Method for characterizing condensate-associated characteristics of compound and use thereof

Abstract

To provide a method for evaluating, for example, characterizing or determining condensate-associated characteristics of a compound, such as a test compound, and an application thereof.SOLUTION: A method for determining partition characteristics of a test compound in a target condensate, a method for determining relative partition characteristics of a test compound in a target condensate, and a method for determining a condensate preference profile of a test compound, are provided. In addition, methods for designing and / or identifying and / or making a compound, or a portion thereof, with desired relative condensate partition characteristics, are provided.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 803,365, filed February 8, 2019, and U.S. Provisional Patent Application No. 62 / 866,526, filed June 25, 2019, the disclosures of each of which are incorporated by reference in their entireties herein.

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

[0003] In addition to membrane-bound organelles such as mitochondria, lysosomes and endoplasmic reticulum, cells contain distinct subcompartments that do not contain membranes between them and their immediate surrounding solution. Many of these membrane-poor molecular assemblies have been shown to form by a process called liquid-liquid phase separation or condensation. During this process, a solution containing biomacromolecules separates into distinct phases: condensates enriched in some of these macromolecules, and the surrounding phase relatively depleted in these macromolecules. Several cellular condensates have been recognized. In addition, phase-separated condensates can form in solution or outside of cells, such as extracellularly (Alberti et al., J Mol Biol, 430(23), 2018, 4806-4820;Muiznieks et al., J Mol Biol, 430(23), 2018, 4741-4753). However, little or nothing is known about the mechanisms that govern the partitioning or exclusion of compounds into or from condensates, or the differences in the partitioning of compounds among various condensates.

[0004] Various condensates are known to be important for modulating cellular processes. For example, condensates can bring molecules together at elevated concentrations to accelerate reactions inside the condensate, or sequester molecules in the condensate, reducing their concentration in the surrounding medium. Abnormal condensate functions have also been implicated in various human diseases, such as neurodegenerative and proliferative disorders (Naumann et al., Nat Commun, 9(1), 2018, 335;Wegmann et al., EMBO J, 37(7), 2018, e98049;Aguzzi et al., Trends Cell Biol, 26(7), 2016, 547-558). However, in addition to a lack of understanding of the mechanisms governing condensate distributing compounds, little or nothing is known about how to identify test compounds that interact with condensates, how to design compounds that improve interactions with condensates, and how such information might be used to improve the treatment of disease. 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] Naumann et al., Nat Commun, 9(1), 2018, 335 [Non-Patent Document 2] Wegmann et al., EMBO J, 37(7), 2018, e98049 [Non-Patent Document 3] Aguzzi et al., Trends Cell Biol, 26(7), 2016, 547-558 Summary of the Invention [Means for solving the problem]

[0006] In some aspects, provided herein is a method for determining the distribution characteristics of a test compound in a target condensate, comprising: (a) combining a test compound with a composition comprising a target condensate and an excess condensate solution; (b) determining the amount of the test compound in the target condensate, thereby determining the distribution characteristics of the test compound in the target condensate. In some embodiments, the method further comprises, prior to step (a), causing the formation of a target condensate.

[0007] In some aspects, provided herein is a method for determining the distribution characteristics of a test compound in a target condensate, comprising: (a) adding a test compound to a composition comprising a target condensate and an excess condensate solution; and (b) determining the amount of the test compound in the target condensate, thereby determining the distribution characteristics of the test compound in the target condensate. In some embodiments, the method further comprises, prior to step (a), causing the formation of a target condensate.

[0008] In some aspects, provided herein is a method for determining the partitioning characteristics of a test compound in a target condensate, the method comprising: (a) causing formation of a target condensate in the presence of a test compound to obtain a composition comprising the target condensate and an excess condensate solution; and (b) determining the amount of the test compound in the target condensate, thereby determining the partitioning characteristics of the test compound in the target condensate.

[0009] In some aspects, provided herein is a method for determining the distribution characteristics of a test compound in a target condensate, comprising: (a) adding the test compound to a composition comprising a precursor molecule; (b) inducing the formation of a target condensate to obtain a composition comprising the target condensate and an excess condensate solution; and (c) determining the amount of the test compound in the target condensate, thereby determining the distribution characteristics of the test compound in the target condensate. In some embodiments, the method further comprises, before step (a), combining the test compound and a precursor composition comprising the precursor molecule. In some embodiments, the method further comprises, before step (a), adding the test compound to a precursor composition comprising the precursor molecule.

[0010] In some embodiments, the method further comprises determining the amount of the test compound in the excess condensate solution. In some embodiments, the amount of the test compound in the target condensate is determined before, simultaneously with, or after the amount of the test compound in the excess condensate solution is determined. In some embodiments, the method further comprises determining the ratio of the amount of the test compound in the target condensate and the amount of the test compound in the excess condensate solution. In some embodiments, the method further comprises separating the target condensate from the excess condensate solution. In some embodiments, the method further comprises identifying the target condensate before the step of determining the amount of the test compound in the target condensate.

[0011] In some embodiments, the dysregulation of the target condensate is associated with disease. In some embodiments, the method further comprises characterizing the target condensate by identifying one or more macromolecules contained therein. In some embodiments, the identifying step comprises determining the amount of one or more macromolecules in the target condensate. In some embodiments, the method further comprises determining the ratio of the amount of the test compound in the target condensate and the amount of one or more macromolecules in the target condensate. In some embodiments, the target condensate comprises a protein that comprises an intrinsically disordered sequence. In some embodiments, the method further comprises labeling the target condensate to visualize the target condensate. In some embodiments, the target condensate is labeled with a radioactive label, a colorimetric label, a chemically reactive label, or a fluorescent label.

[0012] In some embodiments, the composition comprises a cell. In some embodiments, the cell is a microorganism or an animal cell. In some embodiments, the cell comprises a condensate that is determined to be dysregulated. In some embodiments, the cell has one or more characteristics of a neurodegenerative or proliferative disease.

[0013] In some embodiments, the target condensate is a cellular condensate. 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, an intranuclear body, an Oct1 / PTF / transcription (OPT) domain, a paraspeckle, a perinuclear compartment, a PML nucleolus, a PML oncogenic domain, a polycomb body, a processing body, a signaling cluster, a Sam68 nucleolus, a stress granule, or a splicing speckle. In some embodiments, the target condensate is in a cell. In some embodiments, the cell is a microorganism or an animal cell. In some embodiments, the cell has one or more characteristics of a neurodegenerative or proliferative disease. In some embodiments, the excess condensate solution is an intracellular fluid. In some embodiments, the intracellular fluid is a cytosol or a nucleosol.

[0014] In some embodiments, the target condensate is not intracellular. In some embodiments, the target condensate is extracellular condensate. In some embodiments, the excess condensate solution is extracellular fluid. In some embodiments, the extracellular fluid is interstitial fluid.

[0015] In some embodiments, the method is a cell-free assay. In some embodiments, the composition does not include cells. In some embodiments, the composition includes one or more of a macromolecule, a salt, and a buffering agent.

[0016] In some embodiments, the composition comprises two or more target condensates. In some embodiments, the method further comprises repeating the steps of the method for one or more additional condensates.

[0017] In some embodiments, the test compound is a small molecule, a polypeptide or a nucleic acid. In some embodiments, the test compound comprises a test compound label. In some embodiments, the test compound label is a radioactive label, a colorimetric label, a chemically reactive label or a fluorescent label. In some embodiments, the test compound label is a fluorescent label. In some embodiments, the amount of the test compound is determined by detecting the test compound label. In some embodiments, the amount of the test compound is determined by mass spectrometry, liquid chromatography, quantitative fluorescence microscopy and spectroscopy, nuclear magnetic resonance spectroscopy, Raman spectroscopy and / or ultraviolet-visible spectroscopy.

[0018] In some aspects, a method for determining the partitioning properties of a plurality of test compounds in a target condensate is provided herein, comprising performing the method for determining partitioning properties described herein with a plurality of test compounds. In some embodiments, the method further comprises comparing the partitioning properties of a subset or all of the plurality of test compounds in the target condensate. In some embodiments, the method further comprises identifying test compounds that have the same or similar partitioning properties in the target condensate. In some embodiments, the method further comprises identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning properties. In some embodiments, the method further comprises determining the partitioning properties in the target condensate for one or more additional test compounds that include the identified properties. In some embodiments, the method further comprises determining the partitioning properties in the target condensate for one or more additional test compounds that do not include the identified properties.

[0019] In some aspects, the present specification provides a method for determining the relative partitioning properties of a test compound in a target condensate, comprising: (i) determining the partitioning properties of a test compound by carrying out the partitioning properties determining method described herein with a test compound; (ii) determining the partitioning properties of a reference compound by carrying out the partitioning properties determining method described herein with a reference compound; and (iii) calculating the ratio of the partitioning properties determined in (i) and (ii), thereby determining the relative partitioning properties of the test compound in a target condensate.In some embodiments, the test compound comprises a test compound label.In some embodiments, the reference compound is a test compound label.

[0020] In some aspects, provided herein is a method for determining the relative partitioning properties of a plurality of test compounds in a target condensate, comprising: (1) performing the method for determining the relative partitioning properties of the test compounds in a target condensate; and (2) repeating steps (i) and (iii) with a plurality of test compounds. In some embodiments, the method further comprises comparing the relative partitioning properties of a subset or all of the plurality of test compounds in the target condensate. In some embodiments, the method further comprises identifying test compounds that have the same or similar relative partitioning properties in the target condensate. In some embodiments, the method further comprises identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar relative partitioning properties. In some embodiments, the method further comprises determining the relative partitioning properties of one or more additional test compounds in the target condensate that include the identified properties. In some embodiments, the method further comprises determining the relative partitioning properties of one or more additional test compounds that do not include the identified properties in the target condensate.

[0021] In some aspects, a method for determining a condensate preference profile of a test compound is provided herein, comprising: (a) determining the partition profile of the test compound in a first target condensate according to the method disclosed herein; (b) determining the partition profile of the test compound in a second target condensate according to the method disclosed herein; and (c) calculating the ratio of the partition profile of the test compound determined in the first target condensate and the second target condensate, thereby determining the condensate preference profile of the test compound. In some embodiments, the first target condensate and the second target condensate are in the same composition. In some embodiments, the first target condensate and the second target condensate are in different compositions. In some embodiments, the partition profile of the test compound in the first target condensate is determined before, simultaneously with, or after the partition profile of the test compound in the second target condensate is determined.

[0022] In some aspects, a method for determining a condensate preference profile of a test compound is provided herein, comprising: (a) determining the relative partition profile of the test compound in a first target condensate according to the method disclosed herein; (b) determining the relative partition profile of the test compound in a second target condensate according to the method disclosed herein; and (c) calculating the ratio of the partition profile of the test compound determined in the first target condensate and the second target condensate, thereby determining the condensate preference profile of the test compound. In some embodiments, the first target condensate and the second target condensate are in the same composition. In some embodiments, the first target condensate and the second target condensate are in different compositions. In some embodiments, the relative partition profile of the test compound in the first target condensate is determined before, simultaneously with, or after the relative partition profile of the test compound in the second target condensate is determined.

[0023] In some aspects, the present disclosure provides a method for determining the condensate preference profile of a test compound, comprising: (a) adding the test compound to a composition comprising a first target condensate and a second target condensate; (b) determining the amount of the test compound in the first target condensate; (c) determining the amount of the test compound in the second target condensate; and (d) calculating the ratio of the amount of the test compound determined in the first target condensate and the second target condensate, thereby determining the condensate preference profile of the test compound.In some embodiments, the method further comprises, before step (a), causing the formation of the first target condensate and / or the second target condensate.

[0024] In some aspects, the present specification provides a method for determining the condensate preference profile of a test compound, comprising: (a) adding the test compound to a composition comprising precursor molecules; (b) causing the formation of a first target condensate and a second target condensate in the composition; (c) determining the amount of the test compound in the first target condensate; (d) determining the amount of the test compound in the second target condensate; and (e) calculating the ratio of the amount of the test compound determined in the first target condensate and the second target condensate, thereby determining the condensate preference profile of the test compound.In some embodiments, the amount of the test compound in the first target condensate is determined before, simultaneously with, or after the amount of the test compound in the second target condensate is determined.

[0025] In some embodiments, the method further comprises separating the first target condensate and the second target condensate from the composition. In some embodiments, the method further comprises identifying the first target condensate and / or the second target condensate prior to determining the amount of the test compound in the first condensate and / or the second condensate.

[0026] In some embodiments, dysregulation of the first target condensate and / or the second target condensate is associated with a disease. In some embodiments, the method further comprises characterizing the first target condensate and / or the second target condensate by identifying one or more macromolecules contained therein. In some embodiments, the method further comprises labeling the first target condensate and / or the second target condensate to visualize the first target condensate and / or the second target condensate. In some embodiments, the method further comprises labeling the first target condensate and the second target condensate to visualize the first target condensate and the second target condensate. In some embodiments, the first target condensate and the second target condensate are labeled with different labels. In some embodiments, the first target condensate and / or the second target condensate are labeled with a radioactive label, a colorimetric label, a chemically reactive label, or a fluorescent label.

[0027] In some embodiments, the composition comprises a cell. In some embodiments, the cell is a microorganism or an animal cell. In some embodiments, the cell comprises a condensate that is determined to be dysregulated. In some embodiments, the cell has one or more characteristics of a neurodegenerative or proliferative disease.

[0028] In some embodiments, the first target condensate and / or the second target condensate is a cellular condensate.In some embodiments, the first target condensate is a cleavage body, a P granule, a histone locus body, a multivesicular body, a neural RNA granule, a nuclear gem, a nuclear pore, a nuclear speckle, a nuclear stress body, an intranuclear body, an Oct1 / PTF / transcription (OPT) domain, a paraspeckle, a perinuclear compartment, a PML nucleolus, a PML oncogenic domain, a polycomb body, a processing body, a signaling cluster, a viral condensate, a Sam68 nucleolus, a stress granule or a splicing speckle. In some embodiments, the second target 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, an intranuclear body, an Oct1 / PTF / transcription (OPT) domain, a paraspeckle, a perinuclear compartment, a PML nucleolus, a PML oncogenic domain, a polycomb body, a processing body, a signaling cluster, a viral condensate, a Sam68 nucleolus, a stress granule, or a splicing speckle. In some embodiments, the first target condensate and / or the second target condensate is in a cell.

[0029] In some embodiments, the first target condensate and / or the second target condensate is an extracellular condensate.

[0030] In some embodiments, the composition does not comprise cells, hi some embodiments, the composition comprises one or more of a macromolecule, a salt, and a buffering agent.

[0031] In some embodiments, the composition comprises one or more additional target condensates. In some embodiments, the method further comprises repeating the steps of the method for the one or more additional target condensates.

[0032] In some aspects, a method for determining a condensate preference profile of a plurality of test compounds is provided herein, comprising performing the method for determining a condensate preference profile described herein with a plurality of test compounds. In some embodiments, the method further comprises comparing the condensate preference profiles of a subset or all of the plurality of test compounds. In some embodiments, the method further comprises identifying test compounds having the same or similar condensate preference profile. In some embodiments, the method further comprises identifying a property that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profile. In some embodiments, the method further comprises determining a relative partitioning profile for one or more additional test compounds that include the identified property. In some embodiments, the method further comprises determining a relative partitioning profile for one or more additional test compounds that do not include the identified property.

[0033] In some aspects, provided herein is a method for identifying a compound characteristic associated with partitioning of a compound into or out of a condensate, the method comprising the steps of: (a) determining the partitioning characteristics of a plurality of test compounds in a target condensate according to a method described herein; (b) comparing the partitioning characteristics of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds having the same or similar partitioning characteristics in the target condensate; and (d) identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning characteristics.

[0034] In some aspects, provided herein is a method for identifying a compound characteristic associated with partitioning of the compound into a condensate, the method comprising the steps of: (a) determining the partitioning characteristics of a plurality of test compounds in a target condensate according to the methods described herein; (b) comparing the partitioning characteristics of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds having the same or similar partitioning characteristics in the target condensate; and (d) identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning characteristics.

[0035] In some aspects, provided herein is a method for identifying a compound characteristic associated with partitioning of a compound into or out of a condensate, the method comprising the steps of: (a) determining relative partitioning characteristics of a plurality of test compounds in a target condensate according to a method described herein; (b) comparing the relative partitioning characteristics of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds having the same or similar relative partitioning characteristics in the target condensate; and (d) identifying a characteristic that a subset or all of the identified test compounds have in common in addition to the same or similar relative partitioning characteristics.

[0036] In some aspects, provided herein is a method for identifying a compound characteristic associated with the partitioning of the compound into a condensate, the method comprising the steps of: (a) determining the relative partitioning characteristics of a plurality of test compounds in a target condensate according to a method described herein; (b) comparing the relative partitioning characteristics of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds having the same or similar relative partitioning characteristics in the target condensate; and (d) identifying a characteristic that a subset or all of the identified test compounds have in common in addition to the same or similar relative partitioning characteristics.

[0037] In some aspects, provided herein is a method for identifying a compound characteristic associated with partitioning of a compound into or out of a condensate, the method comprising: (a) determining a condensate preference profile for a plurality of test compounds according to a method described herein; (b) comparing the condensate preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar condensate preference profile; and (d) identifying a characteristic that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profile.

[0038] In some aspects, provided herein is a method for identifying a compound characteristic associated with partitioning of the compound into a condensate, the method comprising: (a) determining condensate preference profiles of a plurality of test compounds according to a method described herein; (b) comparing the condensate preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar condensate preference profiles; and (d) identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profile.

[0039] In some aspects, provided herein is a method for designing a compound with desired partitioning properties to or from a target condensate, comprising: (a) determining the partitioning properties of a plurality of test compounds in the target condensate according to the methods described herein; (b) comparing the partitioning properties of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds with the same or similar partitioning properties in the target condensate; (d) identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning properties; and (e) (i) designing a compound that includes the identified properties; or (ii) designing a compound that does not include the identified properties, thereby designing a compound with desired partitioning properties to or from the target condensate. In some embodiments, the step of designing the compound comprises combining a portion that includes the identified properties, thereby imparting the desired partitioning properties to the compound. In some embodiments, the step of designing the compound comprises removing a portion that includes the identified properties, thereby imparting the desired partitioning properties to the compound. In some embodiments, the step of designing the compound comprises modifying a first moiety to a second moiety that comprises the identified property, thereby imparting the desired partitioning property to the compound. In some embodiments, the compound is designed, in whole or in part, using an approach that includes modeling, computer and / or computational based techniques, e.g., bioinformatics, chemoinformatics, and / or artificial intelligence (AI) based techniques.

[0040] In some aspects, provided herein is a method for designing a compound with a desired partitioning property into a target condensate, the method comprising: (a) determining the partitioning property of a plurality of test compounds in the target condensate according to the methods described herein; (b) comparing the partitioning property of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds with the same or similar partitioning property in the target condensate; (d) identifying a property that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning property; and (e) designing a compound that includes the identified property. In some embodiments, the step of designing the compound comprises combining a moiety that includes the identified property, thereby imparting the desired partitioning property to the compound. In some embodiments, the step of designing the compound comprises modifying a first moiety to a second moiety that includes the identified property, thereby imparting the desired partitioning property to the compound. In some embodiments, the compound is designed, in whole or in part, using an approach that includes modeling, computer and / or computation-based techniques, such as bioinformatics, chemoinformatics, and / or artificial intelligence (AI)-based techniques.

