Methods of screening for condensate-associated specificity and uses thereof

Abstract

To provide methods of screening condensate-associated specificity, and uses thereof.SOLUTION: Methods of identifying a compound, such as a test compound, and applications thereof are provided. For example, methods of identifying a compound that preferentially affects, increases, or decreases a level of association of a macromolecule with one or more target condensates or methods of identifying a compound that preferentially causes a macromolecule to associate or disassociate with one or more target condensates are provided. Additionally, methods of designing and / or identifying and / or making a compound, or portion thereof, with a desired characteristic are provided.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 902,316, filed September 18, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to the field of biological condensates. [Background technology]

[0003] Cells contain membrane-bound organelles, such as mitochondria, lysosomes, and the endoplasmic reticulum, which, in part, localize various cellular functions. In addition to membrane-bound organelles, cells contain distinct membrane-free subcompartments between themselves and their immediate surrounding solution. Many of these membrane-free molecular assemblies have been shown to form through a process called liquid-liquid phase separation or condensation, in a manner similar to the partitioning of oil droplets in water. During this process, for example, a solution containing biological macromolecules separates into distinct phases: a condensed high-density phase in which at least some of the biological macromolecules are concentrated, and a surrounding low-density phase. Several cellular condensates have been recognized that play important roles in biology (Banani et al., 2017, Nat Rev Mol Cell Biol,18:285-298).

[0004] Various condensates are known to be important for regulating specific cellular processes in different cell types. Mechanistically, for example, condensates can bring together high concentrations of molecules to promote reactions within the condensate or sequester molecules in the condensate, thereby reducing their concentration in the surrounding medium and preventing their activity. Abnormal condensate function has also been implicated in various human diseases, such as neurodegenerative, proliferative, immune, cardiac, or metabolic disorders (Naumann et al., 2018, Nat Commun, 9(1):335; Wegmann et al., 2018, EMBO J, 37(7):e98049; and Aguzzi et al., 2016, 26(7):547-558). However, there is a lack of understanding of the mechanisms governing the fractionation of single macromolecules into or out of condensates, and little or nothing is known about whether compounds can selectively alter the fractionation of single macromolecules into or out of condensates. All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Banani et al.,2017,Nat Rev Mol Cell Biol,18:285-298 [Non-patent document 2] Naumann et al.,2018,Nat Commun,9(1):335 [Non-patent document 3] Wegmann et al.,2018,EMBO J,37(7):e98049 [Non-patent document 4] Aguzzi et al.,2016,26(7):547-558 Summary of the Invention

[0006] Provided herein are methods for identifying a compound that preferentially affects the level of association between a first macromolecule and one or more target condensates, the method comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; and (b) determining the level of association between the first macromolecule and one or more target condensates and the level of association between at least one additional macromolecule and one or more target condensates, wherein a compound preferentially affects the level of association between the first macromolecule and one or more target condensates if the compound changes the level of each additional macromolecule compared to the first reference level more than the level of each additional macromolecule compared to the reference level of each additional macromolecule. In some embodiments, the compound does not measurably change the level of each additional macromolecule compared to the reference level of each additional macromolecule.

[0007] Also provided herein are methods for identifying a compound that preferentially increases the level of association between a first macromolecule and one or more target condensates, the method comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; and (b) determining the level of association between the first macromolecule and one or more target condensates and at least one additional macromolecule and one or more target condensates, wherein a compound preferentially increases the level of association between the first macromolecule and one or more target condensates if the compound increases the level of each additional macromolecule compared to the first reference level more than the level of each additional macromolecule compared to the reference level of each additional macromolecule. In some embodiments, the compound does not measurably increase the level of each additional macromolecule compared to the reference level of each additional macromolecule.

[0008] Also provided herein are methods for identifying a compound that preferentially reduces the level of association between a first macromolecule and one or more target condensates, the methods comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; and (b) determining the level of association between the first macromolecule and one or more target condensates and at least one additional macromolecule and one or more target condensates, wherein a compound preferentially reduces the level of association between the first macromolecule and one or more target condensates if the compound alters the level of the first macromolecule compared to the first reference level more than the compound reduces the level of each additional macromolecule compared to a reference level for each additional macromolecule. In some embodiments, the compound does not measurably reduce the level of each additional macromolecule compared to the reference level for each additional macromolecule.

[0009] In some embodiments, the first reference level is the level of association of the first macromolecule with one or more reference condensates determined in the absence of the compound. In some embodiments, the reference level of each additional macromolecule is the level of association of each additional macromolecule with one or more reference condensates determined in the absence of the compound.

[0010] Also provided herein is a method for identifying a compound that causes a first macromolecule to preferentially associate with one or more target condensates, the method comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; and (b) determining whether the first macromolecule and at least one additional macromolecule associate with the one or more target condensates, wherein (1) the compound causes the first macromolecule to associate with the one or more target condensates, (2) the compound does not cause each additional macromolecule to associate with the one or more target condensates, and (3) the first macromolecule would not associate with the one or more target condensates in the absence of the compound, then the compound causes the first macromolecule to preferentially associate with the one or more target condensates.

[0011] In some embodiments, the compound (4) causes the first macromolecule to preferentially associate with one or more target condensates when one or more of the at least one additional macromolecule would not associate with the one or more target condensates in the absence of the compound. In some embodiments, the compound (4) causes the first macromolecule to preferentially associate with one or more target condensates when each of the at least one additional macromolecule would not associate with the one or more target condensates in the absence of the compound.

[0012] Also provided herein is a method for identifying a compound that preferentially dissociates a first macromolecule from one or more target condensates, the method comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; and (b) determining whether the first macromolecule and at least one additional macromolecule associate with the one or more target condensates, wherein: (1) the compound causes the first macromolecule not to associate with the one or more target condensates; (2) the compound does not cause each additional macromolecule not to associate with the one or more target condensates; and (3) if the first macromolecule would associate with the one or more target condensates in the absence of the compound, then the compound preferentially dissociates the first macromolecule from the one or more target condensates.

[0013] In some embodiments, the compound (4) causes the first macromolecule to preferentially associate with one or more target condensates when one or more of the at least one additional macromolecule would associate with one or more target condensates in the absence of the compound. In some embodiments, the compound (4) causes the first macromolecule to preferentially associate with one or more target condensates when each of the at least one additional macromolecule would associate with one or more target condensates in the absence of the compound.

[0014] In some embodiments, step (a) comprises contacting a cellular composition with a compound, wherein the cellular composition comprises one or more target condensates, and the method further comprises forming the one or more target condensates prior to step (a). In some embodiments, step (a) comprises contacting a cellular composition with a compound, wherein the one or more target condensates are formed after contacting the cellular composition with the compound, and the method further comprises forming the one or more target condensates.

[0015] In some embodiments, the at least one additional macromolecule is 2 or more, 3 or more, 4 or more, or 5 or more macromolecules, hi some embodiments, the at least one additional macromolecule is 1 to 10 macromolecules.

[0016] In some embodiments, the first macromolecule is abnormally expressed in a disease state. In some embodiments, the association level of the first macromolecule with one or more target condensates in a disease state is altered compared to the association level of the first macromolecule with one or more target condensates in a normal state. In some embodiments, one or more of the at least one additional macromolecule is abnormally expressed in a disease state.

[0017] In some embodiments, the first macromolecule is DNA or RNA.In some embodiments, the first macromolecule is protein.In some embodiments, the first macromolecule comprises a mutation, and changes the association level of the first macromolecule with one or more target condensates compared with a related protein that does not comprise the mutation.In some embodiments, the first macromolecule is FUS or eIF3.

[0018] In some embodiments, one or more of the at least one additional macromolecule is DNA or RNA. In some embodiments, one or more of the at least one additional macromolecule is a protein. In some embodiments, one or more of the at least one additional macromolecule contains a mutation that alters the corresponding level of association with one or more target condensates compared to a related protein that does not contain the mutation. In some embodiments, one or more of the at least one additional macromolecule is FUS, eIF3, G3BP1, FUS and G3BP1, or eIF3 and G3BP1.

[0019] In some embodiments, one or more of the first macromolecule and / or the at least one additional macromolecule is a fusion protein. In some embodiments, one or more of the first macromolecule and / or the at least one additional macromolecule comprises a label. In some embodiments, the method further comprises labeling one or more of the first macromolecule and / or the at least one additional macromolecule. In some embodiments, labeling comprises contacting the cell composition with an antibody or antigen-binding fragment thereof comprising a label. In some embodiments, the label is a radioactive label, a colorimetric label, or a fluorescent label.

[0020] In some embodiments, the cell composition comprises a microorganism or an animal cell. In some embodiments, the cell composition comprises an animal cell. In some embodiments, the animal cell has one or more characteristics of a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease. In some embodiments, the animal cell is a HeLa cell, a HEK293 cell, an induced pluripotent stem cell (iPSC cell), a cardiomyocyte, a muscle cell, a stem cell-derived cell, a neuron, a cancer cell, an immune cell, or an adipocyte.

[0021] In some embodiments, the one or more target condensates are cellular condensates. In some embodiments, the one or more target condensates are nuclear condensates or cytoplasmic condensates. In some embodiments, the cellular condensate is a cleavage body, a p granule, a histone locus body, a multivesicular body, a neuronal RNA granule, a nuclear gem, a nuclear pore, a nuclear speckle, a nuclear stress body, a nucleolus, an Oct1 / PTF / transcription (OPT) domain, a paraspeckle, a juxtanucleolar compartment, a PML nuclear body, a PML oncogenic domain, a polycomb body, a processing body, a Sam68 nuclear structure, a stress granule, or a splicing speckle.

[0022] In some embodiments, the one or more target condensates are a single target condensate, and in some embodiments, the compound does not measurably alter one or more of the size of the target condensate, the location of the target condensate, the surface area of ​​the target condensate, and the dissolution of the target condensate.

[0023] In some embodiments, the one or more target condensates are a plurality of target condensates, and in some embodiments, the compound does not measurably alter one or more of the total number of the plurality of target condensates, the size of the plurality of target condensates, the location of the plurality of target condensates, the surface area of ​​the plurality of target condensates, and the dissolution of the plurality of target condensates.

[0024] In some embodiments, the one or more target condensates are a plurality of target condensates. In some embodiments, the plurality of target condensates is all or a subset of a class of condensates in a portion of a cellular composition. In some embodiments, the plurality of target condensates is all or a subset of a class of condensates in cells in the cellular composition. In some embodiments, the plurality of target condensates is all or a subset of a class of condensates in a portion of cells in the cellular composition. In some embodiments, the portion of a cell is a cytoplasm, a nucleus, or an organelle.

[0025] In some embodiments, the class of condensates includes condensates that comprise specific macromolecules. In some embodiments, the class of condensates includes condensates that are cleavage bodies, the class of condensates includes condensates that are p-granules, the class of condensates includes condensates that are histone locus bodies, the class of condensates includes condensates that are multivesicular bodies, the class of condensates includes condensates that are neuronal RNA granules, the class of condensates includes condensates that are nuclear gems, the class of condensates includes condensates that are nuclear pores, the class of condensates includes condensates that are nuclear speckles, the class of condensates includes condensates that are nuclear stress bodies, the class of condensates includes condensates that are nucleoli, the class of condensates includes Oct1 / PTF / transcription ( The class of condensates includes condensates that are OPT) domains, the class of condensates includes condensates that are paraspeckles, the class of condensates includes condensates that are juxtanucleolar compartments, the class of condensates includes condensates that are PML subnuclear structures, the class of condensates includes condensates that are PML oncogenic domains, the class of condensates includes condensates that are Polycomb bodies, the class of condensates includes condensates that are processing bodies, the class of condensates includes condensates that are Sam68 subnuclear structures, the class of condensates includes condensates that are stress granules, or the class of condensates includes condensates that are splicing speckles.

[0026] Also provided herein is a method for identifying a plurality of compounds that preferentially affect, decrease, or increase the level of association of a first macromolecule with one or more target condensates, or a plurality of compounds that preferentially associate or dissociate a first macromolecule with one or more target condensates, the method comprising performing the method for identifying a compound disclosed herein with a plurality of compounds.

[0027] In some embodiments, the method further includes identifying a feature that a subset or all of the identified compounds have in common in addition to the ability to preferentially affect, decrease, or increase the level of association of a first macromolecule with one or more target condensates, or to preferentially associate or dissociate a first macromolecule with one or more target condensates. In some embodiments, the method further includes performing the compound identification method disclosed herein on one or more additional test compounds that include the identified feature. In some embodiments, the method further includes performing the compound identification method disclosed herein on one or more additional test compounds that do not include the identified feature.

[0028] Also provided herein is a method for identifying a compound characteristic associated with preferentially affecting, decreasing, or increasing the level of association of a first macromolecule with one or more target condensates, or preferentially associating or dissociating a first macromolecule with one or more target condensates, the method comprising: (a) performing a method for identifying a plurality of compounds disclosed herein; and (b) identifying a characteristic that a subset or all of the identified compounds have in common in addition to their ability to preferentially affect, decrease, or increase the level of association of a first macromolecule with one or more target condensates, or their ability to preferentially associate or dissociate a first macromolecule with one or more target condensates.

[0029] Also provided herein is a method for designing a compound that preferentially affects, reduces, or increases the level of association between a first macromolecule and one or more target condensates, or that preferentially associates or dissociates a first macromolecule with one or more target condensates, the method comprising: (a) performing the method for identifying a plurality of compounds disclosed herein; (b) identifying characteristics that a subset or all of the identified compounds have in common in addition to their ability to preferentially affect, reduce, or increase the level of association between a first macromolecule and one or more target condensates, or their ability to preferentially associate or dissociate a first macromolecule with one or more target condensates; and (c) designing a compound that comprises the identified characteristics, thereby designing a compound that preferentially affects, reduces, or increases the level of association between the first macromolecule and one or more target condensates, or that preferentially associates or dissociates a first macromolecule with one or more target condensates.

[0030] Also provided herein are methods for identifying a compound useful for treating a disease in an individual in need thereof, the method comprising: performing a method for identifying a compound disclosed herein, wherein one or more target condensates are associated with the disease; and identifying a compound that preferentially affects, decreases, or increases the level of association between a first macromolecule and one or more target condensates, or that preferentially associates or dissociates a first macromolecule with one or more target condensates, that is useful for treating the disease. In some embodiments, the disease is a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease.

[0031] Also provided herein is a method for identifying a compound that preferentially affects the level of association between a first macromolecule and a first set of one or more target condensates, the method comprising: performing a method for identifying a compound disclosed herein with a first set of one or more target condensates; and performing a method for identifying a compound disclosed herein with a second set of one or more target condensates, wherein a compound preferentially affects the level of association between a first macromolecule and the first set of one or more target condensates if the compound preferentially affects the level of association between the first macromolecule and the first set of one or more target condensates more than the compound preferentially affects the level of association between the first macromolecule and the second set of one or more target condensates. In some embodiments, the compound does not affect the level of association between the first macromolecule and the second set of one or more target condensates.

[0032] Also provided herein is a method for identifying a compound that preferentially increases the level of association between a first macromolecule and a first set of one or more target condensates, the method comprising: performing a method for identifying a compound disclosed herein with the first set of one or more target condensates; and performing a method for identifying a compound disclosed herein with the second set of one or more target condensates, wherein the compound preferentially increases the level of association between the first macromolecule and the first set of one or more target condensates if the compound preferentially increases the level of association between the first macromolecule and the first set of one or more target condensates more than the compound preferentially increases the level of association between the first macromolecule and the second set of one or more target condensates. In some embodiments, the compound does not increase the level of association between the first macromolecule and the second set of one or more target condensates.

[0033] Also provided herein are methods for identifying a compound that preferentially reduces the level of association between a first macromolecule and a first set of one or more target condensates, the methods comprising: performing a method for identifying a compound disclosed herein with the first set of one or more target condensates; and performing a method for identifying a compound disclosed herein with the second set of one or more target condensates, wherein the compound preferentially reduces the level of association between the first macromolecule and the first set of one or more target condensates if the compound preferentially reduces the level of association between the first macromolecule and the first set of one or more target condensates more than the compound preferentially reduces the level of association between the first macromolecule and the second set of one or more target condensates. In some embodiments, the compound does not reduce the level of association between the first macromolecule and the second set of one or more target condensates.

[0034] Also provided herein is a method for identifying a compound that causes a first macromolecule to preferentially associate with a first set of one or more target condensates, the method comprising: performing a method for identifying a compound disclosed herein with a first set of one or more target condensates; and performing a method for identifying a compound disclosed herein with a second set of one or more target condensates, wherein the compound causes the first macromolecule to preferentially associate with the first set of one or more target condensates if the compound causes the first macromolecule to preferentially associate with more than the second set of one or more target condensates.

[0035] Also provided herein is a method for identifying a compound that preferentially dissociates a first macromolecule from a first set of one or more target condensates, the method comprising: performing a method for identifying a compound disclosed herein with a first set of one or more target condensates; and performing a method for identifying a compound disclosed herein with a second set of one or more target condensates, wherein the compound preferentially dissociates the first macromolecule from the first set of one or more target condensates if the compound preferentially associates the first macromolecule with the first set of one or more target condensates more than the compound preferentially associates the first macromolecule with the second set of one or more target condensates.

[0036] In some embodiments, the first and / or second set of one or more target condensates are cellular condensates, hi some embodiments, the first and / or second set of one or more target condensates are nuclear condensates or cytoplasmic condensates.

[0037] In some embodiments, the first and / or second set of one or more target condensates are 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.

[0038] In some embodiments, the first and / or second set of one or more target condensates is a single target condensate.

[0039] In some embodiments, the first and / or second set of one or more target condensates is a plurality of target condensates. In some embodiments, the first and / or second set of one or more target condensates is all or a subset of a class of condensates in a portion of the cellular composition. In some embodiments, the first and / or second set of one or more target condensates is all or a subset of a class of condensates in cells in the cellular composition. In some embodiments, the first and / or second set of one or more target condensates is all or a subset of a class of condensates in a portion of cells in the cellular composition. In some embodiments, the portion of a cell is a cytoplasm, a nucleus, or an organelle.

[0040] In some embodiments, the class of condensates includes condensates that comprise specific macromolecules. In some embodiments, the class of condensates includes condensates that are cleavage bodies, the class of condensates includes condensates that are p-granules, the class of condensates includes condensates that are histone locus bodies, the class of condensates includes condensates that are multivesicular bodies, the class of condensates includes condensates that are neuronal RNA granules, the class of condensates includes condensates that are nuclear gems, the class of condensates includes condensates that are nuclear pores, the class of condensates includes condensates that are nuclear speckles, the class of condensates includes condensates that are nuclear stress bodies, the class of condensates includes condensates that are nucleoli, the class of condensates includes Oct1 / PTF / transcription ( The class of condensates includes condensates that are OPT) domains, the class of condensates includes condensates that are paraspeckles, the class of condensates includes condensates that are juxtanucleolar compartments, the class of condensates includes condensates that are PML subnuclear structures, the class of condensates includes condensates that are PML oncogenic domains, the class of condensates includes condensates that are Polycomb bodies, the class of condensates includes condensates that are processing bodies, the class of condensates includes condensates that are Sam68 subnuclear structures, the class of condensates includes condensates that are stress granules, or the class of condensates includes condensates that are splicing speckles.

[0041] In some embodiments, the class of the first set of one or more target condensates is the same as the class of the second set of one or more target condensates, hi some embodiments, the class of the first set of one or more target condensates is different from the class of the second set of one or more target condensates.

[0042] In some embodiments, the first set of one or more target condensates is in the same cellular composition as the second set of one or more target condensates. In some embodiments, the cellular composition comprises cells comprising the first set of condensates of the one or more target condensates and the second set of one or more target condensates. In some embodiments, the first set of condensates of the one or more target condensates is in a first cellular composition and the second set of one or more target condensates is in a second cellular composition.

[0043] In some embodiments, the first set of condensates of the one or more target condensates is located within cells in the cell composition, and the cells have one or more characteristics of a disease. In some embodiments, the disease is a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease.

[0044] Also provided herein is a method for identifying a plurality of compounds that preferentially affect, decrease, or increase the level of association between a first macromolecule and a first set of one or more target condensates, or a plurality of compounds that preferentially associate or dissociate a first macromolecule with a first set of one or more target condensates, the method comprising performing the method for identifying a compound disclosed herein with a plurality of compounds.

[0045] In some embodiments, the method further includes identifying characteristics that a subset or all of the identified compounds have in common in addition to their ability to preferentially affect, decrease, or increase the level of association of a first macromolecule with a first set of one or more target condensates, or their ability to preferentially associate or dissociate a first macromolecule with a first set of one or more target condensates.

[0046] In some embodiments, the method further comprises performing a compound identification method disclosed herein on one or more additional test compounds that include the identified characteristic. In some embodiments, the method further comprises performing a compound identification method disclosed herein on one or more additional test compounds that do not include the identified characteristic.

[0047] Also provided herein is a method for identifying a compound characteristic associated with preferentially affecting, decreasing, or increasing the level of association of a first macromolecule with a first set of one or more target condensates, or causing a first macromolecule to preferentially associate or dissociate with a first set of one or more target condensates, the method comprising: (a) performing a method for identifying a plurality of compounds disclosed herein; and (b) identifying a characteristic that a subset or all of the identified compounds have in common in addition to their ability to preferentially affect, decrease, or increase the level of association of a first macromolecule with a first set of one or more target condensates, or their ability to associate or dissociate a first macromolecule with a first set of one or more target condensates.

[0048] Also provided herein is a method for designing a compound that preferentially affects, reduces, or increases the level of association between a first macromolecule and a first set of one or more target condensates, or that preferentially associates or dissociates a first macromolecule with the first set of one or more target condensates, the method comprising: (a) performing the method for identifying a plurality of compounds disclosed herein; (b) identifying characteristics that a subset or all of the identified compounds have in common in addition to their ability to preferentially affect, reduce, or increase the level of association between a first macromolecule and the first set of one or more target condensates, or their ability to preferentially associate or dissociate a first macromolecule with the first set of one or more target condensates; and (c) designing a compound that comprises the identified characteristics, thereby designing a compound that preferentially affects, reduces, or increases the level of association between a first macromolecule and the first set of one or more target condensates, or that preferentially associates or dissociates a first macromolecule with the first set of one or more target condensates.

