Solvent induced precipitation (SIP) assays using specific solvent combinations

Abstract

The present invention provides improved methods for investigating protein-ligand binding interactions through the detection of solvent induced shifts of protein precipitation (SIP) using non-purified proteins, protein lysates or intact cells. The solvents used include at least one of NMP, DMA or ethylene glycol and improve reproducibility by avoiding having a high volatility.

Description

[0001] SOLVENT INDUCED PRECIPITATION (SIP) ASSAYS USING SPECIFIC SOLVENT COMBINATIONS

[0002] FIELD OF THE INVENTION

[0003] The present disclosure relates to improved methods investigating protein-ligand binding interactions through the detection of solvent induced shifts of protein precipitation using nonpurified proteins, protein lysates or intact cells. Further, the disclosure relates to improved methods for determining direct binding of a ligand to a non-purified target protein, comprising an incubation of the non-purified proteins, lysates or intact cells with the ligand prior to an incubation of this mixture with a denaturing solvent composition and subsequent separation and quantification of soluble proteins via immunodetection or mass spectrometry.

[0004] BACKGROUND OF THE INVENTION

[0005] The detection of direct binding of ligands to their target proteins is of high importance in biological and medical research. It is of high relevance for the discovery of new bioactive chemical compounds and for their development into effective and safe drugs. Monitoring ligand-protein interactions does not only allow detection and characterization of the main target of a drug, but also allows to get deeper insights into its mode of action. This process is crucial in the early stages of drug discovery where potential therapeutics are screened for their affinity to bind to specific proteins associated with a disease. The identification of ligand interactions with protein targets allows for the discernment of binding dynamics, including the strength and specificity of the interaction. This information is vital to fine-tune the chemical structure of the ligand, to improve its effectiveness and reduce potential side effects. Furthermore, understanding the ligand-protein interaction provides a foundation for elucidating the biological mechanisms involved in the disease.

[0006] Unbiased detection of direct ligand protein interactions in cell lysates or intact cells allows identification of additional protein interactions (so called off-targets), and thereby early detection of potential unwanted adverse effects of a developmental drug candidate. The identification of off-targets involves the recognition of unintended interaction between a drug and proteins other than the primary target. Such interactions can lead to side effects or adverse drug reactions, which are critical considerations for the safety profile of therapeutic agents. Early assessment and identification of off-targets is thus integral to the optimization of drug candidates.

[0007] Interaction of a ligand with a protein often leads to protein stabilization. Therefore, ligand-bound proteins are often less sensitive towards protease-hydrolysis, oxidative denaturation, heat denaturation or chemical denaturation. In the past years, several approaches have been developed to detect these stabilization effects in non-purified protein samples, such as drug affinity target stability assay (DARTS), stability of proteins from rates of oxidation (SPROX), cellular thermal shift assay (CETSA), thermal proteome profiling (TPP) and solvent induced shift assay (SIP).

[0008] DARTS is based on the higher resistance of proteins towards proteolysis upon binding of a ligand and this approach has been successfully used to identify the targets of several chemical compounds including rapamycin and resveratrol (MIMB, 2213, 175-182, 2021). SPROX exploits changes of the thermodynamic protein stability upon ligand binding, which is measured via the oxidation rates of methionine-containing residues as a function of the chemical denaturant concentration (Nat Protoc, 8, 148-161 , 2013). CETSA and TPP are based on the detection of ligand-induced changes in thermal protein stability. This approach was first described by Daniel Molina in 2012 (WO2012 / 143714 A1) and intensively used in drug discovery research to determine ligand-binding to a non-purified target protein. Here, the ligand of interest is incubated with the non-purified protein prior to treatment of sub-samples with different temperatures. Afterwards, the soluble proteins are separated via centrifugation and quantified via immunoblotting (CETSA) or mass spectrometry (TPP) to obtain protein melting curves (Nat Protoc, 10, 1567-1593, 2015). To increase the throughput of TPP while decreasing the costs per sample, protein integral stability alteration (PISA), also referred to as compressed CETSA, was developed (JPR, 18(11), 4027-4037, 2019). In this approach samples heated at different temperatures are pooled after the heat treatment and analyzed together via mass spectrometry. The measurements of protein abundances, which reflect the integral of the proteins melting curve, are subsequently related to a control sample. However, not all proteins show a differential melting behavior upon ligand binding. Additionally, specific precipitation of proteins using temperature requires a level of fine tuning that is often not achieved with current instrumentation. Thermal cyclers normally used for these procedures (as described by Molina in WO2012 / 143714 A1) have specific positional biases, leading to treatment of effective temperatures not equal to the ones reported by the instrumentation. These biases have been described by Maxwell et al (JASMS, 34(6), 1065-1072, 2023) and proven to be challenging to correct.