[0041] In some aspects, provided herein is a method for designing a compound with a desired relative partitioning property to or from a target condensate, comprising: (a) determining the relative partitioning property of a plurality of test compounds in the target condensate according to the method disclosed herein; (b) comparing the relative partitioning property of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds with the same or similar relative partitioning property in the target condensate; (d) identifying a property that a subset or all of the identified test compounds have in common in addition to the same or similar relative partitioning property; and (e) (i) designing a compound that includes the identified property; or (ii) designing a compound that does not include the identified property, thereby designing a compound with a desired relative partitioning property to or from the target condensate. In some embodiments, the step of designing the compound comprises combining a portion that includes the identified property, thereby imparting the desired relative partitioning property to the compound. In some embodiments, the step of designing the compound comprises removing a portion that includes the identified property, thereby imparting the desired partitioning property to the compound. In some embodiments, the step of designing the compound comprises modifying a first moiety to a second moiety that comprises the identified property, thereby imparting the desired partitioning property to the compound. In some embodiments, the compound is designed, in whole or in part, using an approach that includes modeling, computer and / or computational based techniques, e.g., bioinformatics, chemoinformatics, and / or artificial intelligence (AI) based techniques.

[0042] In some aspects, provided herein is a method for designing a compound with a desired relative partitioning property, comprising: (a) determining the relative partitioning property of a plurality of test compounds in a target condensate according to the methods described herein; (b) comparing the relative partitioning property of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds having the same or similar relative partitioning property in the target condensate; (d) identifying a property that a subset or all of the identified test compounds have in common in addition to the same or similar relative partitioning property; and (e) designing a compound that includes the identified property. In some embodiments, the step of designing the compound comprises combining a moiety that includes the identified property, thereby imparting the desired relative partitioning property to the compound. In some embodiments, the step of designing the compound comprises modifying a first moiety to a second moiety that includes the identified property, thereby imparting the desired partitioning property to the compound. In some embodiments, the compound is designed, in whole or in part, using an approach that includes modeling, computer and / or computation-based techniques, such as bioinformatics, chemoinformatics, and / or artificial intelligence (AI)-based techniques.

[0043] In some aspects, provided herein is a method for designing a compound with a desired condensate preference profile, comprising: (a) determining the condensate preference profile of a plurality of test compounds according to the methods disclosed herein; (b) comparing the condensate preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds with the same or similar condensate preference profile; (d) identifying a property that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profile; and (e) (i) designing a compound that includes the identified property; or (ii) designing a compound that does not include the identified property, thereby designing a compound with a desired condensate preference profile. In some embodiments, the method further comprises a step of making a compound. In some embodiments, the step of designing a compound comprises combining a portion that includes the identified property, thereby imparting a desired condensate preference profile to the compound. In some embodiments, the step of designing a compound comprises removing a portion that includes the identified property, thereby imparting a desired partitioning property to the compound. In some embodiments, the step of designing the compound comprises modifying a first moiety to a second moiety that comprises the identified property, thereby imparting the desired partitioning property to the compound. In some embodiments, the compound is designed, in whole or in part, using an approach that includes modeling, computer and / or computational based techniques, e.g., bioinformatics, chemoinformatics, and / or artificial intelligence (AI) based techniques.

[0044] In some aspects, provided herein is a method for designing a compound with a desired condensate preference profile, the method comprising: (a) determining the condensate preference profile of a plurality of test compounds according to the methods described herein; (b) comparing the condensate preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds with the same or similar condensate preference profile; (d) identifying a property, e.g., a chemical moiety or motif, that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profile; and (e) designing a compound that includes the identified property. In some embodiments, the step of designing the compound comprises combining a moiety that includes the identified property, thereby imparting the desired condensate preference profile to the compound. In some embodiments, the step of designing the compound comprises modifying a first moiety to a second moiety that includes the identified property, thereby imparting the desired partitioning property to the compound. In some embodiments, a plurality of compounds designed by the methods described herein are provided. In some embodiments, the compounds are designed, in whole or in part, using approaches that include modeling, computer and / or computational based techniques, such as bioinformatics, chemoinformatics, and / or artificial intelligence (AI) based techniques. In some embodiments, the method further includes making the compound.

[0045] In some aspects, provided herein is a method for screening test compounds for desired partitioning properties from a group of candidate compounds, comprising: (a) determining the partitioning properties of each of the group of candidate compounds; and (b) identifying a test compound having the desired partitioning properties. In some embodiments, the partitioning properties of each of the group of candidate compounds are determined in vitro. In some embodiments, the test compound has suitable partitioning properties that are useful for treating disease in an individual.

[0046] In some aspects, provided herein is a method for identifying a test compound useful for treating a disease in an individual in need thereof, the method comprising: (a) identifying a target condensate associated with the disease; and (b) determining the partitioning properties of a candidate compound in the target condensate; and (c) identifying a test compound having suitable partitioning properties useful for treating the disease.

[0047] In some aspects, the present specification provides a method for determining the distribution characteristics of a test compound in target condensate, comprising: (a) combining a test compound with a composition comprising target condensate and extra condensate solution; (b) obtaining a reference control; (c) using mass spectrometry to measure the MS signal of the test compound or a part thereof in the extra condensate solution; (d) using mass spectrometry to measure the MS signal of the test compound or a part thereof in the reference control; and (e) comparing the MS signal of the test compound from the extra condensate solution and the MS signal of the test compound from the reference control, thereby determining the distribution characteristics of the test compound in target condensate.In some embodiments, the amount of the test compound combined with the composition is 100nM or less, and the amount of precursor molecule in the composition comprising target condensate is about 5μM.

[0048] In some aspects, provided herein is a library comprising a plurality of compounds, each compound of the plurality of compounds comprising the same moiety comprising a property having a desired partitioning property.

[0049] In some aspects, provided herein is a method of designing a test compound with desired partitioning properties, comprising modifying a precursor of the test compound by attaching a moiety to the compound, wherein the moiety comprises a property with the desired partitioning properties.

[0050] Those skilled in the art will appreciate that modifications of the embodiments and details described herein may be made without departing from the scope of the present disclosure. Additionally, while various advantages, aspects and objectives are described with reference to various implementations, the scope of the present disclosure should not be limited by reference to such advantages, aspects and objectives. [Brief description of the drawings]

[0051] [Figure 1] 1 shows an exemplary fluorescence image of the distribution of dyes in the condensate. Dye number 10 is not enriched in the condensate, whereas the other dyes are enriched in the condensate.

[0052] [Diagram 2] 2A and 2B represent the inner and outer regions of an exemplary condensate selected for measuring intensity in the presence (FIG. 2A) and absence (FIG. 2B) of a dye.

[0053] [Diagram 3] Figure 3 shows the ratio of the mean internal to external intensity (I-internal:I-external) determined for each dye in FUS-SNAP condensates from two different days of the experiment. Error bars indicate the standard deviation of two independent experiments.

[0054] [Figure 4] Figure 4 shows the ratio of the mean internal to external intensity (I-internal:I-external) determined for each dye in PGL-3 condensates from two different days of the experiment. Error bars indicate the standard deviation of two independent experiments.

[0055] [Diagram 5] FIG. 5 shows the ratio of the average I-inside:I-outside ratios for each dye in PGL-3 condensates to the ratio of the average I-inside:I-outside ratios for each dye in FUS-SNAP condensates (FUS-SNAP:PGL-3 ratios).

[0056] [Figure 6] FIG. 6 shows fluorescence images of GFP-tagged FUS condensates in the presence or absence of Rhodamine 800 using the FUS-GFP channel, the Rhodamine 800 channel and integration of other images.

[0057] [Figure 7] FIG. 7 shows histograms of the fraction of a compound in the supernatant (outside the condensate) for an exemplary compound using both a fluorescence-based assay and a mass spectrometry-based assay.

[0058] [Figure 8] FIG. 8 shows histograms of the fraction of compound in the supernatant for exemplary compounds evaluated at various compound concentrations.

[0059] [Figure 9] Figures 9A and 9B show histograms of the fraction of a compound of interest outside the condensate measured in a system with a single compound of interest and in a system with a mixture of compounds including the compound of interest. Fluorescein is the compound of interest in Figure 9A. Rhodamine B is the compound of interest in Figure 9B.

[0060] [Figure 10] FIG. 10 shows histograms of the fraction of compound in the supernatant for a particular compound in several multiplexed systems. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] The invention includes, in some aspects, methods and applications thereof for assessing, eg, characterizing or determining, condensation-related properties of a compound, such as a test compound.

[0062] The disclosure of the present application is based in part on the inventors' unique insight into methods for evaluating, e.g., determining or characterizing, condensation-related properties of compounds, which may be useful, for example, for identifying, characterizing and developing compounds or portions thereof capable of desired interactions (including lack of interaction) with condensates. In some embodiments, the desired interactions with condensates result in desired biological activity associated with the compound. In some aspects, the disclosure of the present application is based in part on the inventors' knowledge and discoveries regarding the use of quantitative techniques, such as mass spectrometry, to determine condensation-related properties of compounds, e.g., partitioning properties of test compounds with respect to target condensates. Such methods allow, for example, accurate and reliable determination of partitioning properties of test compounds with respect to target condensates in a high-throughput manner suitable for use in both simple and complex systems. In addition, for example, these methods are hypothesis-free (i.e., do not require known labeled compounds or condensates or components thereof), are compatible with high compound multiplexing, do not require compound enrichment, do not require compound extraction from condensates, can be used in homogeneous and heterogeneous systems, and can be performed with smaller amounts of compound and / or condensate precursor macromolecules, which represent more biologically relevant models and reduce the use of starting materials and reagents. These methods allow for the identification of test compounds or portions thereof, which can be used to guide further identification and / or design of one or more compounds with desired distribution properties, thus providing a method for identifying target compounds with improved efficacy, therapeutic index and / or safety. Identification of test compounds or portions thereof also allows for the advancement of certain drug development applications using such knowledge, for example, development of privileged libraries of compounds with desired distribution properties, as well as modeling and / or computation-based techniques for drug discovery and screening.

[0063] As described herein, the method of evaluating the condensate-related properties of a compound can be applied to many methods and forms thereof, including methods of determining the partitioning properties of a test compound, methods of determining the relative partitioning properties of a test compound, methods of determining the condensate preference profile of a test compound, methods of identifying the properties of a compound associated with condensate association, methods of designing a compound with a desired condensate association, and methods of identifying a compound useful for treating a disease in an individual. Forms of the method described herein include, for example, assays for evaluating the interaction of a single test compound or a portion thereof with a single target condensate, evaluating the interaction of a single test compound or a portion thereof with multiple target condensates, and evaluating the interaction of multiple test compounds or a subset thereof with a single target condensate. As described herein, the unique insights of the inventors allow for the identification, development and optimization of compounds, e.g., pharma- ceutical acceptable compounds, useful for treating a disease in an individual.

[0064] Thus, in some aspects, the present invention includes a method for determining the distribution characteristics of a test compound in a target condensate, comprising the steps of: (a) adding a test compound to a composition comprising a target condensate and excess condensate solution; and (b) determining the amount of the test compound in the target condensate, thereby determining the distribution characteristics of the test compound in the target condensate.

[0065] In another aspect, the invention includes a method for determining the distribution characteristics of a test compound in a target condensate, comprising the steps of: (a) adding a test compound to a composition comprising a precursor molecule; (b) causing the formation of a target condensate to obtain a composition comprising the target condensate and an excess condensate solution; and (c) determining the amount of the test compound in the target condensate, thereby determining the distribution characteristics of the test compound in the target condensate.

[0066] In another aspect, the invention includes a method for determining the distribution characteristics of a plurality of test compounds in a target condensate, the method comprising performing any one or more of the methods described herein using a plurality of test compounds.

[0067] In some embodiments, the present invention includes a method for identifying a compound characteristic associated with the partitioning of the compound into a condensate, the method comprising the steps of: (a) determining the partitioning characteristics of a plurality of test compounds in a target condensate by performing a method for determining the partitioning characteristics of a plurality of test compounds in a target condensate; (b) comparing the partitioning characteristics of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds having the same or similar partitioning characteristics in the target condensate; and (d) identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning characteristics. In some embodiments, the present invention includes a method for designing a compound having desired partitioning properties into a target condensate, the method comprising the steps of: (a) determining the partitioning properties of a plurality of test compounds in a target condensate by performing a method for determining the partitioning properties of a plurality of test compounds in a target condensate; (b) comparing the partitioning properties of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds having the same or similar partitioning properties in the target condensate; (d) identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning properties; and (e) designing a compound that includes the identified properties.

[0068] In another aspect, the present invention includes a method for determining the relative partitioning profile of a test compound in a target condensate, comprising the steps of: (i) determining the partitioning profile of a test compound by performing any one or more of the methods described herein with the test compound; (ii) determining the partitioning profile of a reference compound by performing any one or more of the methods described herein with the reference compound; and (iii) calculating the ratio of the partitioning profiles determined in (i) and (ii), thereby determining the relative partitioning profile of the test compound in the target condensate. In some embodiments, the method further comprises repeating steps (i) and (iii) with a plurality of test compounds. In some embodiments, the present invention includes a method for identifying a compound characteristic associated with the partitioning of the compound into a target condensate, the method comprising the steps of: (a) determining the relative partitioning characteristics of a plurality of test compounds in a target condensate by performing a method for determining the relative partitioning characteristics of a plurality of test compounds in a target condensate; (b) comparing the relative partitioning characteristics of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds having the same or similar relative partitioning characteristics in the target condensate; and (d) identifying a characteristic that a subset or all of the identified test compounds have in common in addition to the same or similar relative partitioning characteristics. In some embodiments, the present invention includes a method for designing a compound having desired relative partition properties, the method comprising the steps of: (a) determining the relative partition properties of a plurality of test compounds in a target condensate by performing a method for determining the relative partition properties of a plurality of test compounds in a target condensate; (b) comparing the relative partition properties of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds having the same or similar relative partition properties in the target condensate; (d) identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar relative partition properties; and (e) designing a compound that includes the identified properties.

[0069] In another aspect, the present invention includes a method for determining a condensate preference profile of a test compound, comprising the steps of: (a) adding the test compound to a composition comprising a first target condensate and a second target condensate; (b) determining the amount of the test compound in the first target condensate; (c) determining the amount of the test compound in the second target condensate; and (d) calculating a ratio of the amounts of the test compound determined in the first target condensate and the second target condensate, thereby determining the condensate preference profile of the test compound.

[0070] In another aspect, the present invention includes a method for determining a condensate preference profile of a test compound, comprising the steps of: (a) adding the test compound to a composition comprising a precursor molecule; (b) causing the formation of a first target condensate and a second target condensate in the composition; (c) determining the amount of the test compound in the first target condensate; (d) determining the amount of the test compound in the second target condensate; and (e) calculating a ratio of the amounts of the test compound determined in the first target condensate and the second target condensate, thereby determining the condensate preference profile of the test compound.

[0071] In other aspects, the invention includes a method for determining a condensate preference profile of a plurality of test compounds, comprising performing any one or more of the methods described herein for determining a condensate preference profile of a test compound using a plurality of test compounds. In some embodiments, the invention includes a method for identifying a compound characteristic associated with the partitioning of the compound into a condensate, comprising: (a) determining a condensate preference profile of a plurality of test compounds by performing a method for determining a condensate preference profile of a plurality of test compounds; (b) comparing the condensate preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar condensate preference profile; and (d) identifying a characteristic that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profile. In some embodiments, the present invention includes a method for designing a compound having a desired condensate preference profile, the method comprising the steps of: (a) determining the condensate preference profiles of a plurality of test compounds by performing a method for determining the condensate preference profiles of a plurality of test compounds; (b) comparing the condensate preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar condensate preference profiles; (d) identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profile; and (e) designing a compound that includes the identified properties. definition

[0072] For purposes of interpreting this specification, the following definitions shall apply and, whenever applicable, terms used in the singular shall include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

[0073] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues can 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 this definition. The term also includes post-translational modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like.

[0074] The terms "comprising," "having," "containing," and "including," as well as other similar forms and their grammatical equivalents, as used herein, are intended to be equivalent in meaning and to be open-ended in that the item or items following any one of these words are not meant to be an exhaustive list of such item or items or to be limited to only the item or items in the list. For example, an article "comprising" components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C, but one or more other components. Thus, "comprising" and similar forms and their grammatical equivalents are intended and understood to include the disclosure of embodiments that "consist essentially of" or "consist of."

[0075] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limit of that range, to the tenth of the unit of the lower limit, as well as any other stated or intervening value within that stated range, is encompassed within the disclosure, unless any specific limit within a stated range is excluded. Where a stated range includes one or both of the limits, ranges excluding either or both of such included limits are also included in the disclosure.

[0076] As used herein, reference to "about" a value or parameter includes (and describes) the variation that is directed to the value or parameter itself. For example, a statement that refers to "about X" includes the statement of "X."

[0077] 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.

[0078] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Methods for evaluating condensate-related properties of compounds

[0079] In some aspects of the present application, methods and applications for evaluating condensate-related properties of compounds, such as test compounds, are provided. In some embodiments, "condensates" as used herein refer to non-membrane encapsulated compartments formed by phase separation (including all stages of phase separation) of one or more proteins and / or other macromolecules.

[0080] Techniques for assessing properties related to condensation of compounds and their application to a number of methods and forms are described in more detail below. In light of the description provided, one of ordinary skill in the art will recognize that several embodiments are possible within the scope and spirit of the disclosure of this application.

[0081] In some aspects, provided herein is a method of determining a partitioning characteristic of a test compound, comprising determining an amount of the test compound in a target condensate. In some embodiments, determining the amount of the test compound in the target condensate thereby determines the partitioning characteristic of the test compound in the target condensate.

[0082] In some aspects, provided herein is a method for determining the relative partitioning properties of a test compound, the method comprising determining an amount of the test compound in a target condensate, determining an amount of a reference compound in the target condensate, and calculating a ratio of the amounts of the test compound and the reference compound in the target condensate.

[0083] In addition, in some aspects, a method for determining a condensate preference profile of a test compound is provided herein, comprising: determining the amount of the test compound in a first target condensate; determining the amount of the test compound in a second target condensate; and calculating the ratio of the amount of the test compound in the first and second target condensates. In some embodiments, the method for determining a condensate preference profile of a test compound comprises: determining the partitioning or relative partitioning characteristic of the test compound in a first target condensate; determining the partitioning or relative partitioning characteristic of the test compound in a second target condensate; and calculating the ratio of the partitioning or relative partitioning characteristic of the test compound in the first and second target condensates.

