Drug discovery assays for screening compounds

Abstract

The present invention provides drug discovery assays to screen for compounds capable of modulating condensates based on the ability of the compounds to alter the condensation propensity of a target of interest, preferably an intrinsically disordered protein.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular, it provides a drug discovery assay that screens compounds that can regulate biomolecular condensates based on the ability of compounds to alter the aggregation tendency of the target under study (preferably an intrinsically disordered protein). Background Technology

[0002] The discovery and study of intrinsically disordered proteins (IDPs) in the late 1990s challenged the central dogma of structure determining function in molecular biology, exemplified by Emil Fisher's "lock and key" model proposed in 1894. IDPs lack well-defined secondary and tertiary structures. Their free energy spectra have multiple minima, allowing for a wide range of conformations (from fully extended to fully folded). A better approach is to define their overall structure as a dynamic set of many discrete conformational states, each with a statistical weight. The structure of IDPs contrasts with that of globular proteins, which have very well-defined global minima in their free energy spectra and their three-dimensional folding, thus fluctuating around an equilibrium position.

[0003] Inherent disorder (ID) is encoded in the primary sequence of proteins. Compared to folded proteins, IDPs exhibit a generally low average hydrophobicity and a high net charge. Protein folding is a spontaneous process. When the hydrophobic core of a protein collapses, a large number of water molecules are released, increasing the entropy of the system. Once the water in a protein is depleted, the enthalpy of interactions between amino acid residues increases (hydrogen bonds and van der Waals interactions). The low hydrophobicity of IDPs leads to weakened hydrophobic interactions, thus preventing them from folding into stable globular structures.

[0004] Globular proteins are primarily involved in processes requiring extensive control of their three-dimensional structure, such as enzymatic reactions. In contrast, intrinsically disordered proteins (IDPs) play a pervasive role in regulatory and signaling processes, as well as in the hubs of protein-protein interactions. For example, intrinsic disorder is prevalent in transcription and transcriptional regulation, particularly in the activation domains of transcription factors. Recently, IDPs have also been shown to participate in biomolecular condensation.

[0005] IDPs can: i) interact with their partners with high specificity and low affinity, meaning their interactions are highly specific and transient, though there are exceptions; ii) respond rapidly to environmental changes; iii) bind to different partners through monovalent and / or multivalent interactions; and iv) bind to multiple partners. In their bound state, IDPs can remain disordered or “fuzzy” or become structured. Most binding events occur via short motifs (called molecular recognition features or short linear motifs, MoRFs) embedded in larger disordered sequences. MoRFs are evolutionarily conserved and generally amphiphilic, allowing for an increase in secondary structure upon binding. To avoid promiscuous binding, IDPs are tightly regulated by post-transcriptional modifications (PTMs) and / or by ensuring appropriate protein levels within the desired timeframe. On the one hand, PTMs can alter the energy spectrum of IDPs, thereby stabilizing or destabilizing secondary structures and leading to transitions between disordered and ordered states. On the other hand, ensuring appropriate IDP levels is important for preventing nonspecific interactions. Cellular concentrations of IDPs are modulated in all production steps, from transcriptional clearance and translation rates to protein degradation, as evidenced by the observed shorter half-life of IDPs compared to structural proteins.

[0006] Dysregulation of IDPs is associated with disease. In fact, the concept of D2 (disorder within disorder) was introduced to emphasize the role of inherent disorder in many diseases, including cancer, neurodegenerative diseases, cardiovascular diseases, and diabetes. For example, the abundance of dysregulated oncogenes c-Myc and cancer suppressor p53 is altered in many types of cancer. Similarly, mutations or dysregulations of some disordered proteins, such as amyloid β-peptide and α-synuclein, respectively contribute to the development of neurodegenerative diseases such as Alzheimer's and Parkinson's.

[0007] IDPs are promising drug targets. They are abundant in the proteome, involved in many important biological functions, and overexpressed in major pathologies. To date, most successful strategies targeting IDPs have circumvented the inherent disorder of this target. Drugs have acted directly on ordered domains of proteins that regulate IDPs, which are inherently disordered, or on enzymes that bind globular chaperones. Only a few strategies directly target the inherently disordered domains for drug therapy. In fact, only two compounds targeting IDPs have entered clinical trials: MSI-1436, targeting tyrosine phosphatase 1B protein, for the treatment of obesity (Trodusquemine, NCT00509132) and cardiovascular disease (Trodusquemine, NCT00005696), and EPI-506, targeting the dysregulated androgen receptor N-terminal transcriptional activation domain (AR-NTD), for the treatment of castration-resistant prostate cancer (Ralaniten, NCT02606123).

[0008] Therefore, it is necessary to find alternative strategies to target IDPs.

[0009] On the other hand, the cytoplasm and nucleoplasm are highly concentrated environments. To achieve spatiotemporal control, cells localize reactions within organelles. Classic organelles are the Golgi apparatus and the endoplasmic reticulum. All of these organelles are membrane-bound, meaning there is a physical separation between the inside and outside of the organelle, and the separation of molecules is regulated by membrane transport mechanisms. However, many other organelles lack physical separation around them but are able to concentrate biochemical processes. Examples include the nucleolus (for ribosome biosynthesis), the Cajal body (for the assembly and / or modification of splicing mechanisms), the PML body (for transcriptional regulation and apoptosis signaling), and stress granules (for mRNA storage during stress).

[0010] Although membraneless condensates differ in composition, subcellular location, and function, they all possess the ability to concentrate biomolecules and are formed by phase transitions. For this reason, they are called biomolecular condensates. Phase transitions have been extensively studied in physics, but their application in biological systems is an important emerging principle in biology. Liquid-liquid phase separation (LLPS) is a special type of phase transition and is by far the most physiologically relevant and significant area of ​​research in the field of condensates. The first evidence that membraneless organelles are liquids and formed by LLPS was reported in a landmark paper in 2009 by Dr. Clifford Brangwynne, Dr. Tony Hyman, and Dr. Frank Jülicher. They characterized P-particles in a line of *Caenorhabditis elegans* and demonstrated that these P-particles behave like liquids: they are spherical, drip-like, surface-wet, and undergo fusion events.

[0011] In cells, non-membrane organelles allow concentration differences to be maintained without the need for energy input. The chemical potentials of the two phases are equal, meaning there is no net diffusion flux between the dense and light phases, but there is a continuous flux of individual molecules between them.

[0012] The principle of LLPS (Liquid-Liquid Synthesis) has been extensively studied in soft matter physics, particularly concerning polymers. LLPS is the process by which a polymer, uniformly distributed in a solvent, spontaneously separates into two coexisting liquid phases: a polymer-rich phase (dense phase) and a polymer-depleted phase (light phase). Figure 8a, adapted from Alberti, S.; Gladfelter, A.; Mittag, T. Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates. Cell 2019, 176(3), 419–434). The negative free energy of stratification is obtained above a certain concentration (saturation concentration or Csat), where enthalpy (when polymer-polymer contact is superior to polymer-solvent contact) and / or entropy (by releasing solvent molecules from the hydrated shell into the lighter phase) increase ( Figure 8 b and 8c, adapted from Brangwynne, CP; Tompa, P.; Pappu, RV Polymer Physics of Intracellular Phase Transitions. Nat. Phys. 2015, 11, 899, and adapted from Posey, AE; Holehouse, AS; Pappu, RV V Phase Separation of Intrinsically Disordered Proteins. Methods Enzymol. 2018, 611, 1–30). LLPS are extremely sensitive to changes in the physicochemical conditions of the environment. Csat can be affected by changes in temperature, ionic strength, pH, post-translational modifications, etc. The physical principles behind polymer LLPS can be explained by the Flory-Huggins theory. However, this theory does not take into account some characteristics of biopolymers relative to polymers (such as sequence effects, the fact that they are not in thermodynamic equilibrium, etc.). For this reason, other mathematical models are needed to fully explain the behavior of phase-separated biopolymers.

[0013] Multivalentity is a key characteristic of LLPS. Multivalent interactions (scaffold-scaffold and scaffold-client) allow for network formation, which is fundamental to LLPS. LLPS can occur when proteins contain multiple interacting motifs with self-binding affinity or affinity for specific ligands. These interacting motifs can be domains linked via disordered linkers that bind to ligands embedded in disordered sequences (e.g., "spacer regions") or motifs (e.g., "stickers"). IDPs play a central role in LLPS because, by definition, they are multivalent proteins that can interact simultaneously with other molecules or with themselves. Other biomolecules capable of LLPS include RNA and DNA, which contain multiple motifs that interact with other nucleic acid molecules or proteins.