[0049] Also provided herein are methods for identifying a compound useful for treating a disease in an individual in need thereof, the method comprising: performing a method for identifying a compound disclosed herein, wherein a first set of one or more target condensates is associated with the disease; and identifying a compound that preferentially affects, decreases, or increases the level of association between a first macromolecule and the first set of one or more target condensates, or that preferentially associates or dissociates a first macromolecule with the first set of one or more target condensates, that is useful for treating the disease. In some embodiments, the disease is a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease. In certain embodiments, for example, the following are provided: (Item 1) 1. A method for identifying a compound that preferentially affects the level of association between a first macromolecule and one or more target condensates, comprising: (a) contacting a cell composition with a compound, (i) the cellular composition comprises the one or more target condensates; and / or (ii) contacting the cellular composition with the compound, wherein simultaneously with and / or after contacting, the one or more target condensates are formed; (b) determining the level of association between the first macromolecule and the one or more target condensates, and the level of association between at least one additional macromolecule and the one or more target condensates; The method, wherein the compound preferentially affects the level of association between the first macromolecule and the one or more target condensates if the compound alters the level of the first macromolecule compared to a first reference level more than the compound alters the level of each additional macromolecule compared to a reference level of each additional macromolecule. (Item 2) 2. The method of claim 1, wherein the compound does not measurably alter the level of each additional macromolecule compared to the reference level of each additional macromolecule. (Item 3) 1. A method for identifying a compound that preferentially increases the level of association between a first macromolecule and one or more target condensates, comprising: (a) contacting a cell composition with a compound, (i) the cellular composition comprises the one or more target condensates; and / or (ii) contacting the cellular composition with the compound, wherein simultaneously with and / or after contacting, the one or more target condensates are formed; (b) determining the level of association between the first macromolecule and the one or more target condensates, and the level of association between at least one additional macromolecule and the one or more target condensates; The method, wherein the compound preferentially increases the level of association between the first macromolecule and the one or more target condensates if the compound increases the level of the first macromolecule compared to a first reference level more than the compound increases the level of each additional macromolecule compared to a reference level of each additional macromolecule. (Item 4) 4. The method of claim 3, wherein the compound does not measurably increase the level of each additional macromolecule compared to the reference level of each additional macromolecule. (Item 5) 1. A method for identifying a compound that preferentially reduces the level of association between a first macromolecule and one or more target condensates, comprising: (a) contacting a cell composition with a compound, (i) the cellular composition comprises the one or more target condensates; and / or (ii) contacting the cellular composition with the compound, wherein simultaneously with and / or after contacting, the one or more target condensates are formed; (b) determining the level of association between the first macromolecule and the one or more target condensates, and the level of association between at least one additional macromolecule and the one or more target condensates; The method, wherein the compound preferentially decreases the level of association between the first macromolecule and the one or more target condensates if the compound alters the level of the first macromolecule compared to a first reference level more than the compound decreases the level of each additional macromolecule compared to a reference level of each additional macromolecule. (Item 6) 6. The method of claim 5, wherein the compound does not measurably decrease the level of each additional macromolecule compared to the reference level of each additional macromolecule. (Item 7) 7. The method of any one of items 1 to 6, wherein the first reference level is an association level of the first macromolecule with one or more reference condensates determined in the absence of the compound. (Item 8) 8. The method of any one of items 1 to 7, wherein the reference level of each additional macromolecule is the association level of each additional macromolecule with one or more reference condensates determined in the absence of the compound. (Item 9) 1. A method for identifying a compound that causes a first macromolecule to preferentially associate with one or more target condensates, comprising: (a) contacting a cell composition with a compound, (i) the cellular composition comprises the one or more target condensates; and / or (ii) contacting the cellular composition with the compound, wherein simultaneously with and / or after contacting, the one or more target condensates are formed; (b) determining whether the first macromolecule and at least one additional macromolecule associate with the one or more target condensates; (1) the compound causes the first macromolecule to associate with the one or more target condensates; (2) the compound does not cause each additional macromolecule to associate with the one or more target condensates; and (3) The method, wherein the compound causes the first macromolecule to preferentially associate with the one or more target condensates if the first macromolecule would not associate with the one or more target condensates in the absence of the compound. (Item 10) (4) The method of item 9, wherein the compound causes the first macromolecule to preferentially associate with the one or more target condensates when one or more of the at least one additional macromolecule would not associate with the one or more target condensates in the absence of the compound. (Item 11) (4) The method of item 9, wherein the compound causes the first macromolecule to preferentially associate with the one or more target condensates when each of the at least one additional macromolecule would not associate with the one or more target condensates in the absence of the compound. (Item 12) 1. A method for identifying a compound that preferentially dissociates a first macromolecule from one or more target condensates, comprising: (a) contacting a cell composition with a compound, (i) the cellular composition comprises the one or more target condensates; and / or (ii) contacting the cellular composition with the compound, wherein simultaneously with and / or after contacting, the one or more target condensates are formed; (b) determining whether the first macromolecule and at least one additional macromolecule associate with the one or more target condensates; (1) the compound prevents the first macromolecule from associating with the one or more target condensates; Let it happen, (2) the compound does not cause each additional macromolecule to not associate with the one or more target condensates; and (3) The method, wherein the compound preferentially dissociates the first macromolecule from the one or more target condensates when the first macromolecule would associate with the one or more target condensates in the absence of the compound. (Item 13) (4) The method of item 12, wherein the compound causes the first macromolecule to preferentially associate with the one or more target condensates when one or more of the at least one additional macromolecule would associate with the one or more target condensates in the absence of the compound. (Item 14) (4) The method of item 12, wherein the compound causes the first macromolecule to preferentially associate with the one or more target condensates when each of the at least one additional macromolecule would associate with the one or more target condensates in the absence of the compound. (Item 15) 15. The method of any one of items 1 to 14, wherein step (a) comprises contacting a cellular composition with a compound, wherein the cellular composition comprises the one or more target condensates, and wherein the method further comprises, prior to step (a), allowing the one or more target condensates to form. (Item 16) 15. The method of any one of items 1 to 14, wherein step (a) comprises contacting a cellular composition with a compound, wherein the one or more target condensates are formed after contacting the cellular composition with the compound, and wherein the method further comprises forming the one or more target condensates. (Item 17) 17. The method of any one of items 1 to 16, wherein the at least one additional macromolecule is two or more, three or more, four or more, or five or more macromolecules. (Item 18) 17. The method of any one of items 1 to 16, wherein the at least one additional macromolecule is 1 to 10 macromolecules. (Item 19) 19. The method of any one of items 1 to 18, wherein the first macromolecule is aberrantly expressed in a disease state. (Item 20) 20. The method of any one of items 1 to 19, wherein the level of association between the first macromolecule and the one or more target condensates in a disease state is altered compared to the level of association between the first macromolecule and the one or more target condensates in a normal state. (Item 21) 21. The method of any one of items 1 to 20, wherein one or more of the at least one additional macromolecule is aberrantly expressed in a disease state. (Item 22) 22. The method according to any one of items 1 to 21, wherein the first macromolecule is DNA or RNA. (Item 23) 23. The method of any one of items 1 to 22, wherein one or more of the at least one additional macromolecule is DNA or RNA. (Item 24) 24. The method of any one of items 1 to 21 or 23, wherein the first macromolecule is a protein. (Item 25) the first macromolecule comprises a mutation, compared to a related protein that does not contain the mutation, 25. The method of claim 24, wherein the level of association between the first macromolecule and the one or more target condensates is altered. (Item 26) 26. The method of item 24 or 25, wherein the first macromolecule is FUS or eIF3. (Item 27) 27. The method of any one of items 1 to 26, wherein one or more of the at least one additional macromolecule is a protein. (Item 28) 28. The method of any one of items 1 to 27, wherein one or more of the at least one additional macromolecule comprises a mutation that alters the corresponding level of association with the one or more target condensates compared to a related protein that does not contain the mutation. (Item 29) 29. The method of any one of items 1 to 28, wherein one or more of the at least one additional macromolecule is FUS, eIF3, G3BP1, FUS and G3BP1, or eIF3 and G3BP1. (Item 30) 30. The method of any one of items 24 to 29, wherein one or more of the first macromolecule and / or the at least one additional macromolecule is a fusion protein. (Item 31) 31. The method of any one of items 1 to 30, wherein one or more of the first macromolecule and / or the at least one additional macromolecule comprises a label. (Item 32) 32. The method of any one of items 1 to 31, further comprising labeling one or more of the first macromolecule and / or the at least one additional macromolecule. (Item 33) 33. The method of claim 32, wherein the labeling comprises contacting the cell composition with an antibody or antigen-binding fragment thereof comprising a label. (Item 34) 34. The method according to any one of items 31 to 33, wherein the label is a radioactive label, a colorimetric label, or a fluorescent label. (Item 35) 35. The method according to any one of items 1 to 34, wherein the cell composition comprises microbial or animal cells. (Item 36) 36. The method of claim 35, wherein the cell composition comprises animal cells. (Item 37) 37. The method of claim 36, wherein the animal cells have one or more characteristics of a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease. (Item 38) 38. The method according to any one of items 35 to 37, wherein the animal cells are HeLa cells, HEK293 cells, induced pluripotent stem cells (iPSC cells), cardiomyocytes, muscle cells, stem cell-derived cells, neurons, cancer cells, immune cells, or adipocytes. (Item 39) 39. The method according to any one of items 1 to 38, wherein the one or more target condensates are cellular condensates. (Item 40) 40. The method according to any one of items 1 to 39, wherein the one or more target condensates are nuclear condensates or cytoplasmic condensates. (Item 41) 40. The method of claim 39, wherein the cellular condensate is a cleavage body, a p-granule, a histone locus body, a multivesicular body, a neuronal RNA granule, a nuclear gem, a nuclear pore, a nuclear speckle, a nuclear stress body, a nucleolus, an Oct1 / PTF / transcription (OPT) domain, a paraspeckle, a juxtanucleolar compartment, a PML nuclear structure, a PML oncogenic domain, a Polycomb body, a processing body, a Sam68 nuclear structure, a stress granule, or a splicing speckle. (Item 42) 42. The method according to any one of items 1 to 41, wherein the one or more target condensates is a single target condensate. (Item 43) Item 43. The method of item 42, wherein the compound does not measurably alter one or more of the size of the target condensate, the location of the target condensate, the surface area of ​​the target condensate, and the dissolution of the target condensate. (Item 44) 42. The method according to any one of items 1 to 41, wherein the one or more target condensates are a plurality of target condensates. (Item 45) 45. The method of claim 44, wherein the plurality of target condensates is all or a subset of a class of condensates in the portion of the cellular composition. (Item 46) 46. ​​The method of claim 44 or 45, wherein the plurality of target condensates is all or a subset of a class of condensates in cells in the cellular composition. (Item 47) 47. The method of any one of items 44 to 46, wherein the plurality of target condensates is all or a subset of a class of condensates in a portion of cells in the cellular composition. (Item 48) 48. The method of claim 47, wherein the portion of the cell is a cytoplasm, a nucleus, or an organelle. (Item 49) 49. The method according to any one of items 45 to 48, wherein the class of condensates comprises condensates containing specific macromolecules. (Item 50) The class of condensates includes condensates that are cleavage bodies, the class of condensates includes condensates that are p-granules, the class of condensates includes condensates that are histone locus bodies, the class of condensates includes condensates that are multivesicular bodies, the class of condensates includes condensates that are neuronal RNA granules, the class of condensates includes condensates that are nuclear gems, the class of condensates includes condensates that are nuclear pores, the class of condensates includes condensates that are nuclear speckles, the class of condensates includes condensates that are nuclear stress bodies, the class of condensates includes condensates that are nucleoli, and the class of condensates includes condensates that are Oct1 / PTF / transcription (OPT) domains. 50. The method of any one of Items 45 to 49, wherein the class of condensates comprises condensates that are paraspeckles, the class of condensates comprises condensates that are juxtanucleolar compartments, the class of condensates comprises condensates that are PML nuclear structures, the class of condensates comprises condensates that are PML oncogenic domains, the class of condensates comprises condensates that are Polycomb bodies, the class of condensates comprises condensates that are processing bodies, the class of condensates comprises condensates that are Sam68 nuclear structures, the class of condensates comprises condensates that are stress granules, or the class of condensates comprises condensates that are splicing speckles. (Item 51) 51. The method of any one of items 44 to 50, wherein the compound does not measurably change one or more of the total number of the plurality of target condensates, the size of the plurality of target condensates, the location of the plurality of target condensates, the surface area of ​​the plurality of target condensates, and the dissolution of the plurality of target condensates. (Item 52) 52. A method for identifying a plurality of compounds that preferentially affect, decrease, or increase the level of association of a first macromolecule with one or more target condensates, or a plurality of compounds that preferentially associate or dissociate the first macromolecule with one or more target condensates, the method comprising carrying out the method of any one of items 1 to 51 using a plurality of compounds. (Item 53) 53. The method of claim 52, further comprising identifying a feature that a subset or all of the identified compounds have in common in addition to the ability to preferentially affect, decrease, or increase the level of association between the first macromolecule and the one or more target condensates, or the ability to preferentially associate or dissociate the first macromolecule with one or more target condensates. (Item 54) 54. The method of claim 53, further comprising performing the method of any one of items 1 to 51 on one or more additional test compounds comprising the identified characteristic. (Item 55) 55. The method of claim 53 or 54, further comprising carrying out the method of any one of items 1 to 51 for one or more additional test compounds that do not contain the identified characteristic. (Item 56) 1. A method for identifying a compound characteristic associated with preferentially affecting, decreasing, or increasing the level of association of a first macromolecule with one or more target condensates, or preferentially associating or dissociating said first macromolecule with one or more target condensates, comprising: (a) carrying out the method described in item 52; and identifying a feature that a subset or all of the identified compounds have in common in addition to their ability to preferentially affect, decrease, or increase the level of association between the first macromolecule and the one or more target condensates, or their ability to preferentially associate or dissociate the first macromolecule with one or more target condensates. (Item 57) 1. A method for designing a compound that preferentially affects, decreases, or increases the level of association of a first macromolecule with one or more target condensates, or causes the first macromolecule to preferentially associate with or dissociate from one or more target condensates, comprising: (a) carrying out the method described in item 52; (b) identifying a feature that a subset or all of the identified compounds have in common in addition to the ability to preferentially affect, decrease, or increase the level of association between the first macromolecule and one or more target condensates, or to preferentially associate or dissociate the first macromolecule with one or more target condensates; (c) designing a compound that contains the identified features; thereby designing a compound that preferentially affects, decreases, or increases the level of association between the first macromolecule and one or more target condensates, or that preferentially associates or dissociates the first macromolecule with one or more target condensates. (Item 58) 1. A method for identifying a compound useful for treating a disease in an individual in need thereof, comprising: 52. Carrying out the method according to any one of items 1 to 51, wherein the one or more targets and wherein the target condensates are associated with the disease. and identifying the compound that preferentially affects, decreases, or increases the level of association between the first macromolecule and the one or more target condensates, or the compound that preferentially associates or dissociates the first macromolecule with one or more target condensates, that is useful for treating the disease. (Item 59) 59. The method of claim 58, wherein the disease is a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease. (Item 60) 1. A method for identifying a compound that preferentially affects the level of association between a first macromolecule and a first set of one or more target condensates, comprising: performing the method according to any one of items 1 to 51 using the first set of one or more target condensates; performing the method of any one of items 1 to 51 with a second set of one or more target condensates, The method, wherein the compound preferentially affects the level of association between the first macromolecule and the first set of one or more target condensates if the compound preferentially affects the level of association between the first macromolecule and the first set of one or more target condensates more than the compound preferentially affects the level of association between the first macromolecule and the second set of one or more target condensates. (Item 61) 61. The method of claim 60, wherein the compound does not affect the level of association between the first macromolecule and the second set of one or more target condensates. (Item 62) 1. A method for identifying a compound that preferentially increases the level of association between a first macromolecule and a first set of one or more target condensates, comprising: performing the method according to any one of items 1 to 51 using the first set of one or more target condensates; performing the method of any one of items 1 to 51 with a second set of one or more target condensates, The method, wherein the compound preferentially increases the level of association between the first macromolecule and the first set of one or more target condensates if the compound preferentially increases the level of association between the first macromolecule and the first set of one or more target condensates more than the compound preferentially increases the level of association between the first macromolecule and the second set of one or more target condensates. (Item 63) 63. The method of claim 62, wherein the compound does not increase the level of association between the first macromolecule and the second set of one or more target condensates. (Item 64) 1. A method for identifying a compound that preferentially reduces the level of association between a first macromolecule and a first set of one or more target condensates, comprising: performing the method according to any one of items 1 to 51 using the first set of one or more target condensates; performing the method of any one of items 1 to 51 with a second set of one or more target condensates, If the compound preferentially reduces the level of association between the first macromolecule and the first set of one or more target condensates more than the compound preferentially reduces the level of association between the first macromolecule and the second set of one or more target condensates, the compound is said to reduce the level of association between the first macromolecule and the first set of one or more target condensates. The method, wherein the level is preferentially reduced. (Item 65) 65. The method of claim 64, wherein the compound does not decrease the level of association between the first macromolecule and the second set of one or more target condensates. (Item 66) 1. A method for identifying a compound that preferentially associates a first macromolecule with a first set of one or more target condensates, comprising: performing the method according to any one of items 1 to 51 using the first set of one or more target condensates; performing the method of any one of items 1 to 51 with a second set of one or more target condensates, The method, wherein the compound causes the first macromolecule to preferentially associate with the first set of one or more target condensates if the compound causes the first macromolecule to preferentially associate with the first set of one or more target condensates more than the compound causes the first macromolecule to preferentially associate with the second set of one or more target condensates. (Item 67) 1. A method for identifying a compound that preferentially dissociates a first macromolecule from a first set of one or more target condensates, comprising: performing the method according to any one of items 1 to 51 using the first set of one or more target condensates; performing the method of any one of items 1 to 51 with a second set of one or more target condensates, The method, wherein the compound preferentially dissociates the first macromolecule from the first set of one or more target condensates when the compound preferentially associates the first macromolecule with the first set of one or more target condensates more than the compound preferentially associates the first macromolecule with the second set of one or more target condensates. (Item 68) 68. The method of any one of items 60 to 67, wherein the first and / or second set of one or more target condensates are cellular condensates. (Item 69) 69. The method of any one of items 60 to 68, wherein the first and / or second set of one or more target condensates are nuclear condensates or cytoplasmic condensates. (Item 70) 69. The method of any one of items 60 to 68, wherein the first and / or second set of one or more target condensates is a cleavage body, a p-granule, a histone locus body, a multivesicular body, a neuronal RNA granule, a nuclear gem, a nuclear pore, a nuclear speckle, a nuclear stress body, a nucleolus, an Oct1 / PTF / transcription (OPT) domain, a paraspeckle, a juxtanucleolar compartment, a PML nuclear structure, a PML oncogenic domain, a Polycomb body, a processing body, a Sam68 nuclear structure, a stress granule, or a splicing speckle. (Item 71) 71. The method of any one of items 60 to 70, wherein the first and / or second set of one or more target condensates is a single target condensate. (Item 72) 71. The method of any one of items 60 to 70, wherein the first and / or second set of one or more target condensates is a plurality of target condensates. (Item 73) 73. The method of any one of items 60-70 or 72, wherein the first and / or second set of one or more target condensates is all or a subset of a class of condensates in the portion of the cellular composition. (Item 74) 74. The method of any one of items 60-70 or 72-73, wherein the first and / or second set of one or more target condensates is all or a subset of a class of condensates in cells in the cellular composition. (Item 75) 75. The method of any one of items 60-70 or 72-74, wherein the first and / or second set of one or more target condensates is all or a subset of a class of condensates in a portion of cells in the cellular composition. (Item 76) 76. The method of claim 75, wherein the portion of the cell is a cytoplasm, a nucleus, or an organelle. (Item 77) 77. The method of any one of items 73 to 76, wherein the class of condensates comprises condensates containing specific macromolecules. (Item 78) The class of condensates includes condensates that are cleavage bodies, the class of condensates includes condensates that are p-granules, the class of condensates includes condensates that are histone locus bodies, the class of condensates includes condensates that are multivesicular bodies, the class of condensates includes condensates that are neuronal RNA granules, the class of condensates includes condensates that are nuclear gems, the class of condensates includes condensates that are nuclear pores, the class of condensates includes condensates that are nuclear speckles, the class of condensates includes condensates that are nuclear stress bodies, the class of condensates includes condensates that are nucleoli, and the class of condensates includes condensates that are Oct1 / PTF / transcription (OPT) domains. 78. The method of any one of Items 73 to 77, wherein the class of condensates comprises condensates that are paraspeckles, the class of condensates comprises condensates that are juxtanucleolar compartments, the class of condensates comprises condensates that are PML nuclear structures, the class of condensates comprises condensates that are PML oncogenic domains, the class of condensates comprises condensates that are Polycomb bodies, the class of condensates comprises condensates that are processing bodies, the class of condensates comprises condensates that are Sam68 nuclear structures, the class of condensates comprises condensates that are stress granules, or the class of condensates comprises condensates that are splicing speckles. (Item 79) 79. The method of any one of items 73 to 78, wherein the class of the first set of one or more target condensates is the same as the class of the second set of one or more target condensates. (Item 80) 79. The method of any one of items 73 to 78, wherein the class of the first set of one or more target condensates is different from the class of the second set of one or more target condensates. (Item 81) 81. The method of any one of items 60 to 80, wherein the first set of one or more target condensates is in the same cell composition as the second set of one or more target condensates. (Item 82) 82. The method of any one of items 60 to 81, wherein the cell composition comprises cells comprising a first set of condensates of the one or more target condensates and a second set of the one or more target condensates. (Item 83) 81. The method of any one of items 60 to 80, wherein the first set of condensates of the one or more target condensates is in a first cellular composition and the second set of the one or more target condensates is in a second cellular composition. (Item 84) 84. The method of any one of items 60 to 83, wherein the first set of condensates of the one or more target condensates is in cells in the cell composition, and the cells have one or more characteristics of a disease. (Item 85) 85. The method of item 84, wherein the disease is a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease. (Item 86) 86. A method for identifying a plurality of compounds that preferentially affect, decrease, or increase the level of association of a first macromolecule with a first set of one or more target condensates, or a plurality of compounds that preferentially associate or dissociate the first macromolecule with the first set of one or more target condensates, the method comprising performing the method of any one of items 60 to 85 using a plurality of compounds. (Item 87) 87. The method of claim 86, further comprising identifying a feature that a subset or all of the identified compounds have in common in addition to the ability to preferentially affect, decrease, or increase the level of association of the first macromolecule with the first set of one or more target condensates, or the ability to preferentially associate or dissociate the first macromolecule with the first set of one or more target condensates. (Item 88) 88. The method of item 87, further comprising carrying out the method of any one of items 60 to 85 for one or more additional test compounds comprising the identified characteristic. (Item 89) 89. The method of claim 87 or 88, further comprising carrying out the method of any one of items 60 to 85 for one or more additional test compounds that do not contain the identified characteristic. (Item 90) 1. A method for identifying a compound characteristic associated with preferentially affecting, decreasing, or increasing the level of association of a first macromolecule with a first set of one or more target condensates, or causing the first macromolecule to preferentially associate with or dissociate from the first set of one or more target condensates, comprising: (a) carrying out the method according to item 86; (b) identifying a feature that a subset or all of the identified compounds have in common in addition to the ability to preferentially affect, decrease, or increase the level of association of the first macromolecule with the first set of one or more target condensates, or the ability to preferentially associate or dissociate the first macromolecule with the first set of one or more target condensates. (Item 91) 1. A method for designing a compound that preferentially affects, decreases, or increases the level of association of a first macromolecule with a first set of one or more target condensates, or causes said first macromolecule to preferentially associate with or dissociate from said first set of one or more target condensates, comprising: (a) carrying out the method according to item 86; (b) identifying a feature that a subset or all of the identified compounds have in common in addition to the ability to preferentially affect, decrease, or increase the level of association of the first macromolecule with the first set of one or more target condensates, or to preferentially associate or dissociate the first macromolecule with the first set of one or more target condensates; (c) designing a compound that contains the identified features; thereby designing a compound that preferentially affects, decreases, or increases the level of association between the first macromolecule and the first set of one or more target condensates, or a compound that preferentially associates or dissociates the first macromolecule with the first set of one or more target condensates. (Item 92) 1. A method for identifying a compound useful for treating a disease in an individual in need thereof, comprising: 86. Carrying out the method of any one of items 60 to 85, wherein the first set of one or more target condensates is associated with the disease; and identifying the compound that preferentially affects, decreases, or increases the level of association of the first macromolecule with the first set of one or more target condensates, or the compound that preferentially associates or dissociates the first macromolecule with the first set of one or more target condensates, which is useful for treating the disease. (Item 93) 93. The method of claim 92, wherein the disease is a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease. [Brief explanation of the drawings]

[0050] [Figure 1] Fluorescence micrographs showing the levels of FUS, eIF3, and G3BP1 in stressed cells with and without treatment with lipoamide. [Figure 2] Fluorescence micrographs showing the levels of FUS, eIF3, and G3BP1 in stressed cells with and without treatment with AZD4547, omipalisib, and TG101209. [Figure 3] Fluorescence micrographs showing the levels of FUS, eIF3, and G3BP1 in stressed cells with and without treatment with GW9508, brefeldin A, AZD3463, and ketanserin. [Figure 4]Fluorescence micrographs showing the levels of FUS, eIF3, and G3BP1 in stressed cells with and without treatment with AZD8931, YM155, and β-lapachone. [Figure 5] Fluorescence micrographs showing the levels of FUS, eIF3, and G3BP1 in stressed cells with and without treatment with lipoamide. [Figure 6] Fluorescence micrographs showing the levels of FUS, eIF3, and G3BP1 in stressed cells with and without treatment with P121, PF-04691502, and TG101209. [Figure 7] Fluorescence micrographs showing the levels of FUS, eIF3, and G3BP1 in stressed cells with and without treatment with coriphosphine O. [Figure 8] Fluorescence micrographs showing the levels of FUS, eIF3, and G3BP1 in stressed cells with and without treatment with YM155, β-lapachone, and bisindolylmaleimide IX. [Figure 9] Fluorescence micrographs showing the levels of FUS, eIF3, and G3BP1 in stressed cells with and without treatment with cediranib. [Figure 10] Fluorescence micrographs showing the levels of FUS, eIF3, and G3BP1 in stressed HeLa cells and stressed human iPSC cells harboring BAC FUS-GFP with and without treatment with maticinib. [Figure 11] FIG. 10 is a plot of dose-response curves of FUS, eIF3, and G3BP1 in stress granules of stressed HeLa cells carrying BAC FUS-GFP treated with YM155. DETAILED DESCRIPTION OF THE INVENTION

[0051] The formation and / or coagulation of abnormal condensates has been associated with some diseases, including neurodegenerative diseases such as ALS. Previously, drug screening for compounds useful in treating diseases associated with abnormal condensates focused on screening for drugs that prevent condensate formation and / or completely dissolve condensates. The disclosure of the present application is based, at least in part, on the inventors' unique insight and unexpected discovery that screens can be developed to identify compounds that selectively alter condensates (e.g., the composition of condensates) without disrupting the condensate as a whole or without disrupting specific other condensates. The methods disclosed herein enable techniques for screening and identifying compounds or portions thereof that have any one or more of the following specificities: selectivity for modulating the inclusion or exclusion of macromolecules in condensates (macromolecule specificity), selectivity for affecting one type of condensate but not another (condensate specificity), and selectivity for affecting one type of tissue but not another (tissue specificity). Identifying and developing compounds that selectively control the behavior of specific macromolecules with respect to condensates, and / or the condensates affected, and / or the tissue types affected, is a powerful strategy for therapeutic intervention to reverse disease phenotypes while minimizing target activity. For example, compounds that can selectively alter condensate composition, specific types of condensates, and / or condensates in specific tissue types may be useful for specifically inhibiting or activating biological pathways, depending on the precise needs associated with treating a disease.

