for FRAP, but with 10% laser power. GFP fluorescence was imaged at 1 Hz and the intensity of the response was analyzed with ImageJ.

[0332] <nmr> To analyze the interaction of compounds with FUS, the compounds were incubated in the presence of 500 μM of compound or an equivalent DMSO solvent control (1%). 1 H- 15 N heteronuclear single quantum coherence NMR and the aforementioned sample conditions 14 We used to express, purify, and analyze the untagged FUS low-complexity domain (1–163).

[0333] By cells 15 To analyze N-lipoamide uptake, HeLa cells expressing FUS-GFP were cultured at 10 6 To simultaneously stress and treat the cells, the medium was diluted with potassium arsenate and 100 μM 15 The medium was replaced with 0.6 mL of N-racemic or R-(+)-lipoamide and left at 37°C for 1 h. High concentrations of compound were used to maximize the signal. The medium was then removed and retained (medium sample), and the cells were washed with approximately 2 mL of PBS before being removed by trypsinization (0.3 mL TrypLE Express (12604013, ThermoFisher) was added, incubated at 37°C for 5 min, and then 0.3 mL medium was added to quench the trypsin). The resuspended cells were retained (cell sample). All samples were frozen at -80°C. No compound (1% DMSO control), 15 N(±)-Lipoamide or 15 Wells were prepared for all combinations of N R-(+)-lipoamide, with or without potassium arsenate, and with or without cells. 1 H detection 15 NEdit 1 Using H-sensitivity enhanced HSQ, 15 N-lipoamide concentrations were quantified (see Supplementary Information). The solvent, pH, and temperature sensitivity of the primary amide proton chemical shifts were determined using dummy samples assembled from appropriate solvents and spiked compounds.

[0334] <Neuron degeneration and regression and axonal transport assay> To analyze axonal degeneration and transport, AH-ALS1-F58 iPS MNs expressing P525L FUS or isogenic controls expressing WT FUS were cultured for 60 days with 2 μM of the compound or an equal volume of DMSO. During this culture period, neurons expressing WT FUS had stable axons, whereas those expressing P525L FUS did not. Axonal degeneration was seen as cell debris accumulating at the exit of axons from the conduit. Experiments and analyses were performed blindly, and axonal degeneration was qualitatively scored using phase-contrast images taken every 10 - 20 days.

[0335] To analyze axonal transport, iPS MNs expressing P525L FUS were treated with 2 μM of the compound or an equal volume of DMSO for 3 days. The incubation time was extended to allow the compound to penetrate and act over the length of the axonal conduit. 2 μM was chosen as the highest concentration at which no toxic effects were observed (qualitatively evaluated). Analysis of axonal transport of liposomes was performed as follows. Liposomes were labeled by adding 50 nM of Lysotracker Red (ThermoFisher) and imaged for 120 seconds at 3 1 / 3 Hz at the proximal or distal end of a silicone conduit containing axons using a Leica DMI6000 inverted microscope equipped with a 100x (NA 1.46) oil immersion objective lens and an Andor iXON 897 EMCCD camera in an incubator chamber (37 °C, 5% CO2). Using particle tracking, the proportion of particles moving at speeds greater than 0.2 μm / second was determined for five video microscopy images. Since each video contained a variable population of non-moving background particles, the data were normalized to the mean proportion of fast-moving lysosomes in DMSO (solvent control) - treated samples for each biological replicate.

[0336] <Protein Aggregation in C.elegans> We used a C. elegans model for stress granule formation and aggregation to analyze the effect of lipoic acid on stress granule protein aggregation in vivo. As previously described, fluorescently labeled PAB-1 forms abundant stress granules and large solid aggregates with aging or under chronic stress. RHO-1 and KIN-19 also aggregate with aging, but neither are RNA-binding or stress granule proteins. Three systems were used: Fluorescently labeled PAB-1 (DCD214:N2;uqIs24[pmyo-2::tagrfp::pab1gene]), 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]) were analyzed as follows, except that DCD13 was maintained at 20°C.

[0337] Worms were exposed to lipoic acid from the L4 larval stage in liquid culture in 96-well plates in a total volume of 50 μL per well of S-Complete (100 mM NaCl, 50 mM potassium phosphate (pH 6), 10 mM potassium citrate, 3 mM MgSO4, 3 mM CaCl2, 5 μg / mL cholesterol, 50 μM ethylenediaminetetraacetic acid (EDTA), 25 μM FeSO4, 10 μM MnCl2, 10 μM ZnSO4, 1 μM CuSO4) supplemented with heat-killed OP50 and 50 μg / mL carbenicillin. A minimum of nine wells, each containing 13 worms, were treated with R-(+)- or S-(-)-lipoic acid or an equal volume of DMSO per experiment. Toxicity was assessed by the number of dead or abnormally small worms.