[0014] Since 2009, numerous studies characterizing biomolecular condensates have been published. Biomolecular condensates are typically heterogeneous in composition. Therefore, the concepts of scaffold and client are useful. Scaffold molecules are those that drive phase separation, while clients are those that dissociate into pre-formed condensates. Biomolecular condensates participate in many cellular functions and require tight control. Dysregulation of the assembly and disintegration of biomolecular condensates appears to be associated with a variety of diseases, including neurodegenerative diseases, cancer, and infectious diseases, although the exact pathological mechanisms remain unclear (e.g., whether the formation and dissolution of aberrant condensates are the cause of disease or its phenotype).

[0015] Biomolecular condensates offer a novel regulatory dimension for IDPs. Therefore, modulating condensates with drugs presents a potential opportunity to target currently untreatable proteins and could contribute to drug pharmacodynamics. However, the druggability of biomolecular condensates remains an underdeveloped area of ​​research.

[0016] This invention provides a novel drug discovery assay that screens compounds capable of modulating aggregates based on their ability to alter the aggregation tendency of the target under study (preferably an intrinsically disordered protein). Summary of the Invention

[0017] In its initial aspect, the present invention relates to a drug discovery assay (hereafter referred to as a Tc assay), preferably a cell-free assay, for screening compounds capable of altering a critical temperature (Tc) at which the target (preferably an intrinsically disordered protein) under investigation forms an aggregate, the assay comprising:

[0018] a. The test compound is combined with a composition containing the target before the formation of aggregates;

[0019] b. By gradually and in a controlled manner increasing the temperature of the solution in a), a target agglomerate is formed in the presence of the test compound, wherein the gradual and controlled increase in the temperature of the solution in a) is carried out at any rate selected from any value of 0.5 to 5 °C / min, more preferably 0.8 to 2 °C / min;

[0020] c. While gradually and controlledly increasing the temperature according to step b), measure the solution in a) by any spectroscopic measurement (preferably by measuring absorbance) to determine whether the test compound alters the reference critical temperature for target agglomeration, wherein the reference critical temperature for target agglomeration is understood as the temperature at which target agglomeration occurs in the absence of the test compound; and

[0021] If a test compound significantly alters the reference critical temperature at which the target forms aggregates, or if the percentage change caused by the test compound is significantly greater than the reference change caused by the reference compound, preferably greater than at least two or three standard deviations of the average activity value of the reference compound, preferably greater than 20%, 25%, 30%, 35%, or 40% of the change caused by the reference compound, then the test compound is selected as a compound capable of altering the aggregation tendency of the target under study.

[0022] In a preferred embodiment of the initial aspect, the Tc determination method includes:

[0023] a. Prior to the formation of aggregates, a multi-stage apparatus is used to allow for the mixing of multiple test compounds in each well of the multi-stage apparatus.

[0024] Compounds from the test compound library are bound to a composition containing the target;

[0025] b. By gradually and in a controlled manner increasing the temperature of multiple devices, a target agglomerate is formed in the presence of the test compound, wherein the gradual and controlled increase in the temperature of the solution in a) is carried out at any rate selected from any value of 0.5 to 5 °C / min (more preferably 0.8 to 2 °C / min);

[0026] c. While gradually and controlledly increasing the temperature according to step b), measure the solution in a) by any spectroscopic measurement (preferably by measuring absorbance) to determine whether the test compound alters the reference critical temperature for target agglomeration, wherein the reference critical temperature for target agglomeration is understood to be the temperature at which target agglomeration occurs in each well in the absence of the test compound; and

[0027] If a test compound significantly alters the reference critical temperature at which the target forms aggregates, or if the percentage change caused by the test compound is significantly greater than the reference change caused by the reference compound, preferably greater than at least two or three standard deviations of the average activity value of the reference compound, preferably greater than 20%, 25%, 30%, 35%, or 40% of the change caused by the reference compound, then the test compound is selected as one of those compounds capable of altering the aggregation tendency of the target under study.

[0028] Another aspect of the invention relates to a drug discovery assay (hereinafter referred to as a critical time value assay), preferably a cell-free assay, for screening compounds capable of altering a critical time value (tc) at which the target (preferably an intrinsically disordered protein) under investigation forms an aggregate, the assay comprising:

[0029] a. The test compound is bound to the composition containing the target in an additional aggregate solution prior to aggregate formation;

[0030] b. By gradually and in a controlled manner increasing the temperature of the solution in a), a target agglomerate is formed in the presence of the test compound, wherein the gradual and controlled increase in the temperature of the solution in a) is carried out at any rate selected from any value of 0.5 to 5 °C / min (more preferably 0.8 to 2 °C / min);

[0031] c. While gradually and controlledly increasing the temperature according to step b), measure the solution in a) by any spectroscopic measurement (preferably by measuring absorbance) to determine whether the test compound changes the reference tc value at which the target aggregates form, wherein the reference tc value is, in the absence of the test compound, the temperature at which the temperature in a) is gradually and controlledly increased.

[0032] The time between the onset of medium solution temperature and the start of aggregation under conditions suitable for target aggregate formation; and

[0033] If the test compound significantly alters the reference tc value when the target forms aggregates, or if the percentage change caused by the test compound is significantly greater than the reference change caused by the reference compound, preferably greater than at least two or three standard deviations of the average activity value of the reference compound, preferably greater than 20%, 25%, 30%, 35%, or 40% of the change caused by the reference compound, then the test compound is selected as a compound capable of altering the aggregation tendency of the target under study.

[0034] The critical time value (tc) is understood as the time from the moment the temperature begins to rise (from the moment the temperature of the solution begins to increase gradually and in a controlled manner) to the moment when coagulation is observed to begin.

[0035] In other preferred embodiments, the critical time value determination method includes:

[0036] a. Prior to the formation of aggregates, a multi-stage apparatus is used to allow for the mixing of multiple test compounds in each well of the multi-stage apparatus.

[0037] Compounds from the test compound library are bound to a composition containing the target;

[0038] b. By gradually and in a controlled manner increasing the temperature of multiple devices, a target agglomerate is formed in the presence of the test compound, wherein the gradual and controlled increase in the temperature of the solution in a) is carried out at any rate selected from any value of 0.5 to 5 °C / min (more preferably 0.8 to 2 °C / min);

[0039] c. While gradually and controlledly increasing the temperature according to step b), measure the absorbance of each well in the multi-device to determine whether the test compound alters the reference tc value for target agglomeration, wherein the reference tc value is the time between the start of gradual and controlled temperature increase in the multi-device and the start of agglomeration in each well under target agglomeration conditions in the absence of the test compound; and

[0040] If a test compound significantly alters the reference tc value at which the target forms aggregates, or if the percentage change caused by the test compound is significantly greater than the reference change caused by the reference, preferably greater than at least two or three standard deviations of the average activity value of the reference compound, preferably greater than 20%, 25%, 30%, 35%, or 40% of the change caused by the reference compound, then the test compound is selected as one of those compounds capable of altering the aggregation tendency of the target under study.

[0041] In any of the foregoing aspects or preferred embodiments thereof, the TC determination method or critical time value determination method further includes labeling the target to visualize the target aggregate, for example, using radioactive labeling, colorimetric labeling or fluorescent labeling.

[0042] In any of the foregoing aspects or preferred embodiments thereof, the composition comprises two or more targets.

[0043] In another preferred embodiment of any of the foregoing aspects or embodiments thereof, the amount of the test compound is determined by mass spectrometry, liquid chromatography and / or ultraviolet-visible spectrophotometry.

[0044] In another preferred embodiment of any of the foregoing aspects or embodiments thereof, the target comprises a protein selected from the list consisting of TARDNA-binding protein 43 (TDP-43), tau protein, fusion sarcoma protein (FUS), hnRNPAI, PGL-3, huntingtin protein, and androgen receptor (AR) or any fragment thereof (including the androgen receptor N-terminal domain (AR-NTD)). Preferably, the target is the androgen receptor N-terminal domain (AR-NTD). More preferably, the target is the androgen receptor N-terminal domain (AR-NTD), and the reference compound is compound EPI-001.

[0045] A further aspect involves methods for ranking or classifying multiple test compounds based on their ability to alter the aggregation tendency of the target under study, methods including:

[0046] a. By using the methods described above or their embodiments, determine the percentage change caused by each compound compared to a reference change; and

[0047] b. Sort or classify the various test compounds according to the percentage change caused by each compound.

