Method for characterizing the association characteristics of condensates of compounds and use thereof

Abstract

Methods and uses thereof for evaluating (e.g., characterizing or determining) the coacervate association characteristics of a compound (e.g., a test compound) are provided. For example, methods for determining the partitioning characteristics of a test compound in a target coacervate, methods for determining the relative partitioning characteristics of a test compound in a target coacervate, and methods for determining the coacervate preference profile of a test compound are provided. In addition, methods for designing and / or identifying and / or preparing compounds or portions thereof having a desired relative coacervate partitioning characteristic are provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 803,365, filed on February 8, 2019, and U.S. Provisional Patent Application No. 62 / 866,526, filed on June 25, 2019, the disclosures of which are hereby incorporated by reference in their entireties. Technical Field

[0003] The present invention relates to the field of biocondensates. Background Art

[0004] In addition to membrane-bound organelles such as mitochondria, lysosomes, and endoplasmic reticulum, cells also contain different subcompartments that do not include a membrane between them and their immediately surrounding solution. Many of these membrane-free molecular assemblies have been shown to be formed by a process called liquid-liquid phase separation or coacervation. In this process, a solution containing biomacromolecules separates into different phases, coacervates that are rich in some of those macromolecules and surrounding phases that are relatively lacking in those macromolecules. A variety of cellular coacervates have been identified. In addition, phase-separated coacervates can be formed extracellularly, such as in solution or outside the cell (Albertiet 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 control the distribution of compounds into coacervates or the exclusion of compounds from coacervates or the differences in the distribution of compounds between various coacervates.

[0005] Various condensates are known to be important for regulating cellular processes. For example, condensates can bring together molecules with increased concentrations to accelerate reactions within the condensate, or can sequester molecules in the condensate, reducing their concentration in the surrounding medium. Abnormal condensate function is also involved in various human diseases, such as neurodegenerative diseases and proliferative diseases (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 the lack of understanding of the mechanisms that control the distribution of compounds to condensates, little or no information is known about how to identify test compounds that interact with condensates, how to design compounds to improve interactions with condensates, and how this information can be used to improve the treatment of diseases.

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

[0007] 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) combining a test compound with a composition comprising a target condensate and an extra-condensate solution; (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. In some embodiments, the method further comprises causing the target condensate to form before step (a).

[0008] In some aspects, provided herein is a method for determining the partitioning characteristics of a test compound in a target coacervate, the method comprising: (a) adding a test compound to a composition comprising a target coacervate and a solution outside the coacervate; (b) determining the amount of the test compound in the target coacervate, thereby determining the partitioning characteristics of the test compound in the target coacervate. In some embodiments, the method further comprises causing the target coacervate to form before step (a).

[0009] In some aspects, the present invention provides a method for determining the distribution characteristics of a test compound in a target coacervate, the method comprising: (a) causing the formation of a target coacervate in the presence of a test compound to obtain a composition comprising a target coacervate and a solution outside the coacervate; and (b) determining the amount of the test compound in the target coacervate, thereby determining the distribution characteristics of the test compound in the target coacervate.

[0010] 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) adding a test compound to a composition comprising a precursor molecule; (b) causing the target condensate to form to obtain a composition comprising a target condensate and a solution outside the condensate; (c) determining the amount of the test compound in the target condensate, thereby determining the partitioning characteristics of the test compound in the target condensate. In some embodiments, the method further comprises combining the test compound with a precursor composition comprising a precursor molecule prior to step (a). In some embodiments, the method further comprises adding the test compound to a precursor composition comprising a precursor molecule prior to step (a).

[0011] In some embodiments, the method further comprises determining the amount of the test compound in the solution outside the coagulation. In some embodiments, the amount of the test compound in the target coagulation is determined before, simultaneously with, or after determining the amount of the test compound in the solution outside the coagulation. In some embodiments, the method further comprises determining a ratio of the amount of the test compound in the target coagulation to the amount of the test compound in the solution outside the coagulation. In some embodiments, the method further comprises separating the target coagulation from the solution outside the coagulation. In some embodiments, the method further comprises identifying the target coagulation before determining the amount of the test compound in the target coagulation.

[0012] In some embodiments, the disorder of the target condensate is associated with a disease. In some embodiments, the method further comprises characterizing the target condensate by identifying one or more macromolecules contained therein. In some embodiments, the identification comprises determining the amount of the 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 to the amount of the one or more macromolecules in the target condensate. In some embodiments, the target condensate comprises a protein comprising 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.

[0013] In some embodiments, the composition comprises a cell. In some embodiments, the cell is a microbial or animal cell. In some embodiments, the cell comprises an aggregate determined to be disordered. In some embodiments, the cell has one or more features of a neurodegenerative disease or a proliferative disease.

[0014] In some embodiments, the target condensate is a cell condensate. In some embodiments, the cell condensate is a lysate, a P- granule, a histone locus body, a multivesicular body, a neuronal RNA granule, a nuclear gem, a nuclear pore, a nuclear plaque, a nuclear stress body, a nucleolus, an Oct1 / PTF / transcription (OPT) domain, a paraspot, a perinuclear chamber, a PML nuclear body, a PML oncogenic domain, a polycomb body, a processing body, a signal transduction cluster, a Sam68 nuclear body, a stress granule or a splicing plaque. 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 features of a neurodegenerative disease or a proliferative disease. In some embodiments, the condensate outside the solution is an intracellular fluid. In some embodiments, the intracellular fluid is a cytosol or a nuclear sol.

[0015] In some embodiments, the target condensate is not in a cell. In some embodiments, the target condensate is an extracellular condensate. In some embodiments, the solution outside the condensate is extracellular fluid. In some embodiments, the extracellular fluid is interstitial fluid.

[0016] In some embodiments, the method is a cell-free assay. In some embodiments, the composition does not comprise cells. In some embodiments, the composition comprises one or more of the following: a macromolecule, a salt, and a buffer.

[0017] In some embodiments, the composition comprises two or more coacervates of interest. In some embodiments, the method further comprises repeating the steps of the method for one or more additional coacervates.

[0018] 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 UV-visible spectrophotometry.

[0019] In some aspects, provided herein is a method for determining the distribution characteristics of multiple test compounds in a target condensate, the method comprising implementing the method for determining the distribution characteristics described herein with multiple test compounds. In some embodiments, the method further comprises comparing the distribution characteristics of a subset or all of the multiple test compounds in the target condensate. In some embodiments, the method further comprises identifying test compounds having the same or similar distribution characteristics in the target condensate. In some embodiments, the method further comprises identifying the characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar distribution characteristics. In some embodiments, the method further comprises determining the distribution characteristics of one or more additional test compounds containing the identified characteristics in the target condensate. In some embodiments, the method further comprises determining the distribution characteristics of one or more additional test compounds that do not contain the identified characteristics in the target condensate.

[0020] In some aspects, provided herein is a method for determining the relative partitioning characteristics of a test compound in a target condensate, the method comprising: (i) determining the partitioning characteristics of the test compound by implementing the method for determining the partitioning characteristics described herein with the test compound; (ii) determining the partitioning characteristics of a reference compound by implementing the method for determining the partitioning characteristics described herein with a reference compound; (iii) calculating the ratio of the partitioning characteristics determined in (i) and (ii), thereby determining the relative partitioning characteristics of the test compound in the target condensate. In some embodiments, the test compound comprises a test compound marker. In some embodiments, the reference compound is a test compound marker.

[0021] In some aspects, provided herein is a method for determining the relative distribution characteristics of a plurality of test compounds in a target condensate, the method comprising: (1) performing a method for determining the relative distribution characteristics of a test compound in a target condensate; (2) repeating steps (i) and (iii) with a plurality of test compounds. In some embodiments, the method further comprises comparing the relative distribution characteristics 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 having the same or similar relative distribution characteristics in the target condensate. In some embodiments, the method further comprises identifying features that a subset or all of the identified test compounds have in common in addition to the same or similar relative distribution characteristics. In some embodiments, the method further comprises determining the relative distribution characteristics of one or more additional test compounds in the target condensate that contain the identified features. In some embodiments, the method further comprises determining the relative distribution characteristics of one or more additional test compounds in the target condensate that do not contain the identified features.

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

[0023] In some aspects, provided herein is a method for determining a coacervate preference profile of a test compound, the method comprising: (a) determining a relative partitioning characteristic of a test compound in a first target coacervate according to a method disclosed herein; (b) determining a relative partitioning characteristic of a test compound in a second target coacervate according to a method disclosed herein; and (c) calculating a ratio of the partitioning characteristics of the test compound determined in the first target coacervate and the second target coacervate, thereby determining a coacervate preference profile of the test compound. In some embodiments, the first target coacervate and the second target coacervate are in the same composition. In some embodiments, the first target coacervate and the second target coacervate are in different compositions. In some embodiments, the relative partitioning characteristics of the test compound in the first target coacervate are determined before, simultaneously with, or after determining the relative partitioning characteristics of the test compound in the second target coacervate.

[0024] In some aspects, provided herein is a method for determining a coacervate preference profile of a test compound, the method comprising: (a) adding a test compound to a composition comprising a first target coacervate and a second target coacervate; (b) determining the amount of the test compound in the first target coacervate; (c) determining the amount of the test compound in the second target coacervate; and (d) calculating the ratio of the amount of the test compound determined in the first target coacervate and the second target coacervate, thereby determining the coacervate preference profile of the test compound. In some embodiments, the method further comprises causing the first target coacervate and / or the second target coacervate to form before step (a).

[0025] In some aspects, provided herein is a method for determining a coacervate preference profile of a test compound, the method comprising: (a) adding a test compound to a composition comprising a precursor molecule; (b) causing a first target coacervate and a second target coacervate to form in the composition; (c) determining the amount of the test compound in the first target coacervate; (d) determining the amount of the test compound in the second target coacervate; (e) calculating a ratio of the amount of the test compound determined in the first target coacervate to the amount of the test compound determined in the second target coacervate, thereby determining the coacervate preference profile of the test compound. In some embodiments, the amount of the test compound in the first target coacervate is determined before, simultaneously with, or after the amount of the test compound in the second target coacervate is determined.

[0026] In some embodiments, the method further comprises separating the first target aggregate and the second target aggregate from the composition. In some embodiments, the method further comprises, before determining the amount of the test compound in the first target aggregate and / or the second target aggregate, identifying the first target aggregate and / or the second target aggregate.

[0027] In some embodiments, the disorder of the first target condensate and / or the second target condensate is associated with a disease. In some embodiments, the method further includes 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 includes 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 includes 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.

[0028] In some embodiments, the composition comprises a cell. In some embodiments, the cell is a microbial or animal cell. In some embodiments, the cell comprises an aggregate determined to be disordered. In some embodiments, the cell has one or more features of a neurodegenerative disease or a proliferative disease.

[0029] In some embodiments, the first target condensate and / or the second target condensate is a cell condensate. In some embodiments, the first target condensate is a lysate, a P-particle, a histone locus body, a multivesicular body, a neuronal RNA granule, a nuclear bud, a nuclear pore, a nuclear plaque, a nuclear stress body, a nucleolus, an Oct1 / PTF / transcription (OPT) domain, a paraplaque, a perinuclear chamber, a PML nuclear body, a PML oncogenic domain, a polycomb body, a processing body, a signal transduction cluster, a viral condensate, a Sam68 nuclear body, a stress granule, or a splicing plaque. In some embodiments, the second target condensate is a lysate, a P-particle, a histone locus body, a multivesicular body, a neuronal RNA granule, a nuclear bud, a nuclear pore, a nuclear plaque, a nuclear stress body, a nucleolus, an Oct1 / PTF / transcription (OPT) domain, a paraplaque, a perinuclear chamber, a PML nuclear body, a PML oncogenic domain, a polycomb body, a processing body, a signal transduction cluster, a viral condensate, a Sam68 nuclear body, a stress granule, or a splicing plaque. In some embodiments, the first target condensate and / or the second target condensate is in a cell.

[0030] In some embodiments, the first target aggregate and / or the second target aggregate is an extracellular aggregate.

[0031] In some embodiments, the composition does not comprise cells. In some embodiments, the composition comprises one or more of: a macromolecule, a salt, and a buffer.

[0032] In some embodiments, the composition comprises one or more additional target coagulants. In some embodiments, the method further comprises repeating the steps of the method for one or more additional target coagulants.

[0033] In some aspects, provided herein is a method for determining a coacervate preference profile for a plurality of test compounds, the method comprising implementing a method for determining a coacervate preference profile as described herein using a plurality of test compounds. In some embodiments, the method further comprises comparing the coacervate 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 coacervate preference profiles. In some embodiments, the method further comprises identifying features that a subset or all of the identified test compounds have in common in addition to the same or similar coacervate preference profiles. In some embodiments, the method further comprises determining the relative distribution characteristics of one or more additional test compounds that contain the identified features. In some embodiments, the method further comprises determining the relative distribution characteristics of one or more additional test compounds that do not contain the identified features.

[0034] In some aspects, provided herein is a method for identifying compound characteristics associated with the partitioning of the compound into or out of a coagulation, the method comprising: (a) determining the partitioning characteristics of a plurality of test compounds in a target coagulation 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 coagulation; (c) identifying test compounds having the same or similar partitioning characteristics in the target coagulation; 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 compound characteristics associated with the partitioning of compounds into agglomerates, the method comprising: (a) determining the partitioning characteristics of multiple test compounds in a target agglomerate according to the methods described herein; (b) comparing the partitioning characteristics of a subset or all of the multiple test compounds in the target compound; (c) identifying test compounds having the same or similar partitioning characteristics in the target agglomerate; 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.

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

[0038] In some aspects, provided herein is a method for identifying compound characteristics that are associated with the partitioning of the compound into or out of a coacervate, the method comprising: (a) determining a coacervate preference profile for a plurality of test compounds according to the methods described herein; (b) comparing the coacervate preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar coacervate 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 coacervate preference profiles.

[0039] In some aspects, provided herein is a method for identifying compound characteristics associated with the partitioning of compounds into condensates, the method comprising: (a) determining condensate preference profiles for a plurality of test compounds according to the methods described herein; (b) comparing the condensate preference profiles for 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 profiles.

[0040] In some aspects, provided herein is a method for designing a compound with a desired partitioning characteristic within or outside a target condensate, the method comprising: (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; (d) identifying a feature that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning characteristics; and (e) (i) designing a compound containing the identified feature; or (ii) designing a compound that does not contain the identified feature, thereby designing a compound with a desired partitioning characteristic within or outside a target condensate. In some embodiments, designing a compound comprises attaching a portion containing the identified feature, thereby conferring a desired partitioning characteristic to the compound. In some embodiments, designing a compound comprises removing a portion containing the identified feature, thereby conferring a desired partitioning characteristic to the compound. In some embodiments, designing a compound comprises changing a first portion to a second portion containing the identified feature, thereby conferring a desired partitioning characteristic to the compound. In some embodiments, compounds are designed in whole or in part using methods that include modeling-based, computer and / or computational techniques, such as bioinformatics-, cheminformatics- and / or artificial intelligence (AI)-based techniques.

[0041] In some aspects, provided herein is a method for designing a compound having a desired partitioning characteristic for a target condensate, the method comprising: (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 a target condensate; (c) identifying test compounds having the same or similar partitioning characteristics in a target condensate; (d) identifying features that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning characteristics; and (e) designing a compound comprising the identified features. In some embodiments, designing a compound comprises attaching a portion comprising the identified features, thereby conferring the desired partitioning characteristics to the compound. In some embodiments, designing a compound comprises changing a first portion to a second portion comprising the identified features, thereby conferring the desired partitioning characteristics to the compound. In some embodiments, the compound is designed in whole or in part using a method comprising a modeling-based, computer- and / or computational technique, such as a technique based on bioinformatics, cheminformatics, and / or artificial intelligence (AI).

[0042] In some aspects, provided herein is a method for designing a compound with a desired relative partitioning characteristic within or outside a target condensate, the method comprising: (a) determining the relative partitioning characteristics of a plurality of test compounds in a target condensate according to the methods disclosed herein; (b) comparing the relative partitioning characteristics of a subset or all of the plurality of test compounds in a target condensate; (c) identifying test compounds having the same or similar relative partitioning characteristics in a target condensate; (d) identifying features that a subset or all of the identified test compounds have in common in addition to the same or similar relative partitioning characteristics; and (e) (i) designing a compound containing the identified features; or (ii) designing a compound that does not contain the identified features, thereby designing a compound with a desired relative partitioning characteristic within or outside a target condensate. In some embodiments, designing a compound comprises attaching a portion containing the identified features, thereby conferring a desired relative partitioning characteristic to the compound. In some embodiments, designing a compound comprises removing a portion containing the identified features, thereby conferring a desired partitioning characteristic to the compound. In some embodiments, designing a compound comprises changing a first portion to a second portion containing the identified features, thereby conferring a desired partitioning characteristic to the compound. In some embodiments, compounds are designed in whole or in part using methods that include modeling-based, computer and / or computational techniques, such as bioinformatics-, cheminformatics- and / or artificial intelligence (AI)-based techniques.