[0084] In some embodiments, the method further comprises repeating the steps of the method for a plurality of test compounds, for example, in some embodiments, the method comprises repeating the steps of the method for at least about any of 2, 3, 4, 5, 10, 15, 20, 25, 40, 50, 75, 100, 250, 500, 1,000, 10,000, 100,000 or more compounds.

[0085] In some embodiments, the method further comprises combining a test compound and a composition comprising a target condensate and an excess condensate solution. In some embodiments, the method further comprises adding a test compound to a composition comprising a target condensate and an excess condensate solution. In some embodiments, the method further comprises causing the formation of a target condensate. In some embodiments, the method further comprises combining a test compound and a precursor composition comprising a condensate precursor molecule, and then causing the formation of a target condensate. In some embodiments, the method further comprises adding a test compound to a composition comprising a condensate precursor molecule, and then causing the formation of a target condensate.

[0086] In some embodiments, the method further comprises combining a test compound and a composition comprising cells.In some embodiments, the method further comprises combining a test compound and a composition comprising cells, and then causing the formation of target condensates.In some embodiments, the method further comprises causing the formation of target condensates in a composition comprising cells, and then combining the test compound and the composition.In some embodiments, the method further comprises allowing the compound to enter cells.

[0087] In some embodiments, the method further comprises adding a test compound to a composition comprising cells. In some embodiments, the method further comprises adding a test compound to a composition comprising cells, and then causing the formation of target condensates. In some embodiments, the method further comprises causing the formation of target condensates in a composition comprising cells, and then adding a test compound to the composition. In some embodiments, the method further comprises allowing the compound to enter cells.

[0088] In some embodiments, the method further comprises a step of separating the target condensate from the excess condensate solution, for example, for the purpose of quantifying the target compound in the condensate or the excess condensate solution. In a cell-free solution, the condensate is typically denser than the excess condensate solution and settles. Thus, in some embodiments, separating the target condensate from the excess condensate solution comprises separating the supernatant from the precipitate. In some embodiments, the method comprises a step of centrifuging the composition. In some embodiments, the method comprises a step of settling the condensate. Techniques for determining properties related to condensates of compounds

[0089] In some aspects, provided herein is a method for determining a condensate-related property of a compound, comprising determining the amount of a test compound that is depleted from an excess condensate solution due to the presence, formation and / or modulation of a target condensate (including determining that there is a lack of depletion). In some embodiments, the method for determining a condensate-related property of a compound comprises the use of a mass spectrometry-based technique (e.g., to determine the amount of a test compound). In some embodiments, the method for determining a condensate-related property of a compound comprises the use of any one or more of liquid chromatography (e.g., HPLC), microscopy, quantitative image analysis, quantitative fluorescence microscopy and spectroscopy, nuclear magnetic resonance spectroscopy, Raman spectroscopy and / or ultraviolet-visible spectroscopy (e.g., to determine the amount of a test compound). In some embodiments, such a method is useful in determining the partitioning properties, relative partitioning properties and / or condensate preference profiles of a test compound in a composition comprising a target condensate and an excess condensate solution.

[0090] In some embodiments, the method includes determining the amount of test compound that is depleted from the system due to the presence and / or formation of the target condensate. In some embodiments, the method includes determining the amount of test compound that is in the excess condensate solution and is not associated with the precursor molecule of the target condensate. In some embodiments, the method includes, for example, determining the amount of test compound associated in the target condensate. In some embodiments, the method includes determining the amount of test compound associated with the precursor molecule of the target condensate. In some embodiments, the amount of test compound that is depleted from the system due to the presence and / or formation of the target condensate is used to determine a condensate-related property, for example, a partitioning property of the test compound with respect to the target condensate.

[0091] In some embodiments, the method includes comparing the MS signal of the test compound or a portion thereof from the excess condensate solution and the MS signal of the test compound or a portion thereof from the reference control, for example, via the ratio of the MS signal of the test compound or a portion thereof from the excess condensate solution and the MS signal of the test compound or a portion thereof from the reference control. In some embodiments, the ratio of the MS signal of the test compound from the excess condensate solution and the MS signal of the test compound from the reference control represents a depletion value. In some embodiments, the depletion value represents the amount of the test compound that is depleted from the system due to the presence and / or formation of the target condensate. In some embodiments, the method further includes obtaining, e.g., measuring, an MS ion signal of the test compound or a portion thereof in the excess condensate solution using a mass spectrometry technique. In some embodiments, the method further includes obtaining, e.g., measuring, an MS ion signal of the test compound or a portion thereof in the reference control using a mass spectrometry technique. In some embodiments, the method further includes combining the test compound and a composition comprising the target condensate and the excess condensate solution. In some embodiments, the method further includes causing the formation of the target condensate in the presence of the test compound to obtain a composition comprising the target condensate and the excess condensate solution. In some embodiments, the method further comprises separating the target condensate in the composition comprising the target condensate and excess condensate solution, e.g., via pelleting the target condensate in the composition. In some embodiments, the method further comprises obtaining, e.g., generating, a reference control.

[0092] In some embodiments, the amount of test compound added to the composition comprising target condensate and excess condensate solution, or their precursors, is based on an amount that allows for determining a relatively small depletion of the total amount of test compound from the excess condensate solution (such as by comparing the measured amount of the compound in the excess condensate and the measured amount of the compound in a reference control). In some embodiments, the amount of test compound added to the composition comprising target condensate and excess condensate solution is based on the amount of target condensate and / or one or more of its precursor molecules in the composition. In some embodiments, the amount of test compound added to the composition comprising target condensate and excess condensate solution is based on the volume of the compound in the target condensate. In some embodiments, the amount of the test compound is about 1 μM or less, e.g., about 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 450 nM or less, 400 nM or less, 350 nM or less, 300 nM or less, 250 nM or less, 200 nM or less, 150 nM or less, 125 nM or less, 100 nM or less, 90 nM or less, or less than about 1 μM. or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less. In some embodiments, the lower limit of the test compound is based on the analytical method used to measure the amount of the test compound.In some embodiments, the amount of precursor molecule in the composition is about 1 μM to about 10 μM and the test compound is about 1 μM or less, e.g., about 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 450 nM or less, 400 nM or less, 350 nM or less, 300 nM or less, 250 nM or less, 200 nM or less, 150 nM or less, 125 nM or less, 100 nM or less. In some embodiments, the antibody is either less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM.

[0093] In some embodiments, the method includes the use of a reference control and / or a method for preparing a reference control. The reference control described herein provides a reference measurement useful for determining the amount of test compound depleted from a system due to the presence and / or formation of a target condensate. In some embodiments, when a composition includes (i) a test compound contained in and associated with a target condensate, (ii) a test compound in a volume of excess condensate solution, such as a concentration, and the composition includes (a) a target condensate, (b) an excess condensate solution, (c) a precursor macromolecule of the target condensate in a volume of excess condensate solution, such as a concentration, and (d) a test compound in a volume of the composition, such as a concentration, the reference control includes a precursor macromolecule of the target condensate in a volume of excess condensate solution, such as a concentration, and a test compound in a volume of the composition, such as a concentration. In some embodiments, the method includes a step of measuring a precursor macromolecule of the target condensate or a portion thereof in a volume of excess condensate solution, such as a concentration, from a composition including the target condensate and the excess condensate solution.

[0094] In some embodiments, the reference control comprises an amount of precursor polymer of the target condensate based on an amount of precursor polymer present in the excess condensate solution after the composition is subjected to pelleting of the target condensate. In some embodiments, the reference control has the same concentration of precursor polymer as the concentration of precursor polymer in the excess condensate solution after the composition is subjected to pelleting of the target condensate. In some embodiments, the reference control comprises a test compound at the same concentration as combined with the composition comprising the target condensate and the excess condensate solution. In some embodiments, the reference control is substantially free of target condensate. In some embodiments, the method further comprises determining the amount of the test compound in the reference control. In some embodiments, the step of determining the amount of the test compound in the reference control comprises measuring the amount of the test compound or a portion thereof in the reference control using a mass spectrometry-based technique.

[0095] A variety of mass spectrometry-based techniques are suitable for the methods described herein. In some embodiments, the mass spectrometry-based technique comprises measuring the MS signal of one or more ion species of one or more compounds, e.g., one or more test compounds. In some embodiments, the MS signal is of one or more ion species of the test compound, such as one or more charge states of the ions of the test compound. In some embodiments, the MS signal is derived from a mass-to-charge (m / z) measurement. In some embodiments, the MS signal is an ionization intensity. In some embodiments, the MS signal is a peak height. In some embodiments, the MS signal is a peak area, e.g., an integral of the signal corresponding to the MS ion signal. In some embodiments, the MS signal is a peak volume, e.g., an integral of the signal corresponding to the MS ion signal. In some embodiments, the MS signal is a cumulative measurement of the measured signal of the ions of the test compound. In some embodiments, the mass spectrometry-based technique comprises a liquid chromatography / mass spectrometry (LC / MS) technique, a liquid chromatography / tandem mass spectrometry (LC / MS) technique, and / or a direct sample introduction technique (e.g., direct spray). In some embodiments, the mass spectrometry based technique comprises gas chromatography / mass spectrometry (GC / MS). In some embodiments, the mass spectrometry based technique comprises an acquisition technique selected from data-dependent acquisition, data-independent acquisition, selected reaction monitoring (SRM), and multiple reaction monitoring (MRM).

[0096] Liquid chromatography techniques contemplated by the present application include methods for separating precursor polymers and / or test compounds that are compatible with mass spectrometry techniques. In some embodiments, the liquid chromatography techniques include high performance liquid chromatography (HPLC) techniques. In some embodiments, the liquid chromatography techniques include high flow rate liquid chromatography techniques. In some embodiments, the liquid chromatography techniques include low flow rate liquid chromatography techniques, e.g., micro flow rate liquid chromatography techniques or nano flow rate liquid chromatography techniques. In some embodiments, the liquid chromatography techniques include online liquid chromatography techniques coupled with a mass spectrometer. In some embodiments, the sample may be introduced into the mass spectrometer using capillary electrophoresis (CE) techniques or electrospray or MALDI techniques. In some embodiments, the sample may be introduced into the mass spectrometer using direct sample introduction techniques. In some embodiments, the mass spectrometry techniques include ionization techniques. Ionization techniques contemplated by the present application include techniques that can charge the precursor polymers and / or test compounds. In some embodiments, the ionization technique is electrospray ionization. In some embodiments, the ionization technique is nanoelectrospray ionization. In some embodiments, the ionization technique is atmospheric pressure chemical ionization. In some embodiments, the ionization technique is atmospheric pressure photoionization. In some embodiments, the ionization technique is matrix-assisted laser desorption ionization (MALDI). In some embodiments, the mass spectrometry technique comprises electrospray ionization, nanoelectrospray ionization, or matrix-assisted laser desorption ionization (MALDI) techniques.

[0097] Mass spectrometers contemplated by the present application that may be coupled to online liquid chromatography techniques include high-resolution mass spectrometers and low-resolution mass spectrometers. Thus, in some embodiments, the mass spectrometer is a time-of-flight (TOF) mass spectrometer. In some embodiments, the mass spectrometer is a quadrupole time-of-flight (Q-TOF) mass spectrometer. In some embodiments, the mass spectrometer is a quadrupole ion trap time-of-flight (QIT-TOF) mass spectrometer. In some embodiments, the mass spectrometer is an ion trap. In some embodiments, the mass spectrometer is a single quadrupole. In some embodiments, the mass spectrometer is a triple quadrupole (QQQ). In some embodiments, the mass spectrometer is an orbitrap. In some embodiments, the mass spectrometer is a quadrupole orbitrap. In some embodiments, the mass spectrometer is a Fourier transform ion cyclotron resonance (FT) mass spectrometer. In some embodiments, the mass spectrometer is a quadrupole Fourier transform ion cyclotron resonance (Q-FT) mass spectrometer. In some embodiments, the mass spectrometry technique includes a positive ion mode. In some embodiments, the mass spectrometry technique comprises a negative ion mode. In some embodiments, the mass spectrometry technique comprises a time-of-flight (TOF) mass spectrometry technique. In some embodiments, the mass spectrometry technique comprises a quadrupole time-of-flight (Q-TOF) mass spectrometry technique. In some embodiments, the mass spectrometry technique comprises an ion mobility mass spectrometry technique. In some embodiments, low resolution mass spectrometry techniques such as ion trap or single or triple quadrupole approaches are suitable.

[0098] In some embodiments, the mass spectrometry-based technique comprises processing the resulting MS signals of the precursor polymer and / or the test compound. In some embodiments, the mass spectrometry-based technique comprises peak detection. In some embodiments, the mass spectrometry-based technique comprises determining ionization intensity. In some embodiments, the mass spectrometry-based technique comprises determining peak height. In some embodiments, the mass spectrometry-based technique comprises determining peak area. In some embodiments, the mass spectrometry-based technique comprises determining peak volume.

[0099] In some embodiments, mass spectrometry based techniques include identifying the test compound.

[0100] Thus, for example, in some embodiments, a method for determining the distribution characteristics of a test compound in a target condensate is provided, which comprises comparing the MS signal of the ion of the test compound from the excess condensate solution and the MS signal of the ion of the test compound from the reference control, thereby determining the distribution characteristics of the test compound in the target condensate.In some embodiments, the method for determining the distribution characteristics of a test compound in a target condensate comprises (a) obtaining, e.g., measuring, the MS signal of the test compound or a part thereof in the excess condensate solution using a mass spectrometry technique; (b) obtaining, e.g., measuring, the MS ion signal of the test compound or a part thereof in the reference control using a mass spectrometry technique; and (c) comparing the MS signal of the ion of the test compound from the excess condensate solution and the MS signal of the ion of the test compound from the reference control, thereby determining the distribution characteristics of the test compound in the target condensate.

[0101] In some embodiments, provided herein are methods for determining condensate-related properties of a small molecule test compound (such as a therapeutic small molecule that is 1,000 Da or smaller and / or satisfies Lipinski's rule of five), comprising determining an amount of the small molecule test compound that is depleted from an excess condensate solution due to the presence, formation and / or modulation of the target condensate (including determining that there is a lack of depletion). In some embodiments, provided are methods for determining a partitioning property of a small molecule test compound in a target condensate, comprising comparing the MS signal of an ion of the small molecule test compound from the excess condensate solution and the MS signal of an ion of the small molecule test compound from a reference control, thereby determining the partitioning property of the small molecule test compound in the target condensate. In some embodiments, a method for determining the distribution characteristics of a small molecule test compound in a target condensate includes the steps of: (a) obtaining, e.g., measuring, an MS signal of the small molecule test compound or a portion thereof in an excess condensate solution using a mass spectrometry technique; (b) obtaining, e.g., measuring, an MS ion signal of the small molecule test compound or a portion thereof in a reference control using a mass spectrometry technique; and (c) comparing the MS signal of the ion of the small molecule test compound from the excess condensate solution and the MS signal of the ion of the small molecule test compound from the reference control, thereby determining the distribution characteristics of the small molecule test compound in the target condensate.

[0102] In some embodiments, provided herein are methods for determining condensate-related properties of a therapeutic compound (such as any or any combination of an exogenous compound, a small molecule, a polypeptide, an oligonucleotide, a nucleic acid, an antibody or fragment thereof, a synthetically produced compound including a compound produced in cell culture, or a compound that is not a condensate precursor polymer), comprising determining the amount of therapeutic compound that is depleted from the excess condensate solution due to the presence, formation, and / or modulation of the target condensate (including determining that there is a lack of depletion). In some embodiments, provided are methods for determining the partitioning properties of a therapeutic compound in a target condensate, comprising comparing the MS signal of an ion of the therapeutic compound from the excess condensate solution and the MS signal of an ion of the therapeutic compound from a reference control, thereby determining the partitioning properties of the therapeutic compound in the target condensate. In some embodiments, a method for determining the distribution characteristics of a therapeutic compound in a target condensate includes the steps of: (a) obtaining, e.g., measuring, an MS signal of the therapeutic compound or a portion thereof in an excess condensate solution using a mass spectrometry technique; (b) obtaining, e.g., measuring, an MS ion signal of the therapeutic compound or a portion thereof in a reference control using a mass spectrometry technique; and (c) comparing the MS signal of the ion of the therapeutic compound from the excess condensate solution and the MS signal of the ion of the therapeutic compound from the reference control, thereby determining the distribution characteristics of the therapeutic compound in the target condensate.

[0103] In some embodiments, a method for determining the distribution characteristics of a test compound in a target condensate comprises the steps of: (a) combining the test compound and a composition comprising the target condensate and an excess condensate solution; (b) obtaining, e.g., preparing, a reference control; (c) measuring the MS signal of the test compound or a portion thereof in the excess condensate solution using a mass spectrometry technique; (d) measuring the MS signal of the test compound or a portion thereof in the reference control using a mass spectrometry technique; and (e) comparing the MS signal of the test compound from the excess condensate solution and the MS signal of the test compound from the reference control, thereby determining the distribution characteristics of the test compound in the target condensate.

[0104] In some embodiments, a method for determining the distribution characteristics of a test compound in a target condensate comprises the steps of: (a) combining the test compound and a composition comprising the target condensate and an excess condensate solution; (b) obtaining, e.g., preparing, a reference control; (c) measuring the MS signal of the test compound or a portion thereof in the excess condensate solution using a mass spectrometry technique; (d) measuring the MS signal of the test compound or a portion thereof in the reference control using a mass spectrometry technique; and (e) comparing the MS signal of the test compound from the excess condensate solution and the MS signal of the test compound from the reference control, thereby determining the distribution characteristics of the test compound in the target condensate.

[0105] In some embodiments, a method for determining the distribution characteristics of a test compound in a target condensate comprises the steps of: (a) combining the test compound and a composition comprising the target condensate and an excess condensate solution; (b) incubating the test compound and the composition; (c) pelleting the target condensate in the composition using a centrifugation technique; (d) obtaining, e.g., preparing, a reference control; (e) measuring the MS signal of the test compound or a portion thereof in the excess condensate solution using a mass spectrometry technique; (f) measuring the MS signal of the test compound or a portion thereof in the reference control using a mass spectrometry technique; and (g) comparing the MS signal of the test compound from the excess condensate solution and the MS signal of the test compound from the reference control, thereby determining the distribution characteristics of the test compound in the target condensate.

[0106] In some embodiments, a method for determining the distribution characteristics of a test compound in a target condensate comprises the steps of: (a) combining the test compound and a composition comprising the target condensate and an excess condensate solution; (b) incubating the test compound and the composition; (c) pelleting the target condensate in the composition using a centrifugation technique; (d) obtaining, e.g., preparing, a reference control; (e) measuring the MS signal of the test compound or a portion thereof in the excess condensate solution using a mass spectrometry technique; (f) measuring the MS signal of the test compound or a portion thereof in the reference control using a mass spectrometry technique; and (g) comparing the MS signal of the test compound from the excess condensate solution and the MS signal of the test compound from the reference control, thereby determining the distribution characteristics of the test compound in the target condensate.