[0048] A further aspect involves a method for selecting or identifying one or more test compounds based on their ability to alter the aggregation tendency of a target under study, the method comprising:

[0049] a. By using any of the foregoing aspects or embodiments thereof, determine the percentage change caused by each compound compared to a reference change; and

[0050] b. Identify the compound with the highest degree of alteration. Attached Figure Description

[0051] Figure 1 a) In PC-3 cells ( CRL-1435 TM (a) Time-resolved fluorescence microscopy of eGFP-AR after activation with 1 nM DHT. (b) Left: Fusion event of the two eGFP-AR condensates in PC-3 cells. (c) Right: FRAP of eGFP-AR condensates.

[0052] Figure 2 a) Phase diagram of AR-NTD condensation under 500 mM NaCl, obtained by apparent absorbance measurement of cloud point temperature (Tc) using turbidity variation assay (TSA). Left: Observation of 50 μM AR-NTD at three ionic intensities (250 mM, 500 mM, and 750 mM NaCl) using differential interference contrast (DIC) microscopy. Right: Tc measurement results by TSA. c) Left: 25 μM 15 N AR-NTD 1 H, 15 N HSQC spectrum. Right: Residue-specific helicity plotted using δ2D algorithm 92, and AR-NTD concentrations (50 μM, 75 μM, and 100 μM) normalized to their intensity at 25 μM. 1 H, 15 The loss of peak intensity in the N HSQC spectrum. The shaded area indicates that the signal is completely lost after focusing.

[0053] Figure 3 a) DIC images of 50 μM AR-NTD samples at different times in 500 mM NaCl. Magnified on the right. b) Left: AR-NTD-L26P mutant fusion event in the sample after incubation for more than 5 minutes. Right: Tc measurements of 50 μM AR-NTD versus 50 μM AR-NTD-L26P in 500 mM NaCl by TSA assay.

[0054] Figure 4 a) Circular dichroism (CD) spectrum of AR-NTD-L26P after TFE titration at 5 °C and percentage of helicity calculated from the CD data (insertion). b) Apparent absorbance measured by TSA at elevated temperature after TFE titration (λ = 340 nm). c) Left: Design of helical break mutants. Right: Their effect on Tc values ​​measured by TSA.

[0055] Figure 5 a) Confocal fluorescence microscopy images of different concentrations of AR-NTD (as shown in the figure) in the absence (top) and presence (bottom) of 75 μM EPI-001 in 20 mM Hepes 7.4, 150 mM NaCl, 10% sucrose, and 2 mM TCEP buffer. In all samples, 1% of AR-NTD was labeled with rhodamine. Scale bar, 5 μm. b) Confocal fluorescence microscopy images of different concentrations of FUS-GFP (as shown in the figure) in the absence (top) and presence (bottom) of 75 μM EPI-001 in 25 mM Tris-HCl 7.4, 150 mM KCl, 2.5% glycerol, and 0.5 mM DTT buffer. Scale bar, 5 μm.

[0056] Figure 6 a) Apparent absorbance (λ = 350 nm) of 30 μM ARNTD in 20 mM Hepes 7.4, 500 mM NaCl, and 2 mM TCEP buffer at elevated temperature, in the absence of (grey) and in the presence of 5 equivalents of EPI-001 (red) or BADGE (green). Dashed lines indicate the onset of agglomeration, and calculated Tc values ​​are shown in inset. b) Tc of different concentrations of AR-NTD in 20 mM Hepes 7.4, 500 mM NaCl, and 2 mM TCEP buffer, determined by TSA assay, in the absence of (grey) and in the presence of 5 equivalents of EPI-001 (red) or BADGE (green). The mean and standard deviation of three independent measurements are plotted.

[0057] Figure 7 Results of molecule screening using agglutination assay. The first two bars show data points corresponding to controls used for activity calculation: negative reference (DMSO) and positive reference (EPI-001, 200 μM). The remaining bars correspond to the activity of the four screening molecules at a screening concentration of 100 μM. An activity cutoff value was set at 25%, which allowed the inactive compound BADGE to be distinguished from the three hit compounds (cyproterone acetate, EPI-001, and EPI-7170). The mean and standard deviation of the three measurements are plotted.

[0058] Figure 8a) A phase diagram is a representation of single-phase and two-phase states (black lines) depending on environmental conditions (such as temperature, pH, ionic strength, etc.). (1) At concentrations below Csat, the system is in a single-phase state. (2) Under any conditions within the two-phase state, the system separates into a light phase (c = cL) and a dense phase (c = cD). (2-4) At increasing concentrations, only the volume fractions of the two phases change relative to each other, but cL and cD remain constant. Adapted from Alberti et al. 2019. b) (1) Below Csat, the polymer is uniformly distributed in the solvent. (2) Above Csat, the system separates into two phases with stronger polymer-polymer interactions. Adapted from Brangwynne et al. 2015. c) There is an unstable region at the interface between the single-phase and two-phase states, where the system separates upon nucleation. At the interface, a metastable, well-mixed state (albeit supersaturated) can persist even if phase separation is thermodynamically favorable. In this case, nucleation is accelerated and determines whether phase separation occurs. Adapted from Posey et al., 2018.

[0059] Figure 9 The results of molecule screening were obtained using agglutination assay, where the amplitude of absorbance was used as a parameter to determine the activity of the test molecules. The first two bars show the data points corresponding to the controls used to calculate the activity: negative reference (DMSO) and positive reference (EPI-001, 200 μM). The remaining bars correspond to the activity of the three screening molecules at a screening concentration of 100 μM. The activity cutoff was set at 25%, which allowed the inactive compound BADGE to be distinguished from the two hit compounds (EPI-001 and EPI-7170). The mean and standard deviation of the three measurements were plotted.

[0060] Figure 10 Phase diagrams for determining additional aggregate solutions based on salt concentration were empirically determined. a) Initial phase diagram with NaCl concentration increments of 200 mM and temperatures ranging from 30°C to 50°C. The temperature at which target protein aggregates formed was determined for each test NaCl condition. At 600 mM NaCl, Tc was within the determined temperature window. b) Fine-tuned phase diagram with NaCl concentration increments of 25 mM and temperatures ranging from 30°C to 50°C. Tc was determined for each test NaCl condition, and regression lines were plotted. Conditions above the regression line allowed for target protein aggregation, while conditions below the regression line kept the target protein in a soluble state.

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[0062] definition

[0063] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly indicates otherwise. Similarly, “or” is used to mean “and / or” unless otherwise stated. Likewise, “comprise,” “comprises,” “comprising,” “include,” “containing,” and “including” are interchangeable and not intended to be restrictive.

[0064] It should be further understood that, where the term “comprising” is used in the description of various embodiments, those skilled in the art will understand that, in some specific instances, the language “consistently consisting of” or “comprises of” can be used instead to describe the embodiments.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While those similar to or equivalent to the methods and materials described herein may be used in the practice of the disclosed methods and compositions, exemplary methods, apparatus, and materials are described herein.

[0066] As used herein, the following words or terms have the following definitions:

[0067] "About" means that the item, parameter, or term is included within a range of plus or minus 10% of the value of the item, parameter, or term.

[0068] As used in this article, “intrinsically disordered protein” is understood as a protein that lacks a defined three-dimensional structure.

[0069] As used in this paper, "altering the aggregation tendency" is understood to mean changing the quantifiable parameters used to describe the properties of aggregates. Specifically, "altering the aggregation tendency" can refer to changing the phase diagram of the system, that is, changing the boundaries between the states on that phase diagram. As an example, for test and reference conditions, when aggregation is observed at different values ​​of specific system parameters (i.e., temperature, protein concentration, salt concentration, or others), this shift in the boundaries between the states can be observed. Conversely, for test and reference conditions, when aggregation is observed at the same values ​​of system parameters, the aggregation tendency does not change.

[0070] As used in this article, "before the formation of aggregates" is understood as a state in which aggregation has not yet occurred, rather than a state in which aggregation is detected after aggregation has been induced by changing system parameters (e.g., changing temperature, salt concentration, pH, or any other parameter that can cause aggregation).

[0071] As used herein, “target” is understood to be any biomolecule, non-limiting examples of which include proteins or nucleic acids that are involved in one or more physiological or pathological processes that can be altered by pharmacological intervention targeting that biomolecule.

[0072] As used herein, “extra-condensate solution” is understood to be a solution containing a buffer, salt, optional reducing agent, and a molecular crowding agent with a predefined salt molar concentration and pH, allowing the initiation of condensation to be observed at the measured temperature.