[0043] In some aspects, provided herein is a method for designing a compound with a desired relative partitioning characteristic, the method comprising: (a) determining the relative partitioning characteristics of a plurality of test compounds in a target condensate according to the 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; (d) identifying features that a subset or all of the identified test compounds have in common in addition to the same or similar relative partitioning characteristics; and (e) designing a compound comprising the identified features. In some embodiments, designing a compound comprises attaching a portion comprising the identified features, thereby conferring the desired relative partitioning characteristics to the compound. In some embodiments, designing a compound comprises changing a first portion to a second portion comprising the identified features, thereby conferring the desired partitioning characteristics to the compound. In some embodiments, the compound is designed in whole or in part using a method comprising a modeling-based, computer- and / or computational technique, such as a technique based on bioinformatics, cheminformatics, and / or artificial intelligence (AI).

[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 profiles 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 profiles; (d) identifying features that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profiles; and (e) (i) designing a compound containing the identified features; or (ii) designing a compound that does not contain the identified features, thereby designing a compound with a desired condensate preference profile. In some embodiments, the method further comprises preparing the compound. In some embodiments, designing a compound comprises attaching a portion containing the identified features, thereby conferring a desired condensate preference profile on the compound. In some embodiments, designing a compound comprises removing a portion containing the identified features, thereby conferring a desired partitioning feature on the compound. In some embodiments, designing a compound comprises changing a first portion to a second portion containing the identified features, thereby conferring a desired partitioning feature on the compound. In some embodiments, compounds are designed in whole or in part using methods that include modeling-based, computer and / or computational techniques, such as bioinformatics-, cheminformatics- and / or artificial intelligence (AI)-based techniques.

[0045] 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 profiles 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 profiles; (d) identifying features that a subset or all of the identified test compounds have in common in addition to the same or similar condensate preference profiles, such as chemical moieties or motifs; and (e) designing compounds comprising the identified features. In some embodiments, designing a compound comprises attaching a portion comprising the identified feature, thereby giving the compound a desired condensate preference profile. In some embodiments, designing a compound comprises changing a first portion to a second portion comprising the identified feature, thereby giving the compound a desired distribution feature. In some embodiments, a plurality of compounds designed by the methods described herein are provided. In some embodiments, the compound is designed in whole or in part using a method comprising a modeling-based, computer- and / or computational technique, such as a technique based on bioinformatics, cheminformatics, and / or artificial intelligence (AI). In some embodiments, the method further comprises preparing the compound.

[0046] In some aspects, provided herein is a method for screening a test compound having a desired partitioning characteristic from a group of candidate compounds, the method comprising: (a) determining the partitioning characteristic of each of the group of candidate compounds; (b) identifying a test compound having a desired partitioning characteristic. In some embodiments, the partitioning characteristic of each of the group of candidate compounds is determined in vitro. In some embodiments, the test compound has a partitioning characteristic suitable for treating a disease in an individual.

[0047] In some aspects, provided herein is a method for identifying a test compound that can be used to treat a disease in an individual in need thereof, the method comprising: (a) identifying a target aggregate associated with the disease; (b) determining a distribution characteristic of a candidate compound in the target aggregate, and (c) identifying a test compound having a distribution characteristic suitable for treating the disease.

[0048] 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) combining a test compound with a composition comprising a target condensate and a solution outside the condensate; (b) obtaining a reference control; (c) measuring the MS signal of the test compound or a portion thereof in the solution outside the condensate 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 solution outside the condensate with the MS signal of the test compound from the reference control, thereby determining the partitioning characteristics of the test compound in the target condensate. In some embodiments, the amount of the test compound combined with the composition is 100 nM or less, and the amount of the precursor molecule in the composition, including in the target condensate, is about 5 μM.

[0049] In some aspects, provided herein are libraries comprising a plurality of compounds, wherein each compound in the plurality of compounds comprises an identical moiety comprising a feature having a desired partitioning characteristic.

[0050] In some aspects, provided herein are methods of designing a test compound having a desired partitioning characteristic, the method comprising modifying a precursor of the test compound by attaching a moiety to the compound, wherein the moiety comprises a feature having the desired partitioning characteristic.

[0051] Those skilled in the art will also appreciate that, without departing from the scope of the present disclosure, the form and details of the embodiments described herein may be modified. In addition, although various advantages, aspects and objects have been described with reference to various embodiments, the scope of the present disclosure should not be limited by mentioning these advantages, aspects and objects. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1An exemplary fluorescent image of dye partitioning in coacervates is shown. Dye #10 is not enriched in coacervates, while the other dyes are enriched in coacervates.

[0053] Figure 2A and 2B Depicted are regions inside and outside of an exemplary coagulant selected to measure dye presence ( Figure 2A ) and does not exist ( Figure 2B ) strength under the circumstances.

[0054] Figure 3 Shown are the ratios of the mean internal intensity to the external intensity (I-in:I-out) determined for each dye in FUS-SNAP aggregates from two different experimental days. Error bars show the standard deviation of these two independent experiments.

[0055] Figure 4 Shown are the ratios of the mean internal intensity to the external intensity (I-in:I-out) determined for each dye in PGL-3 aggregates from two different experimental days. Error bars show the standard deviation of these two independent experiments.

[0056] Figure 5 The ratio of the average I-in:I-out ratio of each dye in FUS-SNAP aggregates to the average I-in:I-out ratio of each dye in PGL-3 aggregates (FUS-SNAP:PGL-3 ratio) is shown.

[0057] Figure 6 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 a merge of other images are shown.

[0058] Figure 7 Shown are histograms of compound fractions in the supernatant (outside of aggregates) for exemplary compounds using both fluorescence-based and mass spectrometry-based assays.

[0059] Figure 8 Shown are histograms of compound fractions in the supernatant for exemplary compounds evaluated at various compound concentrations.

[0060] Fig. 9A and 9B Shown are histograms of the fraction of target compounds outside of coacervates measured in a system with a single target compound and in a system with a mixture of compounds including the target compound. Fig. 9A The target compound in Fig. 9B The target compound in .

[0061] Fig.10 Histograms of compound fractions in the supernatant for certain compounds in several multiplex systems are shown. DETAILED DESCRIPTION

[0062] In some aspects, the invention includes methods and uses thereof for evaluating (eg, characterizing or determining) condensate-associated characteristics of a compound (eg, a test compound).

[0063] The disclosure of the present application is based in part on the inventor's unique insights into methods for assessing (e.g., determining or characterizing) the coacervate association characteristics of a compound, and such methods can be used, for example, to identify, characterize, and develop compounds or portions thereof that are capable of performing desired interactions (including lack of certain interactions) with coacervates. In some embodiments, the desired interaction with coacervates results in the desired biological activity associated with the compound. In some aspects, the disclosure of the present application is based in part on the inventor's discovery and development of quantitative techniques (e.g., mass spectrometry) for determining the coacervate association characteristics of a compound (e.g., the target coacervate partitioning characteristics of a test compound). This method allows, for example, the target coacervate partitioning characteristics of a test compound to be accurately and reliably determined in a high-throughput manner that is applicable to both simple and complex systems. In addition, for example, these methods are hypothesis-free (i.e., no known, labeled compounds or coacervates or components thereof are required), are compatible with a high degree of compound multiplexing, do not require compound enrichment, do not require extraction of compounds from coacervates, can be used for homotypic and heterotypic systems, and can be performed with a smaller amount of compounds and / or coacervate precursor macromolecules, which represents a more biologically relevant model and reduces the use of starting materials and reagents. These methods allow identification of test compounds or portions thereof that can be used to guide further identification and / or design of one or more compounds with a desired partitioning characteristic, thus providing a method for identifying target compounds with improved efficacy, therapeutic index, and / or safety. Identification of test compounds or portions thereof also enables the use of such knowledge to advance certain drug development applications, such as the development of privileged libraries of compounds with a desired partitioning characteristic, and modeling-based and / or computational techniques for drug discovery and screening.

[0064] As described herein, the methods for evaluating the coacervate association characteristics of a compound can be applied to many methods and forms thereof, including methods for determining the partitioning characteristics of a test compound, methods for determining the relative partitioning characteristics of a test compound, methods for determining the coacervate preference profile of a test compound, methods for identifying compound characteristics related to coacervate association, methods for designing compounds with desired coacervate association, and methods for identifying compounds that can be used to treat an individual disease. The forms of the methods described herein include, for example, assays for evaluating the interaction of a single test compound or a portion thereof with a single coacervate of interest, evaluating the interaction of a single test compound or a portion thereof with multiple coacervates of interest, and evaluating the interaction of multiple test compounds or a subset thereof with a single coacervate of interest. As described herein, the unique insights of the inventors enable the identification, development, and optimization of compounds, such as pharmaceutically acceptable compounds, that can be used, for example, to treat an individual disease.

[0065] Thus, in some aspects, the present invention includes a method for determining the distribution characteristics of a test compound in a target coagulant, the method comprising: (a) adding a test compound to a composition comprising a target coagulant and a solution outside the coagulant; and (b) determining the amount of the test compound in the target coagulant, thereby determining the distribution characteristics of the test compound in the target coagulant.

[0066] In other aspects, the present invention includes a method for determining the distribution characteristics of a test compound in a target condensate, the method comprising: (a) adding a test compound to a composition comprising a precursor molecule; (b) causing the target condensate to form to obtain a composition comprising the target condensate and a solution outside the condensate; (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.

[0067] In other aspects, the invention includes methods of determining the partitioning characteristics of a plurality of test compounds in a target agglomerate, the methods comprising performing any one or more of the methods described herein using the plurality of test compounds.

[0068] In some embodiments, the present invention includes a method for identifying compound characteristics associated with the distribution of compounds into agglomerates, the method comprising: (a) determining the distribution characteristics of multiple test compounds in a target agglomerate by implementing a method for determining the distribution characteristics of multiple test compounds in a target agglomerate; (b) comparing the distribution characteristics of a subset or all of the multiple test compounds in the target agglomerate; (c) identifying test compounds having the same or similar distribution characteristics in the target agglomerate; and (d) identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar distribution characteristics. In some embodiments, the present invention includes a method for designing a compound having a desired distribution characteristic for a target condensate, the method comprising: (a) determining the distribution characteristics of multiple test compounds in a target condensate by implementing a method for determining the distribution characteristics of multiple test compounds in a target condensate; (b) comparing the distribution characteristics of a subset or all of the multiple test compounds in the target condensate; (c) identifying test compounds having the same or similar distribution characteristics in the target condensate; (d) identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar distribution characteristics; and (e) designing compounds comprising the identified characteristics.

[0069] In other aspects, the present invention includes a method for determining the relative partitioning characteristics of a test compound in a target condensate, the method comprising: (i) determining the partitioning characteristics of the test compound by performing any one or more methods described herein with the test compound; (ii) determining the partitioning characteristics of the reference compound by performing any one or more methods described herein with the reference compound; (iii) calculating the ratio of the partitioning characteristics determined in (i) and (ii), thereby determining the relative partitioning characteristics 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 compound features associated with the partitioning of a compound into a target condensate, the method comprising: (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 features 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 a desired relative distribution characteristic, the method comprising: (a) determining the relative distribution characteristics of multiple test compounds in a target agglomerate by implementing a method for determining the relative distribution characteristics of multiple test compounds in a target agglomerate; (b) comparing the relative distribution characteristics of a subset or all of the multiple test compounds in the target agglomerate; (c) identifying test compounds having the same or similar relative distribution characteristics in the target agglomerate; (d) identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar relative distribution characteristics; and (e) designing a compound comprising the identified characteristics.

[0070] In other aspects, the present invention includes a method for determining a coagulant preference profile of a test compound, the method comprising: (a) adding a test compound to a composition comprising a first target coagulant and a second target coagulant; (b) determining the amount of the test compound in the first target coagulant; (c) determining the amount of the test compound in the second target coagulant; and (d) calculating the ratio of the amounts of the test compound determined in the first target coagulant and the second target coagulant, thereby determining the coagulant preference profile of the test compound.

[0071] In other aspects, the present invention includes a method for determining a coacervate preference profile of a test compound, the method comprising: (a) adding the test compound to a composition comprising a precursor molecule; (b) causing the formation of a first target coacervate and a second target coacervate in the composition; (c) determining the amount of the test compound in the first target coacervate; (d) determining the amount of the test compound in the second target coacervate; and (e) calculating the ratio of the amount of the test compound determined in the first target coacervate and the second target coacervate, thereby determining the coacervate preference profile of the test compound.

[0072] In other aspects, the invention includes a method for determining a coacervate preference profile for a plurality of test compounds, the method comprising performing any one or more of the methods described herein for determining a coacervate preference profile for a test compound using a plurality of test compounds. In some embodiments, the invention includes a method for identifying compound characteristics associated with the partitioning of the compound into coacervates, the method comprising: (a) determining a coacervate preference profile for a plurality of test compounds by performing a method for determining a coacervate preference profile for a plurality of test compounds; (b) comparing the coacervate preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar coacervate preference profiles; and (d) identifying features that a subset or all of the identified test compounds have in common in addition to the same or similar coacervate preference profiles. In some embodiments, the present invention includes a method for designing a compound having a desired coacervate preference profile, the method comprising: (a) determining the coacervate preference profiles of a plurality of test compounds by implementing a method for determining the coacervate preference profiles of a plurality of test compounds; (b) comparing the coacervate preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar coacervate preference profiles; (d) identifying features that a subset or all of the identified test compounds have in common in addition to the same or similar coacervate preference profiles; and (e) designing a compound comprising the identified features.

[0073] definition

[0074] For the purpose of interpreting this specification, the following definitions shall apply and, whenever appropriate, terms used in the singular shall include the plural and vice versa. In the event of a conflict between any definition set forth below and any document incorporated herein by reference, the set forth definition shall prevail.

[0075] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, including but not limited to peptides, oligopeptides, dimers, trimers and multimers of amino acid residues. The definition includes full-length proteins and fragments thereof. The term also includes post-translational modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, etc.

[0076] The terms "comprising," "having," "including," and "containing," and other similar forms and their grammatical equivalents, as used herein, are intended to be equivalent in meaning and to be open-ended, i.e., any of these words followed by one or more items is not intended to be an exhaustive list of the one or more items, or to be limited to only the listed one or more items. For example, an article "comprising" components A, B, and C may consist of (i.e., contain only) components A, B, and C, or may contain not only components A, B, and C, but also one or more other components. Thus, it is intended and understood that "comprising" and its similar forms and its grammatical equivalents include disclosure of embodiments of "consisting essentially of" or "consisting of."

[0077] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range to one-tenth of the unit of the lower limit (unless the context clearly dictates otherwise) and any other stated or intervening value in that range are included in the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0078] References herein to "about" a value or parameter include (and describe) variations involving the value or parameter itself. For example, descriptions referring to "about X" include descriptions of "X."

[0079] As used herein (including in the appended claims), the singular forms "a," "an," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0080] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0081] Methods for assessing aggregation-related characteristics of compounds

[0082] In some aspects of the present application, methods and uses thereof for evaluating the coacervate association characteristics of compounds such as test compounds are provided. In some embodiments, "coacervate" as described herein refers to a non-membrane enclosed compartment formed by phase separation (including all stages of phase separation) of one or more proteins and / or other macromolecules.

[0083] The following describes in more detail the techniques for evaluating the coacervate association characteristics of compounds and their application to various methods and formats.One skilled in the art will recognize that, based on the description provided, various embodiments are possible within the scope and spirit of the present disclosure.

[0084] In some aspects, provided herein are methods of determining a partitioning characteristic of a test compound, comprising determining the amount of the test compound in a target agglomerate. In some embodiments, determining the amount of the test compound in the target agglomerate determines the partitioning characteristic of the test compound in the target agglomerate.

[0085] In some aspects, provided herein are methods of determining a relative partitioning characteristic of a test compound, comprising determining an amount of the test compound in a target agglomerate, determining an amount of a reference compound in the target agglomerate, and calculating a ratio of the amounts of the test compound and the reference compound in the target agglomerate.

[0086] In addition, in some aspects, provided herein is a method of determining a test compound's condensate preference profile, 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 a ratio of the amount of the test compound in the first and second target condensates. In some embodiments, the method of determining a test compound's condensate preference profile comprises determining a partitioning characteristic or a relative partitioning characteristic of the test compound in a first target condensate, determining a partitioning characteristic or a relative partitioning characteristic of the test compound in a second target condensate, and calculating a ratio of the partitioning characteristics or the relative partitioning characteristics of the test compound in the first and second target condensates.

[0087] 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 2, 3, 4, 5, 10, 15, 20, 25, 40, 50, 75, 100, 250, 500, 1,000, 10,000, 100,000 or more compounds.

[0088] In some embodiments, the method further comprises combining a test compound with a composition comprising a target coacervate and a solution outside the coacervate. In some embodiments, the method further comprises adding a test compound to a composition comprising a target coacervate and a solution outside the coacervate. In some embodiments, the method further comprises causing the target coacervate to form. In some embodiments, the method further comprises combining a test compound with a precursor composition comprising a coacervate precursor molecule, and then causing the target coacervate to form. In some embodiments, the method further comprises adding a test compound to a composition comprising a coacervate precursor molecule, and then causing the target coacervate to form.

[0089] In some embodiments, the method further comprises combining the test compound with a composition comprising cells. In some embodiments, the method further comprises combining the test compound with a composition comprising cells and then causing the formation of a desired condensate. In some embodiments, the method further comprises causing the formation of a desired condensate in a composition comprising cells and then combining the test compound with the composition. In some embodiments, the method further comprises allowing the compound to enter the cell.