[0009] Organic solvents also precipitate proteins. In contrast to heat induced protein precipitation, organic solvents decrease the dielectric constant and compete for protein hydration. Consequently, approaches based on organic solved induced protein precipitation can be advantageous and complementary to temperature-dependent approaches for detection of ligand-protein interactions in non-purified protein samples described before. Xiaolei Zhang et. al have implemented solvent induced precipitation (SIP) to determine the ligand-protein interactions in non-purified protein samples (WO2021 / 098775). For this approach, the heat gradient used in CETSA is replaced by a specific combination of acetone, ethanol and acetic acid, which has a volume ratio acetone: ethanol: acetic acid of 50:50:0.1 and is referred to as AEA. Non-purified proteins are incubated with the ligand of interest and sub-samples are thereafter incubated with different concentrations of AEA at an isothermal temperature. Precipitation curves of proteins are then determined by means of immunoblotting or via mass spectrometry. In analogy to thermal ligand-binding approaches, Van Vranken et al (eLife, 10, e70784, 2021-12) showed, that samples can be pooled and analyzed in a compressed manner via mass spectrometry. Changes in protein stability are then assessed via differences in the integral of their precipitation curves. The application AEA solvent-induced proteome profiling for proteomic quantitation and target discovery of small molecular drugs (called SIPP) has been disclosed in a research paper by Yu Chengli et al. (Proteomics, vol. 23, no. 12, 4 March 2023). This approach was used in cell lysates and has recently also been shown to work in living cells (Dominik Steinbrunn et al., A Proteomic Denaturation Shift Assay for Drug Target Deconvolution [Poster Presentation] EFMC-ISMC 2023, and WO2024 / 261269 A1). However, Van Vranken et al (eLife, 10, e70784, 2021-12) showed decreased reproducibility due to high volatility of the components of the denaturant. High volatility, in this context, indicates the tendency of the components of the denaturant to evaporate. This process thus changes the intended composition of the solvent mixture, causing unwanted changes in the experiment.

[0010] SUMMARY OF THE INVENTION

[0011] The present disclosure relates to improved methods investigating protein-ligand binding interactions through the detection of solvent induced shifts of protein precipitation using nonpurified proteins, protein lysates or intact cells. Further, the disclosure relates to improved methods for determining direct binding of a ligand to a non-purified target protein, comprising an incubation of the non-purified proteins, lysates or intact cells with the ligand prior to an incubation of this mixture with different concentrations of solvent at an isothermal temperature and subsequent separation and quantification of soluble proteins via immunodetection or mass spectrometry. The present disclosure relates to the use of novel solvent combinations comprising small organic molecules to selectively precipitate proteins. This process of precipitation is specifically influenced by interaction between a ligand and target proteins, which affects the physico-chemical interactions of proteins and small organic molecules.