[0073] As used herein, "spectroscopy-based measurements" are understood to measure the energy or intensity of a beam of light or radiation at a specific wavelength. In particular, the measured parameters are based on the absorption, emission, and / or scattering of electromagnetic radiation by atoms, molecules, and / or ions within a substance.

[0074] As used in this paper, the terms "significant / significantly" and "significantly greater" are understood to mean a low probability of occurrence by chance. This significance can be defined when comparing two groups of data through statistical analysis. For example, the statistical term p represents the percentage probability that an outcome occurs randomly. In this case, a dataset is considered significant when p < 0.05 (which corresponds to a 5% probability of obtaining the outcome by chance).

[0075] As used herein, “EPI-001” is understood to be a small molecule that binds to AR-NTDs and is characterized by in vitro and intracellular activity against castration-resistant prostate cancer, and can be represented by the following structure (I):

[0076]

[0077] As used herein, “EPI-001 analogues” are understood to be compounds belonging to the same family as EPI-001, containing two aromatic rings with or without substitutions linked by 1-atom joints (see WO / 2011 / 082488).

[0078] As used herein, “EPI-7170” is understood to be a small molecule that binds to AR-NTDs and is characterized by in vitro and in vivo activity against castration-resistant prostate cancer, and can be represented by the following structure (II):

[0079]

[0080] As used in this article, the "androgen receptor" ("AR") is understood to be a molecule that is a member of the nuclear receptor protein family. The AR binds to androgens and mediates their physiological effects by transcribedly activating androgen response genes. The wild-type AR reference sequence corresponds to Uniprot accession number P10275 in the Uniprot database.

[0081] As used in this article, the "androgen receptor N-terminal domain" is understood to be the domain of amino acids 1-558 located at the N-terminus of the androgen receptor. The androgen receptor N-terminal domain is an inherently disordered domain that is indispensable for AR transcriptional activation.

[0082] As used herein, "multi-device" is understood to be a device capable of processing multiple samples in an automated manner. Specifically, this device is capable of measuring multiple samples without user intervention. More specifically, a multi-device is a plate reader capable of processing multiple samples located on a reading plate.

[0083] As used herein, “tc” (“critical time”) is understood to be the time measured during a screening assay for drug discovery purposes (the subject of the invention described herein), at which the onset of aggregation is observed. Specifically, the tc parameter is measured from the moment the temperature begins to rise or fall. In particular, a tc equal to 0 will interpret the moment the temperature begins to rise or fall.

[0084] As used herein, "agglomeration onset" is understood as the value of a parameter (such as time or temperature) at which agglomeration occurs. Specifically, agglomeration onset can be determined during a measurement process in which these parameters are being gradually and monotonically changed. In this case, agglomeration is observed after, rather than before, the agglomeration onset. To further illustrate, agglomeration onset should be understood as the value of a parameter (such as time or temperature) at which the measured value of agglomeration begins to increase. As the absorbance of the sample, i.e., the measured value of agglomeration, theoretically, the absorbance is equal to zero before agglomeration onset and will be positive after agglomeration onset. In reality, before agglomeration onset, the absorbance may be positive due to the inherent absorbance of the sample, and may have slight fluctuations due to the randomness of the readings. This fluctuation in the absorbance measured value can be expressed as a standard deviation. In the case where the absorbance value is positive before agglomeration onset, the onset of agglomeration can be determined by obtaining the value of a parameter (such as time or temperature) at which the absorbance begins to exceed the background value and the standard deviation of the fluctuation. The significance of the measured value can be calculated using a p-value of 0.01, 0.05, or 0.1.

[0085] As used in this paper, the “critical temperature” or Tc is understood as the temperature at which a given sample is observed to begin condensation during a gradual increase or decrease in temperature.

[0086] As used in this article, "cyproterone acetate (CPA)" is understood as an anti-androgen and progestin drug used to treat androgen-dependent diseases such as acne, hirsutism, premature puberty, and prostate cancer, as a component of feminizing hormone therapy for transgender women, and in contraceptives.

[0087] As used herein, "significantly altering the reference critical temperature at which the target forms aggregates" should be understood as an event in which the critical temperature of the sample changes relative to a reference, characterized by a difference in the critical temperature value greater than the difference resulting from the variability of the measured value. Specifically, this variability is measured by statistical parameters (such as standard deviation), in which case "significantly altering the reference critical temperature at which the target forms aggregates" would mean a change in the critical temperature greater than the standard deviation of the critical temperature of the reference sample. This does not preclude the use of other statistical parameters for this purpose.

[0088] As used herein, “the amplitude of the reference absorbance signal” is understood to be a value describing the difference between the maximum absorbance value measured after sample agglomeration and the background absorbance value or an approximation thereof measured before sample agglomeration (e.g., a value determined by a mathematical function of the absorbance value before agglomeration).

[0089] As used in this article, "constant rate" is understood as a constant change in parameters over time. Specifically, these parameters can be temperature, in which case changing the temperature at a constant rate is understood as a linear change in temperature over time.

[0090] As used in this article, “N / C interaction” is understood as the interaction between amino acids belonging to the N-terminal and C-terminal domains of the androgen receptor.

[0091] To clarify further, "tc" ("critical time") and "critical temperature" (also known as "Tc") are different. The former refers to the time from the moment the temperature begins to rise or fall until the onset of condensation is observed. The latter describes the temperature at which condensation is observed to begin.

[0092] As used in this article, a “phase diagram” is understood as a graph representing the phase states of a system under the influence of two physicochemical parameters (such as protein concentration, salt concentration, pH, and temperature). The system can be in one of two phase states: (i) single-phase, meaning freely diffuse proteins, or (ii) phase-separated, meaning the presence of biomolecular aggregates in the system. Figure 10 b provides a hypothetical example of a phase diagram.

[0093] describe

[0094] This invention provides a novel drug discovery assay that screens compounds capable of modulating aggregates based on their ability to alter the aggregation tendency of the target under study (preferably an intrinsically disordered protein).

[0095] Based on the data presented in Example 1, some observations led us to realize that the androgen receptor (AR) undergoes biomolecular condensation in cells, and that the disordered N-terminal domain (AR-NTD) of the androgen receptor is sufficient to reproduce this process in vitro. Since the AR-NTD is key to receptor condensation, and EPI-001 is known to bind to the AR-NTD, we hypothesize that the mode of action of EPI-001 involves the regulation of AR condensation capacity. From previous experiments, we know that EPI-001 binding enhances the AR-NTD oligomerization process. This is because EPI-001 promotes and stabilizes conformations that readily self-bind. If these findings are translated into the condensation domain, EPI-001 could reduce the saturation concentration (Csat) of AR-NTD by stabilizing the condensed phase and / or readily condensed conformations.

[0096] To quantify the effect of EPI-001 on AR-NTD phase separation, a novel temperature-dependent turbidity change assay (TSA) was performed, which constitutes one of the main aspects of this invention. Specifically, as shown in Example 2, it was determined whether EPI-001 played a role in the aggregation of AR-NTD in vitro. Imaging of AR-NTD at increased concentrations was performed under phase separation conditions (10% sucrose and 150 mM NaCl) in the absence and presence of 75 μM EPI-001. Figure 5 Without EPI-001, AR-NTD formed droplets at approximately 7.5 μM. With EPI-001, AR-NTD decomposed at 2 μM. To quantify the effect of EPI-001 on AR-NTD phase separation, a temperature-dependent turbidity change assay (TSA) was performed. In this assay, the turbidity of the sample was measured as a report of agglomeration, and the temperature at which a given sample decomposed, i.e., the critical temperature (Tc), was determined. When droplets form, they scatter light, causing a sudden increase in turbidity. Therefore, theoretically, a value above 0 absorbance units (AU) in the TSA assay indicates agglomeration. In practice, a reference value before agglomeration is determined as the absorbance of the sample before the temperature rise begins. Values ​​above this reference value will indicate agglomeration. Turbidity was measured as the apparent absorbance at the wavelength where the absorption of other components in the sample is minimal (350 nm in this case). Adding 5 equivalents (eq) of EPI-001 to samples of 30 μM AR-NTD and 500 mM NaCl lowered the Tc of AR-NTD by 10 °C. Figure 6 a). This is related to the imaging data ( Figure 5This is consistent with and reinforces the finding that EPI-001 promotes AR-NTD condensation.