[0052] Thus, in some aspects, provided herein are methods for identifying compounds with any one or more of the following specificities: macromolecule specificity, condensate specificity, and tissue specificity, the methods comprising determining the level of association of a first macromolecule with one or more target condensates, and comparing the association level to a reference level to identify compounds with one or more of those specificities. In some embodiments, the methods identify compounds with macromolecule specificity. In some embodiments, the methods identify compounds with condensate specificity. In some embodiments, the methods identify compounds with tissue (e.g., cell type) specificity. In some embodiments, the methods identify compounds with macromolecule specificity and condensate specificity. In some embodiments, the methods identify compounds with macromolecule specificity and tissue (e.g., cell type) specificity. In some embodiments, the methods identify compounds with condensate specificity and tissue (e.g., cell type) specificity. In some embodiments, the methods identify compounds with macromolecule specificity, condensate specificity, and tissue (or cell type) specificity.

[0053] In other aspects, provided herein are methods for identifying compounds that preferentially affect (e.g., preferentially increase or preferentially decrease) the level of association between a first macromolecule and one or more target condensates.

[0054] Also provided herein are methods for identifying compounds that cause a first macromolecule to preferentially associate with or dissociate from one or more target condensates.

[0055] As described herein, the methods of identifying compounds can be useful, for example, to identify, characterize, and develop compounds or portions thereof that can alter the behavior of macromolecules that have condensates, including for use in treating disease in individuals.

[0056] definition For purposes of interpreting this specification, the following definitions shall apply, and where appropriate, terms used in the singular shall also include the plural and vice versa. In the event that a definition set forth below conflicts with a document incorporated herein by reference, the set forth definition shall control.

[0057] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers 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. The term also includes post-translational modifications of the polypeptide (e.g., glycosylation, sialylation, acetylation, phosphorylation, etc.).

[0058] As used herein, a "cell composition" is a composition comprising at least one cell. Exemplary compositions include a tissue sample or cultured cells.

[0059] As used herein, "condensate" means a non-membrane-enclosed compartment formed by phase separation (including all stages of phase separation) of one or more proteins and / or other macromolecules.

[0060] The terms "comprising," "having," "containing," and "including," and other similar forms and their grammatical equivalents, as used herein, are intended to be equivalent in meaning and are intended to be open-ended in that the item(s) following any one of these words are not meant to be an exhaustive listing of such item(s) or to be limited to only the listed item(s). For example, an article "comprising" components A, B, and C can consist of components A, B, and C (i.e., contain only components A, B, and C), or it can contain not only components A, B, and C, but also one or more other components. Thus, "comprises" and its similar forms, and their grammatical equivalents, are intended and understood to include disclosure of "consisting essentially of" or "consisting of" embodiments.

[0061] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, and between any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0062] Reference herein to "about" a value or parameter includes (and describes) the variation that surrounds that value or parameter. For example, a description that references "about X" includes a description of "X."

[0063] 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.

[0064] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0065] Methods for identifying compounds Provided herein are methods for identifying compounds with any one or more of the following specificities: macromolecule specificity, condensate specificity, and tissue specificity.

[0066] In some embodiments, the identification method includes: (a) contacting a composition with a compound, wherein (i) the composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the composition with the compound; (b) determining the level of association between a first macromolecule and one or more target condensates; and (c) comparing the level of association between the first macromolecule and one or more target condensates to a reference level to identify a compound with one or more of the following specificities: macromolecule specificity, condensate specificity, and tissue specificity. In some embodiments, the composition is a cellular composition (e.g., a cell culture). In some embodiments, the one or more target condensates are present in a particular type of tissue (e.g., a cell type).

[0067] In some embodiments, a method for identifying a compound with macromolecular specificity is provided. In some embodiments, the method includes: (a) contacting a composition with a compound, wherein (i) the composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the composition with the compound; (b) determining an association level between a first macromolecule and one or more target condensates; and (c) comparing the association level between the first macromolecule and one or more target condensates with a reference level to identify the compound with macromolecular specificity, wherein the reference level includes any one or more of: (i) the association level between the first macromolecule and one or more target condensates when the composition is not contacted with the compound; (ii) the association level between one or more other macromolecules and one or more target condensates when the composition is contacted with the compound; or (iii) the association level between one or more other macromolecules and one or more target condensates when the composition is not contacted with the compound. In some embodiments, the reference level is the association level between a second macromolecule and one or more target condensates in the composition. In some embodiments, the reference level is the level of association between the first macromolecule and a reference condensate. In some embodiments, the first macromolecule, one or more target condensates, and the reference condensate are in the same composition. In some embodiments, the one or more target condensates and the reference condensate are in different compositions. In some embodiments, the reference level is the level of association between the first macromolecule and one or more target condensates in a reference system that is not treated with the compound. In some embodiments, the reference system is treated with a vehicle control. In some embodiments, the composition is a cellular composition. In some embodiments, the composition is a cellular composition, and the composition and the reference system comprise the same cell type. In some embodiments, the methods described herein are useful for identifying compounds that exhibit specificity for a single macromolecule (e.g., exhibit an effect and / or activity associated with a single macromolecule in relation to interaction with a condensate). In some embodiments, the methods described herein are useful for identifying compounds that exhibit specificity for a set of macromolecules (e.g., exhibit an effect and / or activity associated with a set of macromolecules in relation to interaction with a condensate).In some embodiments, one or more target condensates are present in a particular type of tissue (e.g., cell type). In some embodiments, the method further includes evaluating the compound for condensate specificity and / or tissue specificity.

[0068] In some embodiments, a method for identifying a compound with condensate specificity is provided, comprising: (a) contacting a composition with a compound, wherein (i) the composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the composition with the compound; (b) determining an association level between a first macromolecule and the one or more target condensates; and (c) comparing the association level between the first macromolecule and the one or more target condensates to a reference level to identify the compound with condensate specificity, wherein the reference level is a reference level that is greater than or equal to (i) the association level between the first macromolecule and the one or more target condensates. The reference level may include any one or more of the following: (i) the level of association between the first macromolecule and another condensate (in the presence or absence of a compound); (ii) the level of association between one or more other macromolecules and another condensate (in the presence or absence of a compound); (iii) the level of association between one or more other macromolecules and one or more target condensates (in the presence or absence of a compound); (iv) characteristics such as size, number, volume, etc. of one or more target condensates (or characteristics such as the distribution, amount, diffusion coefficient, etc. of condensate components); and (v) characteristics such as size, number, volume, etc. of another condensate (or characteristics such as the distribution, amount, diffusion coefficient, etc. of condensate components). In some embodiments, the reference level is the level of association between the second macromolecule and the reference condensate. In some embodiments, the first macromolecule and the second macromolecule are the same. In some embodiments, the first macromolecule and the second macromolecule are different. In some embodiments, the second macromolecule and the reference condensate are in the same composition as the first macromolecule and one or more target condensates. In some embodiments, the composition is a cellular composition. In some embodiments, the composition is a cellular composition, and the composition and the reference system comprise the same cell type. In some embodiments, the method includes (a) contacting the composition with a compound, where (i) the composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the composition with the compound, (b) determining characteristics of the one or more target condensates, and (c) comparing the characteristics of the one or more target condensates to characteristics of a reference condensate to identify a compound with condensate specificity.In some embodiments, the feature of the one or more target condensates is one or more of: (i) the amount of a first macromolecule present in the one or more target condensates, (ii) the size of the one or more target condensates, (iii) the surface area of ​​the one or more target condensates, (iv) the volume of the one or more target condensates, (v) the amount (e.g., total amount) of the one or more target condensates, (vi) the number of the one or more target condensates, and (vii) the number and size of the one or more target condensates. In some embodiments, the feature of the reference condensate is one or more of: (i) the amount of a component (e.g., macromolecule) present in the reference condensate, (ii) the size of the reference condensate, (iii) the surface area of ​​the reference condensate, (iv) the volume of the reference condensate, (v) the amount (e.g., total amount) of the reference condensates, (vi) the number of the reference condensates, and (vii) the number and size of the reference condensates. In some embodiments, the feature being compared is the same feature in both the one or more target condensates and the reference condensate. In some embodiments, the methods described herein are useful for identifying compounds that exhibit specificity for a single condensate type (e.g., exhibit an action and / or activity associated with a single condensate type). In some embodiments, the methods described herein are useful for identifying compounds that exhibit specificity for a set of condensate types (e.g., exhibit an action and / or activity associated with a set of condensate types), such as a set of condensate types that all include a first macromolecule. In some embodiments, one or more target condensates are present in a particular type of tissue (e.g., cell type). In some embodiments, the method further includes evaluating the compound for macromolecule specificity and / or tissue specificity.

[0069] In some embodiments, methods are provided for identifying compounds with tissue (or cell type) specificity.In some embodiments, the method includes: (a) contacting a composition comprising a cell with a compound, wherein (i) the composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the composition with the compound; (b) determining a level of association between a first macromolecule and the one or more target condensates; and (c) comparing the level of association between the first macromolecule and the one or more target condensates to a reference level to identify a compound with tissue specificity, wherein the reference level is: (i) (ii) the level of association of the first macromolecule with one or more target condensates in another cell of a different cell type from the cells of the composition (in the presence or absence of the compound); (iii) the level of association of the first macromolecule with another condensate in another cell of a different cell type from the cells of the composition (in the presence or absence of the compound); (iv) the level of association of one or more other macromolecules with one or more target condensates in the cells of the composition (in the presence or absence of the compound); (v) (vi) the level of association of one or more other macromolecules with one or more target condensates in another cell of a different cell type from the cells of the composition (in the presence or absence of the compound); (vii) the level of association of one or more other macromolecules with one or more condensates in another cell of a different cell type from the cells of the composition (in the presence or absence of the compound); (viii) the size, number, volume, and other characteristics of one or more target condensates in the cells of the composition (or the proportion of condensate components). (ix) characteristics such as size, number, volume, etc. of one or more target condensates in another cell of a cell type different from the cells of the composition (or characteristics such as the distribution, amount, diffusion coefficient, etc. of condensate components); (x) characteristics such as size, number, volume, etc. of another condensate in the cells of the composition (or characteristics such as the distribution, amount, diffusion coefficient, etc. of condensate components); and (xi) characteristics such as size, number, volume, etc. of another condensate in another cell of a cell type different from the cells of the composition (or characteristics such as the distribution, amount, diffusion coefficient, etc. of condensate components).In some embodiments, the reference level is the level of association between the first macromolecule and one or more target condensates in a reference system, the reference system being a model of a tissue (or cell) type different from the composition comprising the one or more target condensates. In some embodiments, the reference system is contacted with the compound. In some embodiments, the composition is a cellular composition. In some embodiments, the composition is a cellular composition, and the composition and the reference system comprise different cell types. In some embodiments, the methods described herein are useful for identifying compounds that exhibit specificity in a single tissue or cell type (e.g., exhibit an effect and / or activity associated with condensates in a single tissue or cell type). In some embodiments, the methods described herein are useful for identifying compounds that exhibit specificity in a set of tissues or cell types (e.g., a set of tissues or cell types that include all of the first macromolecule and one or more target condensates) (e.g., exhibit an effect and / or activity associated with condensates in a set of tissues or cell types). In some embodiments, the one or more target condensates are present in a particular type of tissue (e.g., cell type). In some embodiments, the method further includes evaluating the compound for macromolecule specificity and / or condensate specificity.

[0070] For example, in some embodiments, the methods described herein are useful for identifying compounds that preferentially affect the association of one or more macromolecules (e.g., a first macromolecule, one or more other macromolecules, and / or one or more reference molecules) with one or more target condensates in a first cell type compared to a second cell type (e.g., the association of any macromolecule with a target condensate in a first cell type compared to a second cell type) (hereinafter also referred to as "condensate + cell type selectivity"). In some embodiments, the methods include: 1) comparing the level of association of one or more macromolecules with one or more target condensates in a first cell type composition in the presence of the compound with the level of association of one or more macromolecules with one or more target condensates in a second cell type composition in the presence of the compound; and 2) comparing the level of association of one or more macromolecules with one or more other condensates in the first cell type composition in the presence of the compound with the level of association of one or more macromolecules with one or more other condensates in a second cell type composition in the presence of the compound; A compound is identified as preferentially affecting the association of one or more macromolecules with one or more target condensates in a first cell type compared to a second cell type if a) the level of association of one or more macromolecules with one or more target condensates in the first cell type composition is altered by at least about two-fold or more than the level of association of one or more macromolecules with one or more target condensates in the second cell type composition, and b) the level of association of one or more macromolecules with one or more other condensates in the first cell type composition is unchanged or not significantly altered (e.g., by less than about two-fold) compared to the level of association of one or more macromolecules with one or more other condensates in the second cell type composition.In some embodiments, the method comprises: 1) comparing the level of association between a first macromolecule and one or more target condensates in a first cell type composition in the presence of a compound to the level of association between the first macromolecule and one or more target condensates in a second cell type composition in the presence of the compound; and 2) comparing the level of association between a second macromolecule and one or more target condensates in the first cell type composition in the presence of the compound to the level of association between the second macromolecule and one or more target condensates in the second cell type composition in the presence of the compound; wherein the compound is not identified as having preferential selectivity over a macromolecule type if the compound preferentially affects the association of the first macromolecule and one or more target condensates in the first cell type as well as the extent to which the compound preferentially affects the association of the second macromolecule and one or more target condensates in the first cell type compared to the second cell type.

[0071] In some embodiments, the methods described herein are useful for identifying compounds that preferentially affect the association of a first macromolecule with one or more condensates (e.g., one or more target condensates, one or more other condensates, and / or one or more reference condensates) in a first cell type compared to a second cell type (e.g., the association of a first macromolecule with any condensates in a first cell type compared to a second cell type) (hereinafter also referred to as "macromolecule + cell type selectivity"). In some embodiments, the methods include: 1) comparing the level of association of a first macromolecule with one or more condensates in a first cell type composition in the presence of the compound to the level of association of the first macromolecule with one or more condensates in a second cell type composition in the presence of the compound; and 2) comparing the level of association of one or more other macromolecules with one or more condensates in the first cell type composition in the presence of the compound to the level of association of one or more other macromolecules with one or more condensates in the second cell type composition in the presence of the compound, where a) the association of the first macromolecule with one or more condensates in the first cell type composition is higher than the level of association of the first macromolecule with one or more condensates in the first cell type composition. A compound is identified as preferentially affecting the association of a first macromolecule with one or more condensates in a first cell type compared to a second cell type if a) the level of association of the first macromolecule with one or more condensates in the second cell type composition is altered by at least about two-fold or more than the level of association of the first macromolecule with one or more condensates in the second cell type composition, and b) the level of association of one or more other macromolecules with one or more condensates in the first cell type composition is unchanged or not significantly altered (e.g., less than about two-fold change) compared to the level of association of one or more other macromolecules with one or more condensates in the second cell type composition.

[0072] In some embodiments, the methods described herein include comparing the level of association of a first macromolecule with one or more target condensates to a reference level. As described herein, the reference level provides the necessary comparison to identify whether a compound exhibits any one or more of the following specificities: selectivity for modulating the inclusion or exclusion of a macromolecule in a condensate (macromolecule specificity), selectivity for affecting one type of condensate but not another type of condensate (condensate specificity), and selectivity for affecting one type of tissue but not another type of tissue (tissue specificity). In some embodiments, the association level between a first macromolecule and one or more target condensates is obtained / assessed via any one or more of the following: (i) the amount (e.g., absolute amount, etc.) of the first macromolecule in one or more target condensates, (ii) the amount (e.g., absolute amount, etc.) of the first macromolecule not present in one or more target condensates, (iii) the intensity of the signal from the first macromolecule in one or more target condensates, (iv) the number of one or more target condensates containing the first macromolecule, (v) the size of one or more target condensates containing the first macromolecule, (vi) the surface area of ​​one or more target condensates containing the first macromolecule, (vii) the volume of one or more target condensates containing the first macromolecule, and (viii) the number and size of one or more target condensates containing the first macromolecule. The reference levels described herein can be obtained / assessed in a similar manner as those described for other macromolecules (e.g., first macromolecules) described herein.

[0073] In any of the embodiments described herein, the level of association (e.g., the level of association between a first macromolecule and one or more target condensates) is assessed at multiple compound concentrations. In some embodiments, the method uses information obtained from the analysis at multiple compound concentrations to generate a dose-response curve (IC 50 In some embodiments, the method includes determining the potency (IC) of a compound using a dose-response curve. 50In some embodiments, the method includes determining a dose-response curve (e.g., a reference dose-response curve) obtained for the compound using one or more target condensates. In some embodiments, the method includes identifying a compound with any one or more of the following specificities: macromolecular specificity, condensate specificity, and tissue specificity, and the method includes comparing the potency obtained for the compound using one or more target condensates to a reference potency ...

[0074] In some embodiments, the present invention provides a method for identifying a compound, comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; and (b) determining the level of association between a first macromolecule and one or more target condensates and the level of association between at least one additional macromolecule and one or more target condensates. In some embodiments, the method identifies a compound that preferentially affects the level of association between a first macromolecule and one or more target condensates. In some embodiments, the method identifies a compound that preferentially increases the level of association between a first macromolecule and one or more target condensates. In some embodiments, the method identifies a compound that preferentially decreases the level of association between a first macromolecule and one or more target condensates. In some embodiments, the method identifies a compound useful for treating a disease in an individual in need thereof, wherein one or more target condensates are associated with the disease. In some embodiments, the disease is a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease.

[0075] In some embodiments, the one or more target condensates are only present under disease (or stress) conditions. In some embodiments, the one or more target condensates have an alteration in one or more of the following characteristics under disease (or stress) conditions compared to healthy (or non-stress) conditions: (i) the location of the one or more target condensates, (ii) the distribution of the one or more target condensates and / or their components (e.g., first macromolecules), (iii) the number of the one or more target condensates, (iv) the size of the one or more target condensates, (v) the ratio of the amount of the one or more target condensates to a reference condensate, (vi) a functional activity associated with the one or more target condensates, (vii) the composition of the one or more target condensates, (viii) the co-localization of the one or more target condensates with a biomolecule, (ix) the diffusion coefficient of the one or more target condensate components (e.g., first macromolecules), (x) the stability of the one or more target condensates, (xi) the amount of one or more target condensates. (xii) dissolution or size reduction of the target condensate on the target condensate; (xiii) surface area of ​​one or more target condensates; (xiv) fluidity of one or more target condensates; (xv) solidification of one or more target condensates; (xvi) location of a condensate component (e.g., a first macromolecule); (xvii) amount of a condensate component (e.g., a first macromolecule) or its precursor; (xviii) condensate fractionation of a biomolecule (e.g., a first macromolecule) into one or more target condensates; (xix) functional activity associated with a condensate component (e.g., a first macromolecule); (xx) aggregation of a condensate component (e.g., a first macromolecule); (xxi) post-translational modification state of a condensate component (e.g., a first macromolecule); and (xxii) amount of degradation products of a condensate component (e.g., a first macromolecule). In some embodiments, a first macromolecule has an increased level of association with one or more target condensates under disease (or stress) conditions, and the methods described herein are used to identify compounds that selectively reduce the association of the first macromolecule with one or more target condensates compared to a reference level. For example, the methods described herein are useful for identifying compounds that preferentially reduce the association of the first macromolecule with one or more target condensates compared to the association of another biomolecule (e.g., a condensate component that is not the first macromolecule) with the one or more target condensates.In some embodiments, the methods described herein are useful for identifying compounds that preferentially reduce the association of a first macromolecule with one or more target condensates compared to the association of the first macromolecule with another condensate or the association of a second macromolecule with another condensate. In some embodiments, the methods described herein are useful for identifying compounds that preferentially reduce the association of a first macromolecule with one or more target condensates or another condensate in a diseased (or stressed) tissue or cell type compared to the association of the first macromolecule with one or more target condensates or another condensate in a healthy (or non-stressed) tissue or cell type, or compared to the association of the second macromolecule with one or more target condensates or another condensate in a healthy (or non-stressed) tissue or cell type. In some embodiments, the second macromolecule and the first macromolecule are different. In some embodiments, the second macromolecule is a reference macromolecule (e.g., a macromolecule known to associate with a target condensate or known not to associate with a target condensate or cell / tissue type under healthy or non-stressed conditions).

[0076] Also provided are methods for identifying a plurality of compounds, comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; (b) determining the association level of a first macromolecule with one or more target condensates and the association level of at least one additional macromolecule with one or more target condensates; and (c) performing steps (a) and (b) for the plurality of compounds. In some embodiments, the method is a method for identifying a plurality of compounds that preferentially affect the association level of a first macromolecule with one or more target condensates. In some embodiments, the method is a method for identifying a plurality of compounds that preferentially increase the association level of a first macromolecule with one or more target condensates. In some embodiments, the method is a method for identifying a plurality of compounds that preferentially decrease the association level of a first macromolecule with one or more target condensates. In some embodiments, the method further comprises comparing the association level of the first macromolecule with one or more target condensates upon contact with different compounds. In some embodiments, the method further comprises ranking the identified plurality of compounds based on their effect and / or activity in preferentially increasing (or decreasing) the association level of the first macromolecule with one or more target condensates. In some embodiments, the comparison or ranking is based on the absolute effect and / or activity of the compound in increasing (or decreasing) the association level of the first macromolecule with one or more target condensates. In some embodiments, the comparison or ranking is based on the relative effect and / or activity in preferentially increasing (or decreasing) the association level of the first macromolecule with one or more target condensates compared to the association level of at least one additional macromolecule with one or more target condensates. In some embodiments, the method is for identifying a plurality of compounds useful for treating a disease in an individual in need of treatment, wherein one or more target condensates are associated with the disease. In some embodiments, the disease is a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease.

[0077] In some embodiments, the method further comprises identifying a feature that a subset or all of the identified compounds have in common in addition to their ability to preferentially affect, decrease, or increase the level of association between the first macromolecule and one or more target condensates. In some embodiments, the method further comprises identifying a feature that a subset or all of the identified compounds have in common in addition to their ability to preferentially affect the level of association between the first macromolecule and one or more target condensates. In some embodiments, the method further comprises identifying a feature that a subset or all of the identified compounds have in common in addition to their ability to preferentially decrease the level of association between the first macromolecule and one or more target condensates. In some embodiments, the method further comprises identifying a feature that a subset or all of the identified compounds have in common in addition to their ability to preferentially increase the level of association between the first macromolecule and one or more target condensates. In some embodiments, the method further comprises: (a) contacting the cellular composition with a compound, where (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; and (b) determining, for one or more additional test compounds that comprise the identified feature, the level of association between the first macromolecule and the one or more target condensates and the level of association between at least one additional macromolecule and the one or more target condensates. In some embodiments, the method further comprises performing steps (a) and (b) for one or more additional test compounds that do not comprise the identified feature. In some embodiments, the feature is a compound moiety.

[0078] In some embodiments, the first reference level is the level of association of the first macromolecule with one or more reference condensates determined in the absence of the compound, hi some embodiments, the reference level of each (or at least one) additional macromolecule is the level of association of each (or at least one) additional macromolecule with one or more reference condensates determined in the absence of the compound.

[0079] In some embodiments, a compound preferentially affects the level of association between a first macromolecule and one or more target condensates if the compound changes the level of association between the first macromolecule and one or more target condensates compared to the first reference level more than the compound changes the level of association between each (or at least one) additional macromolecule and one or more target condensates compared to the reference level of each (or at least one) additional macromolecule. In some embodiments, a compound preferentially increases the level of association between a first macromolecule and one or more target condensates if the compound increases the level of association between the first macromolecule and one or more target condensates compared to the first reference level more than the compound increases the level of association between each (or at least one) additional macromolecule and one or more target condensates compared to the reference level of each (or at least one) additional macromolecule. In some embodiments, a compound preferentially reduces the level of association between a first macromolecule and one or more target condensates if the compound reduces the level of association between a first macromolecule and one or more target condensates compared to a first reference level more than if the compound reduces the level of association between each (or at least one) additional macromolecule and one or more target condensates compared to a reference level for each (or at least one) additional macromolecule.