[0090] In some embodiments, the method further comprises adding a test compound to the composition comprising cells. In some embodiments, the method further comprises adding a test compound to the composition comprising cells and then causing the formation of a target condensate. In some embodiments, the method further comprises causing the formation of a target condensate in the composition comprising cells and then adding the test compound to the composition. In some embodiments, the method further comprises allowing the compound to enter the cell.

[0091] In some embodiments, the method further comprises separating the coacervate of interest from the solution outside the coacervate, for example, for the purpose of quantifying the coacervate or the target compound in the solution outside the coacervate. In a cell-free solution, the coacervate is generally denser than the solution outside the coacervate and will precipitate. Therefore, in some embodiments, separating the coacervate of interest from the solution outside the coacervate comprises separating the supernatant from the precipitate. In some embodiments, the method comprises centrifuging the composition. In some embodiments, the method comprises allowing the coacervate to settle.

[0092] Techniques for determining the association characteristics of compound aggregates

[0093] In some aspects, provided herein are methods for determining the association characteristics of condensates of a compound, comprising determining the amount of a test compound consumed (depleted) from a solution outside the condensate due to the presence, formation and / or modulation of a target condensate (including determining the absence of consumption). In some embodiments, the method for determining the association characteristics of condensates of a compound comprises using a mass spectrometry-based technique (e.g., to determine the amount of the test compound). In some embodiments, the method for determining the association characteristics of condensates of a compound comprises using 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 UV-visible spectrophotometry (e.g., to determine the amount of the test compound). In some embodiments, such methods can be used to determine the distribution characteristics, relative distribution characteristics and / or condensate preference profiles of a test compound in a composition comprising a target condensate and a solution outside the condensate.

[0094] In some embodiments, the method includes determining the amount of test compound consumed 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 in solution outside the condensate and not associated with a precursor molecule of the target condensate. In some embodiments, the method includes determining the amount of test compound associated with the target condensate (such as in the target condensate). In some embodiments, the method includes determining the amount of test compound associated with a precursor molecule of the target condensate. In some embodiments, the amount of test compound consumed from the system due to the presence and / or formation of the target condensate is used to determine a condensate association characteristic, such as a target condensate distribution characteristic of the test compound.

[0095] In some embodiments, the method includes comparing the MS signal of the test compound or a portion thereof from the solution outside the condensate with the MS signal of the test compound or a portion thereof from the reference control, such as by the ratio of the MS signal of the test compound or a portion thereof from the solution outside the condensate to 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 solution outside the condensate to the MS signal of the test compound from the reference control represents a consumption value. In some embodiments, the consumption value represents the amount of test compound consumed from the system due to the presence and / or formation of the target condensate. In some embodiments, the method further includes obtaining, such as measuring the MS ion signal of the test compound or a portion thereof in the solution outside the condensate using mass spectrometry. In some embodiments, the method further includes obtaining, such as measuring the MS ion signal of the test compound or a portion thereof in the reference control using mass spectrometry. In some embodiments, the method further includes combining the test compound with a composition comprising the target condensate and the solution outside the condensate. In some embodiments, the method further includes causing the target condensate to form in the presence of the test compound to obtain a composition comprising the target condensate and the solution outside the condensate. In some embodiments, the method further comprises separating the target coacervate in the composition comprising the target coacervate and a solution outside the coacervate, such as by pelleting the target coacervate in the composition. In some embodiments, the method further comprises obtaining (such as generating) a reference control.

[0096] In some embodiments, the amount of test compound added to a composition comprising a coacervate of interest and a solution outside the coacervate or a precursor thereof is based on an amount that enables a relatively small depletion of the total amount of test compound from the solution outside the coacervate to be determined (e.g., by comparing the measurement of the amount of compound outside the coacervate (solution) to the measurement of the amount of compound in a reference control). In some embodiments, the amount of test compound added to a composition comprising a coacervate of interest and a solution outside the coacervate is based on the amount of coacervate of interest and / or one or more precursor molecules thereof in the composition. In some embodiments, the amount of test compound added to a composition comprising a coacervate of interest and a solution outside the coacervate is based on the compound capacity of the coacervate of interest. In some embodiments, the amount of the test compound is about 1 μM or less, such as 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 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 between about 1 μM and 10 μM, and the test compound is about 1 μM or less, such as 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, 1 Any of 25nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 9nM or less, 8nM or less, 7nM or less, 6nM or less, 5nM or less, 4nM or less, 3nM or less, 2nM or less, or 1nM or less.

[0097] 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 that can be used to determine the amount of test compound consumed from the system due to the presence and / or formation of a target condensate. In some embodiments, wherein the composition comprises: (a) a certain amount of target condensate, (b) a solution outside the condensate, (c) a certain amount (such as a concentration) of a precursor macromolecule of the target condensate in the solution outside the condensate, and (d) a certain amount (such as a concentration) of the test compound in the composition, which includes (i) a certain amount of the test compound associated with the target condensate (in the target condensate), and (ii) a certain amount (such as a concentration) of the test compound in the solution outside the condensate, the reference control includes the amount (such as a concentration) of the precursor macromolecule of the target condensate in the solution outside the condensate and the amount (such as a concentration) of the test compound in the composition. In some embodiments, the method includes measuring the amount, such as the concentration, of a precursor macromolecule of the target condensate or a portion thereof in the solution outside the condensate from a composition comprising a target condensate and a solution outside the condensate.

[0098] In some embodiments, the reference control includes a certain amount of precursor macromolecules of the target coacervate, which is based on the amount of precursor macromolecules present in the solution outside the coacervate after the composition has undergone granulation of the target coacervate. In some embodiments, the concentration of precursor macromolecules in the reference control is the same as the concentration of precursor macromolecules in the solution outside the coacervate after the composition has undergone granulation of the target coacervate. In some embodiments, the reference control contains the same test compound concentration as the test compound concentration of the composition combination containing the target coacervate and the solution outside the coacervate. In some embodiments, the reference control is substantially free of the target coacervate. In some embodiments, the method further includes determining the amount of the test compound in the reference control. In some embodiments, determining the amount of the test compound in the reference control includes measuring the amount of the test compound or a portion thereof in the reference control using a mass spectrometry-based technique.

[0099] A variety of mass spectrometry-based techniques are suitable for methods described herein. In some embodiments, mass spectrometry-based techniques include measuring the MS signals of one or more ion species of one or more compounds such as one or more test compounds. In some embodiments, the MS signal is 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 mass-to-charge ratio (m / z) measurement. In some embodiments, the MS signal is ionization intensity. In some embodiments, the MS signal is peak height. In some embodiments, the MS signal is peak area, such as the integral of the signal corresponding to the MS ion signal. In some embodiments, the MS signal is peak volume, such as the integral of the signal corresponding to the MS ion signal. In some embodiments, the MS signal is the cumulative measurement of the measurement signal of the ion of the test compound. In some embodiments, mass spectrometry-based techniques include liquid chromatography / mass spectrometry (LC / MS) techniques, liquid chromatography / tandem mass spectrometry (LC / MS) techniques and / or direct sample introduction techniques (e.g., direct spray). In some embodiments, mass spectrometry-based techniques include gas chromatography / mass spectrometry (GC / MS). In some embodiments, the mass spectrometry-based technique comprises an acquisition technique selected from the group consisting of data-dependent acquisition, data-independent acquisition, selected reaction monitoring (SRM), and multiple reaction monitoring (MRM).

[0100] The liquid chromatography techniques considered in the present application include methods for separating precursor macromolecules and / or test compounds compatible with mass spectrometry. In some embodiments, the liquid chromatography technique includes high performance liquid chromatography (HPLC) technology. In some embodiments, the liquid chromatography technique includes high flow liquid chromatography technology. In some embodiments, the liquid chromatography technique includes low flow liquid chromatography technology, such as micron flow liquid chromatography technology or nano flow liquid chromatography technology. In some embodiments, the liquid chromatography technique includes online liquid chromatography technology connected to a mass spectrometer. In some embodiments, capillary electrophoresis (CE) technology or electrospray or MALDI technology can be used to introduce a sample into a mass spectrometer. In some embodiments, direct sample introduction technology can be used to introduce a sample into a mass spectrometer. In some embodiments, mass spectrometry includes ionization technology. The ionization technology considered in the present application includes technology that can charge precursor macromolecules and / or test compounds. In some embodiments, the ionization technology is electrospray ionization. In some embodiments, the ionization technology is nano electrospray ionization. In some embodiments, the ionization technology 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 includes electrospray ionization, nanoelectrospray ionization, or matrix-assisted laser desorption ionization (MALDI) techniques.

[0101] The mass spectrometers that can be connected with online liquid chromatography technology considered in the present application include high-resolution mass spectrometers and low-resolution mass spectrometers. Therefore, in some embodiments, the mass spectrometer is a time-of-flight (TOL) mass spectrometer. In some embodiments, the mass spectrometer is a quadrupole time-of-flight (Q-TOL) mass spectrometer. In some embodiments, the mass spectrometer is a quadrupole ion trap time-of-flight (QIT-TOL) 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 orbital trap (orbitrap). In some embodiments, the mass spectrometer is a quadrupole orbital trap. In some embodiments, the mass spectrometer is a Fourier conversion ion cyclotron resonance (LT) mass spectrometer. In some embodiments, the mass spectrometer is a quadrupole Fourier conversion ion cyclotron resonance (Q-LT) mass spectrometer. In some embodiments, mass spectrometry includes positive ion mode. In some embodiments, mass spectrometry includes negative ion mode. In some embodiments, mass spectrometry comprises time-of-flight (TOF) mass spectrometry. In some embodiments, mass spectrometry comprises quadrupole time-of-flight (Q-TOF) mass spectrometry. In some embodiments, mass spectrometry comprises ion mobility mass spectrometry. In some embodiments, low-resolution mass spectrometry (such as ion trap) or single or triple quadrupole methods are suitable.

[0102] In some embodiments, mass spectrometry-based techniques include processing the MS signals of the precursor macromolecules and / or test compounds obtained. In some embodiments, mass spectrometry-based techniques include peak detection. In some embodiments, mass spectrometry-based techniques include determining ionization intensity. In some embodiments, mass spectrometry-based techniques include determining peak height. In some embodiments, mass spectrometry-based techniques include determining peak area. In some embodiments, mass spectrometry-based techniques include determining peak volume.

[0103] In some embodiments, mass spectrometry-based techniques include identifying the test compound.

[0104] Thus, for example, in some embodiments, a method for determining a partitioning characteristic of a test compound in a target condensate is provided, the method comprising comparing an MS signal of an ion of the test compound from a solution outside the condensate with an MS signal of an ion of the test compound from a reference control, thereby determining a partitioning characteristic of the test compound in the target condensate. In some embodiments, the method for determining a partitioning characteristic of a test compound in a target condensate comprises: (a) obtaining, such as measuring, an MS signal of a test compound or a portion thereof in a solution outside the condensate using a mass spectrometry technique; (b) obtaining, such as measuring, an MS ion signal of a test compound or a portion thereof in a reference control using a mass spectrometry technique; and (c) comparing the MS signal of an ion of the test compound from a solution outside the condensate with an MS signal of an ion of the test compound from a reference control, thereby determining a partitioning characteristic of the test compound in the target condensate.

[0105] In some embodiments, provided herein are methods for determining the association characteristics of a condensate of a small molecule test compound (e.g., a therapeutic small molecule of 1,000 Da or less and / or that satisfies Lipinski's five-fold rule), comprising determining the amount of the small molecule test compound consumed from a solution outside the condensate due to the presence, formation, and / or modulation of the target condensate (including determining the absence of consumption). In some embodiments, provided are methods for determining the partition characteristics 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 a solution outside the condensate with the MS signal of an ion of the small molecule test compound from a reference control, thereby determining the partition characteristics 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 comprises: (a) obtaining, such as measuring, an MS signal of a small molecule test compound or a portion thereof in a solution outside the condensate using a mass spectrometry technique; (b) obtaining, such as measuring, an MS ion signal of a 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 solution outside the condensate with 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.

[0106] In some embodiments, provided herein are methods for determining the association characteristics of a condensate of a therapeutic compound (such as any one or any combination of the following: 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 by cell culture, or a compound that is not a condensate precursor macromolecule), comprising determining the amount of the therapeutic compound consumed from a solution outside the condensate due to the presence, formation and / or modulation of the target condensate (including determining the absence of consumption). In some embodiments, provided are methods for determining the partitioning characteristics of a therapeutic compound in a target condensate, the methods comprising comparing the MS signals of ions of the therapeutic compound from a solution outside the condensate with the MS signals of ions of the therapeutic compound from a reference control, thereby determining the partitioning characteristics 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: (a) obtaining, such as measuring, an MS signal of a therapeutic compound or a portion thereof in a solution outside the condensate using a mass spectrometry technique; (b) obtaining, such as measuring, an MS ion signal of a 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 solution outside the condensate with 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.

[0107] In some embodiments, a method for determining the distribution characteristics of a test compound in a target condensate comprises: (a) combining a test compound with a composition comprising a target condensate and a solution outside the condensate; (b) obtaining, such as preparing, a reference control; (c) measuring the MS signal of the test compound or a portion thereof in the solution outside the condensate 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 solution outside the condensate with the MS signal of the test compound from the reference control, thereby determining the distribution characteristics of the test compound in the target condensate.

[0108] In some embodiments, a method for determining the distribution characteristics of a test compound in a target condensate comprises: (a) combining a test compound with a composition comprising a target condensate and a solution outside the condensate; (b) obtaining, such as preparing, a reference control; (c) measuring the MS signal of the test compound or a portion thereof in the solution outside the condensate 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 solution outside the condensate with the MS signal of the test compound from the reference control, thereby determining the distribution characteristics of the test compound in the target condensate.

[0109] In some embodiments, a method for determining the distribution characteristics of a test compound in a target agglomerate comprises: (a) combining a test compound with a composition comprising a target agglomerate and a solution outside the agglomerate; (b) incubating the test compound and the composition; (c) pelleting the target agglomerate in the composition using a centrifugation technique; (d) obtaining, such as preparing, a reference control; and (e) measuring the MS signal of the test compound or a portion thereof in the solution outside the agglomerate 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 solution outside the agglomerate with the MS signal of the test compound from the reference control to determine the distribution characteristics of the test compound in the target agglomerate.

[0110] In some embodiments, a method for determining the distribution characteristics of a test compound in a target agglomerate comprises: (a) combining a test compound with a composition comprising a target agglomerate and a solution outside the agglomerate; (b) incubating the test compound and the composition; (c) pelleting the target agglomerate in the composition using a centrifugation technique; and (d) obtaining, such as preparing, a reference control; (e) measuring the MS signal of the test compound or a portion thereof in the solution outside the agglomerate 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 solution outside the agglomerate with the MS signal of the test compound from the reference control to determine the distribution characteristics of the test compound in the target agglomerate.

[0111] In some embodiments of any of the methods or method steps described herein, the method is suitable for determining the association characteristics of a condensate of each of a plurality of test compounds in a single composition comprising a target condensate. For example, in some embodiments, a method is provided comprising: (a) combining a plurality of test compounds with a composition comprising a target condensate and a solution outside the condensate; and (b) comparing the MS signal of an ion of a first test compound from the plurality of test compounds from the solution outside the condensate with 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 from the plurality of test compounds outside the solution outside the condensate is compared with the corresponding MS signal of an ion of each corresponding test compound from a 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 capabilities of the analytical method used to measure the amount of each compound. In some embodiments, the plurality of test compounds comprises at least 5, e.g., 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, an MS signal for each test compound or a portion thereof in a solution outside the condensate using a mass spectrometry technique. In some embodiments, the method further comprises obtaining, e.g., measuring, an MS signal for each test compound or a portion thereof in a reference control using a mass spectrometry technique. In some embodiments, the MS signal for each test compound in a solution outside the condensate or in a reference control is obtained in a single mass spectrometry analysis.

[0112] Test compounds and analysis

[0113] In some embodiments, the test compound is a small molecule, a polypeptide, a peptide mimetic, a lipid or a nucleic acid. In some embodiments, the test compound is an approved compound, such as a compound approved by the United States Food and Drug Administration for medical treatment. In some embodiments, the test compound is a new 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, such as an siRNA, miRNA, mRNA or lnRNA. In some embodiments, the test compound is an siRNA, miRNA or mRNA. In some embodiments, the test compound is a non-naturally occurring compound. In some embodiments, the test compound is a protein.

[0114] In some embodiments, the method herein includes adding two or more test compounds. In some embodiments, the two or more compounds are each selected from any one of small molecules, polypeptides, lipids or nucleic acids. In some embodiments, the two or more test compounds are added sequentially or simultaneously.

[0115] 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 component part 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 can be covalently or non-covalently conjugated to the test compound.

[0116] Methods for determining the amount of a test compound are known. In some embodiments, determining the amount of a test compound includes quantifiably detecting the test compound. In some embodiments, determining the amount of a test compound includes quantifiably detecting a test compound label. In some embodiments, determining the amount of a test compound includes detecting the activity of the test compound and calculating the amount of the test compound required to cause the active amount to be detected. In some embodiments, the amount of the test compound is determined by mass spectrometry, liquid chromatography, and / or ultraviolet-visible spectrophotometry. In some embodiments, the amount of the test compound is determined by fluorescence microscopy. A standard curve can be used to help determine the amount of the test compound. Alternatively or additionally, the amount of the test compound can be compared with a reference compound. In some embodiments, the reference compound is a test compound label.