[0097] Therefore, the above data show that, among other facts, the TSA assay of the present invention constitutes a novel drug discovery assay (preferably cell-free) that allows for the identification of compounds capable of altering the aggregation tendency of the target under study (preferably an inherently disordered protein). Furthermore, as Figure 3 As shown, the ability of TSA assays to identify compounds that can alter the aggregation tendency of the studied target (preferably an intrinsically disordered protein) opens up the possibility of finding improved new inhibitors for such targets.

[0098] Therefore, a first aspect of the present invention relates to a drug discovery assay, preferably a cell-free assay, for screening compounds capable of altering the aggregation tendency of a target under study (preferably an intrinsically disordered protein), the assay comprising:

[0099] a. The test compound is combined with a composition containing a target prior to the formation of aggregates, preferably wherein the target is in an additional aggregate solution prior to the formation of aggregates;

[0100] b. By gradually and in a controlled manner increasing (preferably at a constant rate) the temperature of the solution in a), a target agglomerate is formed in the presence of the test compound, wherein such gradual and controlled increase (preferably at a constant rate) of the temperature of the solution in a) is preferably carried out at any rate selected from 0.5 to 5 °C / min, more preferably 0.8 to 2 °C / min;

[0101] c. While gradually and controlledly increasing the temperature according to step b), measure the solution in a) by any spectroscopic measurement (preferably by measuring absorbance) to determine whether the test compound alters the reference critical temperature for target agglomeration, wherein the reference critical temperature for target agglomeration is understood to be the temperature at which the target agglomerates form in the absence of the test compound, preferably in an additional agglomeration solution; and

[0102] If the test compound significantly alters the reference critical temperature at which the target forms aggregates, or if the percentage change caused by the test compound is significantly greater than the reference change caused by the reference compound, preferably greater than at least two or three standard deviations of the average activity value of the reference compound, preferably greater than about 20%, 25%, 30%, 35%, or 40% of the change caused by the reference compound, then the test compound is selected as a compound capable of altering the aggregation tendency of the target under study.

[0103] In another preferred embodiment of the first aspect of the present invention, the cell-free assay includes:

[0104] a) Prior to the formation of aggregates, a multi-device is used to combine compounds from a variety of test compounds (test compound library) with a composition containing a target in each well of the multi-device, preferably wherein the target is in an additional aggregate solution prior to the formation of aggregates;

[0105] b) By gradually and in a controlled manner increasing (preferably at a constant rate) the temperature of the multiple devices, a target agglomerate is formed in the presence of the test compound. This gradual and controlled increase (preferably at a constant rate) of the temperature of the solution in a) is preferably carried out at any rate selected from 0.5 to 5 °C / min, more preferably 0.8 to 2 °C / min.

[0106] c) While gradually and controlledly increasing the temperature according to step b), measure the solution in a) by any spectroscopic measurement (preferably by measuring absorbance) to determine whether the test compound alters the reference critical temperature for target agglomeration, wherein the reference critical temperature for target agglomeration is understood to be the temperature at which target agglomeration occurs in each well in the absence of the test compound, preferably in an additional agglomeration solution; and

[0107] If a test compound significantly alters the reference critical temperature at which the target forms aggregates, or if the percentage change caused by the test compound is significantly greater than the reference change caused by the reference compound, preferably greater than at least two or three standard deviations of the average activity value of the reference compound, preferably greater than 20%, 25%, 30%, 35%, or 40% of the change caused by the reference compound, then the test compound is selected as one of those compounds capable of altering the aggregation tendency of the target under study.

[0108] Note that, as defined in the examples, the additional agglomerate solution should be understood as a solution containing a buffer, salt, optional reducing agent, and a molecular congestant with a predefined salt molar concentration and pH, allowing the onset of agglomeration to be observed at the assay temperature. More specifically, in a preferred embodiment, the additional agglomerate solution may be different for each protein target and can be assayed as follows:

[0109] The selected protein target is initially used in a solution (“I-solution”) in which the protein has previously been purified, depending on the protein’s manufacturer. The minimum components of this I-solution are determined based on the manufacturer’s experience and typically contain water, a buffer (such as NaP (sodium phosphate)), a salt (such as NaCl), and a reducing agent (such as TCEP (tris(2-carboxyethyl)phosphine)), adjusted to a specific pH determined by the manufacturer based on the isoelectric point of the protein target.

[0110] - In cases where the I-solution is unknown or the protein target is lyophilized and dissolution is unlikely, an I-solution containing the following components can be prepared, with the pH adjusted to be one point higher or lower than the isoelectric point of the target protein:

[0111] ○H2O

[0112] ○ Buffer solution, such as Tris-HCl or NaP, 10mM to 50mM

[0113] ○ Salt, preferably NaCl or KCl, 0 to 500 mM

[0114] ○ Reducing agent, such as TCEP, 1mM

[0115] - Since salt is a key component of the additional aggregate solution that regulates the initiation of aggregate formation of target proteins, the salt concentration of the additional aggregate solution needs to be adjusted empirically. To determine the salt concentration of the additional aggregate solution, the protein in the I- solution should be tested with an initial range of salt concentrations (e.g., NaCl between 0 M and 2 M, in 200 mM increments). For different NaCl conditions, the aggregated state of the protein should be observed at elevated temperatures (e.g., between 30°C and 50°C) using any method known to a person skilled in the art (e.g., microscopy, absorbance measurement, or visual assessment of sample turbidity). The temperature at which aggregates form, Tc, should be noted. For some salt concentrations, the sample will not form aggregates at the test temperature, or conversely, aggregates may appear at the lowest test temperature; both cases should be noted. Based on this, a phase diagram known to a person skilled in the art can be plotted. Figure 10 a). Subsequently, a narrower range of salt concentrations around the detected Tc value within the measurement window should be selected to determine the linear regression line between the tested salt concentration and the observed Tc. Figure 10 b). The regression line and its linear equation (Y = b*X + a, where "Y" is Tc, "X" is the NaCl concentration, "b" is the slope of the line, and "a" is the intercept) represent the phase boundary (the boundary between non-aggregation and aggregation of the target protein).

[0116] Based on the phase diagram and the derived linear equation, the final salt concentration of the additional aggregate solution should be selected as the concentration at the boundary (represented by the regression line) between the 1-phase (non-agglomerated) and 2-phase (agglomerated) states. This temperature corresponds to the median value of the temperature ramp, which will be further used when performing the assay.

[0117] In summary, an additional aggregate solution can be defined as a target-specific solution derived from the I-solution (the solution that stores the protein target), typically comprising a certain molar concentration of buffer and reducing agent, as well as a precise salt concentration empirically determined via an aggregate phase diagram, thereby allowing the determination of the critical salt concentration required to observe target protein aggregation within a given temperature range.

[0118] The appropriate additional aggregate solution determined by the above experience can be easily determined by a technician following the above instructions. As a first example, the AR-NTD protein is initially used in its purified I-solution, i.e., 20 mM NaP buffer, 1 mM TCEP, pH 7.4. The AR-NTD is concentrated to the desired concentration, such as 20 μM, 25 μM, or 30 μM, and the phase diagram is determined by adding NaCl in a multi-stage setting (e.g., a 96-well plate) at concentrations ranging from 0 M to 2 M, in 200 mM increments. The presence or absence of AR-NTD aggregates under these conditions is observed by measuring the absorbance of the sample, increasing from 30 °C to 50 °C. Based on these measurements, an initial phase diagram is obtained, showing whether the system is in a 1-phase state (no aggregates) or a 2-phase state (aggregates present). In the case of AR-NTD, the salinity of the additional aggregate solution should be around 500 mM, because at this concentration of NaCl, aggregates of the target protein occur within the test temperature range, which is 40 °C in this case. Further fine-tuning of the NaCl concentration is required by testing several NaCl concentrations near selected values ​​(e.g., 500 mM, 525 mM, 550 mM, 575 mM, and 600 mM in this case) and observing the aggregation state of AR-NTDs. This fine-tuning of NaCl allows for the plotting of a linear regression line representing the phase boundary. In the case of AR-NTDs, the regression line equation is Y = -0.1301*X + 112.7. This equation should be used to find the precise NaCl concentration at 40 °C (the midpoint of the temperature ramp). Following this procedure, the final additional aggregate solution for AR-NTDs is a 20 mM NaP buffer containing 1 mM TCEP, 558 mM NaCl, and pH 7.4. Following the same procedure, the additional aggregate solution for progesterone receptor (PR) NTDs is determined to be a 20 mM NaP buffer containing 1 mM TCEP, 1158 mM NaCl, and pH 7.4. In this second example, aggregation of the target protein in the initial phase diagram was observed near 1100 mM NaCl. Phase diagram fine-tuning was performed using NaCl concentrations around 1100 mM (1025 mM, 1050 mM, 1075 mM, 1100 mM, and 1125 mM). Observing PR-NTD aggregation under these conditions allowed for the plotting of the fine-tuned phase diagram, with a linear regression line and equation of Y = -0.070*X + 121.1. This equation was used to find the precise NaCl concentration in the additional aggregate solution at 40 °C, which was 1158 mM NaCl.