[0080] In some embodiments, the compound increases the association level of the first macromolecule with one or more target condensates compared to a first reference level. In some embodiments, the compound decreases the association level of the first macromolecule with one or more target condensates compared to a first reference level. In some embodiments, the compound increases or does not measurably change the association level of each (or at least one) additional macromolecule compared to the reference level of each (or at least one) additional macromolecule. In some embodiments, the compound decreases or does not measurably change the association level of each (or at least one) additional macromolecule compared to the reference level of each (or at least one) additional macromolecule. In some embodiments, the compound does not measurably change the association level of each (or at least one) additional macromolecule compared to the reference level of each (or at least one) additional macromolecule. In some embodiments, the compound does not measurably increase the association level of each (or at least one) additional macromolecule compared to the reference level of each (or at least one) additional macromolecule. In some embodiments, the compound does not measurably decrease the association level of each (or at least one) additional macromolecule compared to a reference level of each (or at least one) additional macromolecule, hi some embodiments, the method includes determining a first reference level and a reference level of each (or at least one) additional macromolecule.

[0081] In some embodiments, the compound increases the association level of a first macromolecule with one or more target condensates compared to a first reference level, and the compound does not increase the association level of each (or at least one) additional macromolecule compared to the reference level of each (or at least one) additional macromolecule, or the compound decreases the association level of a first macromolecule with one or more target condensates compared to the first reference level, and the compound does not increase the association level of each (or at least one) additional macromolecule compared to the reference level of each (or at least one) additional macromolecule. In some embodiments, the compound increases the association level of a first macromolecule with one or more target condensates compared to the first reference level, and the compound does not increase the association level of each (or at least one) additional macromolecule compared to the reference level of each (or at least one) additional macromolecule. In some embodiments, the compound reduces the association level of a first macromolecule with one or more target condensates compared to a first reference level, and the compound does not reduce the association level of each (or at least one) additional macromolecule compared to the reference level of each (or at least one) additional macromolecule.

[0082] In some embodiments, a compound preferentially affects the level of association of a first macromolecule with one or more target condensates if the compound changes the level of the first macromolecule relative to the first reference level more than the compound changes the level of each (or at least one) additional macromolecule relative to the reference level of each (or at least one) additional macromolecule. In some embodiments, a compound preferentially increases the level of association of a first macromolecule with one or more target condensates if the compound increases the level of the first macromolecule relative to the first reference level more than the compound changes the level of each (or at least one) additional macromolecule relative to the reference level of each (or at least one) additional macromolecule. In some embodiments, if the compound reduces the level of a first macromolecule compared to the first reference level more than it changes the level of each (or at least one) additional macromolecule compared to the reference level of each (or at least one) additional macromolecule, and the difference is statistically significant, the compound preferentially reduces the association level of the first macromolecule with one or more target condensates. In some embodiments, the difference is statistically significant if p<0.05 (e.g., p<0.025, 0.01, 0.005, or 0.001). Methods for determining statistical significance, such as t-tests, ANOVA, and Fisher's method, are known. The selection of an appropriate test can be determined by one skilled in the art.

[0083] In some embodiments, a compound preferentially affects the level of association of a first macromolecule with one or more target condensates if the compound changes the level of a first macromolecule compared to the first reference level by at least about two-fold (e.g., at least about 2.5-fold, 3-fold, 4-fold, 5-fold, or 10-fold) more than the compound changes the level of each (or at least one) additional macromolecule compared to the reference level of each (or at least one) additional macromolecule. In some embodiments, a compound preferentially increases the level of association of a first macromolecule with one or more target condensates if the compound increases the level of a first macromolecule compared to the first reference level by at least about two-fold (e.g., at least about 2.5-fold, 3-fold, 4-fold, 5-fold, or 10-fold) more than the compound changes the level of each (or at least one) additional macromolecule compared to the reference level of each (or at least one) additional macromolecule. In some embodiments, a compound preferentially decreases the level of association between a first macromolecule and one or more target condensates if the compound decreases the level of a first macromolecule compared to a first reference level by at least about 2-fold (e.g., at least about any of 2.5-fold, 3-fold, 4-fold, 5-fold, or 10-fold) more than the compound changes the level of each (or at least one) additional macromolecule compared to a reference level of each (or at least one) additional macromolecule. In some embodiments, a compound preferentially affects the level of association between a first macromolecule and one or more target condensates if the compound alters the level of association between a first macromolecule and one or more target condensates compared to a first reference level by at least about 0.25-fold, 0.5-fold, 0.75-fold, 1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, or 10-fold or more than the compound alters the level of association between each (or at least one) additional macromolecule and one or more target condensates compared to a reference level for each (or at least one) additional macromolecule.

[0084] In some embodiments, a compound preferentially affects the level of association between a first macromolecule and one or more target condensates if the compound alters the level of association between a first macromolecule and one or more target condensates compared to a first reference level (e.g., by at least about 0.25-fold, 0.5-fold, 0.75-fold, 1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, or 10-fold) more than the compound alters the level of association between a second macromolecule and a reference condensate compared to the reference level. In some embodiments, the first reference level is the level of association between a first macromolecule and one or more target condensates determined in the absence of the compound. In some embodiments, the reference level is the level of association between a second macromolecule and a reference condensate determined in the absence of the compound. In some embodiments, the first macromolecule and the second macromolecule are the same. In some embodiments, the first macromolecule and the second macromolecule are different.

[0085] In some embodiments, a compound preferentially affects the level of association between a first macromolecule and one or more target condensates in a target cell type or target tissue type if the compound alters the level of association between a first macromolecule and one or more target condensates in a target cell type or target tissue type compared to a first reference level (e.g., by at least about 0.25-fold, 0.5-fold, 0.75-fold, 1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, or 10-fold) more than the compound alters the level of association between a first macromolecule and one or more target condensates in a reference cell type or reference tissue type compared to the reference level. In some embodiments, the first reference level is the level of association between a first macromolecule and one or more target condensates in a target cell type or target tissue type determined in the absence of the compound. In some embodiments, the reference level is the level of association between a first macromolecule and one or more target condensates in a reference cell type or reference tissue type determined in the absence of the compound.

[0086] In some embodiments, the level is an absolute amount. In some embodiments, the level is a relative level, such as 1) the amount of a macromolecule compared to the amount of another component of one or more (target or reference) condensates, 2) the amount of a macromolecule associated with one or more (target or reference) condensates compared to the amount of the macromolecule in a cell or another cellular component (e.g., in another condensate or organelle), or 3) the amount of a macromolecule associated with one or more (target or reference) condensates in one tissue or cell type compared to the amount of the macromolecule associated with one or more (target or reference) condensates in another tissue or cell type.

[0087] In some embodiments, the reference is an experimental control. In some embodiments, the reference level of a macromolecule is the association level of the macromolecule with one or more reference condensates. In some embodiments, the one or more target condensates and the one or more reference condensates are located in different parts of a cellular composition. In some embodiments, the one or more target condensates and the one or more reference condensates are located in different parts of a cell. In some embodiments, the one or more target condensates and the one or more reference condensates are located in different cellular compositions. In some embodiments, the cellular composition containing one or more target condensates is a first cellular composition, and the reference level is determined in a second cellular composition. In some embodiments, the reference level is the association level of the macromolecule with one or more target condensates or one or more reference condensates in a cellular composition that has not been contacted with a compound.

[0088] In some embodiments, the reference level is determined in a manner that allows for the evaluation of meaningful results for the compound. For example, in some embodiments, the reference level is determined in a reference cell composition, which is prepared in a manner similar to the cell composition that is contacted with the compound, except that the reference cell composition is not subjected to the compound or the step of contacting with the compound.

[0089] A method for identifying a compound is provided herein, comprising: (a) contacting a first cellular composition with the compound, wherein (i) the first cellular composition comprises a first set of one or more target condensates and / or (ii) the first set of one or more target condensates is formed simultaneously with and / or after contacting the first cellular composition with the compound; (b) determining the level of association between a first macromolecule and the first set of one or more target condensates and the level of association between at least one additional macromolecule and the first set of one or more target condensates; (c) contacting a second cellular composition with the compound, wherein (i) the second cellular composition comprises a second set of one or more target condensates and / or (ii) the second set of one or more target condensates is formed simultaneously with and / or after contacting the second cellular composition with the compound; and (d) determining the level of association between the first macromolecule and the second set of one or more target condensates and the second macromolecule and the second set of one or more target condensates. In some embodiments, the second macromolecule and the first macromolecule are different. In some embodiments, the second macromolecule is one or more of the at least one additional macromolecule.

[0090] Also provided are methods for identifying compounds, comprising: (a) contacting a cellular composition with the compound, wherein (i) the cellular composition comprises a first set of one or more target condensates, and / or (ii) the first and second sets of one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; (b) determining the level of association between a first macromolecule and the first set of one or more target condensates, and at least one additional macromolecule and the first set of one or more target condensates; and (c) determining the level of association between the first macromolecule and the second set of one or more target condensates, and the level of association between the second macromolecule and the second set of one or more target condensates. In some embodiments, the second macromolecule and the first macromolecule are different. In some embodiments, the second macromolecule is one or more of the at least one additional macromolecule.

[0091] In some embodiments, the macromolecule described herein is a first macromolecule. In some embodiments, the macromolecule is not a first macromolecule. In some embodiments, the macromolecule is one or more of at least one additional macromolecule. In some embodiments, the macromolecule is a reference macromolecule (e.g., a macromolecule known to associate with a target condensate, or a macromolecule known not to associate with a target condensate or cell / tissue type under healthy or non-stress conditions). In some embodiments, the macromolecule is a first macromolecule, and the method further comprises determining the level of association of a second macromolecule with a second set of one or more target condensates. In some embodiments, the second macromolecule and the first macromolecule are different. In some embodiments, the second macromolecule is one or more of at least one additional macromolecule.

[0092] In some embodiments, the method is a method for identifying a compound that preferentially affects the association level between a first macromolecule and a first set of one or more target condensates. In some embodiments, the method is a method for identifying a compound that preferentially increases the association level between a first macromolecule and a first set of one or more target condensates. In some embodiments, the method is a method for identifying a compound that preferentially decreases the association level between a first macromolecule and a first set of one or more target condensates. In some embodiments, the method is a method for identifying a compound useful for treating a disease in an individual in need of treatment, wherein the first set of one or more target condensates is associated with the disease. In some embodiments, the disease is a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease.

[0093] In some embodiments, the method is for identifying a plurality of compounds that preferentially affect the level of association between a first macromolecule and a first set of one or more target condensates, the method comprising performing any of the methods described herein on the plurality of compounds. In some embodiments, the method is for identifying a plurality of compounds that preferentially increase the level of association between a first macromolecule and a first set of one or more target condensates, the method comprising performing any of the methods described herein on the plurality of compounds. In some embodiments, the method is for identifying a plurality of compounds that preferentially decrease the level of association between a first macromolecule and a first set of one or more target condensates, the method comprising performing any of the methods described herein on the plurality of compounds. In some embodiments, the method is for identifying a plurality of compounds useful for treating a disease in an individual in need of treatment, the first set of one or more target condensates being associated with the disease, the method comprising performing any of the methods described herein on the plurality of compounds. In some embodiments, the disease is a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease.

[0094] In some embodiments, if the compound affects the level of association between the first macromolecule and the first set of one or more target condensates more than it affects the level of association between the first macromolecule and the second set of one or more target condensates, the compound preferentially affects the level of association between the first macromolecule and the first set of one or more target condensates. In some embodiments, the compound does not affect the level of association between the first macromolecule and the second set of one or more target condensates. In some embodiments, if the compound increases the level of association between the first macromolecule and the first set of one or more target condensates more than it increases the level of association between the first macromolecule and the second set of one or more target condensates, the compound preferentially increases the level of association between the first macromolecule and the first set of one or more target condensates. In some embodiments, the compound does not increase the level of association between the first macromolecule and the second set of one or more target condensates. In some embodiments, a compound preferentially reduces the level of association between a first macromolecule and a first set of one or more target condensates if the compound reduces the level of association between a first macromolecule and a first set of one or more target condensates more than the compound reduces the level of association between a first macromolecule and a second set of one or more target condensates. In some embodiments, a compound does not reduce the level of association between a first macromolecule and a second set of one or more target condensates.

[0095] Also provided herein are methods for identifying a compound, comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; and (b) determining whether a first macromolecule and at least one additional macromolecule associate with the one or more target condensates. In some embodiments, the method is a method for identifying a compound that causes a first macromolecule to associate with or dissociate from one or more target condensates. In some embodiments, the method is a method for identifying a compound that causes a first macromolecule to associate with or dissociate from one or more target condensates. In some embodiments, the method is a method for identifying a compound that causes a first macromolecule to preferentially associate with or dissociate from one or more target condensates. In some embodiments, the method is a method for identifying a compound that causes a first macromolecule to preferentially associate with one or more target condensates. In some embodiments, the method is a method for identifying a compound that preferentially dissociates a first macromolecule with one or more target condensates.In some embodiments, the method is a method for identifying a compound that is useful for treating a disease in an individual in need of treatment, and one or more target condensates are associated with the disease.In some embodiments, the disease is a neurodegenerative disease, a proliferative disease, an immune disease, a cardiovascular disease, or a metabolic disease.

[0096] Also provided herein are methods for identifying a plurality of compounds, the methods comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; (b) determining whether a first macromolecule and at least one additional macromolecule associate with the one or more target condensates; and (c) performing steps (a) and (b) for the plurality of compounds. In some embodiments, the methods are methods for identifying a plurality of compounds that cause a first macromolecule to associate with or dissociate from one or more target condensates. In some embodiments, the methods are methods for identifying a plurality of compounds that cause a first macromolecule to associate with or dissociate from one or more target condensates. In some embodiments, the methods are methods for identifying a plurality of compounds that cause a first macromolecule to dissociate from one or more target condensates. In some embodiments, the methods are methods for identifying a plurality of compounds that cause a first macromolecule to preferentially associate with or dissociate from one or more target condensates. In some embodiments, the method is a method for identifying a plurality of compounds that preferentially associate a first macromolecule with one or more target condensates. In some embodiments, the method is a method for identifying a plurality of compounds that preferentially dissociate a first macromolecule from one or more target condensates. In some embodiments, the method is a method for identifying a plurality of compounds that are useful for treating a disease in an individual in need of treatment, and one or more target condensates are associated with the disease. In some embodiments, the disease is a neurodegenerative disease, a proliferative disease, an immune disease, a cardiovascular disease, or a metabolic disease.

[0097] In some embodiments, the method further comprises identifying a feature that a subset or all of the identified compounds have in common in addition to their ability to preferentially associate or dissociate the first macromolecule with one or more target condensates. In some embodiments, the method further comprises identifying a feature that a subset or all of the identified compounds have in common in addition to their ability to preferentially associate the first macromolecule with one or more target condensates. In some embodiments, the method further comprises identifying a feature that a subset or all of the identified compounds have in common in addition to their ability to preferentially dissociate the first macromolecule with one or more target condensates. In some embodiments, the method further comprises (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; and (b) determining, for one or more additional test compounds comprising the identified feature, whether the first macromolecule and at least one additional macromolecule associate with one or more target condensates. In some embodiments, the method further comprises performing steps (a) and (b) for one or more additional test compounds that do not contain the identified characteristic.

[0098] In some embodiments, the compound causes the first macromolecule to associate with one or more target condensates. In some embodiments, the compound does not cause the first macromolecule to associate with one or more target condensates. In some embodiments, the compound prevents the first macromolecule from associating with one or more target condensates. In some embodiments, the compound does not prevent the first macromolecule from associating with one or more target condensates.

[0099] In some embodiments, the compound causes one or more of the at least one additional macromolecule to associate with one or more target condensates. In some embodiments, the compound causes each additional macromolecule to associate with one or more target condensates. In some embodiments, the compound does not cause one or more of the at least one additional macromolecule to associate with one or more target condensates. In some embodiments, the compound does not cause one or more of the at least one additional macromolecule to not associate with one or more target condensates. In some embodiments, the compound does not cause each additional macromolecule to not associate with one or more target condensates.

[0100] In some embodiments, the first macromolecule will associate with one or more target condensates in the absence of the compound. In some embodiments, the first macromolecule will not associate with one or more target condensates in the absence of the compound. In some embodiments, one or more of the at least one additional macromolecule will associate with one or more target condensates in the absence of the compound. In some embodiments, each additional macromolecule will associate with one or more target condensates in the absence of the compound. In some embodiments, one or more of the at least one additional macromolecule will not associate with one or more target condensates in the absence of the compound. In some embodiments, each additional macromolecule will not associate with one or more target condensates in the absence of the compound. In some embodiments, the first macromolecule and one or more of the at least one additional macromolecule will associate with one or more target condensates in the absence of the compound. In some embodiments, the first macromolecule and each additional macromolecule will associate with one or more target condensates in the absence of the compound. In some embodiments, one or more of the first macromolecule and the at least one additional macromolecule will not associate with one or more target condensates in the absence of the compound, hi some embodiments, the first macromolecule and each additional macromolecule will not associate with one or more target condensates in the absence of the compound.

[0101] In some embodiments, one or more of the at least one additional macromolecule will associate with one or more target condensates in the presence of the compound and will associate with one or more target condensates in the absence of the compound. In some embodiments, each additional macromolecule will associate with one or more target condensates in the presence of the compound and will associate with one or more target condensates in the absence of the compound. In some embodiments, one or more of the at least one additional macromolecule will not associate with one or more target condensates in the presence of the compound and will not associate with one or more target condensates in the absence of the compound. In some embodiments, each additional macromolecule will not associate with one or more target condensates in the presence of the compound and will not associate with one or more target condensates in the absence of the compound.

[0102] In some embodiments, a compound causes a first macromolecule to preferentially associate with one or more target condensates when: (1) the compound causes the first macromolecule to associate with one or more target condensates, (2) the compound causes each (or at least one) additional macromolecule not to associate with one or more target condensates (or causes each (or at least one) additional macromolecule to associate less with one or more target condensates), and (3) the first macromolecule would not associate with one or more target condensates in the absence of the compound. In some embodiments, a compound causes a first macromolecule to preferentially associate with one or more target condensates when: (1) the compound causes the first macromolecule to associate with one or more target condensates; (2) the compound causes each (or at least one) additional macromolecule not to associate with one or more target condensates (or causes each (or at least one) additional macromolecule to associate less with one or more target condensates); and (3) one or more of the first macromolecule and the at least one additional macromolecule would not associate with one or more target condensates in the absence of the compound. In some embodiments, a compound causes a first macromolecule to preferentially associate with one or more target condensates when: (1) the compound causes the first macromolecule to associate with one or more target condensates, (2) the compound causes each (or at least one) additional macromolecule not to associate with one or more target condensates (or causes each (or at least one) additional macromolecule to associate less with one or more target condensates), and (3) the first macromolecule and each additional macromolecule would not associate with one or more target condensates in the absence of the compound.

[0103] In some embodiments, a compound causes a first macromolecule to preferentially dissociate from one or more target condensates when: (1) the compound causes the first macromolecule not to associate with one or more target condensates; (2) the compound does not cause each (or at least one) additional macromolecule to not associate with one or more target condensates (or causes each (or at least one) additional macromolecule to dissociate less from one or more target condensates); and (3) the first macromolecule would associate with one or more target condensates in the absence of the compound. In some embodiments, a compound preferentially dissociates a first macromolecule from one or more target condensates when: (1) the compound prevents the first macromolecule from associating with one or more target condensates; (2) the compound does not prevent each (or at least one) additional macromolecule from associating with one or more target condensates (or causes each (or at least one) additional macromolecule to dissociate less from one or more target condensates); and (3) one or more of the first macromolecule and the at least one additional macromolecule would associate with one or more target condensates in the absence of the compound. In some embodiments, a compound preferentially dissociates a first macromolecule from one or more target condensates when: (1) the compound prevents the first macromolecule from associating with one or more target condensates; (2) the compound does not prevent each (or at least one) additional macromolecule from associating with one or more target condensates (or causes each (or at least one) additional macromolecule to dissociate less from one or more target condensates); and (3) the first macromolecule and each additional macromolecule would associate with one or more target condensates in the absence of the compound.

[0104] In some embodiments, the compound directly causes the first macromolecule to associate with one or more target condensates. In some embodiments, the compound indirectly causes the first macromolecule to associate with one or more target condensates. In some embodiments, the compound directly prevents the first macromolecule from associating with one or more target condensates. In some embodiments, the compound indirectly prevents the first macromolecule from associating with one or more target condensates.

[0105] In some embodiments, the compound directly causes one or more of the at least one additional macromolecule to associate with one or more target condensates. In some embodiments, the compound indirectly causes one or more of the at least one additional macromolecule to associate with one or more target condensates. In some embodiments, the compound directly prevents one or more of the at least one additional macromolecule from associating with one or more target condensates. In some embodiments, the compound indirectly prevents one or more of the at least one additional macromolecule from associating with one or more target condensates.

[0106] In some embodiments, the compound directly causes each additional macromolecule to associate with one or more target condensates. In some embodiments, the compound indirectly causes each additional macromolecule to associate with one or more target condensates. In some embodiments, the compound directly prevents each additional macromolecule from associating with one or more target condensates. In some embodiments, the compound indirectly prevents each additional macromolecule from associating with one or more target condensates.

[0107] In some embodiments, if a macromolecule is determined to associate with one or more target condensates after contacting the cellular composition with the compound and the macromolecule is determined not to associate with one or more reference condensates, the compound causes a macromolecule (e.g., a first macromolecule or an additional macromolecule) to associate with one or more target condensates. In some embodiments, the one or more reference condensates are experimental controls. In some embodiments, the one or more target condensates and the one or more reference condensates are located in different parts of the cellular composition. In some embodiments, the one or more target condensates and the one or more reference condensates are located in different parts of a cell. In some embodiments, the one or more target condensates and the one or more reference condensates are located in different cellular compositions. In some embodiments, the cellular composition comprising one or more target condensates is a first cellular composition, and the one or more reference condensates is a second cellular composition. In some embodiments, the second cellular composition has not been contacted with the compound.

[0108] In some embodiments, the determination of the association of the macromolecule with one or more reference condensates is carried out in a manner that allows for the evaluation of meaningful results for the compound. For example, in some embodiments, the determination of the association of the macromolecule with one or more reference condensates is carried out in a reference cell composition, which is prepared in a manner similar to the cell composition that is contacted with the compound, except that the reference cell composition is not subjected to the compound or a step of contacting the compound.

[0109] In some embodiments, if a macromolecule is determined to associate with one or more target condensates after contacting the cellular composition with the compound, and if it is determined that the macromolecule would not associate with one or more target condensates without contacting the cellular composition with the compound, the compound causes the macromolecule (e.g., the first macromolecule or an additional macromolecule) to associate with one or more target condensates. In some embodiments, the method further includes determining whether the macromolecule associates with one or more target condensates in the absence of the compound. In some embodiments, if a macromolecule is determined to associate with one or more target condensates after contacting the cellular composition with the compound, and the macromolecule does not associate with one or more reference condensates, the compound causes the macromolecule (e.g., the first macromolecule or an additional macromolecule) to associate with one or more target condensates. In some embodiments, the method further includes determining whether the macromolecule associates with one or more reference condensates in the absence of the compound.

[0110] Also provided herein are methods for identifying a compound, comprising: (a) contacting a first cellular composition with the compound, wherein (i) the first cellular composition comprises a first set of one or more target condensates and / or (ii) the first set of one or more target condensates is formed simultaneously with and / or after contacting the first cellular composition with the compound; (b) determining whether a first macromolecule and at least one additional macromolecule associate with the first set of one or more target condensates; (c) contacting a second cellular composition with the compound, wherein (i) the second cellular composition comprises a second set of one or more target condensates and / or (ii) the second set of one or more target condensates is formed simultaneously with and / or after contacting the second cellular composition with the compound; and (d) determining whether the second macromolecule associates with the second set of one or more target condensates. In some embodiments, the second macromolecule and the first macromolecule are the same. In some embodiments, the second macromolecule and the first macromolecule are different. In some embodiments, the second macromolecule is one or more of the at least one additional macromolecule. In some embodiments, the second macromolecule is a reference macromolecule (e.g., a macromolecule known to associate with target condensates or known not to associate with target condensates or cell / tissue types under healthy or non-stress conditions). In some embodiments, the second macromolecule and the first macromolecule are the same, and the method further comprises determining whether a third macromolecule (e.g., different from the first macromolecule and the second macromolecule) associates with a second set of one or more target condensates. In some embodiments, the third macromolecule is one or more of the at least one additional macromolecule.