[0107] In some embodiments of any of the methods or steps of the methods described herein, the method is suitable for determining condensate-related properties for each of a plurality of test compounds in a single composition comprising a target condensate. For example, in some embodiments, a method is provided that includes: (a) combining a plurality of test compounds and a composition comprising a target condensate and an extra condensate solution; and (b) comparing the MS signal of an ion of a first test compound of the plurality of test compounds from the extra condensate solution and the MS signal of an ion of the first test compound from a reference control. In some embodiments, the MS signal of an ion of each test compound of the plurality of test compounds from the extra condensate solution is compared with the MS signal of each ion of each individual test compound from the reference control. In some embodiments, the reference control comprises a plurality of compounds. In some embodiments, the number of compounds in the plurality of test compounds is limited only by the capacity of the analytical method used to measure the amount of each compound. In some embodiments, the plurality of test compounds comprises at least 5 compounds, for example, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1,000 compounds. In some embodiments, the method further comprises obtaining, e.g., measuring, the MS signal of each of the test compounds or portions thereof in the excess condensate solution using a mass spectrometry technique. In some embodiments, the method further comprises obtaining, e.g., measuring, the MS signal of each of the test compounds or portions thereof in the reference control using a mass spectrometry technique. In some embodiments, the MS signal of each of the test compounds in either the excess condensate solution or the reference control is obtained in a single mass spectrometry. Test Compounds and Analysis

[0108] In some embodiments, the test compound is a small molecule, a polypeptide, a peptidomimetic, a lipid, or a nucleic acid. In some embodiments, the test compound is an approved compound, e.g., a compound approved for medical treatment by the U.S. Food and Drug Administration. In some embodiments, the test compound is a novel compound. In some embodiments, the test compound is charged. In some embodiments, the test compound is hydrophobic. In some embodiments, the test compound is hydrophilic. In some embodiments, the test compound is a small molecule. In some embodiments, the small molecule is an alkaloid, a glycoside, a phenazine, a phenol, a polyketide, a terpene, or a tetrapyrrole. In some embodiments, the test compound is an antibody. In some embodiments, the target condensate is an extracellular condensate and the test compound is an antibody. In some embodiments, the test compound is a nucleic acid. In some embodiments, the test compound is an RNA, e.g., an siRNA, an miRNA, an mRNA, or an lnRNA. In some embodiments, the test compound is an siRNA, an miRNA, or an mRNA. In some embodiments, the test compound is a non-naturally occurring compound. In some embodiments, the test compound is a protein.

[0109] In some embodiments, the method herein comprises adding two or more test compounds.In some embodiments, two or more compounds are selected from small molecules, polypeptides, lipids or nucleic acids.In some embodiments, two or more test compounds are added sequentially or simultaneously.

[0110] In some embodiments, the test compound comprises a label. In some embodiments, the label is a radioactive label, a colorimetric label, a luminescent label, a chemically reactive label (such as a moiety of a component used in click chemistry), or a fluorescent label. In some embodiments, the test compound is a small molecule comprising a label. In some embodiments, the test compound is a small molecule comprising a fluorophore. In some embodiments, the test compound is a polypeptide comprising a label. In some embodiments, the test compound is a polypeptide comprising a fluorophore. In some embodiments, the test compound is a nucleic acid comprising a label. In some embodiments, the test compound is a nucleic acid comprising a fluorophore. The test compound label may be covalently or non-covalently conjugated to the test compound.

[0111] Methods for determining the amount of a test compound are known. In some embodiments, determining the amount of a test compound comprises quantitatively detecting the test compound. In some embodiments, determining the amount of a test compound comprises quantitatively detecting a test compound label. In some embodiments, determining the amount of a test compound comprises detecting the activity of the test compound and calculating the amount of test compound required to cause the amount of activity detected. In some embodiments, the amount of a test compound is determined by mass spectrometry, liquid chromatography and / or UV-Vis spectroscopy. In some embodiments, the amount of a test compound is determined by fluorescence microscopy. A calibration curve may be used to assist in determining the amount of a test compound. Alternatively, or in addition, the amount of a test compound may be compared to a reference compound. In some embodiments, the reference compound is a test compound label.

[0112] The methods described herein, including determining the amount of a compound, e.g., a test compound or a reference compound, in the condensate, are expected to encompass direct and indirect techniques for determining the amount of a compound in the condensate. In some embodiments, the amount of a compound in the condensate is determined directly. In some embodiments, the amount of a compound in the condensate is determined indirectly. In some embodiments, the amount of a compound in the condensate is determined via determining the amount of a compound not associated with the condensate, e.g., the amount of a compound in an excess condensate solution. In some embodiments, the amount of a compound in the condensate is determined via determining the amount of a reporter compound. In some embodiments, the reporter compound is associated with the condensate. In some embodiments, the reporter compound is not associated with the condensate.

[0113] The amount of the test compound in the target condensate can be compared with the amount of the test compound in another solution or the amount added to the composition.Thus, in some embodiments, the method further comprises comparing the amount of the test compound in the target condensate with the amount added to the composition, and / or the amount in the extra condensate solution, and / or the amount of the test compound in the cell, and / or the amount of the test compound in the second target condensate.

[0114] In some embodiments, the method further comprises comparing the amount of the test compound in the target condensate to the amount added to the composition, hi some embodiments, the comparing step comprises calculating a ratio or percentage of the amount of the test compound in the target condensate and the amount of the test compound added to the composition.

[0115] In some embodiments, the method further comprises comparing the amount of the test compound in the target condensate with the amount of the test compound in the excess condensate solution. In some embodiments, the comparing step comprises calculating a ratio or percentage of the amount of the test compound in the target condensate and the amount of the test compound in the excess condensate solution. In some embodiments, the method further comprises determining the amount of the test compound in the excess condensate solution. In some embodiments, the amount of the test compound in the target condensate is determined before, simultaneously with, or after the amount of the test compound in the excess condensate solution is determined.

[0116] In some embodiments, the method further comprises comparing the amount of the test compound in the target condensate with the amount of the test compound in the cell. In some embodiments, the comparing comprises calculating a ratio or percentage of the amount of the test compound in the target condensate and the amount of the test compound in the cell. In some embodiments, the method further comprises determining the amount of the test compound in the cell.

[0117] In some embodiments, the method further comprises comparing the amount of the test compound in the target condensate with the amount of the test compound in the second target condensate. In some embodiments, the comparing comprises calculating a ratio or percentage of the amount of the test compound in the target condensate and the amount of the test compound in the second target condensate. In some embodiments, the method further comprises determining the amount of the test compound in the second target condensate. In some embodiments, the amount of the test compound in the first target condensate is determined before, simultaneously with, or after the amount of the test compound in the second target condensate is determined.

[0118] In some embodiments, the method further comprises comparing the amount of the test compound in the target condensate with the amount of one or more macromolecules in the target condensate. In some embodiments, the comparing step comprises calculating the ratio or percentage of the amount of the test compound in the target condensate and the amount of one or more macromolecules in the target condensate. In some embodiments, the method further comprises determining the amount of one or more macromolecules in the target condensate. In some embodiments, the amount of the test compound in the target condensate is determined before, simultaneously with, or after the amount of one or more macromolecules in the target condensate is determined.

[0119] Methods for forming condensates are known and may vary depending on the condensate. For example, condensates can be formed by changing the temperature of the composition, for example by exposing the composition to a lower or higher temperature; changing the salt content in the composition, for example by diluting or adding salt to the composition; increasing the concentration of precursor macromolecules, for example by adding nucleic acid, for example RNA, to the composition; adding or changing a buffering agent in the composition; changing the ionic strength of the composition; changing the pH, for example by changing the pH to less than one unit away from the isoelectric point; or adding a crowding agent, for example PEG or dextran. Some exemplary methods for forming condensates are described in Alberti et al. al., J Mol Biol, 430(23), 2018, 4806-4820, which is incorporated herein by reference. composition

[0120] In some embodiments, the composition comprises a cell. In some embodiments, the target condensate is in a cell. In some embodiments, the excess condensate solution is an intracellular fluid, such as the cytosol or nucleosol.

[0121] In some embodiments, the composition comprises a cell. In some embodiments, the target condensate is not in a cell. In some embodiments, the target condensate is an extracellular condensate, e.g., a condensate in the extracellular matrix. In some embodiments, the extracellular fluid is interstitial fluid or plasma.

[0122] In some embodiments, the cell is a microorganism or an animal cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a neuron. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is or is derived from an induced pluripotent stem cell (iPS cell), a HeLa cell, or a HEK293 cell. In some embodiments, the cell comprises a condensate determined to be dysregulated. In some embodiments, the cell comprises a mutation associated with a disease. In some embodiments, the cell has one or more characteristics of a neurodegenerative or proliferative disease. In some embodiments, the cell is treated with arsenate (and / or another compound known to modulate condensates), a temperature change, or a pH change. In some embodiments, the cell expresses a protein labeled with a fluorescent protein. In some embodiments, the protein is a protein known to be concentrated in target condensates. In some embodiments, the cells express a first protein and a second protein, where the first protein is labeled with a first label, where the first protein is known to be enriched in a first target condensate, the second protein is labeled with a second label, where the second protein is known to be enriched in a second target condensate, and the first label and the second label are distinguishable. In some embodiments, the cells express a first protein and a second protein, where the first protein is labeled with a first fluorescent protein, where the first protein is known to be enriched in a first target condensate, the second protein is labeled with a second fluorescent protein, where the second protein is known to be enriched in a second target condensate, and the first fluorescent protein and the second fluorescent protein are distinguishable.

[0123] In some embodiments, the composition does not include cells. In some embodiments, the composition is cell-free. In some embodiments, the composition includes precursor molecules that are non-phase-separated components that can be incorporated into and / or are incorporated into condensates. Condensates can form in cell-free systems from only a few components. For example, the composition may include a protein or protein fragment, e.g., a portion of a protein or peptide, that can form condensates. In some embodiments, the composition includes a protein or protein fragment that includes a low-complexity domain or an intrinsically disordered sequence. In some embodiments, the composition includes an oligomer or polymer of a nucleic acid, e.g., RNA. In some embodiments, the composition includes a small molecule. In some embodiments, the composition includes a buffering agent. In some embodiments, the composition may also include one or more salts and / or one or more polymeric crowding agents (e.g., polyethylene glycol or dextran).

[0124] In some embodiments, the composition comprises cells that contain precursor molecules. In some embodiments, the composition comprises cells, and the method comprises forming target condensates in the cells. In some embodiments, the method comprises changing the temperature of the cells, e.g., exposing the composition to a lower or higher temperature; changing the salt content of the cells; adding or changing a buffer around the cells; changing the pH of the cells; or adding a crowding agent, e.g., PEG or dextran, to the cells. In some embodiments, the composition comprises cells, and the cells comprise a mutation that causes and / or modifies the target condensate to form the target condensate. In some embodiments, the mutation modifies one or more of the following: the size of the target condensate, the shape of the target condensate, the concentration of one or more components of the target condensate, and the uneven distribution of components within the target condensate. In some embodiments, the composition comprises cells, and the cells comprise a mutation that causes the condensate to form.

[0125] In some embodiments, the composition comprises a plurality of condensates. In some embodiments, the composition comprises at least any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 condensates. In some embodiments, the composition comprises two or more target condensates. In some embodiments, the composition comprises at least any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 target condensates. In some embodiments, the method comprises repeating the steps of the method for one or more additional target condensates, e.g., at least any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 target condensates. In some embodiments, the composition comprises at least any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 condensates of the same condensate type, e.g., condensates having the same composition and / or distribution characteristics. In some embodiments, the composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 different condensate types, e.g., condensates that do not have the same composition and / or distribution characteristics. Condensate

[0126] Many condensates are known in the art. Examples of known condensates include cleavage bodies, P granules, histone locus bodies, multivesicular bodies, neural RNA granules, nuclear gems, nuclear pores, nuclear speckles, nuclear stress bodies, intranuclear bodies, Oct1 / PTF / transcription (OPT) domains, paraspeckles, perinuclear compartments, PML nucleoli, PML oncogenic domains, polycomb bodies, processing bodies, signaling clusters, viral condensates, Sam68 nucleoli, stress granules or splicing speckles. Many condensates can be identified using microscopy. In some embodiments, the method further comprises identifying target condensates.

[0127] The condensate used in the methods described herein may be naturally occurring or non-naturally occurring. For example, in some embodiments, the condensate is naturally occurring. In some embodiments, the condensate is not naturally occurring. In some embodiments, the condensate is artificial. In some embodiments, the condensate is synthetic. In some embodiments, the condensate is semi-synthetic. In some embodiments, the condensate is modified. In some embodiments, the condensate is a modified condensate, where the parent condensate of the modified condensate is modified by adding, removing and / or substituting one or more condensate components.

[0128] In some embodiments where the method described herein evaluates two or more condensates, the first condensate and the second condensate of the two or more condensates may be any combination of condensates. In some embodiments, the first condensate is an altered condensate. In some embodiments, the first condensate is an altered condensate and the second condensate is the parent condensate of the first condensate. In some embodiments, the first condensate is a target condensate in a first system, e.g., a first composition, and the second condensate is a target condensate in a second system, e.g., a second composition. In some embodiments, the first condensate is a normal condensate and the second condensate is a dysregulated condensate.

[0129] In some embodiments, a method is provided for determining a condensate preference profile of a test compound, the method comprising: (a) combining a first composition comprising the test compound and a first target condensate; (b) combining the test compound with a second composition comprising the test compound and a second target condensate; (c) determining the amount of the test compound in the first target condensate; and (d) determining the amount of the test compound in the second target condensate, thereby determining the condensate preference profile of the test compound.

[0130] In some embodiments, a method is provided for determining a condensate preference profile of a test compound, the method comprising the steps of: (a) adding the test compound to a first composition comprising a first target condensate; (b) adding the test compound to a second composition comprising a second target condensate; (c) determining the amount of the test compound in the first target condensate; and (d) determining the amount of the test compound in the second target condensate, thereby determining the condensate preference profile of the test compound.

[0131] The identification of condensates can be aided by the use of labels. For example, dyes or labeled compounds can be added to the condensates. In some embodiments, the dyes or labeled compounds can preferentially enter the target condensates. In some embodiments, the label is a radioactive label, a colorimetric label, a chemically reactive label or a fluorescent label. In some embodiments, the composition comprises a first target condensate and a second target condensate, and the first target condensate and the second target condensate are each labeled with a different label, for example, the first target condensate is labeled with RFP and the second target condensate is labeled with GFP.

[0132] Some condensates may contain specific macromolecules. Thus, in some embodiments, the method further comprises characterizing the macromolecules in the target condensate. In some embodiments, the macromolecule is a protein or a 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 tau, FUS, huntingtin protein, hnRNPA1, TDP43, PGL-3, or fragments or aggregates thereof. In some embodiments, the macromolecule is a nucleic acid, e.g., RNA or DNA. In some embodiments, the macromolecule is RNA.

[0133] In some embodiments, the target condensate is a cellular condensate. Many cellular condensates have been described, and many more are known to form but have not yet been described. In some embodiments, the cellular condensate is a cleavage body, a P granule, a histone locus body, a multivesicular body, a neural RNA granule, a nuclear gem, a nuclear pore, a nuclear speckle, a nuclear stress body, an intranuclear body, an Oct1 / PTF / transcription (OPT) domain, a paraspeckle, a perinuclear compartment, a PML nucleolus, a PML oncogenic domain, a polycomb body, a processing body, a signaling cluster, a viral condensate, a Sam68 nucleolus, a stress granule or a splicing speckle. In some embodiments, the target condensate is in a cell when the amount of the target compound in the target condensate is determined.

[0134] In some embodiments, the target condensate is an extracellular condensate.Extracellular condensates can be formed in biological solutions outside cells, such as extracellular matrix or plasma, to promote reactions or sequester molecules (Muiznieks et al., J Mol Biol, 430(23), 2018, 4741-4753).

[0135] Dysregulation of various condensates may be associated with disease. For example, based on experiments with cellular and acellular condensates, disease-associated mutations in protein fused in sarcoma (FUS) 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., Nat Commun, 9(1), 2018, 335). Thus, in some embodiments, dysregulation of target condensates is associated with disease. In some embodiments, the dysregulation includes changes in one or more of the following: size of the target condensate, shape of the target condensate, concentration of one or more components of the target condensate, and uneven distribution of components within the condensate, e.g., components located in the core instead of the shell of the condensate. In some embodiments, the changes are compared to similar non-dysregulated target condensates. Method for determining a compound characteristic related to the partitioning of a test compound - Patents.com

[0136] In some embodiments, the invention includes methods for identifying properties of a compound that relate to the distribution of the compound or a portion thereof in the condensate, for example, that relate to the surface and / or core of the condensate.

[0137] In some embodiments, the method includes determining the partitioning characteristics of a plurality of test compounds in a target condensate by performing a method disclosed herein, in some embodiments, the method includes identifying or determining a property of the compound or a portion thereof that contributes in whole or in part to the partitioning of the compound or a portion thereof through the condensate.

[0138] In some embodiments, identifying a compound characteristic associated with the partitioning of a compound or a portion thereof having a desired, e.g., similar, partitioning property in the target condensate comprises identifying a common portion or motif of test compounds having a desired, e.g., similar, partitioning property. In some embodiments, the compound characteristic of a compound or a portion thereof is based on one or more of charge and hydrophobicity.

[0139] In some embodiments, the partitioning profile of the compound is based on determining the amount, e.g., relative amount, of the compound in the condensate or comparing with one or more condensates, such as condensate preference profile. For example, in some embodiments, the method includes: (a) determining the partitioning profile of a plurality of test compounds in the target condensate by carrying out the method described herein; and (b) identifying test compounds or a portion thereof that have a desired partitioning profile, e.g., similar partitioning profile, in the condensate. In some embodiments, the method includes: (a) determining the partitioning profile of a plurality of test compounds in the target condensate by carrying out the method described herein; (b) comparing the partitioning profile of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds that have the same or similar partitioning profile in the target condensate; and (d) identifying a property that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning profile.

[0140] In some embodiments, the method includes the steps of (a) determining the relative partitioning properties of a plurality of test compounds in a target condensate by performing a method described herein; and (b) identifying test compounds or a portion thereof that have a desired relative partitioning property in the condensate, e.g., similar relative partitioning properties. In some embodiments, the method includes the steps of (a) determining the relative partitioning properties of a plurality of test compounds in a target condensate by performing a method described herein; (b) comparing the relative partitioning properties of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds that have the same or similar relative partitioning properties in the target condensate; and (d) identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar relative partitioning properties.

[0141] In some embodiments, the method includes: (a) determining the condensate preference profile of a plurality of test compounds in a target condensate by performing the method described herein; and (b) identifying test compounds or portions thereof that have a desired condensate preference profile in the condensate, e.g., a similar condensate preference profile. In some embodiments, the method includes: (a) determining the condensate preference profile of a plurality of test compounds by performing the method described herein; (b) comparing the condensate preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds that have the same or similar condensate preference profile; and (d) identifying a property that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profile.