[0117] It is contemplated that the methods described herein that include determining the amount of a compound, such as a test compound or a reference compound, in a coacervate include direct and indirect techniques for determining the amount of a compound in a coacervate. In some embodiments, the amount of a compound in a coacervate is determined directly. In some embodiments, the amount of a compound in a coacervate is determined indirectly. In some embodiments, the amount of a compound in a coacervate is determined by determining the amount of a compound that is not associated with the coacervate (such as the amount of a compound in a solution outside the coacervate). In some embodiments, the amount of a compound in a coacervate is determined by determining the amount of a reporter compound. In some embodiments, the reporter compound is associated with the coacervate. In some embodiments, the reporter compound is not associated with the coacervate.

[0118] The amount of the test compound in the target condensate can be compared to the amount of the test compound in other solutions or added to the composition. Thus, 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; and / or the amount in the solution outside the condensate; and / or the amount of the test compound in the cell; and / or the amount of the test compound in the second target condensate.

[0119] In some embodiments, the method further comprises comparing the amount of the test compound in the target coacervate to the amount added to the composition. In some embodiments, the comparison comprises calculating a ratio or percentage of the amount of the test compound in the target coacervate to the amount of the test compound added to the composition.

[0120] In some embodiments, the method further comprises comparing the amount of the test compound in the coagulation of interest to the amount of the test compound in the solution outside the coagulation. In some embodiments, the comparison comprises calculating a ratio or percentage of the amount of the test compound in the coagulation of interest to the amount of the test compound in the solution outside the coagulation. In some embodiments, the method further comprises determining the amount of the test compound in the solution outside the coagulation. In some embodiments, the amount of the test compound in the coagulation of interest is determined before, simultaneously with, or after determining the amount of the test compound in the solution outside the coagulation.

[0121] In some embodiments, the method further comprises comparing the amount of the test compound in the target condensate to the amount of the test compound in the cell. In some embodiments, the comparison comprises calculating a ratio or percentage of the amount of the test compound in the target condensate to 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.

[0122] In some embodiments, the method further comprises comparing the amount of the test compound in the target condensate to the amount of the test compound in the second target condensate. In some embodiments, the comparison comprises calculating a ratio or percentage of the amount of the test compound in the target condensate to 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 determining the amount of the test compound in the second target condensate.

[0123] In some embodiments, the method further comprises comparing the amount of the test compound in the target condensate to the amount of one or more macromolecules in the target condensate. In some embodiments, the comparison comprises calculating a ratio or percentage of the amount of the test compound in the target condensate to 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 determining the amount of one or more macromolecules in the target condensate.

[0124] Methods for forming coacervates are known and can vary from coacervate to coacervate. For example, coacervates can be formed by changing the temperature of the composition, such as exposing the composition to lower or higher temperatures; by changing the salt content of the composition, such as diluting the salt in the composition or adding salt to the composition; by increasing the concentration of the precursor macromolecule, such as adding nucleic acids, such as RNA, to the composition; adding or changing a buffer to the composition; changing the ionic strength of the composition; changing the pH, such as changing the pH to less than 1 unit from the isoelectric point; or adding a crowding agent, such as PEG or dextran. Some exemplary methods for forming coacervates are also disclosed in Alberti et al., J Mol Biol, 430(23), 2018, 4806-4820, which is incorporated herein by reference.

[0125] Composition

[0126] In some embodiments, the composition comprises a cell. In some embodiments, the target condensate is in a cell. In some embodiments, the solution outside the condensate is an intracellular fluid, such as a cytosol or a nucleolar sol.

[0127] In some embodiments, the composition comprises cells. In some embodiments, the target aggregate is not in a cell. In some embodiments, the target aggregate is an extracellular aggregate, such as an aggregate in an extracellular matrix. In some embodiments, the extracellular fluid is interstitial fluid or plasma.

[0128] 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 determined disordered aggregate. In some embodiments, the cell comprises a disease-associated mutation. In some embodiments, the cell has one or more features of a neurodegenerative disease or a proliferative disease. In some embodiments, the cell has been treated with arsenate / ester (and / or another compound known to regulate aggregates), temperature changes, or pH changes. 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 a target aggregate. In some embodiments, the cell expresses a first protein and a second protein, wherein the first protein is labeled with a first marker, wherein the first protein is known to be concentrated in a first target aggregate, wherein the second protein is labeled with a second marker, wherein the second protein is known to be concentrated in a second target aggregate, and wherein the first marker and the second marker are distinguishable. In some embodiments, the cell expresses a first protein and a second protein, wherein the first protein is labeled with a first fluorescent protein, wherein the first protein is known to be concentrated in a first target aggregate, wherein the second protein is labeled with a second fluorescent protein, wherein the second protein is known to be concentrated in a second target aggregate, and wherein the first fluorescent protein and the second fluorescent protein are distinguishable.

[0129] In some embodiments, the composition does not include cells. In some embodiments, the composition is cell-free. In some embodiments, the composition includes a precursor molecule, which is a non-phase-separated component that can be incorporated into a coacervate and / or has been incorporated into a coacervate. In a cell-free system, a coacervate can be formed by only a few components. For example, the composition may include a protein or protein fragment that can form a coacervate, such as a part of a protein or peptide. 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 a nucleic acid oligomer or polymer, such as RNA. In some embodiments, the composition includes a small molecule. In some embodiments, the composition includes a buffer. In some embodiments, the composition may also include one or more salts and / or one or more macromolecular crowding agents (e.g., polyethylene glycol or dextran).

[0130] In some embodiments, the composition comprises a cell, which comprises a precursor molecule. In some embodiments, the composition comprises a cell, and the method comprises forming a target condensate in the cell. In some embodiments, the method comprises changing the temperature of the cell, such as exposing the composition to a lower or higher temperature; changing the salt content of the cell; adding or changing a buffer around the cell; changing the pH of the cell; or adding a crowding agent to the cell, such as PEG or dextran. In some embodiments, the composition comprises a cell, and the cell comprises a mutation that causes the formation of a target condensate and / or modifies 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 heterogeneous distribution of components within the target condensate. In some embodiments, the composition comprises a cell, and the cell comprises a mutation that causes the formation of a condensate.

[0131] In some embodiments, the composition comprises a plurality of coacervates. In some embodiments, the composition comprises at least any one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 coacervates. In some embodiments, the composition comprises two or more target coacervates. In some embodiments, the composition comprises at least any one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 target coacervates. In some embodiments, the method comprises repeating the steps of the method for one or more additional target coacervates, such as at least any one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 target coacervates. In some embodiments, the composition comprises at least any one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 coacervates of the same coacervate type, such as coacervates with the same composition and / or distribution properties. In some embodiments, the composition comprises at least any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 different coacervate types, e.g., coacervates that do not have the same composition and / or partitioning properties.

[0132] Condensate

[0133] Many condensates are well known in the art. Examples of known condensates include lysate, P-granules, histone locus bodies, multivesicular bodies, neuronal RNA granules, nuclear leaf buds, nuclear pores, nuclear plaques, nuclear stress bodies, nucleoli, Oct1 / PTF / transcription (OPT) domains, paraspots, perinuclear chambers, PML nuclear bodies, PML oncogenic domains, polycomb bodies, processed bodies, signal transduction clusters, viral condensates, Sam68 nuclear bodies, stress granules or splicing plaques. Many condensates can be identified with microscopy. In some embodiments, the method further includes identifying target condensates.

[0134] The coacervates used in the methods described herein can be naturally occurring or non-naturally occurring. For example, in some embodiments, the coacervates are naturally occurring. In some embodiments, the coacervates are non-naturally occurring. In some embodiments, the coacervates are artificial. In some embodiments, the coacervates are synthetic. In some embodiments, the coacervates are semi-synthetic. In some embodiments, the coacervates are modified. In some embodiments, the coacervates are modified coacervates, wherein the parent coacervate of the modified coacervate is modified by adding, removing and / or replacing one or more coacervate components.

[0135] In some embodiments, where the methods described herein assess two or more coagulants, the first coagulant and the second coagulant of the two or more coagulants can be any combination of coagulants. In some embodiments, the first coagulant is a modified coagulant. In some embodiments, the first coagulant is a modified coagulant and the second coagulant is a parent coagulant of the first coagulant. In some embodiments, the first coagulant is a target coagulant in a first system, such as a first composition, and the second coagulant is a target coagulant in a second system, such as a second composition. In some embodiments, the first coagulant is a normal coagulant and the second coagulant is a disordered coagulant.

[0136] In some embodiments, a method for determining the aggregate preference profile of a test compound is provided, the method comprising: (a) combining the test compound with a first composition comprising a first target aggregate; (b) combining the test compound with a second composition comprising a second target aggregate; (c) determining the amount of the test compound in the first target aggregate; and (d) determining the amount of the test compound in the second target aggregate, thereby determining the aggregate preference profile of the test compound.

[0137] In some embodiments, a method for determining the aggregate preference profile of a test compound is provided, the method comprising: (a) adding the test compound to a first composition comprising a first target aggregate; (b) adding the test compound to a second composition comprising a second target aggregate; (c) determining the amount of the test compound in the first target aggregate; and (d) determining the amount of the test compound in the second target aggregate, thereby determining the aggregate preference profile of the test compound.

[0138] The use of a label can aid in the identification of the coacervate. For example, a dye or a labeled compound can be added to the coacervate. In some embodiments, the dye or labeled compound can preferentially enter the target coacervate. 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 coacervate and a second target coacervate, and the first target coacervate and the second target coacervate are each labeled with a different label, such as the first target coacervate is labeled with RFP and the second target coacervate is labeled with GFP.

[0139] Some condensates may contain specific macromolecules. Therefore, in some embodiments, the method further includes 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 contains a low complexity domain or an intrinsic 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 a fragment or aggregate thereof. In some embodiments, the macromolecule is a nucleic acid, such as RNA or DNA. In some embodiments, the macromolecule is RNA.

[0140] In some embodiments, the target condensate is a cell condensate. A large number of cell condensates have been described, and more large numbers of cell condensates are known to form, but have not yet been described. In some embodiments, the cell condensate is a lysate, a P-granule, a histone locus body, a multivesicular body, a neuronal RNA granule, a nuclear leaf bud, a nuclear pore, a nuclear plaque, a nuclear stress body, a nucleolus, an Oct1 / PTF / transcription (OPT) domain, a paraspot, a perinuclear chamber, a PML nuclear body, a PML oncogenic domain, a polycomb body, a processing body, a signal transduction cluster, a virus condensate, a Sam68 nuclear body, a stress granule or a splicing plaque. In some embodiments, when determining the amount of the target compound in the target condensate, the target condensate is in a cell.

[0141] In some embodiments, the target aggregate is an extracellular aggregate. Extracellular aggregates can be formed in extracellular biological solutions such as extracellular matrix or plasma to promote reactions or sequester molecules (Muiznieks et al., J Mol Biol, 430(23), 2018, 4741-4753).

[0142] Disorders of various condensates can be associated with disease. For example, based on cell and cell-free condensate experiments, disease-associated mutations in the sarcoma (FUS) fusion protein have been shown to lead to abnormal phase separation behavior, which directly promotes the development of the motor neuron disease, amyotrophic lateral sclerosis (ALS) (Naumann et al., Nat Commun, 9(1), 2018, 335). Therefore, in some embodiments, the disorder of the target condensate is associated with disease. In some embodiments, the disorder includes changes in 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; the heterogeneous distribution of components in the condensate, for example, the components are located in the core of the condensate instead of the shell. In some embodiments, the changes are compared with similar non-disordered target condensates.

[0143] Methods for determining compound characteristics associated with the distribution of test compounds

[0144] In some embodiments, the invention includes methods for identifying characteristics of a compound that are associated with partitioning of the compound or a portion thereof within a coacervate, such as association with the surface and / or core of the coacervate.

[0145] In some embodiments, the method comprises determining the partitioning characteristics of a plurality of test compounds in a target agglomerate by performing a method disclosed herein. In some embodiments, the method comprises identifying or determining a property of a compound or portion thereof that fully or partially contributes to the partitioning of the compound or portion thereof with respect to an agglomerate.

[0146] In some embodiments, identifying a partition-related compound signature of a compound or portion thereof having a desired (e.g., similar) partition signature in a target condensate comprises: identifying a common portion or motif of the test compounds having a desired (e.g., similar) partition signature. In some embodiments, the compound signature of a compound or portion thereof is based on one or more of charge and hydrophobicity.

[0147] In some embodiments, the partitioning characteristics of a compound are based on: determining the amount of the compound in the condensate, such as a relative amount, or the amount compared to one or more condensates, such as a condensate preference profile. For example, in some embodiments, the method includes: (a) determining the partitioning characteristics of a plurality of test compounds in a target condensate by implementing the methods described herein; and (b) identifying test compounds or portions thereof having a desired partitioning characteristic, such as a similar partitioning characteristic, in the condensate. In some embodiments, the method includes: (a) determining the partitioning characteristics of a plurality of test compounds in a target condensate by implementing 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 features that a subset or all of the identified test compounds have in common in addition to the same or similar partitioning characteristics.

[0148] In some embodiments, the method includes: (a) determining the relative partitioning characteristics of a plurality of test compounds in a target condensate by implementing the methods described herein; and (b) identifying test compounds or portions thereof having a desired relative partitioning characteristic, such as a similar relative partitioning characteristic, in the condensate. In some embodiments, the method includes: (a) determining the relative partitioning characteristics of a plurality of test compounds in a target condensate by implementing the methods 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 features that a subset or all of the identified test compounds have in common in addition to the same or similar relative partitioning characteristics.

[0149] In some embodiments, the method includes: (a) determining the coagulation preference profiles of a plurality of test compounds in a target coagulation by performing the methods described herein; and (b) identifying test compounds or portions thereof having a desired coagulation preference profile, such as a similar coagulation preference profile, in a coagulation. In some embodiments, the method includes (a) determining the coagulation preference profiles of a plurality of test compounds by performing the methods described herein; (b) comparing the coagulation preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar coagulation preference profiles; and (d) identifying features that a subset or all of the identified test compounds have in common in addition to the same or similar coagulation preference profiles.

[0150] In some embodiments of the methods described herein, identifying a test compound having the same or similar partitioning characteristics, relative partitioning characteristics, or aggregate preference profiles in a target aggregate comprises identifying two or more test compounds that associate with any portion of the target aggregate. In some embodiments of the methods described herein, identifying a test compound having the same or similar partitioning characteristics, relative partitioning characteristics, or aggregate preference profiles in a target aggregate comprises identifying two or more test compounds that do not associate with any portion of the target aggregate.

[0151] In some embodiments, the methods disclosed herein further comprise preparing the compounds identified and / or designed using the methods described herein.

[0152] Methods for designing compounds with desired partitioning characteristics

[0153] In some embodiments, the invention includes methods of designing compounds having desirable partitioning characteristics with respect to a target condensate. In some embodiments, the invention includes methods of designing compounds having desirable partitioning characteristics with respect to a target condensate.

[0154] In some embodiments, the method includes identifying or determining a compound or portion thereof that comprises a desired partitioning characteristic for a target condensate. In some embodiments, the method includes identifying or determining an attribute of a compound or portion thereof that contributes, in whole or in part, to a desired partitioning characteristic for a target condensate. In some embodiments, the method includes identifying or determining a compound or portion thereof that comprises a desired partitioning characteristic for a target condensate. In some embodiments, the method includes identifying or determining an attribute of a compound or portion thereof that contributes, in whole or in part, to a desired partitioning characteristic for a target condensate. In some embodiments, the method includes modifying the identified compound or portion thereof to optimize the desired partitioning characteristic.

[0155] In some embodiments, a method for designing a compound with a desired partitioning characteristic comprises: (a) determining the partitioning characteristic of each of a plurality of test compounds in a target condensate by performing the methods described herein; and (b) identifying one or more of the plurality of test compounds or a portion thereof having a desired (e.g., similar) partitioning characteristic in a target condensate. In some embodiments, the method comprises comparing the partitioning characteristics of a subset or all of the plurality of test compounds in a target condensate. In some embodiments, the method comprises selecting and / or designing a compound or a portion thereof in a plurality of test compounds having a desired partitioning characteristic.

[0156] In some embodiments, a method for identifying compound characteristics associated with a desired distribution characteristic with respect to a target condensate comprises: (a) determining the distribution characteristics of a plurality of test compounds in a target condensate by implementing the methods described herein; (b) comparing the distribution characteristics of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar distribution 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 distribution characteristics.

[0157] In some embodiments, a method for identifying compound characteristics associated with the partitioning of compounds into agglomerates comprises: (a) determining the partitioning characteristics of a plurality of test compounds in a target agglomerate by implementing the methods 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 agglomerate; 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.

[0158] In some embodiments, identifying test compounds or portions thereof having the same or desired (e.g., similar) partitioning characteristics in a target coacervate comprises: identifying common portions or motifs of test compounds having desired (e.g., similar) partitioning characteristics. In some embodiments, the compound characteristics of a compound or portion thereof are based on one or more of charge and hydrophobicity.