[0119] Specific, non-limiting examples of additional aggregate solutions are shown as NaP buffer containing 500 mM NaCl and 1 mM TCEP at pH 7.4, or HEPES buffer containing 150 mM NaCl, 1 mM TCEP, and 10% (v / v) sucrose at pH 7.6.

[0120] In a preferred embodiment of the first aspect of the invention, the gradual and controlled increase in temperature at a constant rate is preferably selected from any constant rate of 0.1 to 5 °C / min, more preferably 0.3 to 3 °C / min, more preferably 0.5 to 1.5 °C / min, still more preferably 0.9 to 1.1 °C / min, and even more preferably by about 1 °C.

[0121] In another preferred embodiment of the first aspect of the invention or any preferred embodiment thereof, the spectroscopic measurement is selected from a list consisting of absorbance, fluorescence, fluorescence polarization, radiation, colorimetry, luminescence or light scattering measurements.

[0122] Another preferred embodiment of the first aspect of the invention or any of its preferred embodiments further includes labeling the target to aid in the visualization of the target aggregates, for example using radioactive labels, colorimetric labels, enzyme fragments, fluorescent labels or other labels.

[0123] In another preferred embodiment of the first aspect of the invention or any preferred embodiment thereof, the composition comprises two or more biomolecules / proteins, at least one of which is a target.

[0124] In another preferred embodiment of the first aspect of the invention or any preferred embodiment thereof, the amount of the test compound may be further determined by, for example, mass spectrometry, liquid chromatography and / or ultraviolet-visible spectrophotometry.

[0125] In another preferred embodiment of the first aspect of the invention or any preferred embodiment thereof, the target is a protein or is contained in a protein selected from the list of TAR DNA-binding protein 43 (TDP-43), tau protein, members of the myc family of transcription factors, sarcoma fusion protein (FUS), hnRNPAI, PGL-3, huntingtin protein, androgen receptor (AR), and other members of nuclear receptor protein families, or any fragment thereof. Preferably, the target is the androgen receptor N-terminal domain (AR-NTD).

[0126] In another preferred embodiment of the first aspect of the invention or any preferred embodiment thereof, the target is the androgen receptor N-terminal domain (AR-NTD), and the reference compound is compound EPI-001 or an analogue thereof. Furthermore, in the context of the invention, the additional aggregate solution contains NaCl and has a pH between 7.0 and 7.8, optionally contains a cryopreservative, and the critical temperature for detecting the AR-NTD is within a measurement window between 30°C and 49°C.

[0127] In an alternative embodiment of the first aspect of the invention or any preferred embodiment thereof, a drug discovery assay (preferably a cell-free assay) for screening compounds capable of altering the aggregation tendency of a target under study (preferably an intrinsically disordered protein) includes measuring the solution in a) of the first aspect of the invention by any spectroscopic measurement (preferably by measuring absorbance) while gradually and controlledly increasing the temperature in step b) according to the first aspect of the invention, to determine whether the test compound alters a reference tc value at which the target forms aggregates, wherein the reference tc value is, in the absence of the test compound, preferably in an additional aggregate solution, the time between the start of gradually and controlled increasing the temperature of the solution in a) and the start of aggregation under the conditions for target aggregate formation; and

[0128] If the test compound significantly alters the reference tc value when the target forms aggregates, or if the percentage change caused by the test compound is significantly greater than the reference change caused by the reference compound, preferably greater than at least two or three standard deviations of the average activity value of the reference compound, preferably greater than about 20%, 25%, 30%, 35%, or 40% of the change caused by the reference compound, then the test compound is selected as a compound capable of altering the aggregation tendency of the target under study.

[0129] Preferably, a cell-free drug discovery assay for screening compounds capable of altering the aggregation tendency of a target under study (preferably an intrinsically disordered protein) includes, according to step b) of the first aspect of the invention, measuring the absorbance of each well in a multi-device while gradually and controlledly increasing the temperature to determine whether one or more test compounds alter a reference tc value for target aggregation, wherein the reference tc value is, preferably in an additional aggregate solution, the time between the start of gradual and controlled temperature increase in the multi-device and the onset of aggregation in each well under target aggregation conditions, in the absence of test compounds; and

[0130] If a test compound significantly alters the reference tc value at which the target forms aggregates, or if the percentage change caused by the test compound is significantly greater than the reference change caused by the reference, preferably greater than at least two or three standard deviations of the average activity value of the reference compound, preferably greater than 20%, 25%, 30%, 35%, or 40% of the change caused by the reference compound, then the test compound is selected as one of those compounds capable of altering the aggregation tendency of the target under study.

[0131] That is, in this alternative embodiment, the tc value is measured instead of the critical temperature reflected in the initial embodiment of the first aspect of the invention.

[0132] In another alternative embodiment of the first aspect of the invention, a cell-free drug discovery assay for screening compounds capable of altering the aggregation tendency of a target under study (preferably an intrinsically disordered protein) includes measuring the solution in step a) of the first aspect of the invention by any spectroscopic measurement (preferably by measuring absorbance) while gradually and controllably increasing the temperature according to step b) of the first aspect of the invention, to determine whether the test compound alters the amplitude of a reference absorbance signal at which the target forms aggregates, wherein the amplitude of the reference absorbance signal is described as the maximum absorbance value measured after sample aggregation and the absorbance value measured before sample aggregation. The value of the difference between the background absorbance value or its approximation (e.g., a value determined by fitting a mathematical function to the absorbance value prior to agglomeration); and the value of the difference between the background absorbance value and its approximation (e.g., a value determined by fitting a mathematical function to the absorbance value prior to agglomeration); and the value of the difference between the background absorbance value and the value of the reference absorbance signal when the test compound significantly alters the amplitude of the reference absorbance signal when the target agglomerates, or the value of the percentage change caused by the test compound is significantly greater than that caused by the reference compound, preferably greater than at least two or three standard deviations of the average activity value of the reference compound, preferably greater than about 20%, 25%, 30%, 35%, or 40% of the change caused by the reference compound, in which case the test compound is selected as a compound capable of altering the agglomeration tendency of the target under study. Note that, with reference to this aspect of the invention, the inventors have shown that this parameter can also be used to select the hit compound (see [link to invention]). Figure 9 ).

[0133] That is, in this alternative embodiment, the amplitude of a reference absorbance signal is measured instead of the critical temperature reflected in the initial embodiment of the first aspect of the invention.

[0134] In another alternative embodiment of the first aspect of the invention or any preferred or alternative embodiment thereof, a cell-free drug discovery assay for screening compounds capable of altering the aggregation tendency of the target under study (preferably an intrinsically disordered protein) comprises measuring the solution in a) of the first aspect of the invention by any spectroscopic-based measurement (preferably by measuring absorbance), but instead of step b) of the first aspect of the invention or any preferred or alternative embodiment thereof, a gradual and controlled increase in temperature is performed by gradually and controlledly increasing the salt in the sample, or by gradually and controlledly increasing the pH of the sample, or by gradually and controlledly increasing the molecular congesting agent in the sample. That is, in this alternative embodiment, instead of step b) of the first aspect of the invention or any preferred or alternative embodiment thereof, a gradual and controlled increase in temperature is performed by gradually and controlledly increasing the salt in the sample, or by gradually and controlledly increasing the pH of the sample, or by gradually and controlledly increasing the molecular congesting agent in the sample.

[0135] A second aspect of the invention relates to a method for ranking or classifying multiple test compounds based on their ability to alter the aggregation tendency of a target under study, the method comprising:

[0136] a. By using the method of the first aspect of the invention or any preferred or alternative embodiment of the first aspect of the invention, determining the percentage change caused by each compound compared to a reference change; and

[0137] b. Sort or classify the various test compounds according to the percentage change caused by each compound.

[0138] Preferably, the second aspect of the method includes determining the activity of the compound in a dose-response manner. Specifically, this includes the ability to determine additional parameters (such as EC50).