[0111] Also provided herein are methods for identifying a compound, comprising: (a) contacting a cellular composition with the compound, wherein (i) the cellular composition comprises a first set of one or more target condensates and / or a second set of one or more target condensates, and / or (ii) the first set of one or more target condensates and / or the second set of one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; (b) determining whether a first macromolecule and at least one additional macromolecule associate with the first set of one or more target condensates; and (c) determining whether a second macromolecule associates with the second set of one or more target condensates. In some embodiments, the second macromolecule and the first macromolecule are the same. In some embodiments, the second macromolecule and the first macromolecule are different. In some embodiments, the second macromolecule is one or more of the at least one additional macromolecule. In some embodiments, the second macromolecule is a reference macromolecule (e.g., a macromolecule known to associate with target condensates or known not to associate with target condensates or cell / tissue types under healthy or non-stress conditions). In some embodiments, the second macromolecule and the first macromolecule are the same, and the method further comprises determining whether a third macromolecule (e.g., different from the first macromolecule and the second macromolecule) associates with a second set of one or more target condensates. In some embodiments, the third macromolecule is one or more of the at least one additional macromolecule.

[0112] In some embodiments, the method is a method for identifying a compound that causes a first macromolecule to associate with a first set of one or more target condensates. In some embodiments, the method is a method for identifying a compound that causes a first macromolecule to dissociate from a first set of one or more target condensates. In some embodiments, the method is a method for identifying a compound that preferentially associates or dissociates a first macromolecule with a first set of one or more target condensates. In some embodiments, the method is a method for identifying a compound that preferentially associates a first macromolecule with a first set of one or more target condensates. In some embodiments, the method is a method for identifying a compound that preferentially dissociates a first macromolecule from a first set of one or more target condensates. In some embodiments, the method is a method for identifying a compound useful for treating a disease in an individual in need of treatment, wherein the first set of one or more target condensates is associated with the disease. In some embodiments, the disease is a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease.

[0113] In some embodiments, the method is for identifying a plurality of compounds that cause a first macromolecule to associate with a first set of one or more target condensates, the method comprising performing any of the methods described herein on the plurality of compounds. In some embodiments, the method is for identifying a plurality of compounds that cause a first macromolecule to dissociate from a first set of one or more target condensates, the method comprising performing any of the methods described herein on the plurality of compounds. In some embodiments, the method is for identifying a plurality of compounds that cause a first macromolecule to preferentially associate with or dissociate from a first set of one or more target condensates, the method comprising performing any of the methods described herein on the plurality of compounds. In some embodiments, the method is for identifying a plurality of compounds that cause a first macromolecule to preferentially associate with a first set of one or more target condensates, the method comprising performing any of the methods described herein on the plurality of compounds. In some embodiments, the method is for identifying a plurality of compounds that cause a first macromolecule to preferentially dissociate from a first set of one or more target condensates, the method comprising performing any of the methods described herein on the plurality of compounds. In some embodiments, the method is for identifying a plurality of compounds useful for treating a disease in an individual in need thereof, wherein a first set of one or more target condensates is associated with the disease, and the method comprises performing any of the methods described herein with the plurality of compounds. In some embodiments, the disease is a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease, and the method comprises performing any of the methods described herein with the plurality of compounds.

[0114] In some embodiments, if the compound causes the first macromolecule to associate with the first set of one or more target condensates more than the compound causes the first macromolecule to associate with the second set of one or more target condensates, the compound preferentially associates the first macromolecule with the first set of one or more target condensates. In some embodiments, if the compound causes the first macromolecule to dissociate from the first set of one or more target condensates more than the compound causes the first macromolecule to dissociate from the first set of one or more target condensates more than the compound causes the first macromolecule to dissociate from the second set of one or more target condensates, the compound preferentially dissociates the first macromolecule from the first set of one or more target condensates.

[0115] Those skilled in the art will readily recognize that cellular processes, including the state of condensates and their components, are dynamic. Accordingly, the methods described herein can involve contacting cells with a compound at any point in the life cycle of one or more target condensates or their components (e.g., various mitotic or non-mitotic phases). For example, the methods can involve contacting a cellular composition with a compound when, for example, one or more target condensates are present in any cellular location, present in any amount, including absence, undergoing a morphological change, such as a change in size or fluidity, or undergoing a change in composition. In some embodiments, the methods can also involve contacting a cellular composition with a compound when one or more target condensate components are present in any cellular location, present in any amount, or have any post-translational modification state.

[0116] compound In some embodiments, the compound is a small molecule, polypeptide, lipid, or nucleic acid. In some embodiments, the compound is an approved compound, such as a compound approved for medical treatment by the U.S. Food and Drug Administration. In some embodiments, the compound is a novel compound. In some embodiments, the compound is a small molecule. In some embodiments, the small molecule is an alkaloid, glycoside, phenazine, phenol, polyketide, terpene, or tetrapyrrole. In some embodiments, the test compound is a nucleic acid. In some embodiments, the compound is an siRNA, miRNA, or mRNA. In some embodiments, the compound is a non-naturally occurring compound. In some embodiments, the compound has a molecular weight of less than about 1,000 Da (e.g., about 500 Da or less). In some embodiments, the compound satisfies Lipinski's Rule of Five. In some embodiments, the compound is a small molecule (e.g., a therapeutic small molecule that is about 1,000 Da or less and / or satisfies Lipinski's Rule of Five). In some embodiments, the compound includes a detectable feature, such as a fluorescent feature. In some embodiments, the compound includes a label, such as a fluorescent label. In some embodiments, the compound is further labeled with a detection tag, such as fluorescein, a fluorescent polypeptide, or a radioisotope label. In some embodiments, the tag does not affect the action and / or activity of the compound, which preferentially causes association / dissociation of a first macromolecule (or a reference macromolecule) with one or more target condensates (or reference condensates). In some embodiments, the label is detectable using microscopy techniques (e.g., fluorescence microscopy techniques).

[0117] In some embodiments, the method herein comprises adding two or more compounds. Thus, in some embodiments, the present invention provides a method for identifying a combination of compounds that preferentially changes (e.g., preferentially increases or preferentially decreases) the association level between a first macromolecule and one or more target condensates, or a combination of compounds that preferentially associates or dissociates a first macromolecule with one or more target condensates. In some embodiments, each of the two or more compounds is selected from a small molecule, a polypeptide, a lipid, or a nucleic acid. In some embodiments, the two or more test compounds are added sequentially or simultaneously. In some embodiments, the two or more test compounds are combined together.

[0118] cell composition The cell compositions disclosed herein are compositions comprising at least one cell. In some embodiments, the cell composition comprises a single cell. In some embodiments, the cell composition comprises at least 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, or more cells. Cell compositions can be obtained from a variety of different sources (e.g., in vivo sources such as body fluid or tissue samples from animals, or in vitro sources such as cultured cells or tissues). Body fluid and tissue samples often contain multiple cell types (e.g., brain tissue can contain neurons, glial cells, and many other types of cells). Thus, in some embodiments, the cell composition comprises at least two, three, four, or five different cell types. The cell composition may also include a medium, such as a cell culture medium. The medium may vary depending on the sample and cell type used. In some embodiments, the cell composition includes one or more of amino acids, vitamins, inorganic salts, glucose, serum, growth factors, hormones, and attachment factors. Exemplary media include Ames' medium, BGJb medium (with or without Fitton-Jackson modifications), Crick medium, CMRL-1066 medium, Fisher's medium, Glasgow minimum essential medium (GMEM), Iscove's modified Dulbecco's medium (IMDM), L-15 medium (Leibovitz's), McCoy's 5A modified medium, NCTC medium, Swim's S-77 medium, Weymouth's medium, William's medium E, Dulbecco's modified Eagle's medium (DMEM), and Eagle's minimum essential medium (EMEM).

[0119] For methods requiring a first cell composition and a second cell composition, any cell composition disclosed herein may be used. In some embodiments, the first cell composition and the second cell composition contain substantially similar components. For example, the first cell composition and the second cell composition may each be a sample from a source cell composition, or each may be prepared by combining the same components using the same method. In some embodiments, the first cell composition and the second cell composition contain substantially different components. For example, the first cell composition and the second cell composition may each be a sample from a different source cell composition, or each may be prepared by combining different components (e.g., different cells or media) or by using components prepared using different methods. In some embodiments, the first cell composition contains healthy or unstressed cells or cells expressing a wild-type macromolecule, and the second cell composition contains diseased (e.g., tumor cells) or stressed cells or cells expressing a mutant macromolecule.

[0120] In some embodiments, the cell is a microorganism or an animal cell. In some embodiments, the cell is an animal cell. In some embodiments, the cell is a human cell. In some embodiments, the cell has one or more characteristics of a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease. In some embodiments, the cell is a HeLa cell, a HEK293 cell, an induced pluripotent stem cell (iPSC cell), a cardiomyocyte, a myocyte, a stem cell-derived cell, a neuron, a cancer cell, an immune cell, or an adipocyte. In some embodiments, the cell is a neuron. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is an induced pluripotent stem cell (iPS cell), a HeLa cell, or a HEK293 cell. When a particular type of cell is described, it is understood to include cells derived from that type of cell unless otherwise specified. For example, a HeLa cell containing a heterologous transgene would be considered a HeLa cell unless otherwise specified.

[0121] In some embodiments, the cells contain condensates determined to be dysregulated. In some embodiments, the cells contain mutations associated with a disease. In some embodiments, the cells have one or more characteristics of a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease. In some embodiments, the cells have been treated with arsenate, a temperature change, or a pH change.

[0122] In some embodiments, the method includes changing the temperature of the cell composition (e.g., exposing the cell composition to a lower or higher temperature), changing the salt content of the cell composition, adding or changing a buffer in the cell composition, changing the pH of the cell composition, or adding a crowding agent (e.g., PEG or dextran) to the cell composition.

[0123] In some embodiments, the cells in the cell composition comprise one or more target condensate-forming mutations and / or one or more target condensate-modifying mutations. In some embodiments, the mutations modify one or more of the following: size of one or more target condensates, shape (e.g., sphericity) of one or more target condensates, surface area of ​​one or more target condensates, concentration of one or more components of one or more target condensates, location of one or more target condensates, number of one or more target condensates, ratio of the amounts of one or more target condensates to a reference condensate, functional activity associated with one or more target condensates, composition of one or more target condensates, colocalization of one or more target condensates with biomolecules, diffusion coefficient of a component of one or more target condensates, stability of one or more target condensates, dissolution or size reduction of one or more target condensates, fluidity of one or more target condensates, solidification of one or more target condensates, location of condensate components, amount of condensate component or its precursor, condensate fractionation of biomolecules into one or more target condensates, functional activity associated with a condensate component, aggregation of a condensate component, post-translational modification state of a condensate component, amount of degradation product of a condensate component, and heterogeneous distribution of a component within one or more target condensates. In some embodiments, the cells in the cell composition contain a mutation that causes condensates to form.

[0124] In some embodiments, the cells express a protein labeled with a fluorescent protein. In some embodiments, the protein is a protein known to be enriched in one or more target condensates. In some embodiments, the cells express a first protein and a second protein, wherein the first protein is labeled with a first label, the first protein known to be enriched in a first set of one or more target condensates, the second protein is labeled with a second label, the second protein known to be enriched in a second set of one or more target condensates, and the first label and the second label are distinguishable. In some embodiments, the cells express a first protein and a second protein, wherein the first protein is labeled with a first fluorescent protein, the first protein known to be enriched in a first set of one or more target condensates, the second protein is labeled with a second fluorescent protein, the second protein known to be enriched in a second set of one or more target condensates, and the first fluorescent protein and the second fluorescent protein are distinguishable. In some embodiments, expression of the protein is inducible or conditional (e.g., using the TetOn system). In some embodiments, expression of the protein is constitutive.

[0125] condensate In some embodiments, the one or more target condensates are 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more target condensates, 25 or more, 50 or more, 75 or more, or 100 or more target condensates. In some embodiments, the one or more target condensates are about 1 to 1,000 condensates (e.g., about 1 to 750, 1 to 500, 1 to 250, 1 to 100, 10 to 500, 10 to 50, 10 to 20, 25 to 1,000, 25 to 500, 25 to 250, 100 to 1,000, or 100 to 500 condensates). In some embodiments, the one or more target condensates are a single condensate type (e.g., a condensate type that contains a common macromolecular component). In some embodiments, the one or more target condensates are a plurality of condensate types, e.g., some condensates of the plurality include the first macromolecular component and some condensates of the plurality do not include the first macromolecular component. In some embodiments, the one or more reference condensates are 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more target condensates, 25 or more, 50 or more, 75 or more, or 100 or more target condensates. In some embodiments, the one or more reference condensates are any of about 1-1000 condensates (1-500, 1-250, 1-100, 10-500, 10-50, 10-20, 25-1000, 25-500, 25-250, 100-1000, or 100-500 condensates). In some embodiments, the one or more reference condensates are a single condensate type (e.g., a condensate type that includes a common macromolecular component). In some embodiments, the one or more reference condensates are a plurality of condensate types, e.g., one type of a plurality of condensates includes a first macromolecular component and one type of a plurality of condensates does not include the first macromolecular component. In some embodiments, the one or more reference condensates and the one or more target condensates do not include a common macromolecular component. In some embodiments, the one or more reference condensates and the one or more target condensates include a common macromolecular component.

[0126] Many condensates are well known in the art, and many condensates can be identified or analyzed using known methods (for exemplary methods, such as microscopy, see Basturea, GN (“Biological Condensates,” MATER METHODS 2019;9:2794)). In some embodiments, the method further includes identifying one or more target condensates. In some embodiments, the method further includes identifying one or more reference condensates. In some embodiments, the one or more target condensates are cellular condensates. In some embodiments, the one or more reference condensates are cellular condensates. In some embodiments, the one or more reference condensates are in vitro condensates. Many cellular condensates have been described, and many more are known to form but have not yet been described or named. In some embodiments, the one or more target condensates are cytoplasmic condensates. In some embodiments, the one or more target condensates are nuclear condensates. In some embodiments, the one or more cellular condensates are 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. In some embodiments, the one or more cellular condensates are not stress granules.

[0127] In some embodiments, the one or more target condensates are a plurality of condensates. In some embodiments, the plurality of target condensates are all or a subset of a class of condensates in a portion of the cellular composition. In some embodiments, the plurality of target condensates are all or a subset of a class of condensates in cells in the cellular composition. In some embodiments, the plurality of target condensates are all or a subset of a class of condensates in a portion of the cells in the cellular composition. In some embodiments, the one or more reference condensates are a plurality of condensates. In some embodiments, the plurality of reference condensates are all or a subset of a class of condensates in a portion of the cellular composition. In some embodiments, the plurality of reference condensates are all or a subset of a class of condensates in cells in the cellular composition. In some embodiments, the plurality of reference condensates are all or a subset of a class of condensates in a portion of the cells in the cellular composition. In some embodiments, the plurality of reference condensates are all or a subset of a class of condensates in an in vitro condensate system.

[0128] In some embodiments, the portion of a cell is the cytoplasm, the nucleus, an organelle, or a non-membrane-bound compartment. In some embodiments, the class of condensates includes condensates that comprise specific macromolecules. In some embodiments, the class of condensates includes condensates that are cleavage bodies, the class of condensates includes condensates that are p-granules, the class of condensates includes condensates that are histone locus bodies, the class of condensates includes condensates that are multivesicular bodies, the class of condensates includes condensates that are neuronal RNA granules, the class of condensates includes condensates that are nuclear gems, the class of condensates includes condensates that are nuclear pores, the class of condensates includes condensates that are nuclear speckles, the class of condensates includes condensates that are nuclear stress bodies, the class of condensates includes condensates that are nucleoli, and the class of condensates includes Oct1 / PTF / transcription ( The class of condensates includes condensates that are OPT) domains, the class of condensates includes condensates that are paraspeckles, the class of condensates includes condensates that are juxtanucleolar compartments, the class of condensates includes condensates that are PML subnuclear structures, the class of condensates includes condensates that are PML oncogenic domains, the class of condensates includes condensates that are Polycomb bodies, the class of condensates includes condensates that are processing bodies, the class of condensates includes condensates that are Sam68 subnuclear structures, the class of condensates includes condensates that are stress granules, or the class of condensates includes condensates that are splicing speckles.

[0129] In some embodiments, the one or more target condensates and the one or more reference condensates are of the same class (or type) of condensate. For example, the one or more target condensates and the one or more reference condensates may contain the same one or more macromolecules, may be formed under the same conditions, may have the same response to a stimulus, may be formed in the same cell / tissue type, and / or may reside at the same location within a cell. In some embodiments, the one or more target condensates and the one or more reference condensates contain the same one or more macromolecules and reside at the same location within a cell. In some embodiments, the class of condensates includes condensates that are cleavage bodies, the class of condensates includes condensates that are p granules, the class of condensates includes condensates that are histone locus bodies, the class of condensates includes condensates that are multivesicular bodies, the class of condensates includes condensates that are neuronal RNA granules, the class of condensates includes condensates that are nuclear gems, the class of condensates includes condensates that are nuclear pores, the class of condensates includes condensates that are nuclear speckles, the class of condensates includes condensates that are nuclear stress bodies, the class of condensates includes condensates that are nucleoli, the class of condensates includes Oct1 / PTF / transcription ( The class of condensates includes condensates that are OPT) domains, the class of condensates includes condensates that are paraspeckles, the class of condensates includes condensates that are juxtanucleolar compartments, the class of condensates includes condensates that are PML subnuclear structures, the class of condensates includes condensates that are PML oncogenic domains, the class of condensates includes condensates that are Polycomb bodies, the class of condensates includes condensates that are processing bodies, the class of condensates includes condensates that are Sam68 subnuclear structures, the class of condensates includes condensates that are stress granules, or the class of condensates includes condensates that are splicing speckles.

[0130] In some embodiments, the one or more target condensates and the one or more reference condensates are different classes (or types) of condensates. For example, the one or more target condensates and the one or more reference condensates may contain one or more different macromolecules, may be formed under different conditions, may have different responses to stimuli, may be formed in different cell / tissue types, may have one or more different characteristics (e.g., shape, fluidity, size, composition, etc.) described herein, and / or may reside in different locations within a cell. In some embodiments, the one or more target condensates and the one or more reference condensates contain one or more of the same macromolecules and reside in different locations within a cell. In some embodiments, the one or more target condensates and the one or more reference condensates contain one or more of the same macromolecules and one or more different macromolecules. In some embodiments, the different macromolecules are variants. For example, in some embodiments, one macromolecule is a wild-type protein and the other macromolecule is a mutant protein. In some embodiments, one macromolecule is a mature form and the other macromolecule is an immature form. In some embodiments, one macromolecule does not have a post-translational modification and the other macromolecule does have a post-translational modification.

[0131] Dysregulation of various condensates can be associated with disease. For example, based on cellular and cell-free condensation experiments, disease-associated mutations in the fused in sarcoma (FUS) protein have been shown to cause abnormal phase separation behavior that directly contributes to the development of the motor neuron disease amyotrophic lateral sclerosis (ALS) (Naumann et al., 2018, Nat Commun, 9(1):335). Thus, in some embodiments, dysregulation of one or more target condensates is associated with disease. In some embodiments, the disease is a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease. In some embodiments, the dysregulation includes changes in one or more of the following: the amount of one or more target condensates, the composition of one or more target condensates, the location of one or more target condensates, the distribution of one or more target condensates, the size of one or more target condensates, the fluidity or coagulation of one or more target condensates, the dissolution of one or more target condensates, the shape of one or more target condensates, the concentration of one or more components of one or more target condensates, the distribution of one or more components of one or more target condensates, the diffusion coefficient of one or more components of one or more target condensates, post-translational modifications of one or more components of one or more target condensates, and uneven distribution of one or more components within one or more target condensates (e.g., components located in the core instead of the shell of the target condensate). In some embodiments, the changes are compared to a similar non-dysregulated condensate.

[0132] In some embodiments, the one or more target condensates are multiple condensates. In some embodiments, the compound does not measurably alter one or more of the following: the total number of the multiple target condensates, the size of the multiple target condensates, the shape of the multiple target condensates, the fluidity or solidification of the multiple target condensates, the location of the multiple target condensates, the surface area of ​​the multiple target condensates, the dissolution of the multiple target condensates, the post-translational modification of one or more components of the multiple target condensates, and the heterogeneous distribution of one or more components within the multiple target condensates.

[0133] In some embodiments, one or more target condensates or reference condensates are a single condensate (e.g., a single condensate type). In some embodiments, the compound does not measurably alter one or more characteristics selected from the following: the number of target condensates and / or reference condensates, the size of the target condensates and / or reference condensates, the location of the target condensates and / or reference condensates, the distribution of the target condensates and / or reference condensates, the surface area of ​​the target condensates and / or reference condensates, the dissolution of the target condensates and / or reference condensates, the shape of the target condensates and / or reference condensates, the fluidity or solidification of the target condensates and / or reference condensates, the post-translational modification of one or more components of the target condensates and / or reference condensates, and the heterogeneous distribution of one or more components within the target condensates and / or reference condensates. In some embodiments, one or more target condensates or reference condensates are a plurality of condensates (e.g., a plurality of condensate types). In some embodiments, the compound does not measurably alter one or more characteristics selected from the following: the number of target condensates and / or reference condensates, the size of the target condensates and / or reference condensates, the location of the target condensates and / or reference condensates, the distribution of the target condensates and / or reference condensates, the surface area of ​​the target condensates and / or reference condensates, the dissolution of the target condensates and / or reference condensates, the shape of the target condensates and / or reference condensates, the fluidity or solidification of the target condensates and / or reference condensates, post-translational modification of one or more components of the target condensates and / or reference condensates, and the heterogeneous distribution of one or more components within the target condensates and / or reference condensates.

[0134] In some embodiments, the location of one or more condensates (e.g., one or more target condensates, one or more reference condensates, a first set of one or more target condensates, a second set of one or more target condensates) is in any part of a cell (e.g., any aspect of the cytoplasm or nucleus of a cell). In some embodiments, the location of one or more condensates is in or based on association with a cytoplasmic organelle or particle. In some embodiments, the location of one or more condensates describes an association of one or more condensates with another cellular feature (e.g., the nucleus or the centroid). In some embodiments, the location of one or more condensates is relative to another cellular feature (e.g., the nucleus or the centroid). In some embodiments, the location of one or more condensates is based on distance to another cellular feature (e.g., the nucleus or the centroid).

[0135] In some embodiments, the number of one or more condensates is the total number of condensates in a cell or a portion of a cell. In some embodiments, the number of one or more condensates is the total number of condensates in a cell portion (e.g., the cytoplasm or nucleus). In some embodiments, the number of one or more condensates is an estimate of the total number of condensates in the cytoplasm (or nucleus) based on measurements of less than the entire cytoplasm (or nucleus). In some embodiments, the number of one or more condensates is the number of condensates in a portion of the cytoplasm (e.g., within a field of view, or in / associated with a cellular feature). In some embodiments, the number of one or more target condensates is reflected by the ratio of the total number of one or more target condensates to the total number of one or more reference condensates. In some embodiments, the ratio of the number of one or more target condensates to the number of one or more reference condensates that do not include the first macromolecule. In some embodiments, the ratio of the number of one or more target condensates to the number of one or more reference condensates is the ratio of the number of one or more target condensates in a portion of the cytoplasm to the number of one or more reference condensates in another portion of the cytoplasm. In some embodiments, the ratio of the number of one or more target condensates to one or more reference condensates is the ratio of the number of one or more target condensates to one or more reference condensates comprising a first macromolecule and located at a certain location (e.g., a nucleus).

[0136] In some embodiments, the distribution of one or more condensates is the distribution of one or more condensates in a portion of the cytoplasm (e.g., a cellular feature or field of view of the cytoplasm). In some embodiments, the distribution of one or more condensates is the distribution of one or more condensates relative to a cellular feature within the cytoplasm (e.g., a nucleus, organelle, or particle). In some embodiments, the distribution of one or more condensates is the distribution of one or more condensates in a portion of the cytoplasm (e.g., a field of view, relative to its location). In some embodiments, the distribution of one or more condensates is based on the distance of each condensate to a reference point. In some embodiments, the distribution of one or more condensates is the distribution of one or more condensates in a portion of the nucleus. The distribution can be uniform or non-uniform.

[0137] In some embodiments, the size of the one or more condensates is based on a maximum condensate transverse dimension measurement (e.g., diameter) of each of the one or more condensates. In some embodiments, the size of the one or more condensates is based on a perimeter of each of the one or more condensates. In some embodiments, the size of the one or more condensates is based on a cross-sectional area of ​​each of the one or more condensates or an image representation thereof (e.g., from a top-down view). In some embodiments, the size of the one or more condensates is based on a volume of each of the one or more condensates. In some embodiments, the size of the one or more condensates is based on an average size of the one or more condensates. In some embodiments, the characteristic associated with the one or more condensates is based on a size distribution (e.g., d5, d10, d90, or d95) of the one or more condensates. In some embodiments, the size of the one or more condensates is determined by a particle size measurement technique (e.g., dynamic light scattering technique).