[0159] In some embodiments, the identified test compound or portion thereof can be used as a basis for identifying and / or designing one or more compounds with a desired distribution characteristic. In some embodiments, the one or more compounds represent a licensed library. In some embodiments, a licensed library comprises a set of one or more compounds comprising a moiety that constitutes the identified compound or portion thereof, which set is a portion or all of the licensed library. In some embodiments, each compound of the licensed library has a distribution characteristic that is similar to each other, such as a distribution characteristic within at least about 20%. In some embodiments, each compound of the licensed library meets or exceeds a threshold distribution characteristic. In some embodiments, the licensed library comprises at least about 10 compounds, such as 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 collection of the licensed library is suitable for administration to an individual. In some embodiments, each compound of the licensed library has a molecular weight of less than 1,000 Da, such as 500 Da or less. In some embodiments, each compound of the licensed library satisfies Lipinski's five-fold rule. In some embodiments, the licensed library comprises compounds present in a single composition. In some embodiments, the licensed library can be used to identify one or more compounds that can be used to target a coacervate of interest, wherein the one or more compounds are identified from the licensed library using one or more traditional drug screening methods.

[0160] In some embodiments, designing a compound comprises adding (e.g., attaching, e.g., covalently attaching) a moiety comprising the identified feature to the compound, thereby imparting the desired partitioning characteristics to the compound. In some embodiments, designing a compound comprises removing a moiety comprising the identified feature, thereby imparting the desired partitioning characteristics to the compound. In some embodiments, the compound is designed in whole or in part using methods comprising modeling-based, computer- and / or computational-based techniques, such as bioinformatics-, cheminformatics- and / or artificial intelligence (AI)-based techniques.

[0161] In some embodiments, the method includes designing a test compound or a portion thereof based on an attached portion, the portion comprising a feature identified by a coacervate association characteristic, such as a chemical structure or motif, thereby conferring a desired partitioning characteristic on the test compound. In some embodiments, the identified feature modulates the coacervate association characteristic of the test compound, such as a partitioning characteristic, in whole or in part, such as increasing or decreasing the degree of partitioning of the test compound in the test coacervate. In some embodiments, the method of designing a test compound includes attaching a portion comprising a feature identified by a coacervate association characteristic 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 portion comprising a feature identified by a coacervate association characteristic. In some embodiments, the method of designing a test compound includes, for a precursor of the test compound, changing a portion to another portion comprising a feature identified by a coacervate association characteristic at any number of positions and / or stereochemical orientations. In some embodiments, the method of designing a test compound includes attaching, removing and / or changing more than one portion comprising a feature identified by a coacervate association characteristic to a precursor of the test compound. In some embodiments, a test compound comprises features that facilitate modulation (eg, attachment, removal, alteration) of a moiety, such as a compound tag (eg, a component moiety used in click chemistry).

[0162] In some embodiments, the identified test compounds or portions thereof (one or more) 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 licensed library. In some embodiments, the methods described herein include designing test compounds or portions thereof based on attaching, removing or altering portions containing features such as chemical structures or motifs identified by condensate association characteristics, thereby conferring the desired biological activity on the test compounds. In some embodiments, the desired biological activity of a precursor compound is increased by adjusting the condensate association characteristics of the precursor compound. In some embodiments, the undesirable biological activity of a precursor compound is reduced by adjusting the condensate association characteristics of the precursor compound.

[0163] In some embodiments, condensate association characteristic data, such as distribution characteristic data, and / or the identified test compound or portion thereof, can be used to establish one or more rule sets. In some embodiments, the one or more rule sets can be used as a basis for identifying or designing one or more compounds (e.g., identifying compounds with desired distribution characteristics based on bioinformatics, cheminformatics, and / or artificial intelligence (AI)) using methods including modeling-based, computer- and / or computational techniques. Computer software for determining and / or applying the one or more rule sets is also provided.

[0164] In some embodiments, the distribution characteristics of a compound are based on: determining the amount of the compound in a coagulation, such as a relative amount, or the amount compared to one or more coagulations, such as a coagulation preference profile. For example, in some embodiments, a method for designing a compound with a desired relative distribution characteristic comprises: determining the relative distribution characteristics of a plurality of test compounds in a coagulation of interest by implementing the methods described herein. In some embodiments, a method for designing a compound with a desired relative distribution characteristic comprises: (a) determining the relative distribution characteristics of a plurality of test compounds in a coagulation of interest by implementing the methods described herein; (b) comparing the relative distribution characteristics of a subset or all of the plurality of test compounds in a coagulation of interest; (c) identifying test compounds having the same or similar relative distribution characteristics in a coagulation of interest; (d) identifying features that a subset or all of the identified test compounds have in common in addition to the same or similar relative distribution characteristics; and (e) designing a compound comprising the identified features. In some embodiments, a method for designing a compound having a desired relative distribution characteristic comprises: (a) determining the relative distribution characteristics of a plurality of test compounds in a target agglomerate by implementing the methods described herein; (b) comparing the relative distribution characteristics of a subset or all of the plurality of test compounds in the target agglomerate; (c) identifying test compounds having the same or similar relative distribution characteristics in the target agglomerate; (d) identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar relative distribution characteristics; and (e) designing a compound that does not contain the identified characteristics.

[0165] In some embodiments, a method for designing a compound having a desired coacervate preference profile comprises determining the coacervate preference profiles of a plurality of test compounds by performing the methods disclosed herein. In some embodiments, a method for designing a compound having a desired coacervate preference profile comprises: (a) determining the coacervate preference profiles of a plurality of test compounds by performing the methods disclosed herein; (b) comparing the coacervate preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar coacervate preference profiles; (d) identifying features that a subset or all of the identified test compounds have in common in addition to the same or similar coacervate preference profiles; and (e) designing a compound comprising the identified features. In some embodiments, a method for designing a compound having a desired coacervate preference profile comprises: (a) determining the coacervate preference profiles of a plurality of test compounds by implementing the methods disclosed herein; (b) comparing the coacervate preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar coacervate preference profiles; (d) identifying features that a subset or all of the identified test compounds have in common in addition to the same or similar coacervate preference profiles; and (e) designing a compound that does not contain the identified features.

[0166] In some embodiments, the methods disclosed herein further comprise preparing the compounds identified and / or designed using the methods described herein.

[0167] Methods for screening test compounds

[0168] In some embodiments, the present invention includes a method for screening a test compound having a desired partitioning characteristic from a group of candidate compounds, the method comprising: (a) determining the partitioning characteristic of each of the group of candidate compounds; and (b) identifying the test compound having the desired partitioning characteristic.

[0169] In some embodiments, the desired partitioning characteristics are based on: a partitioning characteristic associated with a target coagulant. In some embodiments, the desired partitioning characteristics are based on: a partitioning characteristic associated with a plurality of target coagulants. In some embodiments, the method comprises identifying or determining a target coagulant. In some embodiments, the target coagulant is associated with a disease. In some embodiments, the target coagulant associated with a disease is known in the art. In some embodiments, the method comprises obtaining a target coagulant, such as a target coagulant associated with a disease.

[0170] In some embodiments, the distribution characteristics of a group of candidate compounds (such as more than one candidate compound) in the target condensate 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 distribution characteristics of the candidate compounds in the target condensate are determined in vitro. In some embodiments, the distribution characteristics of the candidate compounds in the target condensate are determined in a cell system. In some embodiments, the distribution characteristics of the candidate compounds in the target condensate are determined in a non-cellular system such as a composition comprising the target condensate or its components.

[0171] In some embodiments, identifying a test compound or portion thereof having a desired partitioning characteristic comprises identifying a test compound or portion thereof associated with any portion of a coacervate of interest (e.g., associated with the exterior, surface, and / or core of a coacervate of interest). In some embodiments, a desired partitioning characteristic is a partitioning characteristic suitable for treating a disease in an individual.

[0172] In some embodiments, the invention includes a method for identifying a test compound that can be used to treat a disease in an individual in need thereof. In some embodiments, the method includes: (a) identifying a target aggregate associated with the disease; and (b) determining a partitioning characteristic of a candidate compound in the target aggregate; and (c) identifying a test compound or portion thereof having a partitioning characteristic suitable for treating the disease.

[0173] In some embodiments, the method includes identifying or determining a target coagulant associated with a disease. In some embodiments, the target coagulant associated with a disease is known in the art. In some embodiments, the method includes obtaining a target coagulant associated with a disease. In some embodiments, the method includes identifying or determining one or more target coagulants associated with a disease.

[0174] In some embodiments, the method of screening a test compound comprises: (a) determining the partitioning characteristic of the test compound in the presence of a second compound; and (b) determining the partitioning characteristic of the test compound in the absence of the second compound. In some embodiments, the method further comprises comparing the partitioning characteristic of the test compound in the presence of the second compound with the partitioning characteristic of the test compound in the absence of the second compound. In some embodiments, the method further comprises determining a change in the partitioning characteristic of the test compound in the presence or absence of the second compound.

[0175] In some embodiments, a method of screening a test compound comprises: (a) determining a partitioning characteristic of a second compound in the presence of the test compound; and (b) determining a partitioning characteristic of the second compound in the absence of the test compound. In some embodiments, the method further comprises comparing the partitioning characteristic of the second compound in the presence of the test compound to the partitioning characteristic of the test compound in the absence of the second compound. In some embodiments, the method further comprises determining the ability of the test compound to alter the partitioning characteristic of the second compound.

[0176] In some embodiments, the distribution characteristics of candidate compounds (such as one or more candidate compounds) in the target condensate are determined according to the methods described herein. In some embodiments, the method includes determining the distribution characteristics of the target condensate of multiple candidate compounds. In some embodiments, the multiple candidate compounds are at least about any of 2, 5, 10, 15, 50, 75, 100, 150, 200, 300, 400 or 500 candidate compounds. In some embodiments, the distribution characteristics of candidate compounds in the target condensate are determined in vitro. In some embodiments, the distribution characteristics of candidate compounds in the target condensate are determined in a cell system. In some embodiments, the distribution characteristics of candidate compounds in the target condensate are determined in a non-cellular system such as a composition comprising a target condensate or a component thereof.

[0177] In some embodiments, identifying a test compound or portion thereof having a partitioning characteristic suitable for treating a disease comprises identifying a test compound or portion thereof associated with any portion of the target condensate, such as associated with the exterior, surface and / or core of the target condensate.

[0178] In some embodiments, the methods disclosed herein include identifying, determining, designing and / or screening compounds, such as compounds from a set of candidate compounds, and also include evaluating the compounds based on other parameters of the compounds. In some embodiments, identifying the compounds is based on: evaluating (such as characterizing or determining) the coacervate association characteristics of the compound and one or more other parameters, such as molecular weight and pharmaceutical utility.

[0179] In some embodiments, the methods disclosed herein further comprise preparing the compounds identified, designed and / or screened using the methods described herein.

[0180] Exemplary Embodiments

[0181] The provided implementations include:

[0182] Embodiment 1. A method for determining the distribution characteristics of a test compound in a target agglomerate, the method comprising: (a) adding a test compound to a composition comprising a target agglomerate and a solution outside the agglomerate; and (b) determining the amount of the test compound in the target agglomerate, thereby determining the distribution characteristics of the test compound in the target agglomerate.

[0183] Embodiment 2. The method of embodiment 1, further comprising causing the target condensate to form before step (a).

[0184] Embodiment 3. A method for determining the distribution characteristics of a test compound in a target condensate, the method comprising: (a) adding a test compound to a composition comprising a precursor molecule; (b) causing the target condensate to form to obtain a composition comprising the target condensate and a solution outside the condensate; 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.

[0185] Embodiment 4. The method of any one of embodiments 1-3, further comprising determining the amount of the test compound in the solution outside the coacervate.

[0186] Embodiment 5. The method of embodiment 4, wherein the amount of the test compound in the target coacervate is determined before, simultaneously with, or after the amount of the test compound in the solution outside the coacervate is determined.

[0187] Embodiment 6. The method of embodiment 4 or 5, further comprising determining a ratio of the amount of the test compound in the target coacervate to the amount of the test compound in the solution outside the coacervate.

[0188] Embodiment 7. The method of any one of embodiments 1-6 further comprises separating the target coacervate from the solution outside the coacervate.

[0189] Embodiment 8. The method of any one of embodiments 1-7, further comprising identifying the target aggregate before determining the amount of the test compound in the target aggregate.

[0190] Embodiment 9. The method of any one of embodiments 1-8, wherein the dysregulation of the target aggregate is associated with a disease.

[0191] Embodiment 10. The method of any one of embodiments 1-9, further comprising characterizing the target condensate by identifying one or more macromolecules contained therein.

[0192] Embodiment 11. The method of any one of embodiments 1-10, wherein the target condensate comprises a protein comprising an intrinsically disordered sequence.

[0193] Embodiment 12. The method of any one of embodiments 1-11, further comprising labeling the target aggregate to visualize the target aggregate.

[0194] Embodiment 13. The method of embodiment 12, wherein the target aggregate is labeled with a radioactive label, a colorimetric label, or a fluorescent label.

[0195] Embodiment 14. The method of any one of embodiments 1-13, wherein the composition comprises cells.

[0196] Embodiment 15. The method of embodiment 14, wherein the cell is a microorganism or an animal cell.

[0197] Embodiment 16. The method of embodiment 14 or 15, wherein the cell comprises aggregates that are determined to be dysregulated.

[0198] Embodiment 17. The method of any one of embodiments 14-16, wherein the cell has one or more characteristics of a neurodegenerative disease or a proliferative disease.

[0199] Embodiment 18. The method of any one of embodiments 1-17, wherein the target aggregate is a cell aggregate.

[0200] Embodiment 19. The method of embodiment 18, wherein the cellular condensate is a lysosome, a P-granule, a histone locus body, a multivesicular body, a neuronal RA granule, a nuclear bud, a nuclear pore, a nuclear plaque, a nuclear stress body, a nucleolus, an Oct1 / PTF / transcription (OPT) domain, a paraplaque, a perinucleolar compartment, a PML nuclear body, a PML oncogenic domain, a polycomb body, a processing body, a Sam68 nuclear body, a stress granule or a splicing plaque.

[0201] Embodiment 20. The method of any one of embodiments 1-19, wherein the target condensate is in a cell.

[0202] Embodiment 21. The method of any one of embodiments 1-20, wherein the solution outside the coacervate is intracellular fluid.

[0203] Embodiment 22. The method of embodiment 21, wherein the intracellular fluid is cytosol or nucleosol.

[0204] Embodiment 23. The method of any one of embodiments 1-17, wherein the target aggregates are extracellular aggregates.

[0205] Embodiment 24. The method of any one of Embodiments 23, wherein the extra-coacervate solution is extracellular fluid.

[0206] Embodiment 25. The method of embodiment 24, wherein the extracellular fluid is interstitial fluid.

[0207] Embodiment 26. The method of any one of embodiments 1-13, wherein the composition does not comprise cells.

[0208] Embodiment 27. The method of any one of embodiments 1-26, wherein the composition comprises one or more of: a macromolecule, a salt, and a buffer.

[0209] Embodiment 28. The method of any one of embodiments 1-27, wherein the composition comprises two or more target coagulants.

[0210] Embodiment 29. The method of any one of embodiments 1-28, wherein the method comprises repeating the steps of the method for one or more additional coagulants.

[0211] Embodiment 30. The method of any one of embodiments 1-29, wherein the test compound is a small molecule, a polypeptide, or a nucleic acid.

[0212] Embodiment 31. The method of any one of embodiments 1-30, wherein the test compound comprises a test compound label.

[0213] Embodiment 32. The method of embodiment 31, wherein the test compound label is a radioactive label, a colorimetric label, or a fluorescent label.

[0214] Embodiment 33. The method of embodiment 31 or 32, wherein the test compound label is a fluorescent label.

[0215] Embodiment 34. The method of any one of embodiments 31-33, wherein the amount of the test compound is determined by detecting a test compound label.

[0216] Embodiment 35. The method of any one of embodiments 1-34, wherein the amount of the test compound is determined by mass spectrometry, liquid chromatography, and / or UV-visible spectrophotometry.

[0217] Embodiment 36. A method for determining the partitioning characteristics of a plurality of test compounds in a target agglomerate, the method comprising performing the method of any one of embodiments 1-35 with the plurality of test compounds.

[0218] Embodiment 37. The method of embodiment 36, further comprising comparing the partitioning characteristics of a subset or all of the plurality of test compounds in the target agglomerate.

[0219] Embodiment 38. The method of embodiment 37, further comprising identifying a test compound having the same or similar partitioning characteristics in the target coacervate.

[0220] Embodiment 39. The method of embodiment 38, further comprising identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar assigned characteristics.

[0221] Embodiment 40. The method of embodiment 39, further comprising determining a distribution characteristic of one or more additional test compounds comprising the identified characteristic within the target aggregate.

[0222] Embodiment 41. A method for determining the relative distribution characteristics of a test compound in a target agglomerate, the method comprising: (i) determining the distribution characteristics of the test compound by performing any of the methods of embodiments 1-35 with the test compound; (ii) determining the distribution characteristics of a reference compound by performing any of the methods of embodiments 1-35 with the reference compound; and (iii) calculating the ratio of the distribution characteristics determined in (i) and (ii), thereby determining the relative distribution characteristics of the test compound in the target agglomerate.

[0223] Embodiment 42. The method of embodiment 41, wherein the test compound comprises a test compound label.

[0224] Embodiment 43. The method of embodiment 42, wherein the reference compound is a test compound marker.

[0225] Embodiment 44. A method for determining the relative distribution characteristics of multiple test compounds in a target agglomerate, the method comprising: (1) performing the method of any one of embodiments 41-43; and (2) repeating steps (i) and (iii) using multiple test compounds.

[0226] Embodiment 45. The method of embodiment 44, further comprising comparing the relative distribution characteristics of a subset or all of the plurality of test compounds in the target aggregate.