[0139] A third aspect of the present invention relates to a method for selecting or identifying one or more test compounds based on their ability to alter the aggregation tendency of a target under study, the method comprising:

[0140] a. By using the method of the first aspect of the invention or any preferred or alternative embodiment of the first aspect of the invention, determining the percentage change caused by each compound compared to a reference change; and

[0141] b. Identify the compound with the highest degree of alteration.

[0142] A fourth aspect of the invention relates to a method for preparing one or more test compounds based on the ability of the test compounds to alter the aggregation tendency of a target under study, the method comprising, based on data provided by the method for identifying one or more test compounds according to a third aspect of the invention, preparing a composition comprising the one or more test compounds, wherein the concentration of the one or more test compounds is such that the compounds are capable of altering the aggregation tendency of the target under study.

[0143] The following embodiments are for illustrative purposes only and do not limit the scope of the invention.

[0144] *********************************************************************

[0145] Example

[0146] Example 1. Biomolecular condensation of androgen receptors

[0147] During the course of this invention, it was discovered that the N-terminal domain (AR-NTD) of the androgen receptor underwent biomolecular condensation in vitro and intracellularly.

[0148] Intracellularly, androgen receptors (ARs) aggregate upon binding to the hormone (dihydrotestosterone, DHT). Figure 1 a). In some cell lines, overexpressed eGFP-AR remains soluble in the cytoplasm. The addition of 1 nM DHT causes eGFP-AR to condense into nuclei in the cytoplasm. Based on fluorescence recovery after photobleaching (FRAP) experiments, the AR condensates exhibit liquid-like properties because they are spherical, fuse and release into spheres within seconds, and are highly dynamic. Figure 1 b). 30 to 60 minutes after the hormone increase, eGFP-AR translocates to the nucleus, consistent with the classical AR pathway. In fact, i) AR aggregation occurred after DHT addition, and ii) DHT addition led to... 23 FQNLF 27 The interaction with the AR ligand binding domain (AR-LBD) indicates that the N / C interaction plays a role in AR condensation.

[0149] The purified AR-NTD (AR1-558) underwent LCST-type phase separation in vitro. Figure 2 a). AR-NTD coagulation is driven by hydrophobic and aromatic interactions. This was determined by plotting the dependence of AR-NTD coagulation on the ionic strength of the buffer solution (NaCl concentration). Figure 2b). To determine which regions of the AR-NTD were involved in the oligomerization and phase separation processes, solution nuclear magnetic resonance (NMR) spectroscopy of the light phase was used. Figure 2 c). Obtaining AR-NTD upon increasing protein concentration. 1 H, 15 N HSQC spectrum. The AR-NTD region exhibits a concentration-dependent decrease in signal intensity. 15 The increased lateral relaxation rate of N indicates that concentration leads to more transient long-range interactions between residues. Specifically, the most affected regions correspond to the following motifs: 23 FQNLF 27 Motif, the helix preceding the polyQ chain 54 LLLL 57 The sequence includes the motifs of two partial helices (R1 and R2) in the Tau-5 region, and a highly hydrophobic aromatic region following the polyG chain. The polyQ chain and the R3 helical region in Tau-5 were not detected.

[0150] However, AR-NTD droplets were observed to mature rapidly in vitro (within minutes) as fusion events were prevented, resulting in "worm-like" droplet strings. Figure 3 a) This demonstrates that the liquid-to-solid transition is due to 23 FQNLF 27 Motifs tend to aggregate. In fact, when 23 FQNLF 27 Fiber formation is affected by the Leu to Pro mutation ( 23 FQNPF 27 During interference, the AR-NTD-L26P droplet maintained its liquid properties throughout the experiment. Figure 3 b). This mutation also affects the aggregation tendency of AR-NTD, as the AR-NTD-L26P mutant has a lower tendency for phase separation ( Figure 3 c). Finally, since most of the viscous motifs in AR-NTDs identified by NMR were predicted to be partially helical, we investigated the importance of condensation for the secondary structure tendency of these motifs. This is based on the hypothesis that the dry environment in AR-NTD condensates promotes helical increase of motifs important for AR function and its protein-protein interactions. Indeed, the addition of 2,2,2-trifluoroethanol (TFE) to AR-NTD-L26P increased its helical population ( Figure 4 a) and its cohesive tendency ( Figure 4 b). Furthermore, the abrupt breakage of the additional helical strand in the AR-NTD-L26P helix leads to a reduced tendency for phase separation.

[0151] Interestingly, the three helical motifs (R1, R2, and R3) in Tau-5 had the greatest impact on condensation, indicating that the helicity of the Tau-5 region contributes significantly to AR-NTD condensation. Figure 4 c). These observations led to the “condensation-after-folding” model, in which condensation promotes the recruitment of transcriptional mechanisms and coactivators to AR transcriptocondensates.

[0152] *********************************************************************

[0153] Based on the above data, some observations lead us to recognize that the androgen receptor (AR) undergoes biomolecular condensation within cells, and that the disordered N-terminal domain (AR-NTD) of the androgen receptor is sufficient to reproduce this process in vitro. Since the AR-NTD is key to receptor condensation, and EPI-001 is known to bind to the AR-NTD, we hypothesize that the mode of action of EPI-001 involves the regulation of AR condensation capacity. Previous experiments have shown that EPI-001 binding enhances the oligomerization process of the AR-NTD. This is because EPI-001 promotes and stabilizes conformations that readily bind. If these findings are translated into the condensation domain, EPI-001 may reduce the saturation concentration (Csat) of the AR-NTD by stabilizing the condensed phase and / or readily condensed conformations.

[0154] To quantify the effect of EPI-001 on AR-NTD phase separation, a novel temperature-dependent turbidity change assay (TSA) was performed, which constitutes the main objective of this invention. This assay is fully described in Example 2 below.

[0155] Example 2. In vitro regulation of AR-NTD aggregation by EPI-001

[0156] -EPI-001 reversible binding promotes the aggregation of AR-NTDs.

[0157] First, the effect of EPI-001 on the aggregation of AR-NTD in vitro was determined. Imaging of AR-NTD at increased concentrations was performed under phase-separated conditions (10% sucrose and 150 mM NaCl) in the absence and presence of 75 μM EPI-001. Figure 5Without EPI-001, AR-NTD formed droplets at approximately 7.5 μM. In the presence of EPI-001, AR-NTD stratified at 2 μM. This indicates that EPI-001 promotes AR-NTD aggregation. To assess whether EPI-001 could modulate the aggregation of any protein of interest, we performed the same experiment using sarcoma fusion protein (FUS) as a model. We observed that EPI-001 did not affect the aggregation ability of FUS. Figure 5 b).

[0158] To quantify the effect of EPI-001 on the phase separation of AR-NTD, a temperature-dependent turbidity change assay (TSA) was performed. In this assay, the turbidity of the sample was measured as a report of agglomeration, and the temperature at which a given sample would separate, i.e., the critical temperature, was determined. When droplets form, they scatter light, causing a sudden increase in turbidity. Therefore, values ​​above 0 absorbance units (AU) in the TSA assay indicate agglomeration. Turbidity was measured as the apparent absorbance at the wavelength where absorption by other components in the sample is minimal (350 nm in this case). Adding 5 equivalents (eq) of EPI-001 to 30 μM AR-NTD and 500 mM NaCl samples lowered the Tc of AR-NTD by 10 °C. Figure 5 a). This is related to the imaging data ( Figure 5 This finding is consistent with and reinforces the discovery that EPI-001 promotes AR-NTD aggregation. In contrast, the inactive analogue BADGE only reduced the Tc of AR-NTD by 3°C. Figure 6 We performed TSA assays at different protein concentrations to construct the left arm of the phase diagram. Figure 6 b). We observed that EPI-001, but not BADGE, significantly enhanced AR-NTD coagulation at all tested concentrations.

[0159] Interestingly, in the context of another work, Posey et al. used a multiphase linkage framework to describe the effect of ligands on protein phase separation. Applying their conclusions to this study, the fact that EPI-001 reduces the Tc of AR-NTD is equivalent to establishing that EPI-001 preferentially partitions into the condensed phase of AR-NTD. If the compound interacts equally with both phases, the Tc is unaffected, as was the case with BADGE.

[0160] This embodiment demonstrates, among other things, that the TSA assay constitutes a novel cell-free drug discovery assay that allows for the identification of compounds capable of altering the aggregation tendency of a target under study (preferably an intrinsically disordered protein).