[0142] In some embodiments of the methods described herein, identifying test compounds having the same or similar partitioning characteristics, relative partitioning characteristics, or condensate preference profile in the target condensate comprises identifying two or more test compounds that associate with any portion of the target condensate. In some embodiments of the methods described herein, identifying test compounds having the same or similar partitioning characteristics, relative partitioning characteristics, or condensate preference profile in the target condensate comprises identifying two or more test compounds that do not associate with any portion of the target condensate.

[0143] In some embodiments, the methods disclosed herein further comprise the step of generating the compound identified and / or designed using the methods described herein. Methods for designing compounds with desired partitioning properties

[0144] In some embodiments, the invention includes methods for designing compounds with desired partitioning properties for target condensates.In some embodiments, the invention includes methods for designing compounds with desired partitioning properties for target condensates.

[0145] In some embodiments, the method includes identifying or determining a compound or a portion thereof that includes a desired partitioning property for the target condensate. In some embodiments, the method includes identifying or determining a property of the compound or a portion thereof that contributes in whole or in part to the desired partitioning property for the target condensate. In some embodiments, the method includes identifying or determining a compound or a portion thereof that includes a desired partitioning property for the target condensate. In some embodiments, the method includes identifying or determining a property of the compound or a portion thereof that contributes in whole or in part to the desired partitioning property for the target condensate. In some embodiments, the method includes modifying the identified compound or a portion thereof to optimize the desired partitioning property.

[0146] In some embodiments, the method for designing a compound with a desired partitioning property includes: (a) determining the partitioning property for each of a plurality of test compounds in a target condensate by performing the method described herein; and (b) identifying one or more of the plurality of test compounds or portions thereof that have a desired, e.g., similar, partitioning property in the target condensate. In some embodiments, the method includes comparing the partitioning properties of a subset or all of the plurality of test compounds in the target condensate. In some embodiments, the method includes selecting and / or designing a compound or portion thereof of the plurality of test compounds that have a desired partitioning property.

[0147] In some embodiments, a method for identifying a compound characteristic associated with a desired partitioning property with respect to a target condensate includes the steps of: (a) determining the partitioning properties of a plurality of test compounds in the target condensate by performing a method described herein; (b) comparing the partitioning properties of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar partitioning properties in the target condensate; and (d) identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning properties.

[0148] In some embodiments, a method for identifying a compound characteristic associated with the partitioning of the compound into a condensate includes the steps of: (a) determining the partitioning characteristics of a plurality of test compounds in a target condensate by performing a method described herein; (b) comparing the partitioning characteristics of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar partitioning characteristics in the target condensate; and (d) identifying characteristics that a subset or all of the identified test compounds have in common in addition to their same or similar partitioning characteristics.

[0149] In some embodiments, identifying test compounds or portions thereof that have the same or desired, e.g., similar, partitioning properties in the target condensate comprises identifying a common portion or motif of the test compounds that have the desired, e.g., similar, partitioning properties. In some embodiments, the compound or portion thereof compound properties are based on one or more of charge and hydrophobicity.

[0150] In some embodiments, the identified test compound or a portion thereof can be used as a basis for identifying and / or designing one or more compounds with desired partitioning properties. In some embodiments, the one or more compounds represent a privileged library. In some embodiments, the privileged library includes a set of one or more compounds, the set being a portion or the entirety of the privileged library, including a portion that includes the identified compound or a portion thereof. In some embodiments, each compound of the privileged library has a similar partitioning property, for example, partitioning properties that are at least about 20% apart from each other. In some embodiments, each compound of the privileged library meets or exceeds a partitioning property threshold. In some embodiments, the privileged library comprises at least about 10 compounds, for example, at least about 25 compounds, 50 compounds, 150 compounds, 200 compounds, 250 compounds, 300 compounds, 350 compounds, 400 compounds, 450 compounds, 500 compounds, 1,000 compounds, 1,500 compounds, 2,000 compounds, 2,500 compounds, 3,000 compounds, 3,500 compounds, 4,000 compounds, 4,500 compounds, 5,000 compounds, 10,000 compounds, 20,000 compounds, 30,000 compounds, 40,000 compounds, or 50,000 compounds. In some embodiments, each compound in the set of privileged libraries is suitable for administration to an individual. In some embodiments, each compound in the privileged library has a molecular weight of less than 1,000 Da, for example, 500 Da or less. In some embodiments, each compound in the privileged library satisfies Lipinski's rule of five. In some embodiments, the privileged library comprises compounds present in a single composition.In some embodiments, the privileged library can be used to identify one or more compounds useful for targeting target condensates, and one or more compounds are identified from the privileged library using one or more traditional drug screening methods.

[0151] In some embodiments, designing the compound comprises adding (attaching, e.g., covalently attaching, etc.) a moiety comprising the identified property to the compound, thereby imparting the desired partitioning property to the compound. In some embodiments, designing the compound comprises removing a moiety comprising the identified property, thereby imparting the desired partitioning property to the compound. In some embodiments, the compound is designed, in whole or in part, using an approach that includes modeling, computer and / or computational based techniques, e.g., bioinformatics, chemoinformatics, and / or artificial intelligence (AI) based techniques.

[0152] In some embodiments, the method includes designing a test compound or a portion thereof based on binding a moiety that includes a property identified through a condensate-associated property, e.g., a chemical structure or motif, thereby imparting a desired partitioning property to the test compound. In some embodiments, the identified property modulates, in whole or in part, a condensate-associated property, e.g., a partitioning property, of the test compound, e.g., increases or decreases the extent to which the test compound partitions in the test condensate. In some embodiments, the method of designing a test compound includes binding a moiety that includes a property identified through a condensate-associated property to a precursor of the test compound at any number of positions and / or stereochemical orientations. In some embodiments, the method of designing a test compound includes removing a moiety that includes a property identified through a condensate-associated property to a precursor of the test compound. In some embodiments, the method of designing a test compound includes changing a moiety to another moiety that includes a property identified through a condensate-associated property to a precursor of the test compound at any number of positions and / or stereochemical orientations. In some embodiments, the method of designing a test compound includes binding, removing, and / or changing two or more moieties that include a property identified through a condensate-associated property to a precursor of the test compound. In some embodiments, the test compound comprises a feature that facilitates modulation (binding, removing, changing, etc.) of a moiety such as a compound label (e.g., a moiety of a component used in click chemistry).

[0153] In some embodiments, the identified test compound or a portion thereof can be used as a basis for identifying and / or designing one or more compounds having a desired biological activity. In some embodiments, the one or more compounds represent a privileged library. In some embodiments, the methods described herein include designing a test compound or a portion thereof based on the identified properties via the condensate-associated properties, e.g., binding, removing, or altering a moiety comprising a chemical structure or motif, thereby imparting a desired biological activity to the test compound. In some embodiments, the desired biological activity of the precursor compound is improved by modulating the condensate-associated properties of the precursor compound. In some embodiments, the undesirable biological activity of the precursor compound is reduced by modulating the condensate-associated properties of the precursor compound.

[0154] In some embodiments, the property data associated with the condensate, e.g., partition property data, and / or the identified test compound or a portion thereof, can be used to generate one or more sets of rules. In some embodiments, the one or more sets of rules 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, chemoinformatics, and / or artificial intelligence (AI)-based identification of compounds having desired partition properties. Computer software for determining and / or applying the one or more sets of rules is also provided.

[0155] In some embodiments, the partitioning properties of the compound are based on determining the amount, e.g., relative amount, of the compound in the condensate or comparing it with one or more condensates, such as condensate preference profiles. For example, in some embodiments, the method of designing a compound with a desired relative partitioning property comprises determining the relative partitioning properties of a plurality of test compounds in a target condensate by carrying out a method described herein. In some embodiments, the method of designing a compound with a desired relative partitioning property comprises: (a) determining the relative partitioning properties of a plurality of test compounds in a target condensate by carrying out a method described herein; (b) comparing the relative partitioning properties of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds that have the same or similar relative partitioning properties in the target condensate; (d) identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar relative partitioning properties; and (e) designing a compound that includes the identified properties. In some embodiments, a method for designing a compound having desired relative partition properties includes the steps of: (a) determining the relative partition properties of a plurality of test compounds in a target condensate by performing a method described herein; (b) comparing the relative partition properties of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds having the same or similar relative partition properties in the target condensate; (d) identifying a property that a subset or all of the identified test compounds have in common in addition to the same or similar relative partition properties; and (e) designing a compound that does not contain the identified property.

[0156] In some embodiments, the method for designing a compound with a desired condensate preference profile includes determining the condensate preference profile of a plurality of test compounds by performing the methods disclosed herein. In some embodiments, the method for designing a compound with a desired condensate preference profile includes (a) determining the condensate preference profile of a plurality of test compounds by performing the methods disclosed herein; (b) comparing the condensate preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds with the same or similar condensate preference profile; (d) identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profile; and (e) designing a compound that includes the identified properties. In some embodiments, a method for designing a compound having a desired condensate preference profile includes the steps of: (a) determining the condensate preference profiles of a plurality of test compounds by performing a method disclosed herein; (b) comparing the condensate preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar condensate preference profiles; (d) identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profile; and (e) designing a compound that does not include the identified properties.

[0157] In some embodiments, the methods disclosed herein further comprise the step of generating the compound identified and / or designed using the methods described herein. Methods for screening test compounds

[0158] In some embodiments, the invention includes a method of screening test compounds for desired partitioning properties from a group of candidate compounds, comprising the steps of: (a) determining the partitioning properties of each of the group of candidate compounds; and (b) identifying a test compound having the desired partitioning properties.

[0159] In some embodiments, the desired partition characteristic is based on a partition characteristic associated with a target condensate. In some embodiments, the desired partition characteristic is based on partition characteristics associated with a plurality of target condensates. In some embodiments, the method includes identifying or determining a target condensate. In some embodiments, the target condensate is associated with a disease. In some embodiments, disease-associated target condensates are known in the art. In some embodiments, the method includes obtaining a target condensate, e.g., a disease-associated target condensate.

[0160] In some embodiments, the partitioning characteristics of a candidate compound in a target condensate, for example, a group of two or more candidate compounds, are determined according to the methods described herein. In some embodiments, the group of candidate compounds is at least about any of 2, 5, 10, 15, 50, 75, 100, 150, 200, 300, 400 or 500 candidate compounds. In some embodiments, the partitioning characteristics of a candidate compound in a target condensate are determined in vitro. In some embodiments, the partitioning characteristics of a candidate compound in a target condensate are determined in a cellular system. In some embodiments, the partitioning characteristics of a candidate compound in a target condensate are determined in a non-cellular system, for example, a composition comprising a target condensate or a component thereof.

[0161] In some embodiments, identifying a test compound or portion thereof having a desired partitioning property comprises identifying a test compound or portion thereof that is associated with any portion of the target condensate, e.g., associated with the exterior, surface and / or core of the target condensate. In some embodiments, the desired partitioning property is a suitable partitioning property that is useful for treating a disease in an individual.

[0162] In some embodiments, the present invention includes a method for identifying a test compound useful for treating a disease in an individual in need thereof. In some embodiments, the method includes the steps of (a) identifying a target condensate associated with the disease; and (b) determining the partitioning properties of a candidate compound in the target condensate; and (c) identifying a test compound or a portion thereof that has suitable partitioning properties useful for treating the disease.

[0163] In some embodiments, the method comprises identifying or determining a target condensate associated with a disease. In some embodiments, the target condensate associated with a disease is known in the art. In some embodiments, the method comprises obtaining a target condensate associated with a disease. In some embodiments, the method comprises identifying or determining one or more target condensates associated with a disease.

[0164] In some embodiments, the method of screening a test compound includes: (a) determining the partitioning properties of the test compound in the presence of a second compound; and (b) determining the partitioning properties of the test compound in the absence of the second compound. In some embodiments, the method further includes comparing the partitioning properties of the test compound in the presence of the second compound and the partitioning properties of the test compound in the absence of the second compound. In some embodiments, the method further includes determining a change in the partitioning properties of the test compound in the presence or absence of the second compound.

[0165] In some embodiments, the method of screening a test compound includes: (a) determining the partitioning properties of a second compound in the presence of a test compound; and (b) determining the partitioning properties of a second compound in the absence of a test compound. In some embodiments, the method further includes comparing the partitioning properties of the second compound in the presence of the test compound and the partitioning properties of the second compound in the absence of the test compound. In some embodiments, the method further includes determining the ability of the test compound to change the partitioning properties of the second compound.

[0166] In some embodiments, the partitioning characteristics of the candidate compound, e.g., one or more candidate compounds, in the target condensate are determined according to the methods described herein. In some embodiments, the method includes determining partitioning characteristics of a plurality of candidate compounds for the target condensate. In some embodiments, the plurality of candidate compounds is at least about any of 2, 5, 10, 15, 50, 75, 100, 150, 200, 300, 400, or 500 candidate compounds. In some embodiments, the partitioning characteristics of the candidate compound in the target condensate are determined in vitro. In some embodiments, the partitioning characteristics of the candidate compound in the target condensate are determined in a cellular system. In some embodiments, the partitioning characteristics of the candidate compound in the target condensate are determined in a non-cellular system, e.g., a composition comprising the target condensate or a component thereof.

[0167] In some embodiments, identifying a test compound or portion thereof that has suitable partitioning properties useful for treating a disease includes identifying a test compound or portion thereof that associates with any portion of a target condensate, e.g., that associates with the exterior, surface and / or core of a target condensate.

[0168] In some embodiments, the methods disclosed herein, including identifying, determining, designing and / or screening a compound, e.g., a compound from a group of candidate compounds, further include evaluating the compound based on additional parameters of the compound. In some embodiments, identifying the compound is based on evaluating, e.g., characterizing or determining, a condensation-related property of the compound, as well as one or more additional parameters, e.g., molecular weight and pharmaceutical utility.

[0169] In some embodiments, the methods disclosed herein further comprise the step of producing the compounds identified, designed and / or screened using the methods described herein. Exemplary embodiments

[0170] Among the embodiments provided are the following:

[0171] Embodiment 1. A method for determining the distribution characteristics of a test compound in a target condensate, comprising: (a) adding the test compound to a composition comprising a target condensate and an excess condensate solution; and (b) determining the amount of the test compound in the target condensate, thereby determining the distribution characteristics of the test compound in the target condensate.

[0172] Embodiment 2. The method of embodiment 1, further comprising, prior to step (a), causing formation of a target condensate.

[0173] Embodiment 3. A method for determining the distribution characteristics of a test compound in a target condensate, comprising the steps of: (a) adding the test compound to a composition comprising a precursor molecule; (b) causing the formation of a target condensate to obtain a composition comprising the target condensate and an excess condensate solution; and (c) determining the amount of the test compound in the target condensate, thereby determining the distribution characteristics of the test compound in the target condensate.

[0174] Embodiment 4. The method of any one of embodiments 1 to 3, further comprising determining the amount of the test compound in the excess condensate solution.

[0175] Embodiment 5. The method of embodiment 4, wherein the amount of the test compound in the target condensate is determined before, simultaneously with, or after the amount of the test compound in the excess condensate solution is determined.

[0176] Embodiment 6. The method of embodiment 4 or 5, further comprising determining the ratio of the amount of the test compound in the target condensate and the amount of the test compound in the excess condensate solution.

[0177] Embodiment 7. The method of any one of embodiments 1 to 6, further comprising separating the target condensate from the excess condensate solution.

[0178] Embodiment 8. The method of any one of embodiments 1 to 7, further comprising identifying the target condensate prior to determining the amount of the test compound in the target condensate.

[0179] Embodiment 9. The method of any one of embodiments 1 to 8, wherein dysregulation of target condensates is associated with a disease.

[0180] Embodiment 10. The method of any one of embodiments 1 to 9, further comprising characterizing the target condensates by identifying one or more macromolecules contained therein.

[0181] Embodiment 11 The method of any one of embodiments 1 to 10, wherein the target condensate comprises a protein that comprises an intrinsically disordered sequence.

[0182] Embodiment 12. The method of any one of embodiments 1 to 11, further comprising labeling the target condensates in order to visualize them.

[0183] Embodiment 13. The method of embodiment 12, wherein the target condensate is labeled with a radioactive label, a colorimetric label, or a fluorescent label.

[0184] Embodiment 14 The method of any one of embodiments 1 to 13, wherein the composition comprises cells.

[0185] Embodiment 15. The method of embodiment 14, wherein the cell is a microorganism or an animal cell.

[0186] Embodiment 16 The method of embodiment 14 or 15, wherein the cells contain condensates that are determined to be dysregulated.

[0187] Embodiment 17. The method of any one of embodiments 14 to 16, wherein the cells have one or more characteristics of a neurodegenerative or proliferative disease.

[0188] Embodiment 18. The method of any one of embodiments 1 to 17, wherein the target condensate is a cellular condensate.

[0189] Embodiment 19. The method of embodiment 18, 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, an intranuclear body, an Oct1 / PTF / transcription (OPT) domain, a paraspeckle, a perinuclear compartment, a PML nucleolus, a PML oncogenic domain, a polycomb body, a processing body, a Sam68 nucleolus, a stress granule, or a splicing speckle.

[0190] Embodiment 20. The method of any one of embodiments 1 to 19, wherein the target condensate is in a cell.

[0191] Embodiment 21 The method of any one of embodiments 1 to 20, wherein the excess condensate solution is intracellular fluid.

[0192] Embodiment 22. The method of embodiment 21, wherein the intracellular fluid is a cytosol or a nucleosol.

[0193] Embodiment 23 The method of any one of embodiments 1 to 17, wherein the target condensate is an extracellular condensate.

[0194] Embodiment 24. The method of any of embodiments 23, wherein the excess condensate solution is extracellular fluid.

[0195] Embodiment 25. The method of embodiment 24, wherein the extracellular fluid is interstitial fluid.

[0196] Embodiment 26 The method of any one of embodiments 1 to 13, wherein the composition does not comprise cells.

[0197] Embodiment 27 The method of any one of embodiments 1 to 26, wherein the composition comprises one or more of a polymer, a salt, and a buffering agent.

[0198] Embodiment 28 The method of any one of embodiments 1 to 27, wherein the composition comprises two or more target condensates.

[0199] Embodiment 29. The method of any one of the preceding embodiments, comprising repeating the method steps with one or more additional condensates.

[0200] Embodiment 30 The method of any one of embodiments 1 to 29, wherein the test compound is a small molecule, a polypeptide or a nucleic acid.

[0201] Embodiment 31 The method of any one of embodiments 1 to 30, wherein the test compound comprises a test compound label.

[0202] Embodiment 32 The method of embodiment 31, wherein the test compound label is a radioactive label, a colorimetric label, or a fluorescent label.

[0203] Embodiment 33 The method of embodiment 31 or 32, wherein the test compound label is a fluorescent label.

[0204] Embodiment 34 The method of any one of embodiments 31 to 33, wherein the amount of the test compound is determined by detecting a test compound label.

[0205] Embodiment 35. The method of any one of embodiments 1 to 34, wherein the amount of the test compound is determined by mass spectrometry, liquid chromatography and / or UV-Vis spectroscopy.

[0206] Embodiment 36. A method for determining the partitioning properties of a plurality of test compounds in a target condensate, comprising carrying out a method according to any one of embodiments 1 to 35 using a plurality of test compounds.