[0227] Embodiment 46. The method of embodiment 45, further comprising identifying a test compound having the same or similar relative partitioning characteristics in the target aggregate.

[0228] Embodiment 47. The method of embodiment 46, further comprising identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar relative distribution characteristics.

[0229] Embodiment 48. The method of embodiment 47, further comprising determining the relative distribution characteristics of one or more additional test compounds comprising the identified characteristics in the target aggregate.

[0230] Embodiment 49. A method for determining a coagulant preference profile of a test compound, the method comprising: (a) adding a test compound to a composition comprising a first target coagulant and a second target coagulant; (b) determining the amount of the test compound in the first target coagulant; (c) determining the amount of the test compound in the second target coagulant; and (d) calculating the ratio of the amounts of the test compound determined in the first target coagulant and the second target coagulant, thereby determining the coagulant preference profile of the test compound.

[0231] Embodiment 50. The method of embodiment 49, further comprising causing the formation of the first target coagulant and / or the second target coagulant before step (a).

[0232] Embodiment 51. A method for determining a coacervate preference profile of a test compound, the method comprising: (a) adding a test compound to a composition comprising a precursor molecule; (b) causing the formation of a first target coacervate and a second target coacervate in the composition; (c) determining the amount of the test compound in the first target coacervate; (d) determining the amount of the test compound in the second target coacervate; and (e) calculating the ratio of the amount of the test compound determined in the first target coacervate and the second target coacervate, thereby determining the coacervate preference profile of the test compound.

[0233] Embodiment 52. The method of any one of embodiments 49-51, wherein the amount of the test compound in the first target agglomerate is determined before, simultaneously with, or after the amount of the test compound in the second target agglomerate is determined.

[0234] Embodiment 53. The method of any one of Embodiments 49-52, further comprising separating the first target coagulant and the second target coagulant from the composition.

[0235] Embodiment 54. The method of any one of embodiments 49-53, further comprising identifying the first target aggregate and / or the second target aggregate before determining the amount of the test compound in the first aggregate and / or the second aggregate.

[0236] Embodiment 55. The method of any one of embodiments 49-54, wherein the dysregulation of the first target aggregate and / or the second target aggregate is associated with a disease.

[0237] Embodiment 56. The method of any one of embodiments 49-55, further comprising characterizing the first target coacervate and / or the second target coacervate by identifying one or more macromolecules contained therein.

[0238] Embodiment 57. The method of any one of embodiments 49-56, further comprising labeling the first target condensate and / or the second target condensate to visualize the first target condensate and / or the second target condensate.

[0239] Embodiment 58. The method of embodiment 57, wherein the first target aggregate and the second target aggregate are labeled with different labels.

[0240] Embodiment 59. The method of embodiment 57 or 58, wherein the first target aggregate and / or the second target aggregate is labeled with a radioactive label, a colorimetric label, or a fluorescent label.

[0241] Embodiment 60. The method of any one of embodiments 49-56, wherein the composition comprises cells.

[0242] Embodiment 61. The method of embodiment 60, wherein the cell is a microorganism or an animal cell.

[0243] Embodiment 62. The method of embodiment 60 or 61, wherein the cell comprises aggregates that are determined to be dysregulated.

[0244] Embodiment 63. The method of any one of embodiments 60-62, wherein the cell has one or more characteristics of a neurodegenerative disease or a proliferative disease.

[0245] Embodiment 64. The method of any one of embodiments 49-56, wherein the first target aggregate and / or the second target aggregate is a cell aggregate.

[0246] Embodiment 65. A method as described in embodiment 64, wherein the first target condensate is a lytic body, a P-granule, a histone locus body, a multivesicular body, a neuronal RNA granule, a nuclear bud, a nuclear pore, a nuclear plaque, a nuclear stress body, a nucleolus, an Oct1 / PTF / transcription (OPT) domain, a paraplaque, a perinucleolar compartment, a PML nuclear body, a PML oncogenic domain, a polycomb body, a processing body, a Sam68 nuclear body, a stress granule or a splicing plaque.

[0247] Embodiment 66. The method of embodiment 64 or 65, wherein the second target condensate is a lytic body, a P-granule, a histone locus body, a multivesicular body, a neuronal RNA granule, a nuclear bud, a nuclear pore, a nuclear plaque, a nuclear stress body, a nucleolus, an Oct1 / PTF / transcription (OPT) domain, a paraplaque, a perinucleolar compartment, a PML nuclear body, a PML oncogenic domain, a polycomb body, a processing body, a Sam68 nuclear body, a stress granule, or a splicing plaque.

[0248] Embodiment 67. The method of any one of embodiments 49-66, wherein the first target condensate and / or the second target condensate is in a cell.

[0249] Embodiment 68. The method of any one of embodiments 49-63, wherein the first target aggregate and / or the second target aggregate is an extracellular aggregate.

[0250] Embodiment 69. The method of any one of embodiments 49-59, wherein the composition does not comprise cells.

[0251] Embodiment 70. The method of any one of embodiments 49-69, wherein the composition comprises one or more of: a macromolecule, a salt, and a buffer.

[0252] Embodiment 71. The method of any one of embodiments 49-70, wherein the composition comprises one or more additional target coagulants.

[0253] Embodiment 72. The method of any one of embodiments 49-71, wherein the method comprises repeating the steps of the method for one or more additional target coagulants.

[0254] Embodiment 73. A method for determining the aggregate preference profile of a plurality of test compounds, the method comprising performing the method of any one of embodiments 49-72 with the plurality of test compounds.

[0255] Embodiment 74. The method of embodiment 73, further comprising comparing the aggregate preference profiles of a subset or all of the plurality of test compounds.

[0256] Embodiment 75. The method of embodiment 74, further comprising identifying test compounds having the same or similar aggregate preference.

[0257] Embodiment 76. The method of embodiment 75, further comprising identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar aggregate preference profile.

[0258] Embodiment 77. The method of embodiment 76, further comprising determining a relative partitioning characteristic of one or more additional test compounds comprising the identified characteristic.

[0259] Embodiment 78. A method for identifying compound characteristics associated with the distribution of compounds into agglomerates, the method comprising: (a) determining the distribution characteristics of multiple test compounds in a target agglomerate by performing the method of embodiment 36; (b) comparing the distribution characteristics of a subset or all of the multiple test compounds in the target compounds; (c) identifying test compounds having the same or similar distribution characteristics in the target agglomerate; and (d) identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar distribution characteristics.

[0260] Embodiment 79. A method for identifying compound characteristics associated with the distribution of compounds into a coagulation, the method comprising: (a) determining the relative distribution characteristics of multiple test compounds in a target coagulation by performing the method of embodiment 44; (b) comparing the relative distribution characteristics of a subset or all of the multiple test compounds in the target coagulation; (c) identifying test compounds having the same or similar relative distribution characteristics in the target coagulation; and (d) identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar relative distribution characteristics.

[0261] Embodiment 80. A method for identifying compound characteristics associated with the distribution of compounds into condensates, the method comprising: (a) determining the condensate preference profiles of multiple test compounds by performing the method of embodiment 73; (b) comparing the condensate preference profiles of a subset or all of the multiple test compounds; (c) identifying test compounds with 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 profiles.

[0262] Embodiment 81. A method for designing a compound having a desired distribution characteristic for a target agglomerate, the method comprising: (a) determining the distribution characteristics of a plurality of test compounds in a target agglomerate by performing the method of embodiment 36; (b) comparing the distribution characteristics of a subset or all of the plurality of test compounds in the target agglomerate; (c) identifying test compounds having the same or similar distribution characteristics in the target agglomerate; (d) identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar distribution characteristics; and (e) designing a compound comprising the identified characteristics.

[0263] Embodiment 82. A method for designing compounds with desired relative distribution characteristics, the method comprising: (a) determining the relative distribution characteristics of multiple test compounds in a target agglomerate by performing the method of embodiment 44; (b) comparing the relative distribution characteristics of a subset or all of the multiple test compounds in the target agglomerate; (c) identifying test compounds with the same or similar relative distribution characteristics in the target agglomerate; (d) identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar relative distribution characteristics; and (e) designing compounds containing the identified characteristics.

[0264] Embodiment 83. A method for designing a compound having a desired coagulant preference profile, the method comprising: (a) determining the coagulant preference profiles of a plurality of test compounds by performing the method of embodiment 73; (b) comparing the coagulant preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar coagulant preference profiles; (d) identifying features that a subset or all of the identified test compounds have in common in addition to the same or similar coagulant preference profiles; and (e) designing a compound comprising the identified features.

[0265] Embodiment 84. The method of any one of embodiments 81-83, further comprising preparing the compound.

[0266] Embodiment 85. A method for screening a test compound having a desired partitioning characteristic from a group of candidate compounds, the method comprising: (a) determining the partitioning characteristic of each of the group of candidate compounds; and (b) identifying the test compound having the desired partitioning characteristic.

[0267] Embodiment 86. The method of embodiment 85, wherein the partitioning characteristic of each of the set of candidate compounds is determined in vitro.

[0268] Embodiment 87. The method of embodiment 85 or 86, wherein the test compound has a partitioning characteristic suitable for use in treating a disease in a subject.

[0269] Embodiment 88. A method for identifying a test compound that can be used to treat a disease in an individual in need thereof, the method comprising: (a) identifying a target aggregate associated with the disease; (b) determining the distribution characteristics of a candidate compound in the target aggregate, and (c) identifying a test compound having a distribution characteristic suitable for treating the disease.

[0270] Further exemplary embodiments

[0271] The provided embodiments also include:

[0272] E1. A method for determining the distribution characteristics of a test compound in a target agglomerate, the method comprising: (a) combining a test compound with a composition comprising a target agglomerate and a solution outside the agglomerate; and (b) determining the amount of the test compound in the target agglomerate, thereby determining the distribution characteristics of the test compound in the target agglomerate.

[0273] E2. The method of embodiment E1, further comprising causing the target agglomerate to form before step (a).

[0274] E3. A method for determining the distribution characteristics of a test compound in a target coacervate, the method comprising: (a) causing the formation of a target coacervate in the presence of a test compound to obtain a composition comprising a target coacervate and a solution outside the coacervate; and (b) determining the amount of the test compound in the target coacervate, thereby determining the distribution characteristics of the test compound in the target coacervate.

[0275] E4. The method of embodiment E3, further comprising combining the test compound with a precursor composition comprising the precursor molecule prior to step (a).

[0276] E5. The method of embodiment E3, further comprising adding a test compound to the precursor composition comprising the precursor molecule prior to step (a).

[0277] E6. The method of any one of embodiments E1-E5, further comprising determining the amount of the test compound in the solution outside the coacervate.

[0278] E7. The method of embodiment E6, wherein the amount of the test compound in the coacervate of interest is determined before, simultaneously with, or after the amount of the test compound in the solution outside the coacervate is determined.

[0279] E8. The method of embodiment E6 or E7, further comprising determining a ratio of the amount of the test compound in the target coacervate to the amount of the test compound in the solution outside the coacervate.

[0280] E9. The method of any one of embodiments E1-E8, further comprising separating the target coagulant from the solution outside the coagulant.

[0281] E10. The method of any one of embodiments E1 to E9, further comprising identifying the target aggregate before determining the amount of the test compound in the target aggregate.

[0282] E11. The method of any one of embodiments E1 to E10, wherein the dysregulation of the target aggregate is associated with a disease.

[0283] E12. The method of any one of embodiments E1-E11, further comprising characterizing the target condensate by identifying one or more macromolecules contained therein.

[0284] E13. The method of embodiment E12, wherein the identifying comprises determining the amount of one or more macromolecules in the target coacervate.

[0285] E14. The method of embodiment E13, further comprising determining a ratio of the amount of the test compound in the target coacervate to the amount of one or more macromolecules in the target coacervate.

[0286] E15. The method of any one of embodiments E1 to E14, wherein the target condensate comprises a protein comprising an intrinsically disordered sequence.

[0287] E16. The method of any one of embodiments E1 to E15, further comprising labeling the target aggregates to visualize the target aggregates.

[0288] E17. The method of embodiment E16, wherein the target aggregate is labeled with a radioactive label, a colorimetric label, or a fluorescent label.

[0289] E18. The method of any one of embodiments E1 to E17, wherein the composition comprises cells.

[0290] E19. The method of embodiment E18, wherein the cell is a microorganism or an animal cell.

[0291] E20. The method of embodiment E18 or E19, wherein the cell comprises aggregates that are determined to be dysregulated.

[0292] E21. The method of any one of embodiments E18-E20, wherein the cell has one or more characteristics of a neurodegenerative disease or a proliferative disease.

[0293] E22. The method of any one of embodiments E1-E21, wherein the target aggregate is a cell aggregate.

[0294] E23. The method of embodiment E22, wherein the cellular condensate is a lysosome, a P-granule, a histone locus body, a multivesicular body, a neuronal RA granule, a nuclear bud, a nuclear pore, a nuclear plaque, a nuclear stress body, a nucleolus, an Oct1 / PTF / transcription (OPT) domain, a paraplaque, a perinucleolar compartment, a PML nuclear body, a PML oncogenic domain, a polycomb body, a processing body, a Sam68 nuclear body, a stress granule, or a splicing plaque.

[0295] E24. The method according to any one of embodiments E1 to E23, wherein the target condensate is in a cell.

[0296] E25. The method of embodiment E24, wherein the cell is a microorganism or an animal cell.

[0297] E26. The method of embodiment E24 or E25, wherein the cell has one or more characteristics of a neurodegenerative disease or a proliferative disease.

[0298] E27. The method of any one of embodiments E1 to E26, wherein the solution outside the coacervate is intracellular fluid.

[0299] E28. The method of embodiment E27, wherein the intracellular fluid is cytosol or nucleosol.

[0300] E29. The method of any one of embodiments E1 to E21, wherein the target condensate is not in a cell.

[0301] E30. The method of embodiment E29, wherein the target aggregates are extracellular aggregates.

[0302] E31. The method of any one of embodiments E1-E22 or E29-E30, wherein the solution outside the coacervate is extracellular fluid.

[0303] E32. The method of embodiment E31, wherein the extracellular fluid is interstitial fluid.

[0304] E33. The method of any one of embodiments E1 to E17, wherein the method is a cell-free assay.

[0305] E34. The method of any one of embodiments E1 to E33, wherein the composition comprises one or more of: a macromolecule, a salt, and a buffer.

[0306] E35. The method of any one of embodiments E1 to E34, wherein the composition comprises two or more target coagulants.

[0307] E36. The method of any one of embodiments E1 to E35, wherein the method comprises repeating the steps of the method for one or more additional coagulants.

[0308] 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.

[0309] E38. The method of any one of embodiments E1 to E37, wherein the test compound comprises a test compound label.

[0310] E39. The method of embodiment E38, wherein the test compound label is a radioactive label, a colorimetric label, or a fluorescent label.

[0311] E40. The method of embodiment E38 or E39, wherein the test compound label is a fluorescent label.

[0312] E41. The method of any one of embodiments E38-E40, wherein the amount of the test compound is determined by detecting a test compound label.

[0313] E42. The method of any one of embodiments E1 to E41, wherein the amount of the test compound is determined by mass spectrometry, liquid chromatography and / or UV-visible spectrophotometry.

[0314] E43. A method for determining the partitioning characteristics of a plurality of test compounds in a target agglomerate, the method comprising performing the method of any one of embodiments E1-E42 using the plurality of test compounds.

[0315] E44. The method of embodiment E43, further comprising comparing the partitioning characteristics of a subset or all of the plurality of test compounds in the target agglomerate.

[0316] E45. The method of embodiment E44, further comprising identifying a test compound having the same or similar partitioning characteristics in the target coacervate.

[0317] E46. The method of embodiment E45, further comprising identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar assigned characteristics.

[0318] E47. The method of embodiment E46, further comprising determining a distribution characteristic of one or more additional test compounds comprising the identified characteristic in the target agglomerate.

[0319] E48. The method of embodiment E46 or E47, further comprising determining a distribution characteristic of one or more additional test compounds that do not comprise the identified characteristic in the target aggregate.

[0320] E49. A method for determining the relative distribution characteristics of a test compound in a target agglomerate, the method comprising: (i) determining the distribution characteristics of the test compound by performing any of the methods of embodiments E1-E42 using the test compound; (ii) determining the distribution characteristics of a reference compound by performing any of the methods of embodiments E1-E42 using the reference compound; and (iii) calculating the ratio of the distribution characteristics determined in (i) and (ii) to thereby determine the relative distribution characteristics of the test compound in the target agglomerate.

[0321] E50. The method of embodiment E49, wherein the test compound comprises a test compound label.

[0322] E51. The method of embodiment E50, wherein the reference compound is a test compound marker.

[0323] E52. A method for determining the relative distribution characteristics of multiple test compounds in a target agglomerate, the method comprising: (1) performing the method of any one of embodiments E49-E51; and (2) repeating steps (i) and (iii) using multiple test compounds.

[0324] E53. The method of embodiment E52, further comprising comparing the relative distribution characteristics of a subset or all of the plurality of test compounds in the target agglomerate.

[0325] E54. The method of embodiment E53, further comprising identifying a test compound having the same or similar relative partitioning characteristics in the target coacervate.

[0326] E55. The method of embodiment E54, further comprising identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar relative distribution characteristic.

[0327] E56. The method of embodiment E55, further comprising determining the relative distribution characteristics of one or more additional test compounds comprising the identified characteristics in the target coagulation.