[0138] In some embodiments, the stability of the one or more condensates is the stability of the one or more condensates over time in the presence of cellular activity or in the presence of a compound. In some embodiments, the stability is based, for example, on the maintenance of the size, number, shape, or amount of the one or more condensates.

[0139] In some embodiments, the diffusion coefficient of the one or more condensates is the diffusion coefficient of a component of the one or more condensates (e.g., the first macromolecule). In some embodiments, the diffusion coefficient of the one or more condensates is the diffusion coefficient of a component of the one or more condensates that is not the first macromolecule (e.g., another protein, nucleic acid, or chemical compound).

[0140] In some embodiments, the dissolution or size reduction of one or more condensates is based on the largest condensate cross-dimensional measurement (e.g., diameter) of each of the one or more condensates. In some embodiments, the dissolution or size reduction of one or more condensates is based on the perimeter of each of the one or more condensates. In some embodiments, the dissolution or size reduction of one or more condensates is based on the average size of the one or more condensates. In some embodiments, the dissolution or size reduction of one or more condensates is based on the size distribution (such as d5, d10, d90, or d95) of the one or more condensates.

[0141] In some embodiments, the surface area of ​​the one or more condensates is an estimated surface area based on the perimeter of each of the one or more condensates.

[0142] In some embodiments, the sphericity of the one or more condensates is based on how close each of the one or more condensates is to a perfect sphere. In some embodiments, the sphericity of the one or more condensates is an estimated sphericity based on a cross-sectional or top-down view of each of the one or more condensates. In some examples, the feature associated with the one or more condensates is the shape of each of the one or more RBM20 condensates. In some embodiments, the feature associated with the one or more condensates is a portion of the one or more condensates having a shape type or satisfying a shape parameter.

[0143] In some embodiments, the fluidity and / or solidification of the one or more condensates is based on the manner in which the one or more condensates fuse together and / or the change in structure, size, shape, sphericity, volume, number, and / or surface area of ​​each of the one or more condensates over time. In some embodiments, the fluidity and / or solidification of the one or more condensates is based on fiber formation.

[0144] In some embodiments, the features may be determined based on, for example, any one or more of an evaluation of one or more condensates within a cell or a portion thereof (such as the cytoplasm or nucleus or a portion of a cell), an evaluation of one or more condensates and / or macromolecules within, outside, or at any other location associated with a cell (e.g., the nucleus or a portion of a cell), or an evaluation of another macromolecule (such as another condensate component).

[0145] In some embodiments, determining the feature is based on an evaluation of at least one cell or at least a portion thereof. In some embodiments, the evaluation is an evaluation of a plurality of cells. In some embodiments, the portion of the cell is a field of view (e.g., a microscopic field or a portion thereof). In some embodiments, the portion of the cell is a defined region of an image of the cell or a portion thereof. In some embodiments, the defined region is based on one or more cellular characteristics, e.g., the boundary of the nucleus or cell membrane (e.g., by expression of a fluorescent fusion protein, a nuclear dye, or immunofluorescence (IF) staining). In some embodiments, the defined region is arbitrarily defined, e.g., manually or by software. In some embodiments, determining the feature is based on an iterative assessment. In some embodiments, the iterative assessment is based on multiple portions of an image or multiple images. In some embodiments, determining the feature is based on an average or distribution obtained from two or more portions of an image, two or more images, or two or more portions obtained from at least two or more images.

[0146] In some embodiments, determining the characteristics is based on imaging techniques. In some embodiments, the imaging techniques provide data for assessing one or more condensate- and / or macromolecule-associated characteristics, including the level of condensate-associated macromolecules. In some embodiments, the imaging techniques include obtaining an image of a composition comprising a cell or a portion thereof. In some embodiments, the image is a two-dimensional image. In some embodiments, the image is a three-dimensional image or a rendering thereof. In some embodiments, the imaging techniques are combined with features of another method useful in the methods described herein, such as fluorescence-activated cell sorting (FACS) or fluorescence-activated particle sorting (FAPS) techniques.

[0147] In some embodiments, the methods described herein include imaging a sample or a portion thereof (e.g., a cellular composition) via an imaging technique. In some embodiments, the imaging technique is a fluorescent imaging technique. In some embodiments, the imaging technique includes a fluorescent imaging technique. In some embodiments, the imaging technique includes colorimetric and fluorescent imaging techniques. In some embodiments, the fluorescent imaging technique includes evaluating a fluorescent signal in one or more channels. In some embodiments, the detected light is due to direct labeling of the target (e.g., incorporation or attachment of a label into a compound or macromolecule). In some embodiments, the detected light is due to indirect labeling of the target, such as a labeled probe that specifically binds to a macromolecule (e.g., a labeled anti-antibody or fragment thereof, a nuclear dye, or Annexin V luciferase) that binds to phosphatidylserine (PS) exposed to the outer leaflet of the cell membrane during apoptosis. In some embodiments, the label includes Dendra2, GFP, or mCherry. In some embodiments, determining the characteristic includes an immunofluorescence technique. In some embodiments, the methods described herein include the use of direct and indirect labeling techniques.

[0148] In some embodiments, the method further comprises determining one or more cellular features of the cells, if present (e.g., in addition to condensates and / or macromolecules). One of skill in the art will readily appreciate that cellular features can be determined in several ways. In some embodiments, the method further comprises contacting at least a portion of the composition or cells with a stain, such as a nuclear dye. In some embodiments, the stain is a fluorescent stain. In some embodiments, the stain is a histochemical stain. In some embodiments, the stain is an immune-based stain, such as those used in immunohistochemistry or immunocytochemistry techniques. In some embodiments, the stain allows visualization of cellular features, if present, such as at least a portion of any one or more of the following: plasma or cell membrane, cytoplasm, cytoskeleton, nucleus, endoplasmic reticulum (rough and / or smooth), ribosomes, Golgi apparatus, lysosomes, mitochondria, vacuoles, or centrosomes. In some embodiments, the methods described herein further comprise contacting at least a portion of the cellular composition or cells with a fixative.

[0149] In some embodiments, the feature is determined based on the ratio of the number of cells having one or more target condensates with the feature to the number of cells not having one or more target condensates with the feature, hi some embodiments, the feature is determined based on the number of cells having one or more target condensates with the feature.

[0150] In some embodiments, the characteristic is determined over a period of time (e.g., at two or more time points). In some embodiments, determining the characteristic includes assessing a change in the characteristic over a period of time. In some embodiments, the characteristic, such as the association level, is assessed during the life of the condensate. For example, as discussed herein, because condensates are dynamic and change over time, the characteristic is assessed at a time point at which the effect of the compound on the condensate can be accurately measured. In some embodiments, the characteristic, such as the association level, is assessed during the life of the cell (e.g., during or outside mitosis).

[0151] In some embodiments, the features are determined using one or more measurements and / or techniques. In some embodiments, multiple features associated with one or more condensates and / or polypeptides are determined using one or more measurements and / or techniques.

[0152] In some embodiments, the methods described herein include techniques for visualizing, analyzing, and / or quantifying, for example, macromolecules and / or their precursors. Such techniques are well known to those of skill in the art. For example, microscopy techniques for visualizing proteins, such as fluorescently labeled proteins, including those compatible with cellular systems, are encompassed herein. Also encompassed herein are mass spectrometry (MS) techniques for analyzing protein composition (e.g., by the cross-linking MS technique "XL-MS"), including studying post-translational modifications, protein quantification, and condensate composition. Also encompassed herein are functional assays for assessing cellular processes. Also encompassed herein are enrichment and / or isolation techniques, such as centrifuge techniques for isolating cellular fractions or affinity-based techniques for isolating proteins or nucleic acids. In some embodiments, the techniques evaluate a feature in one or more cells or in a defined region(s) of one or more cells. In some embodiments, the techniques evaluate one or more of intensity, area, and condensate number in one or more cells or in a defined region(s) of one or more cells. In some embodiments, the techniques evaluate the number of cells with or without the feature. In some embodiments, the technique evaluates the number of condensates in cells with or without features. In some embodiments, the method includes using z-scores to evaluate the assay and its results. Z-scores and their uses are known in the art. See, e.g., Zhang et al., J Biomol Screen, 1999.

[0153] In some embodiments, the location of the one or more condensates is determined by assessing the presence, absence, or level of one or more condensates in or associated with at least a portion of a cell, such as a cellular feature (e.g., the cytoplasm or nucleus). In some embodiments, determining the location of the one or more condensates comprises determining a cellular feature.

[0154] In some embodiments, the number of one or more condensates is determined by assessing the total number of condensates in the cell, such as the cytoplasm. In some embodiments, the number of one or more condensates is determined by assessing the number of condensates in a portion of the cell (e.g., the cytoplasm), such as in a field of view. In some embodiments, the number of one or more condensates is determined by estimating the total number of condensates in the cell or a portion thereof (e.g., the cytoplasm) based on measurements of less than the total number of cells.

[0155] In some embodiments, the size of the one or more condensates is determined by assessing the largest condensate cross-dimensional measurement (e.g., diameter) of each of the one or more condensates. In some embodiments, the size of the one or more condensates is determined by assessing the perimeter of each of the one or more condensates. In some embodiments, the size of the one or more condensates is determined by assessing the cross-sectional area of ​​each of the one or more condensates or an image representation thereof (e.g., from a top-down view). In some embodiments, the size of the one or more condensates is determined by a particle size measurement technique (e.g., dynamic light scattering technique).

[0156] In some embodiments, dissolution or reduction in size of one or more condensates is determined based on a change in the structure of each of the one or more condensates over time in the presence of cellular activity or in the presence of a compound.

[0157] In some embodiments, the surface area of ​​one or more condensates is determined based on estimating the surface area using a measured parameter (e.g., perimeter, largest condensate cross-dimensional measurement) of each of the one or more condensates.

[0158] In some embodiments, the method includes contacting a cellular composition with a compound, wherein the cellular composition comprises one or more target condensates, and the method further includes forming the one or more target condensates prior to step (a). In some embodiments, the method includes contacting a cellular composition with a compound, wherein the one or more target condensates are formed after contacting the cellular composition with the compound, and the method further includes forming the one or more target condensates. Methods for forming condensates are known. For example, cellular stress can cause the formation of stress granules. Examples of cellular stress include arsenate treatment, temperature change, or pH change. Thus, in some embodiments, forming the one or more target condensates includes contacting the cellular composition with arsenate, acid, or base, or changing the temperature of the cellular composition.

[0159] For methods requiring a first set of one or more target condensates and a second set of one or more target condensates, any of the target condensates disclosed herein may be used. In some embodiments, the first set of one or more target condensates and the second set of one or more target condensates are of different classes of condensates (e.g., any of the classes of condensates disclosed herein).

[0160] In some embodiments, the method includes analyzing one or more images to evaluate characteristics of the condensate. In some embodiments, the analysis includes mapping cell boundaries or boundaries of portions of cells (e.g., organelles). In some embodiments, the analysis includes mapping condensate boundaries. In some embodiments, the analysis of the images is completed and / or facilitated by analysis software. In some embodiments, one or more images are compared, for example, in a time course study. In some embodiments, the analysis includes measuring the intensity of signals (e.g., fluorescent fusion protein, IF staining, or luminescence signals) of condensates, macromolecules, and / or compounds (e.g., compounds co-localized with condensates and / or macromolecules). In some embodiments, the analysis further includes calculating the enrichment of the measured signals (e.g., measured signals within condensate boundaries).

[0161] In some embodiments, colocalization of condensate components (e.g., a first macromolecule and a second macromolecule) is determined by assessing the presence, absence, or level of a component (or compound) in or associated with a condensate. In some embodiments, the assessment, such as measuring, is performed directly or indirectly. In some embodiments, the condensate components are assessed using a mass spectrometry technique (e.g., APEX or XL-MS). In some embodiments, the condensate components are assessed using a FAPS technique. In some embodiments, the assessing involves the use of a labeling technique (e.g., directly attaching a label to a condensate component or using immunolabeling such as used in IF techniques). In some embodiments, the condensate is isolated and / or enriched, and then the presence, absence, or level of a component in or associated with the condensate is assessed. In some embodiments, the condensate is not isolated and / or enriched, e.g., from other cellular components, but is assessed, e.g., in situ. In some embodiments, the condensate or cells containing the condensate are fixed prior to assessment. In some embodiments, the condensate or cells containing the condensate are not fixed prior to assessment. In some embodiments, the amount of components in the condensates can be quantified using techniques known in the art, such as imaging, mass spectrometry, Western blot, immunoprecipitation (IP), immunofluorescence (IF) staining, in situ, FISH, Northern blot, or qPCR.

[0162] macromolecule In some embodiments, the macromolecule (e.g., the first macromolecule, one or more of the at least one additional macromolecule, or the reference macromolecule) is a nucleic acid or a protein. In some embodiments, the macromolecule (e.g., the first macromolecule, one or more of the at least one additional macromolecule, or the reference macromolecule) is a protein or protein fragment. In some embodiments, the protein or protein fragment comprises a low complexity domain or an intrinsically disordered sequence. In some embodiments, the macromolecule is a transcription factor or an RNA-binding protein. In some embodiments, the macromolecule is a nucleic acid, such as RNA or DNA. In some embodiments, the macromolecule is RNA.

[0163] In some embodiments, the first macromolecule is FUS or eIF3. In some embodiments, the at least one additional macromolecule is FUS, eIF3, G3BP1, FUS and G3BP1, or eIF3 and G3BP1. In some embodiments, the first macromolecule is FUS and the at least one additional macromolecule is eIF3, G3BP1, or eIF3 and G3BP1. In some embodiments, the first macromolecule is eIF3 and the at least one additional macromolecule is FUS, G3BP1, or FUS and G3BP1.

[0164] In some embodiments, the first macromolecule comprises a mutation that alters the level of association of the first macromolecule with one or more target condensates compared to a related macromolecule that does not comprise the mutation. In some embodiments, one or more of the at least one additional macromolecule comprises a mutation that alters the corresponding level of association of the first macromolecule with one or more target condensates compared to a related macromolecule that does not comprise the mutation. In some embodiments, each of the at least one additional macromolecule comprises a mutation that alters the corresponding level of association of the first macromolecule with one or more target condensates compared to a related macromolecule that does not comprise the mutation. In some embodiments, the first macromolecule comprises a mutation that alters the level of association of the first macromolecule with one or more target condensates compared to a related macromolecule that does not comprise the mutation. In some embodiments, the first macromolecule does not have altered association (or has altered association by up to about two-fold) with one or more target condensates compared to a related macromolecule that does not comprise the mutation. In some embodiments, the first macromolecule comprises a mutation that alters the level of association of the second macromolecule with one or more target condensates. In some embodiments, the first macromolecule and the second macromolecule are different. In some embodiments, the first macromolecule comprises a mutation that alters the level of association of the first macromolecule with one or more target condensates in only one or more cell types (e.g., neurons) or tissue types compared to the association of the first macromolecule with one or more target condensates in another cell type (e.g., cardiomyocytes) or tissue type. In some embodiments, the mutation is associated with a disease, such as a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease.

[0165] In some embodiments, the macromolecule (e.g., one or more of the first macromolecule or the at least one additional macromolecule) is aberrantly expressed in a disease state or a stress state. In some embodiments, the first macromolecule is aberrantly expressed in a disease state or a stress state. In some embodiments, one or more of the at least one additional macromolecule is aberrantly expressed in a disease state or a stress state. In some embodiments, each of the at least one additional macromolecule is aberrantly expressed in a disease state. In some embodiments, the level of association of the macromolecule with one or more target condensates in a disease state or a stress state is altered compared to the level of association of the macromolecule with one or more target condensates in a normal (e.g., healthy or non-stressed) state. In some embodiments, the level of association of the first macromolecule with one or more target condensates in a disease state or a stress state is altered compared to the level of association of the first macromolecule with one or more target condensates in a normal state. In some embodiments, the level of association of one or more of the at least one additional macromolecule with one or more target condensates in a disease state or stress state is altered compared to the level of association of one or more of the at least one additional macromolecule with one or more target condensates in a normal state. In some embodiments, the level of association of each of the at least one additional macromolecule with one or more target condensates in a disease state or stress state is altered compared to the level of association of each of the at least one additional macromolecule with one or more target condensates in a normal state. In some embodiments, the level of association of a first macromolecule with one or more target condensates in a disease state or stress state is altered (e.g., at least about two-fold altered) compared to the level of association of the first macromolecule with one or more target condensates in a normal state, while the level of association of one or more of the at least one additional macromolecule with one or more target condensates in a disease state or stress state is unchanged (or, e.g., altered by less than two-fold) compared to the level of association of one or more of the at least one additional macromolecule with one or more target condensates in a normal state.

[0166] In some embodiments, the at least one additional macromolecule is 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more macromolecules. In some embodiments, the at least one additional macromolecule is 2 or more macromolecules. In some embodiments, the at least one additional macromolecule is 3 or more macromolecules. In some embodiments, the at least one additional macromolecule is 5 or more macromolecules. In some embodiments, the at least one additional macromolecule is 1 to 10 macromolecules (e.g., 1 to 3, 2 to 5, 2 to 9, 3 to 8, 4 to 7, 5 to 10, or 7 to 10 macromolecules). In some embodiments, the at least one additional macromolecule is a single macromolecule. In some embodiments, the at least one additional macromolecule is 2, 3, 4, 5, 6, 7, 8, 9, or 10 macromolecules. In some embodiments, the at least one additional macromolecule is 2 macromolecules. In some embodiments, the at least one additional macromolecule is 3 macromolecules. In some embodiments, the at least one additional macromolecule is 5 macromolecules.

[0167] In some embodiments, one or more of the first macromolecule and / or the at least one additional macromolecule is a fusion protein. In some embodiments, the fusion protein comprises a label. In some embodiments, the label is a fluorescent protein or a luminescent protein. In some embodiments, the first macromolecule is a first fusion protein comprising a first label, and one of the at least one additional macromolecule is a second fusion protein comprising a second label. In some embodiments, the at least one additional macromolecule is a plurality of macromolecules. In some embodiments, the plurality comprises a first fusion protein comprising a first label and a second fusion protein comprising a second label. In some embodiments, the first macromolecule is a first fusion protein comprising a first label, and the plurality comprises a second fusion protein comprising a second label and a third fusion protein comprising a third label. In some embodiments, the first label and the second label are distinguishable. In some embodiments, the first label, the second label, and the third label are distinguishable.

[0168] In some embodiments, one or more of the first macromolecule and / or the at least one additional macromolecule are tagged with a label (e.g., fluorescein). Additionally, or alternatively, the macromolecule may be labeled by contacting the cellular composition with a label. Thus, in some embodiments, the method further comprises contacting the cellular composition with a label. In some embodiments, the method further comprises labeling one or more of the first macromolecule and / or the at least one additional macromolecule. In some embodiments, labeling comprises contacting the cellular composition with an antibody or antigen-binding fragment thereof comprising a label. In some embodiments, the label is a radioactive label, a colorimetric label, or a fluorescent label.

[0169] In some embodiments, the method includes contacting the cell composition with a fixative. Exemplary fixatives include formaldehyde, glutaraldehyde, and paraformaldehyde. In some embodiments, the method includes contacting the cell composition with a permeabilization agent. Exemplary permeabilization agents include a saponin compound, methanol, acetone, or a detergent (e.g., Triton® X-100).

[0170] How to characterize a compound Also provided herein are methods of identifying compound characteristics, such as moieties that are responsible, in whole or in part, for any one or more of the following specificities: macromolecule specificity, condensate specificity, and tissue specificity. In some embodiments, the method of identifying compound characteristics comprises evaluating a plurality of compounds according to the methods described herein, and then identifying compound characteristics that include a subset or all of the plurality of compounds, wherein the subset or all of the plurality of compounds exhibit similar specificities.

[0171] In some aspects, the methods described herein are used in screening to assay a library of compounds. In some aspects, the methods described herein are used in screening to assay a library of condensates. In some aspects, the methods described herein are used in screening to assay a library of cells (e.g., disease model cell lines). In some aspects, the methods described herein are used in screening to assay a library of cells, the library of cells comprising cells with different mutations in macromolecules, such as disease-associated mutations. In some embodiments, the methods described herein comprise evaluating two or more compounds in a single system, such as a composition comprising cells.

[0172] In some aspects, the methods described herein are formatted for any level of throughput (eg, high throughput, medium throughput, or low throughput).

[0173] In some embodiments, the methods described herein further comprise evaluating the identified compound using a second cell-based assay, e.g., in a cell composition of a similar disease type (e.g., breast cancer) but with a different mutation (e.g., a different allele). In some embodiments, the methods described herein further comprise evaluating the identified compound using an in vitro assay.

[0174] In some embodiments, the methods described herein further comprise determining the amount of the compound in the cell, or portion thereof, or one or more target condensates. In some embodiments, determining the amount of the compound comprises quantitatively detecting the compound. In some embodiments, determining the amount of the compound comprises quantitatively detecting a label of the compound. In some embodiments, determining the amount of the compound comprises detecting the activity of the compound and calculating the amount of compound required to cause the detected amount of activity (e.g., cause dissociation of the macromolecule from one or more target condensates). In some aspects, the amount of the compound is determined by mass spectrometry, liquid chromatography, and / or UV-visible spectrophotometry. In some embodiments, the amount of the compound is determined by fluorescence microscopy. A standard curve may be used to aid in determining the amount of the compound.

[0175] In some embodiments, provided herein are methods for identifying characteristics of compounds associated with preferentially affecting the level of association between a first macromolecule and one or more target condensates, the methods comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; (b) determining the level of association between the first macromolecule and one or more target condensates and the level of association between at least one additional macromolecule and one or more target condensates; (c) performing steps (a) and (b) for a plurality of compounds; and (d) identifying characteristics that a subset or all of the identified compounds have in common in addition to their ability to preferentially affect the level of association between the first macromolecule and one or more target condensates. Also provided herein is a method for identifying a compound characteristic associated with preferentially affecting the level of association between a first macromolecule and a first set of one or more target condensates, the method comprising performing the steps of the method disclosed herein: identifying a plurality of compounds that preferentially affect the level of association between a first macromolecule and a first set of one or more target condensates; and identifying a characteristic that a subset or all of the identified compounds have in common in addition to their ability to preferentially affect the level of association between the first macromolecule and the first set of one or more target condensates.

[0176] Also provided herein is a method for identifying a characteristic of a compound associated with preferentially reducing the level of association of a first macromolecule with one or more target condensates, comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; (b) determining the level of association of the first macromolecule with one or more target condensates and the level of association of at least one additional macromolecule with one or more target condensates; (c) performing steps (a) and (b) for a plurality of compounds; and (d) identifying a characteristic that a subset or all of the identified compounds have in common in addition to their ability to preferentially reduce the level of association of the first macromolecule with one or more target condensates. Also provided herein is a method for identifying a compound characteristic associated with preferentially reducing the level of association between a first macromolecule and a first set of one or more target condensates, the method comprising performing the steps of the method disclosed herein: identifying a plurality of compounds that preferentially reduce the level of association between a first macromolecule and a first set of one or more target condensates; and identifying a characteristic that a subset or all of the identified compounds have in common in addition to their ability to preferentially reduce the level of association between the first macromolecule and the first set of one or more target condensates.

[0177] Also provided herein is a method for identifying a characteristic of a compound associated with preferentially increasing the level of association of a first macromolecule with one or more target condensates, comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; (b) determining the level of association of the first macromolecule with one or more target condensates and the level of association of at least one additional macromolecule with one or more target condensates; (c) performing steps (a) and (b) for a plurality of compounds; and (d) identifying a characteristic that a subset or all of the identified compounds have in common in addition to their ability to preferentially increase the level of association of the first macromolecule with one or more target condensates. Also provided herein is a method for identifying a compound characteristic associated with preferentially increasing the level of association between a first macromolecule and a first set of one or more target condensates, the method comprising performing the steps of the method disclosed herein: identifying a plurality of compounds that preferentially increase the level of association between a first macromolecule and a first set of one or more target condensates; and identifying a characteristic that a subset or all of the identified compounds have in common in addition to their ability to preferentially increase the level of association between the first macromolecule and the first set of one or more target condensates.

[0178] Also provided herein is a method for identifying a compound characteristic associated with preferentially associating a first macromolecule with one or more target condensates, comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; (b) determining whether the first macromolecule and at least one additional macromolecule associate with one or more target condensates; (c) performing steps (a) and (b) for a plurality of compounds; and (d) identifying a characteristic that a subset or all of the identified compounds have in common in addition to the ability to preferentially associate the first macromolecule with one or more target condensates. Also provided herein is a method for identifying compound characteristics associated with preferentially associating a first macromolecule with a first set of one or more target condensates, the method comprising performing the steps of the method disclosed herein: identifying a plurality of compounds that preferentially associate a first macromolecule with one or more target condensates; and identifying characteristics that a subset or all of the identified compounds have in common in addition to their ability to preferentially associate a first macromolecule with the first set of one or more target condensates.