[0207] Embodiment 37. The method of embodiment 36, further comprising comparing the partitioning properties of a subset or all of the multiple test compounds in the target condensate.

[0208] Embodiment 38 The method of embodiment 37, further comprising identifying test compounds that have the same or similar partitioning properties in the target condensate.

[0209] Embodiment 39. The method of embodiment 38, further comprising identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning properties.

[0210] Embodiment 40. The method of embodiment 39, further comprising determining the partitioning properties in the target condensate for one or more additional test compounds that comprise the identified properties.

[0211] Embodiment 41. A method for determining a relative partition profile of a test compound in a target condensate, comprising the steps of: (i) determining the partition profile of the test compound by performing the method of any one of embodiments 1 to 35 using the test compound; (ii) determining the partition profile of a reference compound by performing the method of any one of embodiments 1 to 35 using the reference compound; and (iii) calculating the ratio of the partition profiles determined in (i) and (ii), thereby determining the relative partition profile of the test compound in the target condensate.

[0212] Embodiment 42 The method of embodiment 41, wherein the test compound comprises a test compound label.

[0213] Embodiment 43 The method of embodiment 42, wherein the reference compound is a test compound label.

[0214] Embodiment 44. A method for determining the relative partitioning properties of a plurality of test compounds in a target condensate, comprising: (1) carrying out a method according to any one of embodiments 41 to 43; and (2) repeating steps (i) and (iii) with a plurality of test compounds.

[0215] Embodiment 45. The method of embodiment 44, further comprising the step of comparing the relative partitioning properties of a subset or all of the plurality of test compounds in the target condensate.

[0216] Embodiment 46 The method of embodiment 45, further comprising identifying test compounds that have the same or similar relative partitioning properties in the target condensate.

[0217] Embodiment 47. The method of embodiment 46, further comprising identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar relative partitioning properties.

[0218] Embodiment 48. The method of embodiment 47, further comprising determining the relative partitioning properties in the target condensate for one or more additional test compounds that comprise an identified property.

[0219] Embodiment 49. A method for determining a condensate preference profile of a test compound, comprising the steps of: (a) adding the test compound to a composition comprising a first target condensate and a second target condensate; (b) determining an amount of the test compound in the first target condensate; (c) determining an amount of the test compound in the second target condensate; and (d) calculating a ratio of the amounts of the test compound determined in the first target condensate and the second target condensate, thereby determining the condensate preference profile of the test compound.

[0220] Embodiment 50. The method of embodiment 49, further comprising, prior to step (a), causing formation of a first target condensate and / or a second target condensate.

[0221] Embodiment 51. A method for determining a condensate preference profile of a test compound, comprising the steps of: (a) adding the test compound to a composition comprising a precursor molecule; (b) causing the formation of a first target condensate and a second target condensate in the composition; (c) determining an amount of the test compound in the first target condensate; (d) determining an amount of the test compound in the second target condensate; and (e) calculating a ratio of the amounts of the test compound determined in the first target condensate and the second target condensate, thereby determining the condensate preference profile of the test compound.

[0222] Embodiment 52. The method of any one of embodiments 49 to 51, wherein the amount of the test compound in the first target condensate is determined before, simultaneously with, or after the amount of the test compound in the second target condensate is determined.

[0223] Embodiment 53 The method of any one of embodiments 49 to 52, further comprising separating the first target condensate and the second target condensate from the composition.

[0224] Embodiment 54. The method of any one of embodiments 49 to 53, further comprising identifying the first target condensate and / or the second target condensate prior to determining the amount of the test compound in the first condensate and / or the second condensate.

[0225] Embodiment 55 The method of any one of embodiments 49 to 54, wherein dysregulation of the first target condensate and / or the second target condensate is associated with a disease.

[0226] Embodiment 56. The method of any one of embodiments 49 to 55, further comprising characterizing the first target condensate and / or the second target condensate by identifying one or more macromolecules contained therein.

[0227] Embodiment 57. The method of any one of embodiments 49 to 56, further comprising labeling the first target condensate and / or the second target condensate for visualizing the first target condensate and / or the second target condensate.

[0228] Embodiment 58 The method of embodiment 57, wherein the first target condensate and the second target condensate are labeled with different labels.

[0229] Embodiment 59. The method of embodiment 57 or 58, wherein the first target condensate and / or the second target condensate is labeled with a radioactive label, a colorimetric label or a fluorescent label.

[0230] Embodiment 60 The method of any one of embodiments 49 to 56, wherein the composition comprises cells.

[0231] Embodiment 61. The method of embodiment 60, wherein the cell is a microorganism or an animal cell.

[0232] Embodiment 62 The method of embodiment 60 or 61, wherein the cells contain condensates that are determined to be dysregulated.

[0233] Embodiment 63. The method of any one of embodiments 60 to 62, wherein the cells have one or more characteristics of a neurodegenerative or proliferative disease.

[0234] Embodiment 64. The method of any one of embodiments 49 to 56, wherein the first target condensate and / or the second target condensate is a cellular condensate.

[0235] Embodiment 65. The method of embodiment 64, wherein the first 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, an intranuclear body, an Oct1 / PTF / transcription (OPT) domain, a paraspeckle, a perinuclear compartment, a PML nucleolus, a PML oncogenic domain, a polycomb body, a processing body, a Sam68 nucleolus, a stress granule, or a splicing speckle.

[0236] Embodiment 66. The method of embodiment 64 or 65, wherein the second 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, an intranuclear body, an Oct1 / PTF / transcription (OPT) domain, a paraspeckle, a perinuclear compartment, a PML nucleolus, a PML oncogenic domain, a polycomb body, a processing body, a Sam68 nucleolus, a stress granule, or a splicing speckle.

[0237] Embodiment 67. The method of any one of embodiments 49 to 66, wherein the first target condensate and / or the second target condensate is in a cell.

[0238] Embodiment 68 The method of any one of embodiments 49 to 63, wherein the first target condensate and / or the second target condensate is an extracellular condensate.

[0239] Embodiment 69. The method of any one of embodiments 49 to 59, wherein the composition does not comprise cells.

[0240] Embodiment 70. The method of any one of embodiments 49 to 69, wherein the composition comprises one or more of a polymer, a salt, and a buffering agent.

[0241] Embodiment 71. The method of any one of embodiments 49 to 70, wherein the composition comprises one or more additional target condensates.

[0242] Embodiment 72. The method of any one of embodiments 49 to 71, wherein the method comprises repeating the steps of the method for one or more additional target condensates.

[0243] Embodiment 73. A method for determining a condensate preference profile for a plurality of test compounds, comprising carrying out a method according to any one of embodiments 49 to 72 using the plurality of test compounds.

[0244] Embodiment 74 The method of embodiment 73, further comprising comparing the condensate preference profiles of a subset or all of the plurality of test compounds.

[0245] Embodiment 75 The method of embodiment 74, further comprising identifying test compounds having the same or similar condensate preference profile.

[0246] Embodiment 76 The method of embodiment 75, further comprising identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profile.

[0247] Embodiment 77. The method of embodiment 76, further comprising determining the relative partitioning properties for one or more additional test compounds that comprise the identified properties.

[0248] Embodiment 78. A method for identifying a compound characteristic associated with the partitioning of the compound into a condensate, comprising the steps of: (a) determining the partitioning characteristics of a plurality of test compounds in a target condensate by performing the method of embodiment 36; (b) comparing the partitioning characteristics of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds having the same or similar partitioning characteristics in the target condensate; and (d) identifying characteristics that a subset or all of the identified test compounds have in common in addition to their same or similar partitioning characteristics.

[0249] Embodiment 79. A method for identifying a compound characteristic associated with the partitioning of the compound into a condensate, comprising the steps of: (a) determining the relative partitioning characteristics of a plurality of test compounds in a target condensate by performing the method described in embodiment 44; (b) comparing the relative partitioning characteristics of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds having the same or similar relative partitioning characteristics in the target condensate; and (d) identifying a characteristic that a subset or all of the identified test compounds have in common in addition to their same or similar relative partitioning characteristics.

[0250] Embodiment 80. A method for identifying a compound characteristic associated with partitioning of the compound into a condensate, comprising the steps of: (a) determining condensate preference profiles of a plurality of test compounds by performing the method of embodiment 73; (b) comparing the condensate preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar condensate preference profiles; and (d) identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profile.

[0251] Embodiment 81. A method for designing a compound having desired partitioning properties for a target condensate, comprising the steps of: (a) determining the partitioning properties of a plurality of test compounds in the target condensate by performing the method described in embodiment 36; (b) comparing the partitioning properties of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds having the same or similar partitioning properties in the target condensate; (d) identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning properties; and (e) designing a compound that includes the identified properties.

[0252] Embodiment 82. A method for designing a compound having desired relative partition properties, comprising the steps of: (a) determining the relative partition properties of a plurality of test compounds in a target condensate by performing the method described in embodiment 44; (b) comparing the relative partition properties of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds having the same or similar relative partition properties in the target condensate; (d) identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar relative partition properties; and (e) designing a compound comprising the identified properties.

[0253] Embodiment 83. A method for designing a compound having a desired condensate preference profile, comprising the steps of: (a) determining the condensate preference profiles of a plurality of test compounds by performing the method described in embodiment 73; (b) comparing the condensate preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar condensate preference profile; (d) identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profile; and (e) designing a compound that includes the identified properties.

[0254] Embodiment 84 The method of any one of embodiments 81 to 83, further comprising making the compound.

[0255] Embodiment 85. A method for screening test compounds for desired partitioning properties from a group of candidate compounds, comprising the steps of: (a) determining the partitioning properties of each of the group of candidate compounds; and (b) identifying a test compound having the desired partitioning properties.

[0256] Embodiment 86 The method of embodiment 85, wherein the partitioning properties of each of the group of candidate compounds are determined in vitro.

[0257] Embodiment 87 The method of embodiment 85 or 85, wherein the test compound has suitable distribution properties that are useful for treating a disease in an individual.

[0258] Embodiment 88. A method for identifying a test compound useful for treating a disease in an individual in need thereof, comprising the steps of: (a) identifying a target condensate associated with the disease; (b) determining the partitioning properties of a candidate compound in the target condensate; and (c) identifying a test compound having suitable partitioning properties useful for treating the disease. Further Exemplary Embodiments

[0259] The embodiments provided also include the following.

[0260] E1. A method for determining the distribution characteristics of a test compound in a target condensate, comprising: (a) combining the test compound and a composition comprising target condensate and excess condensate solution; (b) determining the amount of the test compound in the target condensate, thereby determining the distribution characteristics of the test compound in the target condensate.

[0261] E2. The method of embodiment E1, further comprising, prior to step (a), causing formation of a target condensate.

[0262] E3. A method for determining the partitioning characteristics of a test compound in a target condensate, comprising: (a) causing the formation of a target condensate in the presence of a test compound to obtain a composition comprising the target condensate and an excess condensate solution; and (b) determining the amount of the test compound in the target condensate, thereby determining the partitioning characteristics of the test compound in the target condensate.

[0263] E4. The method of embodiment E3, further comprising, prior to step (a), combining the test compound and a precursor composition comprising the precursor molecule.

[0264] E5. The method of embodiment E3, further comprising, prior to step (a), adding a test compound to a precursor composition comprising the precursor molecule.

[0265] E6. The method of any one of embodiments E1 to E5, further comprising determining the amount of the test compound in the excess condensate solution.

[0266] E7. The method of embodiment E6, wherein the amount of the test compound in the target condensate is determined before, simultaneously with, or after the amount of the test compound in the excess condensate solution is determined.

[0267] E8. The method of embodiment E6 or E7, further comprising determining the ratio of the amount of the test compound in the target condensate and the amount of the test compound in the excess condensate solution.

[0268] E9. The method of any one of embodiments E1 to E8, further comprising separating the target condensate from excess condensate solution.

[0269] E10. The method of any one of embodiments E1 to E9, further comprising identifying the target condensate prior to determining the amount of the test compound in the target condensate.

[0270] E11. The method of any one of embodiments E1 to E10, wherein dysregulation of target condensates is associated with disease.

[0271] E12. The method of any one of embodiments E1 to E11, further comprising characterizing the target condensates by identifying one or more macromolecules contained therein.

[0272] E13. The method of embodiment E12, wherein the identifying step comprises determining the amount of one or more macromolecules in the target condensate.

[0273] E14. The method of embodiment E13, further comprising determining a ratio of the amount of the test compound in the target condensate and the amount of one or more macromolecules in the target condensate.

[0274] E15. The method of any one of embodiments E1 to E14, wherein the target condensate comprises a protein comprising an intrinsically disordered sequence.

[0275] E16. The method of any one of embodiments E1 to E15, further comprising labeling the target condensates to visualize them.

[0276] E17. The method of embodiment E16, wherein the target condensate is labeled with a radioactive label, a colorimetric label, or a fluorescent label.

[0277] E18. The method of any one of embodiments E1 to E17, wherein the composition comprises cells.

[0278] E19. The method of embodiment E18, wherein the cell is a microbial or animal cell.

[0279] E20. The method of embodiment E18 or E19, wherein the cells comprise condensates that are determined to be dysregulated.

[0280] E21. The method of any one of embodiments E18 to E20, wherein the cells have one or more characteristics of a neurodegenerative or proliferative disease.

[0281] E22. The method of any one of embodiments E1 to E21, wherein the target condensate is a cellular condensate.

[0282] E23. The method of embodiment E22, wherein the cellular condensates are cleavage bodies, P granules, histone locus bodies, multivesicular bodies, neuronal RNA granules, nuclear gems, nuclear pores, nuclear speckles, nuclear stress bodies, intranuclear bodies, Oct1 / PTF / transcription (OPT) domains, paraspeckles, perinuclear compartments, PML nucleoli, PML oncogenic domains, polycomb bodies, processing bodies, Sam68 nucleoli, stress granules, or splicing speckles.

[0283] E24. The method of any one of embodiments E1 to E23, wherein the target condensate is in a cell.

[0284] E25. The method of embodiment E24, wherein the cell is a microbial or animal cell.

[0285] E26. The method of embodiment E24 or E25, wherein the cells have one or more characteristics of a neurodegenerative or proliferative disease.

[0286] E27. The method of any one of embodiments E1 to E26, wherein the excess condensate solution is intracellular fluid.

[0287] E28. The method of embodiment E27, wherein the intracellular fluid is cytosol or nucleosol.

[0288] E29. The method of any one of embodiments E1 to E21, wherein the target condensate is not in a cell.

[0289] E30. The method of embodiment E29, wherein the target condensate is an extracellular condensate.

[0290] E31. The method of any one of embodiments E1 to E22 or E29 to E30, wherein the excess condensate solution is extracellular fluid.

[0291] E32. The method of embodiment E31, wherein the extracellular fluid is interstitial fluid.

[0292] E33. The method of any one of embodiments E1 to E17, which is a cell-free assay.

[0293] E34. The method of any one of embodiments E1-E33, wherein the composition comprises one or more of a polymer, a salt, and a buffering agent.

[0294] E35. The method of any one of embodiments E1 to E34, wherein the composition comprises two or more target condensates.

[0295] E36. The method of any one of embodiments E1 to E35, comprising repeating the method steps with one or more additional condensates.

[0296] E37. The method of any one of embodiments E1 to E36, wherein the test compound is a small molecule, a polypeptide, or a nucleic acid.

[0297] E38. The method of any one of embodiments E1 to E37, wherein the test compound comprises a test compound label.

[0298] E39. The method of embodiment E38, wherein the test compound label is a radioactive label, a colorimetric label, or a fluorescent label.

[0299] E40. The method of embodiment E38 or E39, wherein the test compound label is a fluorescent label.

[0300] E41. The method of any one of embodiments E38 to E40, wherein the amount of the test compound is determined by detecting a test compound label.

[0301] E42. The method of any one of embodiments E1-E41, wherein the amount of the test compound is determined by mass spectrometry, liquid chromatography and / or UV-Vis spectroscopy.

[0302] E43. A method for determining the partitioning properties of a plurality of test compounds in a target condensate, comprising carrying out the method of any one of embodiments E1 to E42 with the plurality of test compounds.

[0303] E44. The method of embodiment E43, further comprising comparing the partitioning properties of a subset or all of the multiple test compounds in the target condensate.

[0304] E45. The method of embodiment E44, further comprising identifying test compounds having the same or similar partitioning properties in the target condensate.

[0305] E46. The method of embodiment E45, further comprising identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning properties.

[0306] E47. The method of embodiment E46, further comprising determining the partitioning properties in the target condensate for one or more additional test compounds that comprise the identified properties.

[0307] E48. The method of embodiment E46 or E47, further comprising determining the partitioning properties in the target condensate for one or more additional test compounds that do not contain the identified property.

[0308] E49. A method for determining the relative partitioning properties of a test compound in a target condensate, comprising the steps of: (i) determining the partitioning properties of the test compound by performing the method of any one of embodiments E1 to E42 using the test compound; (ii) determining the partitioning properties of a reference compound by performing the method of any one of embodiments E1 to E42 using the reference compound; and (iii) calculating the ratio of the partitioning properties determined in (i) and (ii), thereby determining the relative partitioning properties of the test compound in the target condensate.

[0309] E50. The method of embodiment E49, wherein the test compound comprises a test compound label.

[0310] E51. The method of embodiment E50, wherein the reference compound is a test compound label.

[0311] E52. A method for determining the relative partitioning properties of a plurality of test compounds in a target condensate, comprising: (1) performing a method according to any one of embodiments E49 to E51; and (2) repeating steps (i) and (iii) with a plurality of test compounds.

[0312] E53. The method of embodiment E52, further comprising comparing the relative partitioning properties in the target condensate of a subset or all of the plurality of test compounds.

[0313] E54. The method of embodiment E53, further comprising identifying test compounds having the same or similar relative partitioning properties in the target condensate.

[0314] E55. The method of embodiment E54, further comprising identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar relative partitioning properties.

[0315] E56. The method of embodiment E55, further comprising determining the relative partitioning properties in the target condensate for one or more additional test compounds that comprise the identified properties.

[0316] E57. The method of embodiment E55 or E56, further comprising determining the relative partitioning properties in the target condensate for one or more additional test compounds that do not contain the identified property.

[0317] E58. A method for determining a condensate preference profile of a test compound, comprising: (a) determining a distribution profile of the test compound in a first target condensate using a method described in any one of embodiments E1 to E42; (b) determining a distribution profile of the test compound in a second target condensate using a method described in any one of embodiments E1 to E42; and (c) calculating a ratio of the distribution profiles of the test compound determined in the first target condensate and the second target condensate, thereby determining the condensate preference profile of the test compound.

[0318] E59. The method of embodiment E58, wherein the first target condensate and the second target condensate are in the same composition.

[0319] E60. The method of embodiment E58, wherein the first target condensate and the second target condensate are in different compositions.

[0320] E61. The method of any one of embodiments E58 to E60, wherein a partitioning characteristic of the test compound in a first target condensate is determined before, simultaneously with, or after a partitioning characteristic of the test compound in a second target condensate is determined.