[0328] E57. The method of embodiment E55 or E56, further comprising determining the relative distribution characteristics of one or more additional test compounds that do not comprise the identified characteristics in the target aggregate.

[0329] E58. A method for determining a coagulation preference profile of a test compound, the method comprising: (a) determining a distribution characteristic of the test compound in a first target coagulation using the method of any one of embodiments E1-E42; (b) determining a distribution characteristic of the test compound in a second target coagulation using the method of any one of embodiments E1-E42; and (c) calculating a ratio of the distribution characteristics of the test compound determined in the first target coagulation and the second target coagulation, thereby determining the coagulation preference profile of the test compound.

[0330] E59. The method of embodiment E58, wherein the first target coagulant and the second target coagulant are in the same composition.

[0331] E60. The method of embodiment E58, wherein the first target coagulant and the second target coagulant are in different compositions.

[0332] E61. The method of any one of embodiments E58 to E60, wherein the partitioning characteristic of the test compound in the first target agglomerate is determined before, simultaneously with, or after the partitioning characteristic of the test compound in the second target agglomerate is determined.

[0333] E62. A method for determining a coagulation preference profile of a test compound, the method comprising: (a) determining a relative distribution characteristic of the test compound in a first target coagulation using the method of any one of embodiments E49-E51; (b) determining a relative distribution characteristic of the test compound in a second target coagulation using the method of any one of embodiments E49-E51; and (c) calculating a ratio of the distribution characteristics of the test compound determined in the first target coagulation and the second target coagulation, thereby determining the coagulation preference profile of the test compound.

[0334] E63. The method of embodiment E62, wherein the first target coagulant and the second target coagulant are in the same composition.

[0335] E64. The method of embodiment E62, wherein the first target coagulant and the second target coagulant are in different compositions.

[0336] E65. The method of any one of embodiments E62 to E64, wherein the relative partitioning characteristic of the test compound in the first target agglomerate is determined before, simultaneously with, or after the relative partitioning characteristic of the test compound in the second target agglomerate is determined.

[0337] E66. The method of any one of embodiments E58-E65, further comprising labeling the first target condensate and the second target condensate to visualize the first target condensate and the second target condensate.

[0338] E67. The method of embodiment E66, wherein the first target condensate and the second target condensate are labeled with different labels.

[0339] E68. The method of any one of embodiments E58-E67, wherein the method comprises repeating the steps of the method for one or more additional target coagulants.

[0340] E69. A method for determining the aggregate preference profile of a plurality of test compounds, the method comprising performing the method of any one of embodiments E58-E68 with the plurality of test compounds.

[0341] E70. The method of embodiment E69, further comprising comparing the aggregate preference profiles of a subset or all of the plurality of test compounds.

[0342] E71. The method of embodiment E70, further comprising identifying a test compound having the same or similar aggregate preference profile.

[0343] E72. The method of embodiment E71, further comprising identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar aggregate preference profile.

[0344] E73. The method of embodiment E72, further comprising determining a relative distribution characteristic of one or more additional test compounds comprising the identified characteristic.

[0345] E74. The method of embodiment E72 or E73, further comprising determining the relative distribution characteristics of one or more additional test compounds that do not comprise the identified characteristics.

[0346] E75. A method for identifying compound characteristics associated with the distribution of compounds into or out of a coagulation, the method comprising: (a) determining the distribution characteristics of multiple test compounds in a target coagulation by performing the method of embodiment E43; (b) comparing the distribution characteristics of a subset or all of the multiple test compounds in the target compound; (c) identifying test compounds having the same or similar distribution characteristics in the target coagulation; and (d) identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar distribution characteristics.

[0347] E76. A method for identifying compound characteristics associated with the distribution of compounds into or out of a coagulation, the method comprising: (a) determining the relative distribution characteristics of multiple test compounds in a target coagulation by performing the method of embodiment E52; (b) comparing the relative distribution characteristics of a subset or all of the multiple test compounds in the target coagulation; (c) identifying test compounds having the same or similar relative distribution characteristics in the target coagulation; and (d) identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar relative distribution characteristics.

[0348] E77. A method for identifying compound characteristics that are associated with the assignment of compounds into or out of a coagulant, the method comprising: (a) determining the coagulant preference profiles of a plurality of test compounds by performing the method of embodiment E69; (b) comparing the coagulant preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar coagulant 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 coagulant preference profiles.

[0349] E78. A method for designing compounds having desired distribution characteristics within or outside a target condensate, the method comprising: (a) determining the distribution characteristics of multiple test compounds in a target condensate by performing the method of embodiment E43; (b) comparing the distribution characteristics of a subset or all of the multiple test compounds in the target condensate; (c) identifying test compounds having the same or similar distribution characteristics in the target condensate; (d) identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar distribution characteristics; and (e) (i) designing compounds containing the identified characteristics; or (ii) designing compounds that do not contain the identified characteristics, thereby designing compounds having desired distribution characteristics within or outside the target condensate.

[0350] E79. A method for designing compounds having desired relative distribution characteristics within or outside a target condensate, the method comprising: (a) determining the relative distribution characteristics of multiple test compounds in a target condensate by performing the method of embodiment E52; (b) comparing the relative distribution characteristics of a subset or all of the multiple test compounds in the target condensate; (c) identifying test compounds having the same or similar relative distribution characteristics in the target condensate; (d) identifying characteristics that a subset or all of the identified test compounds have in common in addition to the same or similar relative distribution characteristics; and (e) (i) designing compounds containing the identified characteristics; or (ii) designing compounds that do not contain the identified characteristics, thereby designing compounds having desired relative distribution characteristics within or outside the target condensate.

[0351] E80. A method for designing a compound having a desired coagulation preference profile, the method comprising: (a) determining the coagulation preference profiles of a plurality of test compounds by performing the method of embodiment E69; (b) comparing the coagulation preference profiles of a subset or all of the plurality of test compounds; (c) identifying test compounds having the same or similar coagulation preference profiles; (d) identifying features that a subset or all of the identified test compounds have in common in addition to the same or similar coagulation preference profiles; and (e) (i) designing a compound containing the identified features; or (ii) designing a compound that does not contain the identified features, thereby designing a compound having a desired coagulation preference profile.

[0352] E81. The method of any one of embodiments E78-E80, further comprising preparing the compound.

[0353] E82. A method for screening a test compound having a desired partitioning characteristic from a group of candidate compounds, the method comprising: (a) determining the partitioning characteristic of each of the group of candidate compounds; and (b) identifying the test compound having the desired partitioning characteristic.

[0354] E83. The method of embodiment E82, wherein the partitioning characteristic of each of the set of candidate compounds is determined in vitro.

[0355] E84. The method of embodiment E82 or E83, wherein the test compound has a partitioning characteristic suitable for use in treating a disease in the individual.

[0356] E85. A method for identifying a test compound that can be used to treat a disease in an individual in need thereof, the method comprising: (a) identifying a target aggregate associated with the disease; and (b) determining the distribution characteristics of a candidate compound in the target aggregate, and (c) identifying a test compound having a distribution characteristic suitable for treating the disease.

[0357] E86. A method for determining the distribution characteristics of a test compound in a target condensate, the method comprising: (a) combining a test compound with a composition comprising a target condensate and a solution outside the condensate; (b) obtaining a reference control; (c) measuring the MS signal of the test compound or a portion thereof in the solution outside the condensate using mass spectrometry; (d) measuring the MS signal of the test compound or a portion thereof in the reference control using mass spectrometry; and (e) comparing the MS signal of the test compound from the solution outside the condensate with the MS signal of the test compound from the reference control, thereby determining the distribution characteristics of the test compound in the target condensate.

[0358] E87. The method of E86, wherein the amount of the test compound combined with the composition is 100 nM or less and the amount of the precursor molecule in the composition, including in the target condensate, is about 5 μM.

[0359] E88. A library comprising a plurality of compounds, wherein each compound in the plurality of compounds comprises the same portion comprising a feature having a desired distribution characteristic.

[0360] E89. A method of designing a test compound having a desired partitioning characteristic, the method comprising modifying a precursor of the test compound by attaching a moiety to the compound, wherein the moiety comprises a feature having the desired partitioning characteristic.

[0361] Those skilled in the art will recognize that many embodiments are possible within the scope and spirit of the present disclosure.The present disclosure is further illustrated by the following examples, which should not be construed as limiting the present disclosure in scope or spirit to the specific procedures described therein.

[0362] Example

[0363] Example 1

[0364] Characterization of one or more compounds in solution-based coacervates

[0365] Aggregates 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 aggregates is diluted into a buffer that simulates physiological salt conditions.

[0366] Alternatively, a solution containing one or more proteins capable of forming coacervates is diluted into a buffer and a crowding agent is added. In a specific embodiment, the protein capable of forming coacervates is combined 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.

[0367] The test compound is added to the solution either before or after coacervate formation. When added after coacervate formation, the solution is incubated to allow the test compound to partition.

[0368] The density of the coacervate is generally greater than the surrounding solution, thus allowing the coacervate to settle. In some cases, the coacervate is imaged. For example, the coacervate is visualized using a dye or labeled protein known to be concentrated in the coacervate.

[0369] In some cases, the supernatant is removed from the coagulant. In some cases, the supernatant is analyzed to determine the amount of compound present.

[0370] The coacervate is analyzed to determine the amount of compound present. In some cases, the ratio of compound in the coacervate to compound in the supernatant is calculated.

[0371] Example 2

[0372] Characterization of dye compounds in solution-based FUS and PGL-3 coacervates

[0373] A variety of exemplary test compounds were assayed using the exemplary methods to determine partitioning characteristics, relative partitioning characteristics, and aggregate preference profiles. For this example, the exemplary test compound used was a dye; however, the test compound is not limited to a dye. The entire procedure was performed twice in two independent experiments on different days and in different orders.

[0374] Sample preparation

[0375] 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 produce 11.765 μM dye in 1.1765% DMSO. 17 μl of dye was distributed in a 384-well non-binding plate (Greiner).

[0376] Table 1. Dyes tested

[0377]

[0378]

[0379]

[0380]

[0381]

[0382] Thaw SNAP-tagged FUS protein or untagged PGL-3 protein at room temperature for at least 10 minutes and change the buffer to partition buffer (PB: 50mM Tris pH 7.25, 500mM KCl, 5% glycerol, and 1mM DTT). The protein was then diluted to 33.3μM in PB. 3μl of protein solution was added to a 384-well plate to start phase separation, resulting in 20μl of 5μM protein and 10μM dye in 75mM KCl, 0.75% glycerol, 20mM Tris, 1mM DTT and 1% DMSO, or for a no-dye control reaction, 20μl of 5μM protein in 75mM KCl, 0.75% glycerol, 20mM Tris, 1mM DTT.

[0383] Imaging

[0384] Images of the samples were acquired 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 according to the specific fluorescence emission intensity of each dye. The exposure time range was 1-1000ms. Appropriate filter settings were used to match the excitation and emission spectra of the corresponding dyes. The channels used for each dye are shown in Table 1. Three images were taken per well. Exemplary images are shown in Figure 1 For all applied imaging conditions, the background signal of the protein droplets in the absence of dye was recorded.

[0385] Image and data analysis

[0386] Using the image analysis software Fiji, the fluorescence intensity inside an exemplary target condensate (internal intensity, I-in) and in the area next to the exemplary target condensate (external intensity, I-out) was manually measured for each image. An example of the measured area is shown in Figure 2A A total of three I-in and three I-out values ​​were measured for each dye in each experiment and averaged. The I-in and I-out values ​​of FUS-SNAP aggregates measured in one day of experiment are shown in Table 2 and Table 3, respectively. The I-in and I-out values ​​of PGL-3 aggregates measured in one day of experiment are shown in Table 4 and Table 5, respectively. All values ​​were background corrected based on background images (no dye control) taken under the same microscopy conditions. An exemplary background image is shown in Figure 2B and the measured I-in or I-out background measurement values ​​are shown in Table 3-5.

[0387] Table 2. Dye measurements inside FUS-SNAP condensates.

[0388]

[0389]

[0390] Table 3. Dye measurements on the exterior of FUS-SNAP aggregates.

[0391]

[0392] Table 4. Dye measurements inside PGL-3 coacervates

[0393]

[0394]

[0395] Table 5. Dye measurements on the exterior of PGL-3 coacervates.

[0396]

[0397] The ratio of the average intensity measured inside the target aggregate and outside the target aggregate was calculated by dividing I-in by I-out, although I-out divided by I-in could also be used. The I-in:I-out ratios determined from two different experimental days were then averaged. Table 6 shows the daily and average I-in:I-out ratios. The average I-in:I-out ratios for FUS-SNAP aggregates are shown in Figure 3 and the average I-in:I-out ratio of PGL-3 aggregates is shown in Figure 4 The standard deviation between the two experiments is given by Figure 3 and Figure 4 The error bars in represent .

[0398] The ratios of FUS-SNAP I-in:I-out to PGL-3 I-in:I-out were also calculated and are shown in Tables 6 and Figure 5 middle.

[0399] Table 6. Calculated dye intensity ratios

[0400]

[0401] All compounds tested showed a more preferential partitioning into FUS-SNAP aggregates relative to aggregates formed with PGL-3. Compounds #1 (DAPI), #11 (fluorescein-12-UTP), #16 (rhodamine B), #17 (rhodamine 101 inner salt), and #18 (rhodamine 800) had an I-in:I-out ratio of 4 or higher for FUS-SNAP aggregates. Compounds #11, #16, #17, and #18 preferentially partitioned into FUS-SNAP aggregates relative to PGU-3 with a ratio of 3 or higher.

[0402] Example 3

[0403] Colocalization of partitioned compounds and FUS in FUS condensates

[0404] To verify that the compounds partitioned into coacervates in Example 2 were partitioned into coacervates containing FUS, dye #18 (Rhodamine 800) with an average I-in:I-out ratio of 9.3 was further evaluated.

[0405] Sample preparation

[0406] FUS-GFP aggregates were prepared using dye 18 as described in Example 2 for FUS-SNAP.

[0407] Imaging

[0408] Images of the samples were acquired 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 based on the specific fluorescence emission intensity of each dye. The exposure time range was 1-1000ms. Appropriate filter settings were used to match the excitation and emission spectra of the dyes and GFP. Control images of condensates containing FUS-GFP and without dye were acquired.

[0409] result

[0410] The resulting image is shown in Figure 6 GFP-tagged FUS was detected in condensates in the presence or absence of the dye. Dye 18 was detected in condensates, and FUS protein and the dye colocalized in condensates. For the no-dye control, no fluorescence was detected in condensates when images were acquired with the Rhodamine 800 channel.

[0411] Example 4

[0412] Determining the distribution characteristics of a group of compounds using mass spectrometry

[0413] A mass spectrometry-based method was established to measure the partitioning characteristics of exemplary test compounds. The measurements from the mass spectrometry-based method were compared with the measurements obtained using the fluorescence-based assay disclosed in Example 2.

[0414] method

[0415] Prepare protein buffer (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. Prepare 20 μl aliquots of 70 μM FUS-SNAP protein stock solution in protein buffer. Freeze and store FUS-SNAP protein stock aliquots before use. Prepare dilution buffer (14.7 mM Tris pH 7.25 and 1 mM DTT).

[0416] Thaw aliquots of 70 μM FUS-SNAP protein stock solution at room temperature for about 10 minutes. The protein stock solution aliquots are clear with no visible precipitate. Filter the thawed protein stock solution using a centrifugal filter (Millipore, UFC30VV00). In brief, aliquots of the protein stock solution are added to a centrifugal field and centrifuged at 20,000 rcf for 1 minute at room temperature. Collect the flow-through. Measure the protein concentration of the effluent, and prepare a 33.3 μM FUS-SNAP solution by diluting the filtered effluent and dilution buffer.

[0417] In PCR tubes or PCR strips (8-well; AXYGEN TM 40 μl test reactions were prepared in 8-Strip PCR Tubes, 0.2 mL) with: (i) 34 μL dilution buffer with a given concentration of test compound and with DMSO to obtain a final DMSO concentration of 1%, and (ii) 6 μL of 33.3 μM FUS-SNAP filtered protein solution. The final concentration of the 40 μL reaction was 5 μM FUS-SNAP, 75 mM KCl, 20 mM Tris, 0.75% glycerol, 1 mM DTT, 1% DMSO; and for reactions containing test compound, the desired test compound concentration.

[0418] A 40 μl reference reaction was prepared as described for the test reactions, but no test compound was added at this stage.

[0419] The test and reference reactions were incubated at room temperature for 15 minutes. It is expected that in the test reaction, condensed high-density droplets (mM protein concentration) of FUS-SNAP protein (condensed phase) are present in the environment of a lower concentration solution (μM protein concentration) of FUS-SNAP dilute phase. The test compound may or may not be distributed into the condensed phase, resulting in a decrease in the concentration of the outside (test compound in the dilute phase) and an increase in the concentration of the inside (test compound in the droplet). The volume of the high-density droplets is estimated to be at least 1000 times smaller than the total reaction volume, for example, 20nL condensed phase in 20μL reaction.

[0420] After the 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 20° C. cooled centrifuge. When PCR strips were used, the rotor used was an Eppendorf F-45-48-5-PCR. 35 μL of supernatant was removed from each tube and transferred to a new tube, taking care not to disturb the pellet containing the condensates.