[0338] Forty-eight hours after switching L4 from 20 °C to 25 °C (day 2 of adulthood), extensive aggregation of fluorescently labeled PAB-1 and RHO-1 occurred in the pharyngeal muscles, and KIN-19 aggregated throughout the body. After fixation with 2 mM levamisole, aggregation was scored using a fluorescence stereomicroscope (Leica M165 FC, Plan Apo 2.0x objective lens). For PAB-1, aggregates mainly occurred in the terminal bulbs of the pharynx, and aggregation was scored as high (>10 aggregates per animal) or low (<10). For RHO-1, aggregates in the isthmus of the pharynx were scored, and aggregation was scored as high (>50% of the isthmus), medium (<50%), or low (no aggregation). For KIN-19, aggregates occurred throughout the body wall muscles, and aggregation was scored as high (aggregates in the head, middle, and tail), medium (aggregates in the head and middle >15), or low (aggregates in the head or middle >15). High-magnification images were obtained using a Leica SP8 confocal microscope equipped with an HC Plan Apo CS2 63x (NA 1.40) oil immersion objective lens and a Leica HyD hybrid detector. tagRFP::PAB-1 was detected in the emission range of 565 - 650 nm using 555 nm as the excitation light. Representative confocal images are shown as maximum Z-stack projections.

[0339] <Motor Deficits in D. melanogaster> All Drosophila stocks were maintained using standard cornmeal media in a temperature-controlled 25 °C incubator with a 12-hour light / dark cycle. w1118, UAS-eGFP, and D42-GAL4 were obtained from the Bloomington Stock Center. UAS-FUS WT, UAS-FUS P525L, and UAS-FUS R521C have been previously described by Anderson et al. (Hum. Mol. Genet. 27, 1366 - 1381, 2018).

[0340] Climbing assays were performed as previously described. Briefly, flies expressing FUS, eGFP, or w1118 were grown in the presence or absence of (±)-α-lipoic acid (0.43 mM or 2.15 mM diluted in ethanol), (R)-(+)-α-lipoic acid (0.43 mM or 2.15 mM diluted in ethanol), (S)-(-)-α-lipoic acid (0.43 mM or 2.15 mM diluted in ethanol), (±)-α-lipoamide (0.43 mM diluted in DMSO), or PP242 / Torkinib (10 μM or 50 μM diluted in DMSO), then anesthetized, placed in a vial, and allowed to acclimate to a new vial for 15 min. For each fly genotype, the vial was tapped three times on the pedestal of the laboratory bench, and the flies' movements climbing the wall of the vial were recorded by a video camera. The percentage of flies that climbed 4 cm in 30 s was recorded. Statistical analyses were performed in GraphPad Prism 6 using either Student's t test or one-way ANOVA with Tukey's or Dunnett's multiple comparison test.

[0341] <Racemic (±) and 15 Synthesis and characterization of N R-(+)-lipoamide 15 N(±)-Lipoamide>

[0342] [ka]

[0343] (±)-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 h. The solution was diluted with EtOAc (100 mL) and washed with H2O (100 mL) and saturated aqueous NaHCO3 (100 mL). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The resulting NHS ester, trimethylamine (1.1 mL, 7.89 mmol), and 15 NH4Cl (500 mg, 9.36 mmol) was dissolved in DCM (20 mL) and the mixture was stirred for 20 h. The solution was diluted with DCM (100 mL) and washed with H2O (100 mL), saturated aqueous NaHCO3 (100 mL), and again with H2O (100 mL, twice). The organic layer was dried over MgSO4, filtered off, and concentrated under reduced pressure to give the crude product. 15 The N-lipoamide was obtained, which was further purified by silica gel column chromatography (DCM:MeOH=30:1). The solvent was removed under reduced pressure to give a yellow solid. 15 N-lipoamide was obtained. Yield: 554.2 mg (51%). 1 H NMR (400 MHz, chloroform-d) δ 5.62 (d, J = 31.3 Hz, 1H, NH cis ), 5.37(d, J = 31.0 Hz, 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 showed that 15 It is suggested that N labeling is about 99%. IR: 3352cm -1 , 3176cm -1 (CONH2), 2937cm -1 , 2898cm -1 , 2865cm -1 , 2783cm -1 (CH), 1746cm -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℃, Rf=0.60 (DCM:MeOH=20:1).