[0321] E62. A method for determining a condensate preference profile of a test compound, comprising the steps of: (a) determining a relative partition profile of the test compound in a first target condensate using the method of any one of embodiments E49 to E51; (b) determining a relative partition profile of the test compound in a second target condensate using the method of any one of embodiments E49 to E51; and (c) calculating a ratio of the partition profiles of the test compound determined in the first target condensate and the second target condensate, thereby determining the condensate preference profile of the test compound.

[0322] E63. The method of embodiment E62, wherein the first target condensate and the second target condensate are in the same composition.

[0323] E64. The method of embodiment E62, wherein the first target condensate and the second target condensate are in different compositions.

[0324] E65. The method of any one of embodiments E62 to E64, wherein the relative partition profile of the test compound in the first target condensate is determined before, simultaneously with, or after the relative partition profile of the test compound in the second target condensate is determined.

[0325] E66. The method of any one of embodiments E58 to E65, further comprising labeling the first target condensate and the second target condensate to visualize the first target condensate and the second target condensate.

[0326] E67. The method of embodiment E66, wherein the first target condensate and the second target condensate are labeled with different labels.

[0327] E68. The method of any one of embodiments E58 to E67, wherein the method comprises repeating the steps of the method for one or more additional target condensates.

[0328] E69. A method for determining the condensate preference profile of a plurality of test compounds, comprising performing the method of any one of embodiments E58 to E68 with the plurality of test compounds.

[0329] E70. The method of embodiment E69, further comprising comparing the condensate preference profiles of a subset or all of the plurality of test compounds.

[0330] E71. The method of embodiment E70, further comprising identifying test compounds having the same or similar condensate preference profile.

[0331] E72. The method of embodiment E71, further comprising identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profile.

[0332] E73. The method of embodiment E72, further comprising determining the relative partitioning properties for one or more additional test compounds that comprise the identified properties.

[0333] E74. The method of embodiment E72 or E73, further comprising determining the relative partitioning properties for one or more additional test compounds that do not contain the identified property.

[0334] E75. A method for identifying a compound characteristic associated with partitioning of a compound into or out of a condensate, comprising the steps of: (a) determining the partitioning characteristics of a plurality of test compounds in a target condensate by performing the method of embodiment E43; (b) comparing the partitioning characteristics of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds having the same or similar partitioning characteristics in the target condensate; and (d) identifying a characteristic that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning characteristics.

[0335] E76. A method for identifying a property of a compound related to partitioning of the compound into or out of a condensate, comprising the steps of: (a) determining relative partitioning properties of a plurality of test compounds in a target condensate by performing the method of embodiment E52; (b) comparing the relative partitioning properties of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds having the same or similar relative partitioning properties in the target condensate; and (d) identifying a property that a subset or all of the identified test compounds have in common in addition to their same or similar relative partitioning properties.

[0336] E77. A method for identifying a property of a compound associated with partitioning of the compound into or out of a condensate, comprising the steps of: (a) determining a condensate preference profile of a plurality of test compounds by performing the method of embodiment E69; (b) comparing the condensate preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar condensate preference profile; and (d) identifying a property that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profile.

[0337] E78. A method for designing a compound having desired partitioning properties into or out of a target condensate, comprising the steps of: (a) determining the partitioning properties of a plurality of test compounds in the target condensate by performing the method of embodiment E43; (b) comparing the partitioning properties of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds having the same or similar partitioning properties in the target condensate; (d) identifying a property that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning properties; and (e) (i) designing a compound that includes the identified property; or (ii) designing a compound that does not include the identified property, thereby designing a compound having desired partitioning properties into or out of the target condensate.

[0338] E79. A method for designing a compound having a desired relative partitioning property to or from a target condensate, comprising the steps of: (a) determining the relative partitioning property of a plurality of test compounds in the target condensate by performing the method of embodiment E52; (b) comparing the relative partitioning property of a subset or all of the plurality of test compounds in the target condensate; (c) identifying test compounds having the same or similar relative partitioning property in the target condensate; (d) identifying a property that a subset or all of the identified test compounds have in common in addition to the same or similar relative partitioning property; and (e) (i) designing a compound that includes the identified property; or (ii) designing a compound that does not include the identified property, thereby designing a compound having a desired relative partitioning property to or from the target condensate.

[0339] E80. A method for designing a compound having a desired condensate preference profile, comprising the steps of: (a) determining the condensate preference profiles of a plurality of test compounds by performing the method of embodiment E69; (b) comparing the condensate preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar condensate preference profiles; (d) identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profile; and (e) (i) designing a compound that includes the identified properties; or (ii) designing a compound that does not include the identified properties, thereby designing a compound having the desired condensate preference profile.

[0340] E81. The method of any one of embodiments E78 to E80, further comprising making the compound.

[0341] E82. A method for screening test compounds for desired partitioning properties from a group of candidate compounds, comprising: (a) determining the partitioning properties of each of the group of candidate compounds; and (b) identifying a test compound having the desired partitioning properties.

[0342] E83. The method of embodiment E82, wherein the partitioning properties of each of the group of candidate compounds are determined in vitro.

[0343] E84. The method of embodiment E82 or E83, wherein the test compound has suitable distribution properties that are useful for treating the disease in an individual.

[0344] E85. A method for identifying a test compound useful for treating a disease in an individual in need thereof, comprising the steps of: (a) identifying a target condensate associated with the disease; and (b) determining the partitioning properties of a candidate compound in the target condensate; and (c) identifying a test compound having suitable partitioning properties useful for treating the disease.

[0345] E86. A method for determining the distribution characteristics of a test compound in a target condensate, comprising the steps of: (a) combining the test compound and a composition comprising the target condensate and an excess condensate solution; (b) obtaining a reference control; (c) measuring the MS signal of the test compound or a portion thereof in the excess condensate solution using a mass spectrometry technique; (d) measuring the MS signal of the test compound or a portion thereof in the reference control using a mass spectrometry technique; and (e) comparing the MS signal of the test compound from the excess condensate solution and the MS signal of the test compound from the reference control, thereby determining the distribution characteristics of the test compound in the target condensate.

[0346] E87. The method of embodiment E86, wherein the amount of the test compound combined with the composition is 100 nM or less, and the amount of precursor molecule in the composition comprising the target condensate is about 5 μM.

[0347] E88. A library comprising a plurality of compounds, each compound of the plurality of compounds comprising the same moiety comprising a feature having a desired partitioning property.

[0348] E89. A method for designing a test compound having desired partitioning properties, comprising modifying a precursor of the test compound by attaching a moiety to the compound, wherein the moiety comprises a property having the desired partitioning properties.

[0349] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the disclosure of this application. The 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. EXAMPLES

[0350] Example 1 Characterization of one or more compounds in solution-based condensates The condensates are formed in solution, for example, a solution containing a high concentration of salt and a high concentration of one or more proteins capable of forming condensates is diluted into a buffer that mimics physiological salt conditions.

[0351] Alternatively, a solution containing one or more proteins capable of forming aggregates is diluted in a buffer and a crowding agent is added. In a specific embodiment, the protein capable of forming aggregates is mixed with 10% dextran in a buffer containing 25 mM Tris-HCl (pH 7.4), 150 mM KCl, 2.5% glycerol and 0.5 mM DTT.

[0352] The test compound is added to the solution either before or after condensate formation. If added after condensate formation, the solution is incubated to allow partitioning of the test compound.

[0353] The density of the condensates is typically higher than the surrounding solution, resulting in the condensates settling. In some cases, the condensates are imaged, for example using dyes or labeled proteins known to be concentrated in the condensates to make them visible.

[0354] In some instances, a supernatant liquid is removed from the condensate. In some instances, the supernatant liquid is analyzed to determine the amount of compound present.

[0355] The condensate is analyzed to determine the amount of compound present, and in some instances, the ratio of the compound in the condensate to the compound in the supernatant is calculated. Example 2 Characterization of pigment compounds in solution-based FUS and PGL-3 condensates

[0356] Various exemplary test compounds are assayed using the exemplary method to determine partitioning characteristics, relative partitioning characteristics and condensation preference profile.For this example, the exemplary test compound used is a dye, however, test compounds are not limited to dyes.The entire procedure is carried out twice in two independent experiments on different days, in different orders. Sample preparation

[0357] All dye stocks were stored as 1 mM solutions in 100% DMSO. The dyes used are shown in Table 1. The dyes were dissolved in dilution buffer (DB: 14.7 mM Tris, pH 7.25, 1 mM DTT) to give 11.765 μM dye in 1.1765% DMSO. 17 μl of dye was dispensed into a 384-well non-binding plate (Greiner). Table 1. Assay dyes [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0358] Either SNAP-tagged FUS protein or untagged PGL-3 protein was thawed at room temperature for at least 10 minutes and the buffer exchanged into partitioning buffer (PB: 50 mM Tris pH 7.25, 500 mM KCl, 5% glycerol, and 1 mM DTT). Protein was then diluted to 33.3 μM in PB. 3 μl of protein solution was added to a 384-well plate to initiate phase separation, resulting in 20 μl of 5 μM protein and 10 μM dye in 75 mM KCl, 0.75% glycerol, 20 mM Tris, 1 mM DTT, and 1% DMSO, or 20 μl of 5 μM protein in 75 mM KCl, 0.75% glycerol, 20 mM Tris, 1 mM DTT for the no dye control reaction. Imaging

[0359] Images of the samples were obtained using a spinning disk confocal microscope. A 60x oil high NA objective was used to capture the condensate droplets. The laser power was set to 20% and the exposure time was adjusted to the fluorescence emission intensity specific for each dye. The exposure time ranged from 1 to 1000 ms. Appropriate filter settings were used to tune to the excitation and emission spectra of each dye. The channels used for each dye are shown in Table 1. 3 images were acquired per well. Exemplary images are shown in Figure 1. The background signal of the protein droplets in the absence of dye was recorded for all applied imaging conditions. Image and data analysis

[0360] Fluorescence intensity was measured inside one exemplary target condensate per image (inner intensity, I-in) and in the area next to an exemplary target condensate (outer intensity, I-out) by manually using the image analysis software Fiji. An example representing the measured area is shown in Figure 2A. Three I-in and three I-out values ​​were measured overall for each dye in each experiment and averaged. The I-in and I-out values ​​measured for FUS-SNAP condensates on day 1 of the experiment are shown in Tables 2 and 3, respectively. The I-in and I-out values ​​measured for PGL-3 condensates on day 1 of the experiment are shown in Tables 4 and 5, respectively. All values ​​were background corrected based on a background image (no dye control) acquired under the same microscope conditions. An exemplary background image is shown in Figure 2B and the measured I-in or I-out background measurements are shown in Tables 3-5. Table 2. Measurements of dyes inside FUS-SNAP condensates [Table 2-1] [Table 2-2] Table 3. Measurements of dyes outside FUS-SNAP condensates [Table 3] Table 4. Measurements of pigments inside PGL-3 condensates [Table 4] Table 5. Measurements of pigments outside the PGL-3 condensate [Table 5-1] [Table 5-2]

[0361] The ratio of the average intensities measured in the target condensate and outside the target condensate was calculated by dividing I-in by I-out, although I-out by I-in could also be used. The I-in:I-out ratios determined from the two different days of the experiment were then averaged. The individual days and average I-in:I-out ratios are shown in Table 6. The average I-in:I-out ratios for FUS-SNAP condensates are shown in Figure 3, and the average I-in:I-out ratios for PGL-3 condensates are shown in Figure 4. The standard deviation between the two experiments is represented by the error bars in Figures 3 and 4.

[0362] The ratio of FUS-SNAP I-internal:I-external to PGL-3 I-internal:I-external was also calculated and is shown in Table 6 and FIG. Table 6. Calculated ratios of dye intensities [Table 6-1] [Table 6-2] N / A: Not applicable as the error was too high due to low signal of the dye

[0363] All compounds tested appear to partition preferentially into FUS-SNAP condensates over those formed by PGL-3. Compounds #1 (DAPI), #11 (fluorescein-12-UTP), #16 (rhodamine B), #17 (rhodamine 101 inner salt) and #18 (rhodamine 800) have I-inside:I-outside ratios for FUS-SNAP condensates of 4 or higher. Compounds #11, #16, #17 and #18 partitioned preferentially into FUS-SNAP condensates at ratios of 3 or higher compared to PGL-3. Example 3 Colocalization of partitioned compounds and FUS in FUS condensates

[0364] To confirm the compounds partitioned into the condensates in Example 2, condensates containing FUS, dye number 18 (rhodamine 800), which had an average I-internal:I-external ratio of 9.3, were further evaluated. Sample preparation

[0365] FUS-GFP condensates were prepared using dye 18 as described for FUS-SNAP in Example 2. Imaging

[0366] Images of the samples were obtained using a spinning disk confocal microscope. A 60x oil high NA objective was used to capture droplets of condensate. The laser power was set to 20% and the exposure time was adjusted to the fluorescence emission intensity specific for the respective dye. Exposure times ranged from 1 to 1000 ms. Appropriate filter settings were used to tune to the excitation and emission spectra of the dye and GFP. Control images of condensates containing FUS-GFP without dye were acquired. result

[0367] The images obtained are shown in Figure 6. GFP-tagged FUS was detected in the condensates in the presence or absence of the dye. Dye 18 was detected in the condensates, and the FUS protein and the dye colocalized in the condensates. For the no dye control, no fluorescence was detected in the condensates when images were obtained using the Rhodamine 800 channel. Example 4 Determining the partitioning properties of a set of compounds using mass spectrometry assays

[0368] A mass spectrometry-based method was developed to measure the partitioning properties of exemplary test compounds. Measurements from the mass spectrometry-based method were compared to those obtained using the fluorescence-based assay disclosed in Example 2. method

[0369] A protein buffer was prepared (50 mM Tris, pH 7.25, 500 mM KCl, 1 mM DTT and 5% glycerol). The high salt concentration of the protein buffer prevents phase separation of macromolecules, e.g., proteins, contained therein. A 20 μl aliquot of a 70 μM stock solution of FUS-SNAP protein was prepared in the protein buffer. The aliquot of the stock solution of FUS-SNAP protein was frozen and stored until use. A dilution buffer was prepared (14.7 mM Tris, pH 7.25 and 1 mM DTT).

[0370] An aliquot of 70 μM FUS-SNAP protein stock solution was thawed at room temperature for approximately 10 minutes. The aliquot of protein stock solution appeared clear with no visible precipitate. The thawed protein stock solution was filtered using a centrifuge filter (Millipore, UFC30VV00). Briefly, the aliquot of protein stock solution was added to a filed centrifuge and centrifuged at 20,000 rcf for 1 minute at room temperature. The flow-through was collected. The protein concentration of the flow-through was measured and a solution of 33.3 μM FUS-SNAP was prepared by diluting the filtered flow-through and dilution buffer.

[0371] 40 μL test reactions were prepared in PCR tubes or PCR strips (8 wells; AXYGEN™ 8-strip PCR tubes, 0.2 mL) using: (i) 34 μL of dilution buffer with test compound at a given concentration and DMSO to obtain a final DMSO concentration of 1%, and (ii) 6 μL of 33.3 μM FUS-SNAP filtered protein solution. The final concentrations of the 40 μL reactions were: 5 μM FUS-SNAP, 75 mM KCl, 20 mM Tris, 0.75% glycerol, 1 mM DTT, 1% DMSO for reactions containing test compound, desired test compound concentration.

[0372] A 40 μL reference reaction was prepared as described for the test compounds, but no test compounds were added at this stage.

[0373] The test and reference reactions were incubated at room temperature for 15 minutes. Within the test reaction, condensed high-density droplets (mM protein concentration) of FUS-SNAP protein (condensed phase) are expected to exist in an environment of a less concentrated solution of FUS-SNAP dilute phase (μM protein concentration). The test compound may or may not partition into the condensed phase, causing a reduction in concentration outside (test compound in dilute phase) and an increase in concentration inside (test compound in droplet). The volume of the high-density droplet is estimated to be at least 1000 times smaller than the total volume of the reaction, e.g., 20 nL of condensed phase in a 20 μL reaction.

[0374] After a 15 minute incubation period, the test and reference reactions were processed to separate the condensates (high density droplets) from the supernatant (dilute phase). Briefly, the test and reference reactions were centrifuged at 10,000 rcf for 10 minutes in a refrigerated centrifuge at 20° C. When using PCR strips, the rotor used was an Eppendorf F-45-48-5-PCR. 35 μL of the supernatant from each tube was removed and carefully transferred to a new tube without disturbing the pellet containing the condensates.

[0375] For the reference reaction, a known amount of the test compound is added to the tube containing the supernatant (containing proteins not associated with condensate), so that the final compound concentration is the same as the initial concentration of the compound in the test reaction. A reference reaction can also be formed by measuring the amount of protein in the supernatant of the test reaction and reproducing the supernatant solution based on the information obtained. The mass spectrometry signal of the test reaction was calibrated using a known amount of compound added to the supernatant from the reference reaction.

[0376] In addition, compounds were extracted from the test reactions to directly measure the amount of compound in the condensates formed. The supernatant containing an amount of test compound was separated from the pellet containing the condensed protein. Briefly, the lid of each PCR tube was removed with scissors and each PCR tube was placed upside down in a 1.5 mL Eppendorf tube. The tubes were then centrifuged at 2,000 rcf for 5 seconds in a standard tabletop centrifuge. If PCR strips were used, the PCR strips can be plugged upside down in an Eppendorf Microplate 96 / V-PP (cat. no. 951040188) and centrifuged at 4,500 rcf for 1 minute. Compounds were then isolated from each pellet using a solution of ACN:MeOH (1:1). Samples may be sonicated if necessary.

[0377] The resulting samples from the test and reference reactions were analyzed by mass spectrometry. The ratio between the integrals of the corresponding MS signals of the relevant supernatant test and reference reactions reflected the depletion of the test compound due to partitioning into the formed condensates.

[0378] The partitioning properties of the test compounds were also determined using the fluorescence-based assay method disclosed in Example 2. result

[0379] Rhodamine 101 (Rho101), Rhodamine 800 (Rho808), Rhodamine B (Rho B), Rhodamine 123 (Rho123) and fluorescein were evaluated using both the fluorescence-based and mass spectrometry-based assays as discussed above. As shown in Figure 7, the two test methods produced partitioning property measurements that were in good agreement for all test compounds. Using the measurements, the ratio of the condensate-associated compound concentration to the non-condensate-associated compound concentration was calculated for each compound. A volume fraction of condensate of 1 / 1000 relative to the external phase was used. The results are presented in Table 7. Table 8. Ratio of compound concentration inside to compound concentration outside [Table 8]

[0380] In addition, pelleted condensate samples were processed to extract and quantitate the test compounds therein, and the results demonstrated that the sum of the amount of test compound in the condensate and the amount of test compound in the supernatant was consistent with the total amount of test compound added to the test reaction (data not shown).