[0179] Also provided herein is a method for identifying a characteristic of a compound associated with preferentially dissociating a first macromolecule from one or more target condensates, comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; (b) determining whether the first macromolecule and at least one additional macromolecule dissociate from the one or more target condensates; (c) performing steps (a) and (b) for a plurality of compounds; and (d) identifying a characteristic that a subset or all of the identified compounds have in common in addition to the ability to preferentially dissociate the first macromolecule from one or more target condensates. Also provided herein is a method for identifying a compound characteristic associated with preferentially dissociating a first macromolecule from a first set of one or more target condensates, the method comprising performing the steps of the method disclosed herein: identifying a plurality of compounds that preferentially dissociate a first macromolecule from one or more target condensates; and identifying a characteristic that a subset or all of the identified compounds have in common in addition to their ability to preferentially dissociate the first macromolecule from the first set of one or more target condensates.

[0180] In some embodiments, provided herein are methods for identifying a characteristic of a compound associated with preferentially affecting the level of association of a first macromolecule with a first set of one or more condensates relative to one or more other sets of one or more condensates (e.g., a second set of one or more condensates), the method comprising: (a) contacting a cellular composition with the compound, wherein (i) the cellular composition comprises the first set of one or more condensates and one or more other sets of one or more condensates (e.g., the second set of one or more condensates), and / or (ii) simultaneously with and / or after contacting the cellular composition with the compound, the first set of one or more condensates and one or more other sets of one or more condensates. (b) contacting a plurality of compounds to form one or more condensates with the first macromolecule and one or more other sets of one or more condensates (e.g., a second set of one or more condensates); (b) determining the level of association between the first macromolecule and the first set of one or more condensates and the level of association between the first macromolecule and each other set of one or more condensates (e.g., a second set of one or more condensates); (c) performing steps (a) and (b) for a plurality of compounds; and (d) identifying characteristics that a subset or all of the identified compounds have in common in addition to their ability to preferentially affect the level of association between the first macromolecule and the first set of one or more condensates. In some embodiments, the first (or other) set of one or more condensates are of the same type, e.g., all are stress granules. In some embodiments, the first (or other) set of one or more condensates share a common macromolecule. In some embodiments, the method further includes determining the level of association between one or more additional macromolecules and the first set of one or more condensates and the level of association between the one or more additional macromolecules and each other set of one or more condensates. In some embodiments, the method further comprises determining the level of association of the first macromolecule with the reference condensate and / or the level of association of one or more additional macromolecules with the reference condensate.

[0181] In some embodiments, provided herein are methods for identifying a characteristic of a compound that is associated with preferentially affecting the level of association of a first macromolecule with one or more target condensates in a first set of one or more cell / tissue types compared to one or more other sets of one or more cell / tissue types, comprising: (a) contacting a first set of one or more cellular compositions comprising one or more cell / tissue types and one or more other sets of one or more cellular compositions comprising one or more cell / tissue types with a compound, wherein (i) each cellular composition comprises one or more target condensates, and / or (ii) simultaneously with and / or after contacting each cellular composition with the compound, one or more target condensates are associated with one or more target condensates. (b) determining the level of association between the first macromolecule and the one or more target condensates in a first set of one or more cellular compositions and the level of association between the first macromolecule and the one or more target condensates in each other set of one or more cellular compositions; (c) performing steps (a) and (b) for a plurality of compounds; and (d) identifying characteristics that a subset or all of the identified compounds have in common in addition to their ability to preferentially affect the level of association between the first macromolecule and the one or more target condensates in the first set of one or more cellular compositions comprising one or more cell / tissue types. In some embodiments, the method further includes determining the level of association between the one or more additional macromolecules and the one or more target condensates in the first set of one or more cellular compositions and the level of association between the one or more additional macromolecules and the one or more target condensates in each other set of one or more cellular compositions. In some embodiments, the method further comprises determining a level of association between the first macromolecule and the reference condensate in a first set of one or more cellular compositions and a level of association between the first macromolecule and the reference condensate in each other set of one or more cellular compositions. In some embodiments, the method further comprises determining a level of association between the one or more additional macromolecules and the reference condensate in the first set of one or more cellular compositions and a level of association between the one or more additional macromolecules and the reference condensate in each other set of one or more cellular compositions.

[0182] In some embodiments, the methods described herein can further identify one or more characteristics that a subset or all of the identified compounds have in common that alter one or more of the following: (i) the location of one or more target condensates, (ii) the distribution of one or more target condensates and / or their components (e.g., first macromolecules), (iii) the number of one or more target condensates, (iv) the size of one or more target condensates, (v) the ratio of the amount of one or more target condensates to a reference condensate, (vi) a functional activity associated with one or more target condensates, (vii) the composition of one or more target condensates, (viii) the co-localization of one or more target condensates with a biomolecule, (ix) the diffusion coefficient of one or more target condensate components (e.g., first macromolecules), (x) the stability of one or more target condensates. (xi) dissolution or size reduction of one or more target condensates; (xii) surface area of ​​one or more target condensates; (xiii) sphericity of one or more target condensates; (xiv) fluidity of one or more target condensates; (xv) solidification of one or more target condensates; (xvi) location of a condensate component (e.g., a first macromolecule); (xvii) amount of a condensate component (e.g., a first macromolecule) or its precursor; (xviii) condensate fractionation of a biomolecule (e.g., a first macromolecule) into one or more target condensates; (xix) functional activity associated with a condensate component (e.g., a first macromolecule); (xx) aggregation of a condensate component (e.g., a first macromolecule); (xxi) post-translational modification state of a condensate component (e.g., a first macromolecule); and (xxii) amount of degradation products of a condensate component (e.g., a first macromolecule).

[0183] Methods for designing compounds Also provided herein are methods for designing compounds with one or more of the following specificities: macromolecule specificity, condensate specificity, and tissue specificity. In some embodiments, the design method comprises selecting and / or constructing one or more moieties with the desired specificity.

[0184] In some embodiments, a method for designing a compound that preferentially affects the level of association between a first macromolecule and one or more target condensates includes: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; (b) determining the level of association between the first macromolecule and one or more target condensates and the level of association between at least one additional macromolecule and one or more target condensates; (c) performing steps (a) and (b) for a plurality of compounds; (d) identifying characteristics that a subset or all of the identified compounds have in common in addition to their ability to preferentially affect the level of association between the first macromolecule and one or more target condensates; and (e) designing a compound that includes the identified characteristics, thereby designing a compound that preferentially affects the level of association between the first macromolecule and one or more target condensates.

[0185] Also provided herein is a method for designing a compound that preferentially reduces the level of association between a first macromolecule and one or more target condensates, comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; (b) determining the level of association between the first macromolecule and one or more target condensates and the level of association between at least one additional macromolecule and one or more target condensates; (c) performing steps (a) and (b) for a plurality of compounds; (d) identifying characteristics that a subset or all of the identified compounds have in common in addition to their ability to preferentially reduce the level of association between the first macromolecule and one or more target condensates; and (e) designing a compound that comprises the identified characteristics, thereby preferentially reducing the level of association between the first macromolecule and one or more target condensates.

[0186] Also provided herein is a method for designing a compound that preferentially increases the level of association between a first macromolecule and one or more target condensates, comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; (b) determining the level of association between the first macromolecule and the one or more target condensates and the level of association between at least one additional macromolecule and the one or more target condensates; (c) performing steps (a) and (b) for a plurality of compounds; (d) identifying characteristics that a subset or all of the identified compounds have in common in addition to their ability to preferentially increase the level of association between the first macromolecule and one or more target condensates; and (e) designing a compound that comprises the identified characteristics, thereby designing a compound that preferentially increases the level of association between the first macromolecule and one or more target condensates.

[0187] Also provided herein is a method for designing a compound that preferentially associates a first macromolecule with one or more target condensates, comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; (b) determining whether the first macromolecule and at least one additional macromolecule associate with one or more target condensates; (c) performing steps (a) and (b) with a plurality of compounds; (d) identifying characteristics that a subset or all of the identified compounds have in common in addition to their ability to preferentially associate the first macromolecule with one or more target condensates; and (e) designing a compound that includes the identified characteristics, thereby designing a compound that preferentially associates the first macromolecule with one or more target condensates.

[0188] Also provided herein is a method for designing a compound that preferentially dissociates a first macromolecule from one or more target condensates, comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises one or more target condensates and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; (b) determining whether the first macromolecule and at least one additional macromolecule dissociate from the one or more target condensates; (c) performing steps (a) and (b) with a plurality of compounds; (d) identifying characteristics that a subset or all of the identified compounds have in common in addition to their ability to preferentially dissociate the first macromolecule from one or more target condensates; and (e) designing a compound that includes the identified characteristics, thereby designing a compound that preferentially dissociates the first macromolecule from one or more target condensates.

[0189] In some embodiments, provided herein are methods for identifying a characteristic of a compound associated with preferentially affecting the level of association of a first macromolecule with a first set of one or more condensates relative to one or more other sets of one or more condensates (e.g., a second set of one or more condensates), the method comprising: (a) contacting a cellular composition with the compound, wherein (i) the cellular composition comprises the first set of one or more condensates and one or more other sets of one or more condensates (e.g., the second set of one or more condensates), and / or (ii) simultaneously with and / or after contacting the cellular composition with the compound, the first set of one or more condensates and one or more other sets of one or more condensates (e.g., the second set of one or more condensates). (b) contacting a plurality of compounds to form a first set of one or more condensates; (b) determining the level of association of the first macromolecule with a first set of one or more condensates and the level of association of the first macromolecule with each other set of one or more condensates (e.g., a second set of one or more condensates); (c) performing steps (a) and (b) for a plurality of compounds; (d) identifying characteristics that a subset or all of the identified compounds have in common in addition to their ability to preferentially affect the level of association of the first macromolecule with the first set of one or more condensates; and (e) designing compounds that include the identified characteristics, thereby preferentially affecting the level of association of the first macromolecule with the first set of one or more condensates. In some embodiments, the first (or other) set of one or more condensates are of the same type, e.g., all are stress granules. In some embodiments, the first (or other) set of one or more condensates share a common macromolecule.

[0190] In some embodiments, provided herein are methods for identifying a characteristic of a compound associated with preferentially affecting the level of association of a first macromolecule with one or more target condensates in a first set of one or more cell / tissue types compared to one or more other sets of one or more cell / tissue types, the method comprising: (a) contacting a first set of one or more cellular compositions comprising the one or more cell / tissue types and one or more other sets of one or more cellular compositions comprising the one or more cell / tissue types with a compound, wherein (i) each cellular composition comprises one or more target condensates, and / or (ii) one or more target condensates are formed simultaneously with and / or after contacting each cellular composition with the compound; and (b) contacting a first set of one or more cellular compositions with one or more target condensates. (c) performing steps (a) and (b) for a plurality of compounds; (d) identifying characteristics that a subset or all of the identified compounds have in common in addition to their ability to preferentially affect the level of association of the first macromolecule with one or more target condensates in the first set of one or more cellular compositions comprising one or more cell / tissue types; and (e) designing compounds that comprise the identified characteristics, thereby preferentially affecting the level of association of the first macromolecule with one or more target condensates in the first set of one or more cellular compositions comprising one or more cell / tissue types.

[0191] In some embodiments, the methods described herein further include identifying one or more characteristics that a subset or all of the identified compounds have in common in that they alter one or more of the following: (i) the location of one or more target condensates, (ii) the distribution of one or more target condensates and / or their components (e.g., first macromolecules), (iii) the number of one or more target condensates, (iv) the size of one or more target condensates, (v) the ratio of the amount of one or more target condensates to a reference condensate, (vi) a functional activity associated with one or more target condensates, (vii) the composition of one or more target condensates, (viii) the co-localization of one or more target condensates with a biomolecule, (ix) the diffusion coefficient of one or more target condensate components (e.g., first macromolecules), (x) the stability of one or more target condensates. (xi) dissolution or size reduction of one or more target condensates, (xii) surface area of ​​one or more target condensates, (xiii) sphericity of one or more target condensates, (xiv) fluidity of one or more target condensates, (xv) solidification of one or more target condensates, (xvi) location of a condensate component (e.g., a first macromolecule), (xvii) amount of a condensate component (e.g., a first macromolecule) or its precursor, (xviii) condensate fractionation of a biomolecule (e.g., a first macromolecule) into one or more target condensates, (xix) functional activity associated with a condensate component (e.g., a first macromolecule), (xx) aggregation of a condensate component (e.g., a first macromolecule), (xxi) post-translational modification state of a condensate component (e.g., a first macromolecule), and (xxii) amount of degradation products of a condensate component (e.g., a first macromolecule). In some embodiments, the methods described herein further include designing compounds that further comprise one or more of the above-identified characteristics (in addition to preferentially affecting the level of association between the macromolecule and one or more target condensates), thereby designing compounds that not only preferentially affect the level of association between the macromolecule and one or more target condensates, but also can alter one or more of the above-mentioned characteristics of the condensate / condensate component.

[0192] In some embodiments, the methods described herein can be used to develop one or more rule sets based on the achieved desired change in the association of a macromolecule with one or more target condensates (and / or the desired change in one or more condensate / condensate component characteristics described above). In some embodiments, the one or more rule sets can be used as a basis for identifying and / or designing one or more compounds using approaches including modeling, computer- and / or computation-based techniques (e.g., bioinformatics, cheminformatics, and / or artificial intelligence (AI)-based identification) of compounds with the desired modulation of one or more characteristics described herein. Also provided is computer software for determining and / or applying one or more rule sets.

[0193] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present disclosure. The present disclosure is further illustrated by the following examples, which should not be construed as limiting the disclosure in scope or spirit to the specific procedures described therein.

[0194] Illustrative Embodiments The provided embodiments include the following:

[0195] Embodiment 1. A method for identifying a compound that preferentially affects the level of association between a first macromolecule and one or more target condensates, comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises the one or more target condensates, and / or (ii) the one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; and (b) determining the level of association between the first macromolecule and the one or more target condensates and the level of association between at least one additional macromolecule and the one or more target condensates, wherein the compound preferentially affects the level of association between the first macromolecule and the one or more target condensates if the compound alters the level of each additional macromolecule compared to a reference level of each additional macromolecule more than the level of the first macromolecule compared to a first reference level.

[0196] Embodiment 2. The method of embodiment 1, wherein said compound does not measurably alter said level of each additional macromolecule compared to said reference level of each additional macromolecule.

[0197] Embodiment 3. A method for identifying a compound that preferentially increases the level of association between a first macromolecule and one or more target condensates, comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises the one or more target condensates, and / or (ii) the one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; and (b) determining the level of association between the first macromolecule and the one or more target condensates and the level of association between at least one additional macromolecule and the one or more target condensates, wherein the compound preferentially increases the level of association between the first macromolecule and the one or more target condensates if the compound increases the level of the first macromolecule compared to a first reference level more than the level of each additional macromolecule compared to a reference level of each additional macromolecule.

[0198] Embodiment 4. The method of embodiment 3, wherein the compound does not measurably increase the level of each additional macromolecule compared to the reference level of each additional macromolecule.

[0199] Embodiment 5. A method for identifying a compound that preferentially reduces the level of association between a first macromolecule and one or more target condensates, comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises the one or more target condensates, and / or (ii) the one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; and (b) determining the level of association between the first macromolecule and the one or more target condensates and the level of association between at least one additional macromolecule and the one or more target condensates, wherein the compound preferentially reduces the level of the first macromolecule and the one or more target condensates if the compound alters the level of the first macromolecule compared to a first reference level more than reduces the level of each additional macromolecule compared to a reference level of each additional macromolecule.

[0200] Embodiment 6 The method of embodiment 5, wherein the compound does not measurably decrease the level of each additional macromolecule compared to the reference level of each additional macromolecule.

[0201] Embodiment 7. The method of any one of embodiments 1 to 6, wherein the first reference level is an association level of the first macromolecule with one or more reference condensates determined in the absence of the compound.

[0202] Embodiment 8. The method of any one of embodiments 1-7, wherein the reference level of each additional macromolecule is the level of association of each additional macromolecule with one or more reference condensates determined in the absence of the compound.

[0203] Embodiment 9. A method for identifying a compound that causes a first macromolecule to preferentially associate with one or more target condensates, comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises the one or more target condensates, and / or (ii) the one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; and (b) determining whether a first macromolecule and at least one additional macromolecule associate with the one or more target condensates, wherein (1) the compound causes the first macromolecule to associate with the one or more target condensates, (2) the compound does not cause each additional macromolecule to associate with the one or more target condensates, and (3) if the first macromolecule would not associate with the one or more target condensates in the absence of the compound, then the compound causes the first macromolecule to preferentially associate with the one or more target condensates.

[0204] Embodiment 10. (4) The method of embodiment 9, wherein the compound causes the first macromolecule to preferentially associate with the one or more target condensates if one or more of the at least one additional macromolecule would not associate with the one or more target condensates in the absence of the compound.

[0205] Embodiment 11. (4) The method of embodiment 9, wherein the compound causes the first macromolecule to preferentially associate with the one or more target condensates when each of the at least one additional macromolecule would not associate with the one or more target condensates in the absence of the compound.

[0206] Embodiment 12. A method for identifying a compound that preferentially dissociates a first macromolecule from one or more target condensates, comprising: (a) contacting a cellular composition with a compound, wherein (i) the cellular composition comprises the one or more target condensates, and / or (ii) the one or more target condensates are formed simultaneously with and / or after contacting the cellular composition with the compound; and (b) determining whether a first macromolecule and at least one additional macromolecule associate with the one or more target condensates, wherein: (1) the compound causes the first macromolecule not to associate with the one or more target condensates; (2) the compound does not cause each additional macromolecule not to associate with the one or more target condensates; and (3) if the first macromolecule would associate with the one or more target condensates in the absence of the compound, then the compound preferentially dissociates the first macromolecule from the one or more target condensates.

[0207] Embodiment 13. (4) The method of embodiment 12, wherein the compound causes the first macromolecule to preferentially associate with the one or more target condensates if one or more of the at least one additional macromolecule would associate with the one or more target condensates in the absence of the compound.

[0208] Embodiment 14. (4) The method of embodiment 12, wherein the compound causes the first macromolecule to preferentially associate with the one or more target condensates when each of the at least one additional macromolecule would associate with the one or more target condensates in the absence of the compound.

[0209] Embodiment 15. The method of any one of embodiments 1 to 14, wherein step (a) comprises contacting a cellular composition with a compound, wherein the cellular composition comprises the one or more target condensates, and wherein the method further comprises, prior to step (a), allowing the one or more target condensates to form.

[0210] Embodiment 16. The method of any one of embodiments 1 to 14, wherein step (a) comprises contacting a cellular composition with a compound, wherein the one or more target condensates are formed after contacting the cellular composition with the compound, and wherein the method further comprises forming the one or more target condensates.

[0211] Embodiment 17. The method of any one of embodiments 1 to 16, wherein the at least one additional macromolecule is two or more, three or more, four or more, or five or more macromolecules.

[0212] Embodiment 18. The method of any one of embodiments 1 to 16, wherein the at least one additional macromolecule is 1 to 10 macromolecules.

[0213] Embodiment 19. The method of any one of embodiments 1 to 18, wherein the first macromolecule is aberrantly expressed in a disease state.

[0214] Embodiment 20. The method of any one of embodiments 1 to 19, wherein the level of association between the first macromolecule and the one or more target condensates in a disease state is altered compared to the level of association between the first macromolecule and the one or more target condensates in a normal state.

[0215] Embodiment 21 The method of any one of embodiments 1 to 20, wherein one or more of the at least one additional macromolecule is aberrantly expressed in a disease state.

[0216] Embodiment 22. The method of any one of embodiments 1 to 21, wherein the first macromolecule is DNA or RNA.

[0217] Embodiment 23. The method of any one of embodiments 1 to 22, wherein one or more of the at least one additional macromolecule is DNA or RNA.

[0218] Embodiment 24. The method of any one of embodiments 1 to 21 or 23, wherein the first macromolecule is a protein.

[0219] Embodiment 25. The method of embodiment 24, wherein the first macromolecule comprises a mutation that alters the level of association between the first macromolecule and the one or more target condensates compared to a related protein that does not contain the mutation.

[0220] Embodiment 26 The method of embodiment 24 or 25, wherein the first macromolecule is FUS or eIF3.

[0221] Embodiment 27. The method of any one of embodiments 1 to 26, wherein one or more of the at least one additional macromolecule is a protein.

[0222] Embodiment 28. The method of any one of embodiments 1 to 27, wherein one or more of the at least one additional macromolecule comprises a mutation that alters the corresponding level of association with the one or more target condensates compared to a related protein that does not comprise the mutation.

[0223] Embodiment 29. The method of any one of embodiments 1 to 28, wherein one or more of the at least one additional macromolecule is FUS, eIF3, G3BP1, FUS and G3BP1, or eIF3 and G3BP1.

[0224] Embodiment 30. The method of any one of embodiments 24 to 29, wherein one or more of the first macromolecule and / or the at least one additional macromolecule is a fusion protein.

[0225] Embodiment 31. The method of any one of embodiments 1 to 30, wherein one or more of the first macromolecule and / or the at least one additional macromolecule comprises a label.

[0226] Embodiment 32. The method of any one of embodiments 1 to 31, further comprising labeling one or more of the first macromolecule and / or the at least one additional macromolecule.

[0227] Embodiment 33. The method of embodiment 32, wherein said labeling comprises contacting said cell composition with an antibody or antigen-binding fragment thereof comprising a label.

[0228] Embodiment 34. The method of any one of embodiments 31 to 33, wherein the label is a radioactive label, a colorimetric label, or a fluorescent label.

[0229] Embodiment 35. The method of any one of embodiments 1 to 34, wherein the cell composition comprises microbial or animal cells.

[0230] Embodiment 36 The method of embodiment 35, wherein the cell composition comprises animal cells.

[0231] Embodiment 37. The method of embodiment 36, wherein the animal cells have one or more characteristics of a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease.

[0232] Embodiment 38. The method of any one of embodiments 35 to 37, wherein the animal cell is a HeLa cell, a HEK293 cell, an induced pluripotent stem cell (iPSC cell), a cardiomyocyte, a muscle cell, a stem cell-derived cell, a neuron, a cancer cell, an immune cell, or an adipocyte.

[0233] Embodiment 39. The method of any one of embodiments 1 to 38, wherein the one or more target condensates are cellular condensates.

[0234] Embodiment 40. The method of any one of embodiments 1 to 39, wherein the one or more target condensates are nuclear condensates or cytoplasmic condensates.

[0235] Embodiment 41. The method of embodiment 39, wherein the cellular condensate is a cleavage body, a p-granule, a histone locus body, a multivesicular body, a neuronal RNA granule, a nuclear gem, a nuclear pore, a nuclear speckle, a nuclear stress body, a nucleolus, an Oct1 / PTF / transcription (OPT) domain, a paraspeckle, a juxtanucleolar compartment, a PML nuclear structure, a PML oncogenic domain, a Polycomb body, a processing body, a Sam68 nuclear structure, a stress granule, or a splicing speckle.

[0236] Embodiment 42. The method of any one of embodiments 1 to 41, wherein the one or more target condensates is a single target condensate.

[0237] Embodiment 43. The method of embodiment 42, wherein the compound does not measurably alter one or more of the size of the target condensate, the location of the target condensate, the surface area of ​​the target condensate, and the dissolution of the target condensate.

[0238] Embodiment 44. The method of any one of embodiments 1 to 41, wherein the one or more target condensates is a plurality of target condensates.

[0239] Embodiment 45. The method of embodiment 44, wherein the plurality of target condensates is all or a subset of a class of condensates in the portion of the cellular composition.

[0240] Embodiment 46 The method of embodiment 44 or 45, wherein the plurality of target condensates is all or a subset of a class of condensates in cells in the cellular composition.

[0241] Embodiment 47. The method of any one of embodiments 44 to 46, wherein the plurality of targeted condensates is all or a subset of a class of condensates in a portion of cells in the cellular composition.

[0242] Embodiment 48 The method of embodiment 47, wherein the portion of the cell is a cytoplasm, a nucleus, or an organelle.

[0243] Embodiment 49. The method of any one of embodiments 45 to 48, wherein the class of condensates comprises condensates containing specific macromolecules.