[0344] 15 N R-(+)-Lipoamide

[0345] [ka]

[0346] 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 h. The solution was diluted with EtOAc (100 mL) and washed with H2O (100 mL) and saturated aqueous NaHCO3 (100 mL). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The resulting NHS ester, trimethylamine (1.1 mL, 7.89 mmol), and 15 NH4Cl (500 mg, 9.36 mmol) was dissolved in DCM (20 mL) and the mixture was stirred for 20 h. The solution was diluted with DCM (100 mL) and washed with H2O (100 mL), saturated aqueous NaHCO3 (100 mL), and again with H2O (100 mL, twice). The organic layer was dried over MgSO4, filtered off, and concentrated under reduced pressure to give the crude product. 15 The N-lipoamide was obtained, which was further purified by silica gel column chromatography (DCM:MeOH=30:1). The solvent was removed under reduced pressure to give a yellow solid. 15 N(R)-lipoamide was obtained. Yield: 554.5 mg (51%). 1 H NMR (400 MHz, chloroform-d) δ 5.56 (d, J = 4.1 Hz, 1H, NH cis ), 5.34(d, J = 4.7 Hz, 1H, NH trans ), 3.60(ddt,1H,SSCH), 3.30-3.03(m,2H,SSCH2), 2.61-2.35(m,1H,SSCH2CH trans ), 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 showed that 15 It is suggested that N labeling is about 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℃, Rf=0.60 (DCM:MeOH=20:1), [α] 25 +105.6 (c=2.0, CHCl3).

[0347] Lipoamide 1 To confirm the assignment of the H NMR spectrum, density functional theory (DFT) calculations were performed. Gaussian098 was used to generate an optimized structure of lipoamide, from which the shielding tensors were calculated to determine the isotropic and anisotropic components. The B3LYP density functional and the 6-31G(d) basis set were used for the DFT calculations. 72 DFT calculations confirmed that the cis-amide proton 13 should have a larger chemical shift than the trans-amide proton 14 (Figure 13A), and that the proton attached to carbon 3, which is closest to the proton attached to carbon 2, should have a larger chemical shift than the other protons on carbon 3 (Figure 13A).

[0348] < 15 N lipoamide ( 15 N) 1 H NMR 1 H detection 15 NEdit 1 H sensitivity enhanced HSQ NMR (herein ( 15 N) 1 H) spectra were acquired on a small-bore Varian liquid spectrometer equipped with a room temperature probe and operating at a static magnetic field strength of 14.1 T.

[0349] Free induction decays were recorded with an acquisition time of 0.0624 and 1000 sweeps at 8 kHz with a return delay of 1 s. In total, 10,000 transients were collected, with a total experiment time of 3 h 1 min. 15 The J coupling between N was determined to be 96 Hz, so the 1 / 4J transfer time in the INEPT portion of the pulse sequence was set to 2.6 ms. With this setting, 15 N Ammonia and ammonium ions are not detected. 15 When N-lipoamide is chemically modified (e.g., covalently bonded to the apoenzyme), 15 N) 1 The H NMR spectrum shows a large change. Similarly, the peaks in the cell samples broadened significantly when lipoamide dissolves in the phospholipid membrane. Neither was observed, consistent with the free diffusion of lipoamide.

[0350] Under the same conditions (ionic strength, buffer composition, temperature, pH, etc.), the integrated NMR signal intensity is proportional to the concentration 73 Chemical exchange is expected since the amide proton of lipoamide is unstable in water. 74 To ensure that appropriate conditions were selected, the ELISA was performed in different solvents (chloroform-d, HO, culture medium, culture medium containing 3 mM KAsO2, and a 50:50 mixture of culture medium containing 3 mM KAsO2 and EDTA-trypsin). 15 N) 1 H NMR spectra were obtained (Figure 13B, C). Only in pure water and chloroform-d did both amide proton resonances have comparable signal intensities. In other solvents, the signal of the cis-amide proton was reduced, suggesting chemical exchange (Figure 13B). Therefore, the trans-amide proton was used to quantify the concentration. To measure the temperature and pH sensitivity of the trans-amide proton signal, the ( 15 N) 1 H spectra were acquired at different temperatures (Figure S13D) and pH (Figure S13E). Under high temperature and high pH conditions, both amide protons showed chemical exchange, with the trans-amide protons being less affected (Figure S13D, E). To investigate whether lipoamide decomposes over time, the trans-amide proton signal was observed at 37 °C and 10 °C for 10 h. The signal intensity decayed slowly at 37 °C, but not at 10 °C (Figure S13F), suggesting slow hydrolysis to produce ammonia. Therefore, at 10 °C and below pH 8.6, the integrated signal from the trans-amide proton resonance was 15 It is a good indicator of N-lipoamide concentration.