[0161] Example 3. In vitro regulation of AR-NTD aggregation by EPI-001 analogues

[0162] Newly acquired knowledge about the ability of EPI-001 to enhance AR aggregation and the ability of TSA assays to screen for targets (preferably intrinsically disordered proteins) that alter the aggregation tendency of the studied, opens up the possibility of finding improved new inhibitors targeting the androgen receptor N-terminal domain (AR-NTD) for the treatment of castration-resistant prostate cancer (CRPC).

[0163] For this purpose, we further tested the TSA assay of the present invention to determine whether molecules known to be used to treat androgen-dependent diseases (such as cyproterone acetate or EPI-001 analogs, such as EPI-7170) can be identified as inhibitors targeting the androgen receptor N-terminal domain (AR-NTD) for the treatment of castration-resistant prostate cancer (CRPC) by using the TSA assay of the present invention.

[0164] In this sense, Figure 7 In the middle, the first two bars show the data points corresponding to the control used to calculate activity:

[0165] -Negative Reference–DMSO

[0166] -Positive reference-200μM EPI-001

[0167] The remaining columns correspond to the activity of the four screening molecules at a screening concentration of 100 μM. The activity cutoff value was set to 25%, which allowed the non-hit compound BADGE to be distinguished from the three hit compounds (cyproterone acetate, EPI-001, and EPI-7170).

[0168] In short, to quantitatively rank the efficacy of the test compounds in enhancing AR-NTD aggregation in vitro and compare them with the efficacy of EPI-001, we measured the cloud point temperature (Tc) of each sample at a constant protein concentration (100 μM). We then calculated the percentage change caused by each compound compared to the reference change caused by 200 μM EPI-001. Next, we plotted the normalized change against compound concentration.

[0169] In summary, this example demonstrates that the ability of TSA assays to identify compounds that can alter the aggregation tendency of the studied target (preferably an intrinsically disordered protein) opens the possibility of finding improved new inhibitors targeting the androgen receptor N-terminal domain (AR-NTD) for the treatment of castration-resistant prostate cancer (CRPC).

Claims

1. A drug discovery assay for screening compounds capable of altering a critical temperature (Tc) at which the target under study forms aggregates, wherein the assay is a cell-free assay, the target is an intrinsically disordered protein, and the assay comprises: a. The test compound is combined with a composition containing the target prior to the formation of the aggregate; b. By gradually and in a controlled manner increasing the temperature of the solution in a), target agglomerates are formed in the presence of the test compound, wherein such gradual and controlled increase in the temperature of the solution in a) is carried out at any rate selected from any value from 0.5 to 5 °C / min; c. While gradually and controlledly increasing the temperature according to step b), measure the solution in a) by arbitrary spectroscopic measurements to determine whether the test compound alters the reference critical temperature at which the target forms the agglomerates, wherein the reference critical temperature at which the target forms the agglomerates is understood to be the temperature at which the target forms the agglomerates in the absence of the test compound; and The test compound is selected as a compound capable of altering the aggregation tendency of the target if it significantly changes the reference critical temperature at which the target forms the aggregates, or if the percentage change caused by the test compound is significantly greater than the reference change caused by the reference compound.

2. The determination method according to claim 1, wherein the determination method comprises: a. Prior to forming the aggregate, a multi-device is used to combine a variety of test compounds, i.e., compounds from a test compound library, with the composition containing the target in each well of the multi-device; b. By gradually and in a controlled manner increasing the temperature of the multiple devices, a target agglomerate is formed in the presence of the test compound, wherein this gradual and controlled increase in the temperature of the solution in a) is carried out at any rate selected from any value from 0.5 to 5 °C / min; c. While gradually and controlledly increasing the temperature according to step b), measure the solution in a) by arbitrary spectroscopic measurements to determine whether the test compound alters the reference critical temperature at which the target forms the aggregates, wherein the reference critical temperature at which the target forms the aggregates is understood as the temperature at which the target forms the aggregates in each well in the absence of the test compound; and The test compound is selected as one of those compounds capable of altering the aggregation tendency of the target if it significantly changes the reference critical temperature at which the target forms the aggregates, or if the percentage change caused by the test compound is significantly greater than the reference change caused by the reference compound.

3. A drug discovery assay for screening compounds capable of altering a critical time value (tc) at which the target under study forms an aggregate, wherein the assay is a cell-free assay, the target is an intrinsically disordered protein, and the assay comprises: a. The test compound is combined with a composition containing the target prior to the formation of the aggregate; b. By gradually and in a controlled manner increasing the temperature of the solution in a), target agglomerates are formed in the presence of the test compound, wherein such gradual and controlled increase in the temperature of the solution in a) is carried out at any rate selected from any value from 0.5 to 5 °C / min; c. While gradually and controlledly increasing the temperature according to step b), measure the solution in a) by any spectroscopic measurement to determine whether the test compound alters the reference tc value for target agglomeration, wherein the reference tc value is the time between the start of gradually and controlled increasing the solution temperature in a) and the start of agglomeration under conditions where the test compound is absent; and If the test compound significantly alters the reference tc value for the formation of the aggregates by the target, or if the percentage change caused by the test compound is significantly greater than the reference change caused by the reference compound, then the test compound is selected as a compound capable of altering the aggregation tendency of the target under study. The critical time value (tc) is understood as the time from the moment the temperature begins to rise, i.e., from the moment the temperature of the solution in a) is gradually and controlledly increased, until the onset of coagulation is observed.

4. The determination method according to claim 3, wherein the determination method comprises: a. Prior to forming the aggregate, a multi-device is used to combine a variety of test compounds, i.e., compounds from a test compound library, with the composition containing the target in each well of the multi-device; b. By gradually and in a controlled manner increasing the temperature of the multiple devices, a target agglomerate is formed in the presence of the test compound, wherein this gradual and controlled increase in the temperature of the solution in a) is carried out at any rate selected from any value from 0.5 to 5 °C / min; c. While gradually and controlledly increasing the temperature according to step b), measure the absorbance of each well of the multi-device to determine whether the test compound alters the reference tc value for target agglomeration, wherein the reference tc value is the time between the start of gradually and controlled increasing the temperature of the multi-device and the start of agglomeration in each well under the condition that the target agglomerates in the absence of the test compound; and The test compound is selected as one of those compounds capable of altering the aggregation tendency of the target if it significantly changes the reference tc value when the target forms the aggregate, or if the percentage change caused by the test compound is significantly greater than the reference change caused by the reference compound.

5. The determination method according to any one of claims 1 to 3, wherein the solution in a) is measured by measuring absorbance.

6. The determination method according to any one of claims 1 to 4, wherein if the percentage change caused by the test compound is greater than at least two or three standard deviations of the average activity value of the reference compound, the test compound is selected as a compound capable of altering the aggregation tendency of the target under study.

7. The determination method according to any one of claims 1 to 4, wherein if the percentage change caused by the test compound is greater than 20%, 25%, 30%, 35%, or 40% of the change caused by the reference compound, then the test compound is selected as a compound capable of altering the aggregation tendency of the target under study.

8. The determination method according to any one of claims 1 to 4, further comprising labeling the target with a radioactive marker, a colorimetric marker, or a fluorescent marker to visualize the target aggregate.

9. The assay according to any one of claims 1 to 4, wherein the composition comprises two or more targets.

10. The determination method according to any one of claims 1 to 4, wherein the amount of the test compound is determined by mass spectrometry, liquid chromatography and / or ultraviolet-visible spectrophotometry.

11. The assay according to any one of claims 1 to 4, wherein the target comprises a protein selected from the list consisting of TAR DNA-binding protein 43 (TDP-43), tau protein, fusion sarcoma protein (FUS), hnRNPAI, PGL-3, huntingtin protein, and androgen receptor (AR) or any fragment thereof including the androgen receptor N-terminal domain (AR-NTD).

12. The assay according to claim 11, wherein the target is the androgen receptor N-terminal domain (AR-NTD).

13. The assay according to claim 12, wherein the target is the androgen receptor N-terminal domain (AR-NTD), and the reference compound is compound EPI-001.

14. A method for ranking or classifying said multiple test compounds based on their ability to alter the aggregation tendency of a target under study, said method comprising: a. Determining the percentage change caused by each compound compared to a reference change by using the determination method according to any one of claims 1 to 13; as well as b. Sort or classify the various test compounds according to the percentage change caused by each compound.

15. A method for selecting or identifying one or more test compounds based on their ability to alter the aggregation tendency of a target under study, the method comprising: a. Determining the percentage change caused by each compound compared to a reference change by using the determination method according to any one of claims 1 to 13; as well as b. Identify the compound with the highest degree of alteration.

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