[0244] Embodiment 50. The class of condensates includes condensates that are cleavage bodies, the class of condensates includes condensates that are p-granules, the class of condensates includes condensates that are histone locus bodies, the class of condensates includes condensates that are multivesicular bodies, the class of condensates includes condensates that are neuronal RNA granules, the class of condensates includes condensates that are nuclear gems, the class of condensates includes condensates that are nuclear pores, the class of condensates includes condensates that are nuclear speckles, the class of condensates includes condensates that are nuclear stress bodies, the class of condensates includes condensates that are nucleoli, and the class of condensates includes Oct1 / PTF / transcription (OPT) domains. The method of any one of embodiments 45 to 49, wherein the condensates comprise condensates that are paraspeckles, the class of condensates comprises condensates that are juxtanucleolar compartments, the class of condensates comprises condensates that are PML nuclear structures, the class of condensates comprises condensates that are PML oncogenic domains, the class of condensates comprises condensates that are Polycomb bodies, the class of condensates comprises condensates that are processing bodies, the class of condensates comprises condensates that are Sam68 nuclear structures, the class of condensates comprises condensates that are stress granules, or the class of condensates comprises condensates that are splicing speckles.

[0245] Embodiment 51. The method of any one of embodiments 44 to 50, wherein the compound does not measurably alter one or more of the total number of the plurality of target condensates, the size of the plurality of target condensates, the location of the plurality of target condensates, the surface area of ​​the plurality of target condensates, and the dissolution of the plurality of target condensates.

[0246] Embodiment 52. A method for identifying a plurality of compounds that preferentially affect, decrease, or increase the level of association of a first macromolecule with one or more target condensates, or a plurality of compounds that preferentially associate or dissociate the first macromolecule with one or more target condensates, comprising performing the method of any one of embodiments 1 to 51 using a plurality of compounds.

[0247] Embodiment 53. The method of embodiment 52, further comprising identifying a feature that a subset or all of the identified compounds have in common in addition to the ability to preferentially affect, decrease, or increase the level of association of the first macromolecule with the one or more target condensates, or to preferentially associate or dissociate the first macromolecule with one or more target condensates.

[0248] Embodiment 54. The method of embodiment 53, further comprising performing the method of any one of embodiments 1 to 51 on one or more additional test compounds comprising the identified characteristic.

[0249] Embodiment 55. The method of embodiment 53 or 54, further comprising performing the method of any one of embodiments 1 to 51 on one or more additional test compounds that do not contain the identified characteristics.

[0250] Embodiment 56. A method for identifying a compound characteristic associated with preferentially affecting, decreasing, or increasing the level of association of a first macromolecule with one or more target condensates, or causing the first macromolecule to preferentially associate or dissociate with one or more target condensates, comprising: (a) performing the method of embodiment 52; and (b) identifying a characteristic that a subset or all of the identified compounds have in common in addition to their ability to preferentially affect, decrease, or increase the level of association of the first macromolecule with one or more target condensates, or their ability to preferentially associate or dissociate the first macromolecule with one or more target condensates.

[0251] Embodiment 57. A method for designing a compound that preferentially affects, reduces, or increases the level of association of a first macromolecule with one or more target condensates, or causes the first macromolecule to preferentially associate or dissociate with one or more target condensates, comprising: (a) performing the method of embodiment 52; (b) identifying a feature that a subset or all of the identified compounds have in common in addition to their ability to preferentially affect, reduce, or increase the level of association of the first macromolecule with one or more target condensates, or their ability to preferentially associate or dissociate the first macromolecule with one or more target condensates; and (c) designing a compound that comprises the identified feature, thereby designing a compound that preferentially affects, reduces, or increases the level of association of the first macromolecule with one or more target condensates, or causes the first macromolecule to preferentially associate or dissociate with one or more target condensates.

[0252] Embodiment 58. A method for identifying a compound useful for treating a disease in an individual in need thereof, comprising carrying out the method of any one of embodiments 1 to 51, wherein the one or more target condensates are associated with the disease; and identifying the compound that preferentially affects, decreases, or increases the level of association between the first macromolecule and the one or more target condensates, or the compound that preferentially associates or dissociates the first macromolecule with one or more target condensates, as being useful for treating the disease.

[0253] Embodiment 59. The method of embodiment 58, wherein the disease is a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease.

[0254] Embodiment 60. A method for identifying a compound that preferentially affects the level of association between a first macromolecule and a first set of one or more target condensates, comprising: performing the method of any one of embodiments 1 to 51 with the first set of one or more target condensates; and performing the method of any one of embodiments 1 to 51 with a second set of one or more target condensates, wherein the compound preferentially affects the level of association between the first macromolecule and the first set of one or more target condensates if the compound preferentially affects the level of association between the first macromolecule and the first set of one or more target condensates more than if the compound preferentially affects the level of association between the first macromolecule and the first set of one or more target condensates.

[0255] Embodiment 61 The method of embodiment 60, wherein the compound does not affect the level of association between the first macromolecule and the second set of one or more target condensates.

[0256] Embodiment 62. A method for identifying a compound that preferentially increases the level of association between a first macromolecule and a first set of one or more target condensates, comprising: performing the method of any one of embodiments 1 to 51 using the first set of one or more target condensates; and performing the method of any one of embodiments 1 to 51 using a second set of one or more target condensates, wherein the compound preferentially increases the level of association between the first macromolecule and the first set of one or more target condensates if the compound preferentially increases the level of association between the first macromolecule and the first set of one or more target condensates more than the compound preferentially increases the level of association between the first macromolecule and the second set of one or more target condensates.

[0257] Embodiment 63 The method of embodiment 62, wherein the compound does not increase the level of association between the first macromolecule and the second set of one or more target condensates.

[0258] Embodiment 64. A method for identifying a compound that preferentially reduces the level of association between a first macromolecule and a first set of one or more target condensates, comprising: performing the method of any one of embodiments 1 to 51 with the first set of one or more target condensates; and performing the method of any one of embodiments 1 to 51 with a second set of one or more target condensates, wherein the compound preferentially reduces the level of association between the first macromolecule and the first set of one or more target condensates if the compound preferentially reduces the level of association between the first macromolecule and the first set of one or more target condensates more than the compound preferentially reduces the level of association between the first macromolecule and the second set of one or more target condensates.

[0259] Embodiment 65. The method of embodiment 64, wherein the compound does not decrease the level of association between the first macromolecule and the second set of one or more target condensates.

[0260] Embodiment 66. A method for identifying a compound that causes a first macromolecule to preferentially associate with a first set of one or more target condensates, comprising: performing the method of any one of embodiments 1 to 51 with the first set of one or more target condensates; and performing the method of any one of embodiments 1 to 51 with a second set of one or more target condensates, wherein the compound causes the first macromolecule to preferentially associate with the first set of one or more target condensates if the compound causes the first macromolecule to preferentially associate with the first set of one or more target condensates more than the compound causes the first macromolecule to preferentially associate with the second set of one or more target condensates.

[0261] Embodiment 67. A method for identifying a compound that preferentially dissociates a first macromolecule from a first set of one or more target condensates, comprising: performing the method of any one of embodiments 1 to 51 with the first set of one or more target condensates; and performing the method of any one of embodiments 1 to 51 with a second set of one or more target condensates, wherein the compound preferentially dissociates the first macromolecule from the first set of one or more target condensates if the compound preferentially associates the first macromolecule with the first set of one or more target condensates more than the compound preferentially associates the first macromolecule with the second set of one or more target condensates.

[0262] Embodiment 68. The method of any one of embodiments 60 to 67, wherein the first and / or second set of one or more target condensates are cellular condensates.

[0263] Embodiment 69: The method of any one of embodiments 60 to 68, wherein the first and / or second set of one or more target condensates are nuclear condensates or cytoplasmic condensates.

[0264] Embodiment 70. The method of any one of embodiments 60 to 68, wherein the first and / or second set of one or more target condensates are 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.

[0265] Embodiment 71. The method of any one of embodiments 60 to 70, wherein the first and / or second set of one or more target condensates is a single target condensate.

[0266] Embodiment 72. The method of any one of embodiments 60 to 70, wherein the first and / or second set of one or more target condensates is a plurality of target condensates.

[0267] Embodiment 73. The method of any one of embodiments 60-70 or 72, wherein the first and / or second set of one or more target condensates is all or a subset of a class of condensates in a portion of the cellular composition.

[0268] Embodiment 74. The method of any one of embodiments 60-70 or 72-73, wherein the first and / or second set of one or more target condensates is all or a subset of a class of condensates in cells in the cellular composition.

[0269] Embodiment 75. The method of any one of embodiments 60-70 or 72-74, wherein the first and / or second set of one or more target condensates is all or a subset of a class of condensates in a portion of cells in the cellular composition.

[0270] Embodiment 76 The method of embodiment 75, wherein the portion of the cell is a cytoplasm, a nucleus, or an organelle.

[0271] Embodiment 77. The method of any one of embodiments 73 to 76, wherein the class of condensates comprises condensates containing specific macromolecules.

[0272] Embodiment 78. The class of condensates includes condensates that are cleavage bodies, the class of condensates includes condensates that are p-granules, the class of condensates includes condensates that are histone locus bodies, the class of condensates includes condensates that are multivesicular bodies, the class of condensates includes condensates that are neuronal RNA granules, the class of condensates includes condensates that are nuclear gems, the class of condensates includes condensates that are nuclear pores, the class of condensates includes condensates that are nuclear speckles, the class of condensates includes condensates that are nuclear stress bodies, the class of condensates includes condensates that are nucleoli, and the class of condensates includes Oct1 / PTF / transcription (OPT) domains. The method of any one of embodiments 73 to 77, wherein the condensates comprise condensates that are paraspeckles, the class of condensates comprises condensates that are juxtanucleolar compartments, the class of condensates comprises condensates that are PML nuclear structures, the class of condensates comprises condensates that are PML oncogenic domains, the class of condensates comprises condensates that are Polycomb bodies, the class of condensates comprises condensates that are processing bodies, the class of condensates comprises condensates that are Sam68 nuclear structures, the class of condensates comprises condensates that are stress granules, or the class of condensates comprises condensates that are splicing speckles.

[0273] Embodiment 79. The method of any one of embodiments 73 to 78, wherein the class of the first set of one or more target condensates is the same as the class of the second set of one or more target condensates.

[0274] Embodiment 80. The method of any one of embodiments 73 to 78, wherein the class of the first set of one or more target condensates is different from the class of the second set of one or more target condensates.

[0275] Embodiment 81. The method of any one of embodiments 60 to 80, wherein the first set of one or more target condensates is in the same cellular composition as the second set of one or more target condensates.

[0276] Embodiment 82. The method of any one of embodiments 60 to 81, wherein the cell composition comprises cells comprising a first set of condensates of the one or more target condensates and a second set of the one or more target condensates.

[0277] Embodiment 83. The method of any one of embodiments 60 to 80, wherein a first set of condensates of the one or more target condensates is in a first cellular composition and a second set of the one or more target condensates is in a second cellular composition.

[0278] Embodiment 84. The method of any one of embodiments 60 to 83, wherein the first set of condensates of the one or more target condensates is within cells in the cell composition, and the cells have one or more characteristics of a disease.

[0279] Embodiment 85. The method of embodiment 84, wherein the disease is a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease.

[0280] Embodiment 86. A method for identifying a plurality of compounds that preferentially affect, decrease, or increase the level of association of a first macromolecule with a first set of one or more target condensates, or a plurality of compounds that preferentially associate or dissociate the first macromolecule with the first set of one or more target condensates, comprising performing the method of any one of embodiments 60 to 85 using a plurality of compounds.

[0281] Embodiment 87. The method of embodiment 86, further comprising identifying a feature that a subset or all of the identified compounds have in common in addition to the ability to preferentially affect, decrease, or increase the level of association of the first macromolecule with the first set of one or more target condensates, or to preferentially associate or dissociate the first macromolecule with the first set of one or more target condensates.

[0282] Embodiment 88. The method of embodiment 87, further comprising performing the method of any one of embodiments 60 to 85 on one or more additional test compounds comprising the identified characteristic.

[0283] Embodiment 89. The method of embodiment 87 or 88, further comprising performing the method of any one of embodiments 60 to 85 on one or more additional test compounds that do not contain the identified characteristics.

[0284] Embodiment 90. A method for identifying a compound characteristic associated with preferentially affecting, decreasing, or increasing the level of association of a first macromolecule with a first set of one or more target condensates, or causing the first macromolecule to preferentially associate or dissociate with the first set of one or more target condensates, comprising: (a) performing the method of embodiment 86; and (b) identifying a characteristic that a subset or all of the identified compounds have in common in addition to their ability to preferentially affect, decrease, or increase the level of association of the first macromolecule with the first set of one or more target condensates, or their ability to preferentially associate or dissociate the first macromolecule with the first set of one or more target condensates.

[0285] Embodiment 91. A method for designing compounds that preferentially affect, decrease, or increase the level of association between a first macromolecule and a first set of one or more target condensates, or compounds that cause the first macromolecule to preferentially associate or dissociate with the first set of one or more target condensates, comprising: (a) performing the method of embodiment 86; and (b) determining whether a subset or all of the identified compounds preferentially affect, decrease, or increase the level of association between the first macromolecule and the first set of one or more target condensates. The method includes (a) identifying a common feature in addition to the ability to increase the association level or to preferentially associate or dissociate the first macromolecule with the first set of one or more target condensates; and (c) designing a compound that comprises the identified feature, thereby preferentially affecting, decreasing, or increasing the association level of the first macromolecule with the first set of one or more target condensates, or designing a compound that preferentially associates or dissociates the first macromolecule with the first set of one or more target condensates.

[0286] Embodiment 92. A method for identifying a compound useful for treating a disease in an individual in need thereof, comprising carrying out the method of any one of embodiments 60 to 85, wherein the first set of one or more target condensates is associated with the disease; and identifying the compound that preferentially affects, decreases, or increases the level of association of the first macromolecule with the first set of one or more target condensates, or the compound that causes the first macromolecule to preferentially associate with or dissociate from the first set of one or more target condensates, as being useful for treating the disease.

[0287] Embodiment 93. The method of embodiment 92, wherein the disease is a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, or a metabolic disease. [Example]

[0288] Example 1 Screening for compounds that cause changes in condensed protein levels Stress granules are condensates that form in the cytoplasm of cells under stress. Several proteins are known to be enriched in stress granules, including FUS, eukaryotic translation initiation factor 3 (eIF3), and G3BP1. FUS is involved in pre-mRNA maturation, and various mutations in FUS have been associated with ALS. Although FUS is found in stress granules, it is not thought to be essential for stress granule formation. Like FUS, eIF3 is also not thought to be essential for stress granule formation. As its name suggests, eIF3 is a eukaryotic translation initiation factor but is also involved in translation recycling. In contrast, G3BP1 is thought to initiate stress granule formation and is potentially important for maintaining its structure. G3BP1 is an RNA-binding protein and a component of the Ras signaling pathway. These three proteins were tracked in cells to determine whether compounds could preferentially exclude one of these proteins from stress granules without dissolving them.

[0289] method Three cell lines (HeLa Kyoto, HeLa containing a bacterial artificial chromosome (BAC) encoding FUS-GFP, and induced pluripotent stem cells (iPS) expressing FUS-GFP) were grown separately in culture. One of the cell lines was individually exposed to 1200 compounds from a compound library at concentrations of 1 μM, 5 μM, or 25 μM for 1 h. For some compounds, this procedure was repeated for one or both of the other cell lines. Cells not exposed to compounds (DMSO-only vehicle treatment) served as controls. Cells were then exposed to arsenate stress for 1 h using a final concentration of 2 mM potassium arsenate (Sigma A6631, diluted in PBS) to stimulate stress granule formation.

[0290] After stress treatment, cells were fixed with 3.7% formaldehyde (FA) at room temperature for 15 minutes. Subsequently, cells were washed using a plate washer, permeabilized (using 0.1% Triton X / PBS at room temperature for 10 minutes), and blocked (using 0.2% fish skin gelatin / PBS for 30 minutes). For cells expressing FUS-GFP, GFP fluorescence was directly observed. For other protein markers of interest, antibodies were used: anti-FUS (Sigma AMAb90549, 1:500), anti-eIF3 (Santa Cruz sc-137214, 1:500), and anti-G3BP1 (Invitrogen) antibodies. PA5-29455, 1:4000), and a combination of appropriate secondary antibodies (Life Technologies, Alexa 594 and 647) were applied to visualize FUS, eIF3, and G3BP1 proteins in the cells.

[0291] Images of the samples were acquired using a spinning disk confocal microscope with confocal fluorescence at 405 / 488 / 561 / 640 nm. Images were taken using a 40x objective. The gain was set to 1.4, and binning was set to 1. The exposure time was adjusted to the specific fluorescence emission intensity for each label. The exposure time ranged from 100 to 300 ms. Appropriate filter settings were used to match the excitation and emission spectra of the labels. Image acquisition was automated, and the resulting data were analyzed using KNIME (Berthold et al., 2008, Data Analysis, Machine Learning and Applications. Studies in Classification, Data Analysis, and Knowledge Organization. Springer, Berlin, Heidelberg) and CellProfiler (Carpenter et al., 2006, Genome Biol 7:R100). For each image, 25 imaging parameters were extracted, including droplet number, area, and shape of the marker signal. For each imaging parameter, the Z' value for the whole plate was calculated according to the following formula: Z'=1-[3*(SD(pos)+SD(neg)) / (Avg(pos)-Avg(neg))] where SD(pos) is the standard deviation of the positive control, SD(neg) is the standard deviation of the negative control, Avg(pos) is the mean of the positive control, and Avg(neg) is the mean of the negative control.

[0292] Additionally, for each test compound, a z-score was calculated using the following formula:

number

number

[0293] result In the absence of compound, FUS, eIF3, and G3BP1 all appeared to colocalize in the same condensates in all three cell lines tested during stress (see Figures 1-4, without compound treatment). Furthermore, many of the compounds did not appear to alter the localization or levels of FUS, eIF3, and G3BP1. Certain compounds were found that appeared to completely dissolve stress granules. Figures 1 and 2 show exemplary images, and Table 1 lists the compounds used in Figures 1 and 2. No compounds were found to selectively exclude G3BP1 from stress granules. Whenever G3BP1 was found not to be localized to stress granules after treatment with a compound, neither FUS nor eIF3 were found to be localized to stress granules after treatment. In contrast, at least four compounds were found that caused partial or complete exclusion of FUS from stress granules but did not reduce the levels of the other proteins assayed or appear to dissolve stress granules. See Figure 3 for exemplary images. Table 1 lists the compounds used in Figure 3. Also, in contrast to G3BP1, at least three compounds were found that caused partial or complete exclusion of eIF3 from stress granules but did not reduce the levels of other proteins assayed or appear to dissolve stress granules. See Figure 4 for example images. Table 1 lists the compounds used in Figure 4. [Table 1-1] [Table 1-2]

[0294] Example 2 This example demonstrates a method for identifying compounds with macromolecular and tissue specificity.

[0295] method The method used in this example was similar to that discussed in Example 1. Briefly, three cell lines (HeLa Kyoto, HeLa containing a bacterial artificial chromosome (BAC) encoding FUS-GFP, and induced pluripotent stem cells (iPS) expressing FUS-GFP) were grown separately in culture. 1200 compounds from the compound library were individually exposed to one of the cell lines for 1 hour at concentrations of 1 μM, 5 μM, or 25 μM. For some compounds, this procedure was repeated for one or both of the other cell lines. Hits from the screen were further characterized by treating them with 0.0021 μM, 0.0062 μM, 0.017 μM, 0.065 μM, 0.131 μM, 0.196 μM, 0.52 μM, 3.3 μM, 10 μM, and 30 μM of the compounds, and the resulting dose-response data were analyzed using a four-parameter logistic equation to determine compound potency (IC 50 The α- and β-catenin-dependent stress responses were calculated using the same method as in Example 1. Cells not exposed to compounds (DMSO-only vehicle treatment) were used as a control. Cells were then exposed to arsenate stress for 1 hour using a final concentration of 2 mM potassium arsenate (Sigma A6631, diluted in PBS) to stimulate the formation of stress granules.

[0296] After stress treatment, cells were fixed with 3.7% formaldehyde (FA) at room temperature for 15 minutes. Subsequently, cells were washed using a plate washer, permeabilized (using 0.1% Triton X / PBS at room temperature for 10 minutes), and blocked (using 0.2% fish skin gelatin / PBS for 30 minutes). For cells expressing FUS-GFP, GFP fluorescence was directly observed. For other protein markers of interest, antibodies were used: anti-FUS (Sigma AMAb90549, 1:500), anti-eIF3 (Santa Cruz sc-137214, 1:500), and anti-G3BP1 (Invitrogen) antibodies. PA5-29455, 1:4000), and a combination of appropriate secondary antibodies (Life Technologies, Alexa 594 and 647) were applied to visualize FUS, eIF3, and G3BP1 proteins in the cells.

[0297] Images of the samples were acquired using a spinning disk confocal microscope with confocal fluorescence at 405 / 488 / 561 / 640 nm. Images were taken using a 40x (primary screening) and a 20x (potency testing) air objective. The gain was set to 1.4, and binning was set to 1. The exposure time was adjusted to the specific fluorescence emission intensity for each label. The exposure time ranged from 100 to 300 ms. Appropriate filter settings were used to match the excitation and emission spectra of the labels. Image acquisition was automated, and the resulting data were analyzed using KNIME (Berthold et al., 2008, Data Analysis, Machine Learning and Applications. Studies in Classification, Data Analysis, and Knowledge Organization. Springer, Berlin, Heidelberg), CellProfiler (Carpenter et al., 2006, Genome Biol 7:R100), or, in the case of efficacy studies, Harmony (PerkinElmer, Waltham MA) and Vault (Collaborative Drug Discovery, Burlingame CA). For each image, 25 imaging parameters were extracted, including droplet number, area, and shape of the marker signal. For each imaging parameter, the Z' value for the entire plate was calculated according to the following formula: Z'=1-[3*(SD(pos)+SD(neg)) / (Avg(pos)-Avg(neg))] where SD(pos) is the standard deviation of the positive control, SD(neg) is the standard deviation of the negative control, Avg(pos) is the mean of the positive control, and Avg(neg) is the mean of the negative control.

[0298] Additionally, for each test compound, a z-score was calculated using the following formula:

number

number

[0299] Compounds were classified as "hits" if the Z' for the plate was at least 0.1 and at least two imaging parameters had a z-score ≧3.0 standard deviations from the parameter median.

[0300] Use a four-parameter logistic equation for the measurements and analysis of the number of cytoplasmic drops per cell to determine the number of screening hits (inhibitory concentration; IC 50 Potency calculations were performed (similar to IC ). Briefly, the % effect of compound treatment was first plotted as a function of the log10 (molar concentration) of compound. The data were then fit to a four-parameter logistic equation via least squares to determine the 50% inhibition constant (IC 50 ) was calculated. %Effect = (Measured value - Negative control) / (Positive control - Negative control)*100 Logistic equation: y=lower+(upper-lower) / (1+10^((logIC 50 -x)*HS)) During the ceremony, x=log 10 (molar concentration of compound) lower = dose response lower baseline upper = upper baseline of dose response HS = Hill slope of dose response

[0301] result In the absence of compound, FUS, eIF3, and G3BP1 all appeared to colocalize in the same condensates in all three cell lines tested during stress (see Figures 5-10, without compound treatment). As shown in Figures 5-10, many of the compounds did not appear to alter the localization or levels of FUS, eIF3, and G3BP1. Certain compounds were found to completely dissolve stress granules. Table 2 lists the compounds used in Figures 5-10 and their macromolecular specificities. No compound was found to selectively exclude G3BP1 from stress granules. Whenever G3BP1 was found not to be localized to stress granules after treatment with a compound, neither FUS nor eIF3 were found to be localized to stress granules after treatment. In contrast, one compound was found to cause the exclusion of FUS from stress granules but did not reduce the levels of the other proteins assayed or appear to dissolve stress granules (see, for example, Figure 7). Also, in contrast to G3BP1, at least three compounds were found to cause partial or complete elimination of eIF3 but did not reduce the levels of other proteins assayed or appear to dissolve stress granules (see, e.g., Figure 8). One compound was found to cause elimination of both eIF3 and FUS but not G3BP1 (see Figure 9). One compound was also observed to selectively eliminate FUS in human iPSC cells but not in HeLa BAC FUS-GFP cells (see Figure 10).

[0302] The selectivity of a compound for the exclusion of a given protein compared to other proteins can be further quantified by calculating its potency for the exclusion of a specific protein compared to other proteins (50% inhibition constant (IC 50)). Figure 11 includes an example dose-response curve and potency values ​​for YM155, showing the exclusion of eIF3 at lower concentrations of the compound compared to FUS and G3BP1. This information provides additional means for comparison, e.g., dose-response curves and / or potency values, to identify compound specificity. [Table 2-1] [Table 2-2]

Claims

[Claim 1] The invention described in the specification.

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