[0351] The cellular uptake was measured by comparing the signal intensity S of trans-amide protons of lipoamide obtained in the absence (-cells, sample i, Figure 2A) and presence (+cells, sample ii, Figure 2A) of HeLa cells. The measured fractional uptake U was obtained by the following equation: JPEG0007654540000003.jpg20145

[0352] The amount (mol) of added lipoamide (add) is distributed between the intracellular (cell) and extracellular (out) environments after uptake, which can be expressed as a concentration c and a volume V. JPEG0007654540000004.jpg13145

[0353] The total volume of the cell is V cell =V1N cell where V1 is the volume of a single cell, N cell is the number of cells. V cell < <V add Therefore, V add = V0. Fractional uptake can also be written in terms of these concentrations and volumes. JPEG0007654540000005.jpg20145

[0354] Rearranged, the amount of lipoamide added and the measured fractional uptake U give an equation that describes the intracellular and extracellular concentrations: JPEG0007654540000006.jpg41145

[0355] HeLa cells were placed in a radius of 10 -5 mV1 = 4.19 x 10 -15 m 3 In our experiments, we approximate the spheres of N cell =10 6 , c add = 100 μM, and V add = 600 μL.

[0356] Although no evidence of peak broadening associated with dissolution into phospholipid membranes was observed, in principle the decrease in signal intensity upon uptake could be due to uptake into membranes rather than the cytoplasm.

[0357] Calculations show that this is unlikely. The number of phospholipid molecules in the cell membrane is proportional to the footprint A of each lipid molecule. L =0.5 nm 2 It can be estimated from 75 Assuming a spherical cell, the surface area of ​​one cell is A1 = 1.3 × 10 -9 m 2 Therefore, the total number of phospholipids on the molecule taken up by the cell, N uptake =c cell V cell N A (N A is the Avogadro constant). The ratio of lipoamide to lipid molecules, R, is given by the following equation: JPEG0007654540000007.jpg20145

[0358] For the experimentally observed average value of U = 0.35 (i.e., 35% uptake), one would expect R = 4.9, i.e., 4.9 lipoamide molecules per plasma membrane lipid molecule. Even though the plasma membrane is not the only membrane in the cell, if it represented 10% of the total phospholipids, roughly one lipoamide molecule would be required for every two phospholipid molecules.< / nmr>

Claims

1. 1. A method for identifying a compound that modulates one or more condensate-associated characteristics, including a condensate-associated molecule, comprising: (a) contacting a cellular composition that includes one or more condensates or that is capable of forming one or more condensates with the compound; and (b) determining a characteristic associated with the one or more condensates; modulation of said characteristic indicates that said compound modulates a characteristic associated with said one or more condensates relative to a reference; determining the characteristic associated with the one or more condensates is based on (i) a number of condensates that contain the condensate-associated molecule, (ii) a size of the one or more condensates, and (iii) a distribution of the one or more condensates. method.

2. 2. The method of claim 1 , wherein determining the characteristic associated with the one or more condensates is based on any one or more of: (i) the location of the one or more condensates; (ii) the surface area of ​​the one or more condensates; (iii) the composition of the one or more condensates; (iv) the fluidity of the one or more condensates; (v) the solidification of the one or more condensates; (vi) the dissolution of the one or more condensates; (vii) the presence and / or amount of fibril formation; (viii) the location of the condensate-associated molecules; (ix) the partitioning of the condensate-associated molecules into the one or more condensates; and (x) the aggregation of the condensate-associated molecules.

3. The method of claim 1 or 2, wherein the one or more condensates are present within one or more cells in the cellular composition.

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

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

6. The condensate forming conditions include (i) an oxidative stressor, (ii) a mitochondrial electron transport chain inhibitor, (iii) a thermal stressor, (iv) an osmotic stressor, The method according to claim 4 or 5, characterized in that the stressor is any one or more of: (v) a hyperosmotic stressor; and (vi) inhibition of glycolysis.

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

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

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

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

11. The method according to any one of claims 1 to 10, wherein the cells in the cell composition express a condensate-associated molecule.

12. The method according to any one of claims 1 to 11, characterized in that the cell composition comprises HeLA cells, iPSC cells or iPSC MN cells.

13. The method of any one of claims 1 to 12, further comprising imaging at least a portion of the cellular composition.

14. The method of any one of claims 1 to 13, further comprising contacting at least a portion of the cell composition with a fixative.

15. The method of any one of claims 1 to 14, further comprising contacting at least a portion of the cellular composition with a staining agent.

16. The method of any one of claims 1 to 15, further comprising subjecting at least a portion of said cellular composition to DNA damaging conditions.

17. 17. The method of claim 16, wherein the DNA damaging condition is laser irradiation.

18. 18. The method of any one of claims 1 to 17, further comprising evaluating the identified compound using at least one of a motor neuron degeneration and regression assay, a motor neuron trafficking assay, a filter binding assay, a fibrillation assay, a viability assay, a toxicity assay, or an animal model motor function deficit assay.

19. A method for identifying a compound useful for treating amyotrophic lateral sclerosis (ALS), comprising: identifying a compound according to any one of the methods of claims 1 to 18, wherein the cell composition comprises an ALS model; method.

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