[0421] For the reference reaction, a known amount of the test compound is added to a tube containing the supernatant (which contains proteins that are not associated with the coacervate) so that the final compound concentration is the same as the initial compound concentration 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 duplicating the supernatant based on the information obtained. The known amount of compound added to the supernatant from the reference reaction is used to calibrate the mass spectrometry signal of the test reaction.

[0422] In addition, compounds are extracted from the test reaction to directly measure the amount of compounds in the formed coacervate. The supernatant containing a certain amount of test compound is separated from the pellet containing the coagulated protein. In short, the cap of each PCR tube is removed with scissors and each PCR tube is inverted in a 1.5mL Eppendorf tube. The tube is then centrifuged at 2,000rcf for 5 seconds on a standard desktop centrifuge. When using PCR strips, the PCR strips can be inserted inverted into Eppendorf Microplate 96 / V-PP (Cat#951040188) and centrifuged at 4,500rcf for 1 minute. Subsequently, the compound is separated from each pellet using a solution of ACN:MeOH (1:1). The sample can be ultrasonically treated as needed.

[0423] Samples from the test and reference reactions were analyzed by mass spectrometry. The ratio between the integrals of the corresponding MS signals between the relevant supernatant test and reference reactions reflects the depletion of the test compound due to partitioning into the formed aggregates.

[0424] The fluorescence-based assay disclosed in Example 2 was also used to determine the partitioning characteristics of the test compounds.

[0425] result

[0426] Rhodamine 101 (Rho 101), Rhodamine 800 (Rho 808), Rhodamine B (Rho B), Rhodamine 123 (Rho 123), and fluorescein were evaluated using a fluorescence-based assay and a mass spectrometry-based assay as described above. Figure 7As shown, the partition characteristic measurements produced by these two test methods are very consistent for all compounds tested. Using this measurement, the ratio of the concentration of compound associated with coacervate to the concentration of compound not associated with coacervate was calculated for each compound. A coacervate volume fraction of 1 / 1000 relative to the external phase was used. The results are provided in Table 7.

[0427] Table 8. Ratios of internal compound concentrations to external compound concentrations

[0428] Compound MS-based assays Fluorescence-based assays Rho 101 >8,000 1,500 Rho 800 >2,000 1,000 Rho B 666 666 Fluorescein 1-100 1 Rho 123 1-100 1

[0429] In addition, the pelleted coacervate samples were processed to extract and quantify the test compound therein. The results showed that the sum of the test compound amount in the coacervate and the test compound amount in the supernatant was consistent with the total amount of test compound added to the test reaction (data not shown).

[0430] As demonstrated herein, mass spectrometry-based assays are robust and sensitive assays for quantitatively determining the partitioning characteristics of compounds. The technique can be used as a high throughput screen. Such mass spectrometry-based assays achieve certain additional advantages. For example, mass spectrometry-based assays are not limited to fluorescent compounds, are an assumption-free technique (i.e., the identity of the test compound does not need to be known prior to the assay), are not limited by fluorescence quenching that may occur inside the droplet, and, as demonstrated in Example 5, can be easily multiplexed.

[0431] Example 5

[0432] This example evaluates the effect of using different concentrations of test compound in the test reaction on the measurement of the test compound in the supernatant.

[0433] Test reactions and reference controls were prepared as described in Example 4. Fluorescein (which was identified as not partitioning into FUS aggregates) and Rhodamine 101 (which was identified as partitioning into FUS aggregates) were evaluated in separate test reactions at 0.01 μM, 0.1 μM, 1 μM, and 10 μM.

[0434] result

[0435] like Figure 8 As shown, fluorescein does not partition into FUS aggregates at any of the concentrations tested, and the partitioning characteristics of fluorescein are independent of concentration. Rhodamine 101 was observed to partition into FUS-aggregates at lower compound concentrations (see Figure 8 ; 0.01 μM and 0.1 μM). Data represent mean ± SD, N = 3 technical replicates. Figure 8As shown, the partitioning characteristics of reduced compound concentrations have improved detection efficiency. When the amount of compound partitioning into the agglomerates may be small, using smaller amounts of test compound added to the test reaction can allow detection of small changes in supernatant compound concentration relative to a reference control.

[0436] Example 6

[0437] Multiplexed mass spectrometry consumption assay

[0438] This example demonstrates the use of the mass spectrometry based assay disclosed in Example 4 to study a system containing multiple test compounds.

[0439] method

[0440] 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, were used to create 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.

[0441] result

[0442] like Fig. 9A As shown, the measured fraction of external fluorescein of FUS-SNAP aggregates in the single compound test reactions was consistent with the measured fraction of external fluorescein of FUS-SNAP aggregates in the presence of the mixture of the four compounds. Fig. 9B As shown, the measured fraction of external Rho B on FUS-SNAP condensates for single compound test reactions is consistent with the measured fraction of external Rho B on FUS-SNAP condensates in the presence of a mixture of the four compounds. This suggests that mass spectrometry-based techniques can be used for multiplexed assays, which, for example, can enable higher throughput and / or study of more complex systems.

[0443] Example 7

[0444] Leveraging multiplexing of mass spectrometry-based assays

[0445] This example further demonstrates the utility of the mass spectrometry-based assay disclosed in Example 4 for studying systems containing multiple test compounds.

[0446] method

[0447] FUS-SNAP protein reactants and reference reactants were prepared as described in Example 4. For the test reactants, fluorescein and rhodamine 101 were measured individually, and three pools of compounds were also measured. The first pool had 10 compounds (and included fluorescein), the second pool had 15 compounds (and included fluorescein and rhodamine 101), and the third pool had 20 compounds (and included fluorescein and rhodamine 101). All samples were prepared and measured in triplicate. All samples were analyzed using the mass spectrometry-based assay described in Example 4.

[0448] result

[0449] like Fig.10 As shown, the assay, which measures, for example, the depletion of a test compound in the supernatant due to the presence of aggregates, is able to measure the partitioning characteristics of individual compounds independently of the compound pool size. These results demonstrate that compounds can be mixed in large pool sizes and still allow for measurements comparable to single compound partitioning characteristics measurements evaluated individually. Thus, the mass spectrometry-based assay described herein enables high throughput screening.

Claims

1. A method for determining the partitioning characteristics of a small molecule test compound in a target condensate, the method comprising: (a) combining the small molecule test compound with a composition comprising the target coacervate and a solution outside the coacervate; (b) separating the target coagulant from the solution outside the coagulant; (c) determining the amount of the small molecule test compound in the target coacervate by mass spectrometry based on the amount of the test compound in the solution outside the coacervate, The partitioning characteristics of the small molecule test compound in the target aggregate are thereby determined.

2. The method of claim 1, further comprising causing the target agglomerate to form prior to step (a).

3. A method for determining the partitioning characteristics of a small molecule test compound in a target condensate, the method comprising: (a) causing the target coacervate to form in the presence of the small molecule test compound to obtain a composition comprising the target coacervate and a solution outside the coacervate; and (b) separating the target coagulant from the solution outside the coagulant; and (c) determining the amount of the small molecule test compound in the target coacervate by mass spectrometry based on the amount of the test compound in the solution outside the coacervate, The partitioning characteristics of the test compound in the target aggregate are thereby determined.

4. The method of claim 3, further comprising combining the small molecule test compound with a precursor composition comprising a precursor molecule prior to step (a).

5. The method of claim 3, further comprising adding the small molecule test compound to a precursor composition comprising a precursor molecule prior to step (a). 6 . The method of claim 1 , wherein the amount of the small molecule test compound in the target coacervate is determined before, simultaneously with, or after the amount of the small molecule test compound in the solution outside the coacervate is determined.

7. The method according to any one of claims 1-6, further comprising determining a ratio of the amount of the small molecule test compound in the target coacervate to the amount of the small molecule test compound in the solution outside the coacervate.

8. The method according to any one of claims 1 to 7, further comprising separating the target coagulant from the coagulant-external solution.

9. The method of any one of claims 1-8, further comprising identifying the target aggregate prior to determining the amount of the small molecule test compound in the target aggregate.

10. The method of any one of claims 1-9, wherein the dysregulation of the target aggregate is associated with a disease.

11. The method according to any one of claims 1 to 10, further comprising characterizing the target condensate by identifying one or more macromolecules contained therein.

12. The method of claim 11, wherein the identifying comprises determining the amount of the one or more macromolecules in the target coacervate. 13 . The method of claim 12 , further comprising determining a ratio of the amount of the small molecule test compound in the target coacervate to the amount of the one or more macromolecules in the target coacervate.

14. The method of any one of claims 1-13, wherein the target condensate comprises a protein comprising an intrinsically disordered sequence.

15. The method according to any one of claims 1-14, further comprising labeling the target aggregate to visualize the target aggregate.

16. The method of claim 15, wherein the target aggregate is labeled with a radioactive label, a colorimetric label, or a fluorescent label.

17. The method of any one of claims 1-16, wherein the composition comprises cells.

18. The method of claim 17, wherein the cell is a microorganism or an animal cell.

19. The method of claim 17 or 18, wherein the cells comprise aggregates that are determined to be dysregulated.

20. The method of any one of claims 17-19, wherein the cell has one or more characteristics of a neurodegenerative disease or a proliferative disease.

21. The method of any one of claims 1-20, wherein the target aggregate is a cell aggregate.

22. The method of claim 21, wherein the cellular condensate is a lysosome, a P-granule, a histone locus body, a multivesicular body, a neuronal RA granule, a nuclear bud, a nuclear pore, a nuclear plaque, a nuclear stress body, a nucleolus, an Oct1 / PTF / transcription (OPT) domain, a paraplaque, a perinucleolar compartment, a PML nuclear body, a PML oncogenic domain, a polycomb body, a processing body, a Sam68 nuclear body, a stress granule, or a splicing plaque.

23. The method of any one of claims 1-22, wherein the target condensate is in a cell.

24. The method of claim 23, wherein the cell is a microorganism or an animal cell.

25. The method of claim 23 or 24, wherein the cell has one or more characteristics of a neurodegenerative disease or a proliferative disease.

26. The method of any one of claims 1-25, wherein the extra-coacervate solution is intracellular fluid.

27. The method of claim 26, wherein the intracellular fluid is cytosol or nucleosol.

28. The method of any one of claims 1-20, wherein the target aggregate is not in a cell.

29. The method of claim 28, wherein the target aggregates are extracellular aggregates.

30. The method of any one of claims 1-20, 28 or 29, wherein the extra-coacervate solution is extracellular fluid.

31. The method of claim 30, wherein the extracellular fluid is interstitial fluid.

32. The method of any one of claims 1-16, wherein the method is a cell-free assay.

33. The method of any one of claims 1-32, wherein the composition comprises one or more of the following: a macromolecule, a salt, and a buffer.

34. The method of any one of claims 1-33, wherein the composition comprises two or more target coagulants.

35. The method of any one of claims 1-34, wherein the method comprises repeating the steps of the method for one or more additional coagulants.

36. The method of any one of claims 1-35, wherein the small molecule test compound comprises a test compound label.

37. The method of claim 36, wherein the test compound label is a radioactive label, a colorimetric label, or a fluorescent label.

38. The method of claim 36 or 37, wherein the test compound label is a fluorescent label.

39. The method of any one of claims 36-38, wherein the amount of the test compound is determined by detecting the test compound marker.

40. A method for determining the partitioning characteristics of a plurality of small molecule test compounds in a target agglomerate, the method comprising performing the method according to any one of claims 1 to 39 with the plurality of small molecule test compounds.

41. The method of claim 40, further comprising comparing the partitioning characteristics of a subset or all of the plurality of small molecule test compounds in the target aggregate.

42. The method of claim 41, further comprising identifying small molecule test compounds having the same or similar partitioning characteristics in the target aggregates.

43. The method of claim 42, further comprising identifying characteristics that a subset or all of the identified small molecule test compounds have in common in addition to the same or similar assigned characteristics.

44. The method of claim 43, further comprising determining a partitioning signature of one or more additional small molecule test compounds comprising the identified signature within the target aggregate.

45. The method of claim 43 or 44, further comprising determining the partitioning characteristics of one or more additional small molecule test compounds that do not contain the identified characteristics within the target aggregate.

46. ​​A method for determining the partitioning characteristics of a small molecule test compound in a target condensate, the method comprising: (a) combining the small molecule test compound with a composition comprising the target coacervate and a solution outside the coacervate; (b) obtaining a reference control; (c) measuring the MS signal of the small molecule test compound or a portion thereof in the solution outside the condensate using mass spectrometry; (d) measuring the MS signal of the small molecule test compound or a portion thereof in the reference control using mass spectrometry; and (e) comparing the MS signal of the small molecule test compound from the solution outside the coacervate with the MS signal of the test compound from the reference control, The partitioning characteristics of the small molecule test compound in the target aggregate are thereby determined.

47. The method of claim 46, wherein the amount of the small molecule test compound combined with the composition is 100 nM or less and the amount of the precursor molecule included in the target condensate in the composition is about 5 μM.

48. A method for determining the relative partitioning characteristics of a small molecule test compound in a target condensate, the method comprising: (i) determining the partitioning characteristic of the small molecule test compound by performing the method according to any one of claims 1-39, 46 or 47 with the small molecule test compound; (ii) determining the partitioning characteristic of a reference compound by performing a method according to any one of claims 1 to 39, 46 or 47 with the reference compound; and (iii) calculating the ratio of the distribution characteristics determined in (i) and (ii), The relative partitioning characteristics of the small molecule test compound in the target aggregate are thereby determined.

49. The method of claim 48, wherein the small molecule test compound comprises a test compound label.

50. The method of claim 49, wherein the reference compound is a marker for the test compound.

51. A method for determining the relative distribution characteristics of a plurality of small molecule test compounds in a target condensate, the method comprising: (1) carrying out the method according to any one of claims 48 to 50; and (2) Repeating steps (i) and (iii) using the plurality of small molecule test compounds.

52. The method of claim 51, further comprising comparing the relative partitioning characteristics of a subset or all of the plurality of small molecule test compounds in the target aggregate.

53. The method of claim 52, further comprising identifying small molecule test compounds having the same or similar relative partitioning characteristics in the target condensate.

54. The method of claim 53, further comprising identifying characteristics that a subset or all of the identified small molecule test compounds have in common in addition to the same or similar relative distribution characteristics.

55. The method of claim 54, further comprising determining the relative partitioning characteristics of one or more additional small molecules of a test compound comprising the identified characteristics within the target aggregate.

56. The method of claim 54 or 55, further comprising determining the relative partitioning characteristics of one or more additional small molecule test compounds that do not contain the identified characteristics in the target aggregate.

57. A method for determining an aggregate preference profile of a small molecule test compound, the method comprising: (a) determining the partitioning characteristics of the small molecule test compound in the first target aggregate using the method according to any one of claims 1-39, 46 or 47; (b) determining the partitioning characteristics of the small molecule test compound in the second target condensate using the method according to any one of claims 1-39, 46 or 47; and (c) calculating a ratio of the partitioning characteristics of the small molecule test compound determined in the first target agglomerate and the second target agglomerate, The aggregate preference profile of the small molecule test compound is thereby determined.

58. The method of claim 57, wherein the first target coagulant and the second target coagulant are in the same composition.

59. The method of claim 57, wherein the first target coagulant and the second target coagulant are in different compositions.

60. The method of any one of claims 57-59, wherein the partitioning characteristic of the small molecule test compound in the first target agglomerate is determined before, simultaneously with, or after the partitioning characteristic of the small molecule test compound in the second target agglomerate is determined.

61. A method for determining an aggregate preference profile of a small molecule test compound, the method comprising: (a) determining the relative distribution characteristics of the small molecule test compound in the first target aggregate using the method according to any one of claims 48-50; (b) determining the relative distribution characteristics of the small molecule test compound in the second target condensate using the method according to any one of claims 48-50; and (c) calculating a ratio of the partitioning characteristics of the small molecule test compound determined in the first target agglomerate and the second target agglomerate, The aggregate preference profile of the small molecule test compound is thereby determined.

62. The method of claim 61, wherein the first target coagulant and the second target coagulant are in the same composition.

63. The method of claim 61, wherein the first target coagulant and the second target coagulant are in different compositions.

64. The method according to any one of claims 61-63, wherein the relative distribution characteristics of the small molecule test compound in the first target agglomerate are determined before, simultaneously with, or after the relative distribution characteristics of the small molecule test compound in the second target agglomerate are determined.

65. The method of any one of claims 57-64, further comprising labeling the first target condensate and the second target condensate to visualize the first target condensate and the second target condensate.

66. The method of claim 65, wherein the first target agglomerate and the second target agglomerate are labeled with different labels.

67. The method of any one of claims 57-66, wherein the method comprises repeating the steps of the method for one or more additional target agglomerates.

68. A method for determining the aggregate preference profile of a plurality of small molecule test compounds, the method comprising performing the method of any one of claims 57-67 with the plurality of small molecule test compounds.

69. The method of claim 68, further comprising comparing aggregate preference profiles of a subset or all of the plurality of small molecule test compounds.

70. The method of claim 69, further comprising identifying small molecule test compounds having the same or similar aggregate preference profile.

71. The method of claim 70, further comprising identifying features that a subset or all of the identified small molecule test compounds have in common in addition to the same or similar aggregate preference profile.

72. The method of claim 71, further comprising determining the relative distribution characteristics of one or more additional small molecule test compounds comprising the identified characteristics.

73. The method of claim 71 or 72, further comprising determining the relative distribution characteristics of one or more additional small molecule test compounds that do not contain the identified characteristics.

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