[0381] As demonstrated herein, mass spectrometry-based assay is a robust and sensitive assay for quantitatively determining the partitioning properties of compounds.This technique is useful as high-throughput screening.Certain additional means are understood for this mass spectrometry-based assay.For example, mass spectrometry-based assay is not limited to fluorescent compounds, is a hypothesis-free technique (i.e., the identity of test compound does not need to be known before assay), is not limited to fluorescence quenching that may occur inside droplets, and can be easily multiplexed, as demonstrated in Example 5. Example 5

[0382] This example evaluates the effect of using different concentrations of test compound in the test reactions on the measurement of the test compound in the supernatant.

[0383] Test reactions and reference controls were prepared as described in Example 4. Fluorescein (identified as not partitioning into FUS condensates) and rhodamine 101 (identified as partitioning into FUS condensates) were evaluated in separate test reactions at 0.01 μM, 0.1 μM, 1 μM and 10 μM. result

[0384] As shown in FIG. 8, fluorescein does not partition into FUS condensates at any of the concentrations tested, and the partitioning properties of fluorescein are concentration independent. Rhodamine 101 was observed to partition into FUS condensates at lower compound concentrations (see FIG. 8; 0.01 μM and 0.1 μM). Data represent mean ± SD, N=3 technical replicates. As illustrated in FIG. 8, the efficiency of detection of partitioning properties improves for reduced compound concentrations. Because the amount of compound that partitions into the condensate may be small, using a smaller amount of test compound than added to the test reaction may allow detection of small changes in supernatant compound concentration compared to the reference control. Example 6 Multiplexed mass spectrometry depletion assay

[0385] This example demonstrates the use of the mass spectrometry-based assay disclosed in Example 4 on a study system containing multiple test compounds. method

[0386] FUS-SNAP protein reactions and reference reactions were prepared as described in Example 4. For the test reactions, a set of four compounds including fluorescein and rhodamine B was used to generate test reactions with single compounds at concentrations of 0.01 μM, 0.1 μM and 1 μM, as well as test reactions with a mixture of all four compounds. All samples were prepared in triplicate. All samples were analyzed using the mass spectrometry-based assay described in Example 4. result

[0387] As shown in Figure 9A, the fraction of fluorescein measured outside the FUS-SNAP condensate for the single compound test reactions matched the fraction of fluorescein measured outside the FUS-SNAP condensate in the presence of the four compound mixture. As shown in Figure 9B, the fraction of rhodamine B (Rho B) measured outside the FUS-SNAP condensate for the single compound test reactions matched the fraction of rhodamine B measured outside the FUS-SNAP condensate in the presence of the four compound mixture. This demonstrates that mass spectrometry based techniques can be used for multiplex assays, for example, allowing high throughput and / or study of more complex systems. Example 7 Multiplexing using mass spectrometry-based assays

[0388] This example further demonstrates the use of the mass spectrometry-based assay disclosed in Example 4 for a study system containing multiple test compounds. method

[0389] FUS-SNAP protein reactions and reference reactions were prepared as described in Example 4. For the test reactions, fluorescein and rhodamine 101 were assayed individually, and three sets of pooled compounds were also assayed. The first pool had 10 compounds (including fluorescein), the second pool had 15 compounds (including fluorescein and rhodamine 101), and the third pool had 20 compounds (including fluorescein and rhodamine 101). All samples were prepared and assayed in triplicate. All samples were analyzed using the mass spectrometry-based assay described in Example 4. result

[0390] As shown in Figure 10, the described assay, which measures, for example, the depletion of test compounds in the supernatant due to the presence of condensates, can measure the partitioning properties of individual compounds, independent of the pool size of the compounds. These results demonstrate that compounds can be mixed in large pool sizes and still be comparable to partitioning properties of single compounds evaluated individually. Thus, the mass spectrometry-based assay described herein allows for high-throughput screening. In certain embodiments, for example, the following items are provided: (Item 1) 1. A method for determining the partitioning properties of a test compound in a target condensate, comprising: (a) combining said test compound and a composition comprising said target condensate and excess condensate solutions; (b) determining the amount of said test compound in said target condensate thereby determining the partitioning characteristic of the test compound in the target condensate. (Item 2) 2. The method of claim 1, further comprising, prior to step (a), causing formation of the target condensate. (Item 3) 1. A method for determining the partitioning properties of a test compound in a target condensate, comprising: (a) causing formation of the target condensate in the presence of the test compound to obtain a composition comprising the target condensate and an excess condensate solution; and (b) determining the amount of said test compound in said target condensate thereby determining the partitioning characteristic of the test compound in the target condensate. (Item 4) 4. The method of claim 3, further comprising the step of combining the test compound and a precursor composition comprising a precursor molecule prior to step (a). (Item 5) 4. The method of claim 3, further comprising the step of adding the test compound to a precursor composition comprising a precursor molecule prior to step (a). (Item 6) 6. The method of any one of items 1 to 5, further comprising the step of determining the amount of the test compound in the excess condensate solution. (Item 7) The method according to claim 6, wherein the amount of the test compound in the target condensate is determined before, simultaneously with, or after the amount of the test compound in the excess condensate solution is determined. 8. The method according to item 6 or 7, further comprising the step of determining the ratio of the amount of test compound in the target condensate and the amount of test compound in the excess condensate solution. (Item 9) 9. The method according to any one of items 1 to 8, further comprising a step of separating the target condensate from the excess condensate solution. (Item 10) 10. The method according to any one of items 1 to 9, further comprising a step of identifying the target condensate prior to the step of determining the amount of the test compound in the target condensate. (Item 11) 11. The method of any one of items 1 to 10, wherein dysregulation of the target condensate is associated with a disease. (Item 12) 12. The method according to any one of items 1 to 11, further comprising a step of characterizing the target condensates by identifying one or more macromolecules contained therein. (Item 13) 13. The method of claim 12, wherein the identifying step comprises determining the amount of the one or more macromolecules in the target condensate. (Item 14) 14. The method of claim 13, further comprising determining the ratio of the amount of the test compound in the target condensate and the amount of the one or more macromolecules in the target condensate. (Item 15) 15. The method of any one of items 1 to 14, wherein the target condensate comprises a protein comprising an intrinsically disordered sequence. (Item 16) 16. The method of any one of items 1 to 15, further comprising a step of labeling the target condensate in order to visualize the target condensate. (Item 17) 17. The method of claim 16, wherein the target condensate is labeled with a radioactive label, a colorimetric label or a fluorescent label. (Item 18) 18. The method of any one of items 1 to 17, wherein the composition comprises a cell. (Item 19) 19. The method of claim 18, wherein the cell is a microorganism or an animal cell. (Item 20) 20. The method of claim 18 or 19, wherein the cell comprises a condensate that is determined to be dysregulated. (Item 21) 21. The method of any one of items 18 to 20, wherein the cells have one or more characteristics of a neurodegenerative or proliferative disease. (Item 22) The method according to any one of claims 1 to 21, wherein the target condensate is a cellular condensate. 23. The method of claim 22, wherein the cellular condensates are cleavage bodies, P granules, histone locus bodies, multivesicular bodies, neuronal RNA granules, nuclear gems, nuclear pores, nuclear speckles, nuclear stress bodies, intranuclear bodies, Oct1 / PTF / transcription (OPT) domains, paraspeckles, perinuclear compartments, PML nucleoli, PML oncogenic domains, polycomb bodies, processing bodies, Sam68 nucleoli, stress granules, or splicing speckles. (Item 24) 24. The method of any one of items 1 to 23, wherein the target condensate is in a cell. (Item 25) 25. The method of claim 24, wherein the cell is a microorganism or an animal cell. (Item 26) 26. The method of claim 24 or 25, wherein the cells have one or more characteristics of a neurodegenerative or proliferative disease. (Item 27) 27. The method according to any one of the preceding claims, wherein the excess condensate solution is intracellular fluid. (Item 28) 28. The method of claim 27, wherein the intracellular fluid is a cytosol or a nucleosol. (Item 29) 22. The method of any one of items 1 to 21, wherein the target condensate is not in a cell. (Item 30) 30. The method of claim 29, wherein the target condensate is an extracellular condensate. (Item 31) 31. The method of any one of items 1 to 22 or 29 to 30, wherein the excess condensate solution is extracellular fluid. (Item 32) 32. The method of claim 31, wherein the extracellular fluid is interstitial fluid. (Item 33) 18. The method according to any one of items 1 to 17, which is a cell-free assay. (Item 34) 34. The method of any one of the preceding claims, wherein the composition comprises one or more of a polymer, a salt and a buffering agent. (Item 35) 35. The method of any one of the preceding claims, wherein the composition comprises two or more target condensates. (Item 36) 36. The method according to any one of the preceding claims, comprising repeating said steps of the method with one or more additional condensates. (Item 37) 37. The method of any one of items 1 to 36, wherein the test compound is a small molecule, a polypeptide or a nucleic acid. (Item 38) 38. The method of any one of items 1 to 37, wherein the test compound comprises a test compound label. (Item 39) 39. The method of claim 38, wherein the test compound label is a radioactive label, a colorimetric label or a fluorescent label. (Item 40) 40. The method of claim 38 or 39, wherein the test compound label is a fluorescent label. (Item 41) 41. The method according to any one of items 38 to 40, wherein the amount of the test compound is determined by detecting the test compound label. (Item 42) 42. The method according to any one of the preceding items, wherein the amount of the test compound is determined by mass spectrometry, liquid chromatography and / or UV-Vis spectroscopy. (Item 43) 43. A method for determining the partitioning properties of a plurality of test compounds in a target condensate, comprising carrying out the method according to any one of items 1 to 42 with a plurality of test compounds. (Item 44) 44. The method of claim 43, further comprising the step of comparing the partitioning properties of a subset or all of the plurality of test compounds in the target condensate. (Item 45) 45. The method of claim 44, further comprising identifying test compounds having the same or similar partitioning properties in the target condensate. (Item 46) 46. ​​The method of claim 45, further comprising identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning properties. (Item 47) 47. The method of claim 46, further comprising determining the partitioning properties in the target condensate for one or more additional test compounds that have the identified properties. (Item 48) 48. The method of claim 46 or 47, further comprising determining the partitioning properties in the target condensate for one or more additional test compounds not containing the identified properties. (Item 49) 1. A method for determining the relative partitioning properties of a test compound in a target condensate, comprising: (i) determining the partitioning property of the test compound by carrying out the method according to any one of items 1 to 42 with the test compound; (ii) determining the partitioning properties of a reference compound by carrying out the method according to any one of items 1 to 42 with said reference compound; and (iii) calculating the ratio of the partition characteristics determined in (i) and (ii). thereby determining the relative partitioning properties of the test compound in the target condensate. (Item 50) 50. The method of claim 49, wherein the test compound comprises a test compound label. (Item 51) 51. The method of claim 50, wherein the reference compound is the test compound label. (Item 52) 1. A method for determining the relative partitioning properties of a plurality of test compounds in a target condensate, comprising: (1) carrying out the method according to any one of items 49 to 51; and (2) repeating steps (i) and (iii) with a plurality of test compounds. A method comprising: (Item 53) 53. The method of claim 52, further comprising the step of comparing the relative partitioning properties in the target condensate of a subset or all of the plurality of test compounds. (Item 54) 54. The method of claim 53, further comprising identifying test compounds having the same or similar relative partitioning properties in the target condensate. (Item 55) 55. The method of claim 54, further comprising identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar relative partitioning properties. (Item 56) 57. The method of claim 55, further comprising determining the relative partitioning properties in the target condensate for one or more additional test compounds that have the identified properties. 57. The method of claim 55 or 56, further comprising determining the relative partitioning properties in the target condensate for one or more additional test compounds not containing the identified property. (Item 58) 1. A method for determining a condensation preference profile of a test compound, comprising: (a) determining the partitioning properties of a test compound in a first target condensate using a method according to any one of items 1 to 42; (b) determining the partitioning properties of the test compound in the second target condensate using the method according to any one of items 1 to 42; and (c) calculating a ratio of the partitioning properties of the test compound determined in the first target condensate and the second target condensate. thereby determining the condensate preference profile of the test compound. (Item 59) Item 59. The method of item 58, wherein the first target condensate and the second target condensate are in the same composition. (Item 60) Item 59. The method of item 58, wherein the first target condensate and the second target condensate are in different compositions. (Item 61) 61. The method of any one of items 58 to 60, wherein the partitioning characteristic of the test compound in the first target condensate is determined before, simultaneously with, or after the partitioning characteristic of the test compound in the second target condensate is determined. (Item 62) 1. A method for determining a condensation preference profile of a test compound, comprising: (a) determining the relative partitioning properties of a test compound in a first target condensate using a method according to any one of items 49 to 51; (b) determining the relative partitioning properties of the test compound in the second target condensate using the method according to any one of items 49 to 51; and (c) calculating a ratio of the partitioning properties of the test compound determined in the first target condensate and the second target condensate. thereby determining the condensate preference profile of the test compound. (Item 63) Item 63. The method of item 62, wherein the first target condensate and the second target condensate are in the same composition. (Item 64) Item 63. The method of item 62, wherein the first target condensate and the second target condensate are in different compositions. (Item 65) 65. The method of any one of items 62 to 64, wherein the relative partitioning profile of the test compound in the first target condensate is determined before, simultaneously with, or after the relative partitioning profile of the test compound in the second target condensate is determined. (Item 66) 66. The method of any one of items 58 to 65, further comprising a step of labeling the first target condensate and the second target condensate to visualize the first target condensate and the second target condensate. (Item 67) Item 67. The method of item 66, wherein the first target condensate and the second target condensate are labeled with different labels. (Item 68) 68. The method of any one of items 58 to 67, comprising repeating said steps of the method for one or more additional target condensates. (Item 69) Item 70. A method for determining a condensate preference profile for a plurality of test compounds, comprising carrying out the method according to any one of items 58 to 68 using the plurality of test compounds. 70. The method of claim 69, further comprising the step of comparing the condensate preference profiles of a subset or all of the plurality of test compounds. (Item 71) 71. The method of claim 70, further comprising identifying test compounds having the same or similar condensate preference profile. (Item 72) 72. The method of claim 71, further comprising identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profile. (Item 73) 73. The method of claim 72, further comprising determining the relative partitioning properties for one or more additional test compounds that have the identified properties. (Item 74) 74. The method of claim 72 or 73, further comprising determining the relative partitioning properties for one or more additional test compounds that do not contain the identified property. (Item 75) 1. A method for identifying a property of a compound that is related to partitioning of the compound into or out of a condensate, comprising: (a) determining the partitioning properties of a plurality of test compounds in a target condensate by carrying out the method described in item 43; (b) comparing the partitioning characteristics of a subset or all of said plurality of test compounds among a target compound; (c) identifying test compounds that have the same or similar partitioning properties in the target condensate; and (d) identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning properties; A method comprising: (Item 76) 1. A method for identifying a property of a compound that is related to partitioning of the compound into or out of a condensate, comprising: (a) determining the relative partitioning properties of a plurality of test compounds in a target condensate by carrying out the method described in item 52; (b) comparing the relative partitioning properties of a subset or all of said plurality of test compounds in said target condensate; (c) identifying test compounds that have the same or similar relative partitioning properties in the target condensate; and (d) identifying properties that a subset or all of the identified test compounds have in common in addition to their same or similar relative partitioning properties. A method comprising: (Item 77) 1. A method for identifying a property of a compound that is related to partitioning of the compound into or out of a condensate, comprising: (a) determining condensate preference profiles of a plurality of test compounds by performing the method described in item 69; (b) comparing the condensate preference profiles of a subset or all of said plurality of test compounds; (c) identifying test compounds having the same or similar condensate preference profile; and (d) identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profile. A method comprising: (Item 78) A method for designing a compound having desired partitioning properties into or out of a target condensate, comprising the steps of: (a) determining the partitioning properties of a plurality of test compounds in the target condensate by carrying out the method according to item 43; (b) comparing the partitioning characteristics of a subset or all of said plurality of test compounds in said target condensate; (c) identifying test compounds that have the same or similar partitioning properties in the target condensate; (d) identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning properties; and (e)(i) designing a compound that comprises the identified property; or (ii) designing a compound that does not contain the identified property. thereby designing a compound having said desired partitioning properties into or out of said target condensate. (Item 79) A method for designing a compound having desired relative partitioning properties into or out of a target condensate, comprising the steps of: (a) determining the relative partitioning properties of a plurality of test compounds in the target condensate by carrying out the method of item 52; (b) comparing the relative partitioning properties of a subset or all of said plurality of test compounds in said target condensate; (c) identifying test compounds that have the same or similar relative partitioning properties in said target condensates; (d) identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar relative partitioning properties; and (e)(i) designing a compound that comprises the identified property; or (ii) designing a compound that does not contain the identified property. thereby designing a compound having said desired relative partitioning properties to or from said target condensate. (Item 80) 1. A method for designing a compound having a desired condensate preference profile, comprising: (a) determining condensate preference profiles of a plurality of test compounds by performing the method described in item 69; (b) comparing the condensate preference profiles of a subset or all of said plurality of test compounds; (c) identifying test compounds having the same or similar condensate preference profile; (d) identifying properties that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profile; and (e)(i) designing a compound that comprises the identified property; or (ii) designing a compound that does not contain the identified property. thereby designing a compound having said desired condensate preference profile. (Item 81) 81. The method of any one of items 78 to 80, further comprising the step of making the compound. (Item 82) 1. A method for screening test compounds for desirable partitioning properties from a group of candidate compounds, comprising: (a) determining the partitioning characteristics of each of said group of candidate compounds; and (b) identifying said test compound having said desired partitioning characteristics. The method includes: (Item 83) 83. The method of claim 82, wherein the partitioning property of each of the group of candidate compounds is determined in vitro. (Item 84) 84. The method of claim 82 or 83, wherein the test compound has suitable distribution properties that are useful for treating a disease in an individual. (Item 85) 1. A method for identifying a test compound useful for treating a disease in an individual in need thereof, comprising: (a) identifying a target condensate associated with the disease; and (b) determining the partitioning properties of a candidate compound in the target condensate; and (c) identifying said test compounds that have suitable distribution properties useful for treating said disease. The method includes: (Item 86) 1. A method for determining the partitioning properties of a test compound in a target condensate, comprising: (a) combining said test compound and a composition comprising said target condensate and excess condensate solutions; (b) obtaining a reference control; (c) measuring the MS signal of said test compound or a portion thereof in said excess condensate solution using a mass spectrometry technique; (d) measuring the MS signal of the test compound or a portion thereof in the reference control using mass spectrometry techniques; and (e) comparing the MS signal of the test compound from the excess condensate solution and the MS signal of the test compound from the reference control. thereby determining the partitioning characteristic of the test compound in the target condensate. (Item 87) 87. The method of claim 86, wherein the amount of the test compound combined with the composition is 100 nM or less, and the amount of precursor molecule in the composition containing the target condensate is about 5 μM. (Item 88) A library comprising a plurality of compounds, each compound of said plurality of compounds comprising the same portion comprising a property having a desired partitioning characteristic. (Item 89) A method for designing a test compound having desired partitioning properties, comprising modifying a precursor of the test compound by attaching a moiety to the compound, the moiety comprising a property having the desired partitioning properties.

Claims

[Claim 1] The invention described in this specification.

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