[0012] As summarized above, current methodologies using changes in protein solubility to identify protein-ligand interactions were deficient in several key points. Thermal shift analysis suffers from a low capability of controlling temperature treatments. Solvent denaturation methods had low reproducibility due to limitation in the chemical composition of the solvent. In contrast, the inventors have developed novel denaturing solvent compositions which can be used to reproducibility determine the binding of a ligand in both cell lysates and in living cells. The inventors have specifically focused in developing denaturing solvent compositions with high ease to handle and producing greater shifts compared to state of the art. The assays using these denaturing solvent compositions can be used in a wide range of samples from different biological origins, and generic, to study a variety of protein-ligand combinations. Proteins precipitate in response to increased concentration of the denaturing solvent combination in predictable and reproducible fashion. Increases or decreases in stability, upon solvent addition of a protein with the ligand compared to a protein without the ligand indicates a ligand-protein binding event. Altered stability can be evaluated by increased or decreased amounts of soluble putative target protein between the protein solutions with and without ligand.

[0013] The present disclosure is concerned with the analysis of any protein sample using novel denaturing solvent compositions from the inventors. Impure samples comprised of a native composition of protein of bodily fluids, lysed cells or living cells, are a suitable analyte. In a similar manner, partially purified protein samples originating from deep fractionation, protein enrichment or other kind of biological purification methods are also suitable starting material for the application of the differential protein precipitation in the presence of a ligand-protein interaction. As such, any combination of the above-mentioned material applies mutatis mutandis to the method herein described.

[0014] BRIEF DESCRIPTION OF THE FIGURES

[0015] Figure 1 : Schematic workflow of the solvent-induced precipitation (SIP) assay.

[0016] Figure 2: Evaluation of the stabilization effect of different denaturing solvent combinations on the interaction of Talmapimod with MAPK14. HEK293 cells were treated with 50 pM Talmapimod (SCIO-469) for 60 min followed by treatment with the denaturing solvent composition at a final concentration of 0, 1 , 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 % (v / v) for 20 min at 37 °C. Afterwards, cells were lysed, and soluble proteins were separated via centrifugation and quantified via capillary electrophoresis. To obtain precipitation curves, band intensities of samples treated with denaturing solvent were related to the respective sample treated with PBS (0 % (v / v) denaturing solvent composition), which was set to 100 %. A: Acetone, ethanol and acetic acid (volume ratio 50:50:0.1 , v / v / v). B: NMP, ethylene glycol and acetic acid (volume ratio 50:50:0.1 , v / v / v). C: NMP, 2-methylpropan-2-ol and acetic acid (volume ratio 50:50:0.1 , v / v / v). D: NMP, dimethylacetamide and acetic acid (volume ratio 50:50:0.1 , v / v / v). E: 2-methylpropan- 2-ol, dimethylacetamide and acetic acid (volume ratio 50:50:0.1 , v / v / v ). F: Ethanol and acetic acid (volume ratio 100:0.1 , v / v). G: NMP, propan-2-ol (isopropanol) and acetic acid (volume ratio 50:50:0.1 , v / v / v). Figure 3: Evaluation of talmapimod target identification using the SIP-MS technology with NMP:2-methylpropan-2-ol:acetic acid (volume ratio 50:50:0.1 v / v / v) as denaturing solvent combination. HEK293 cells were treated with 10 pM Talmapimod for 15 min followed by treatment with of NMP:2-methylpropan-2-ol:acetic acid or AEA at concentrations of 6 %, 9 %, 12 % or 15 % (v / v) for 20 min at 37 °C. Afterwards, cells were lysed, soluble proteins were separated via centrifugation and quantified by means of mass spectrometry. To obtain significantly (de-) stabilized proteins, protein intensities of Talmapimod treated samples were related to protein intensities of DMSO treated samples. A: Evaluation of assays reproducibility. B: Significant stabilization of the Talmapimod target MAPK14. C: Comparison of AEA and of NMP:2-methylpropan-2-ol: acetic acid induced MAPK14 stabilization.

[0017] Figure 4: Evaluation of talmapimod target identification using the SIP-MS technology with NMP:ethylene glyol: acetic acid (volume ratio 50:50:0.1 v / v / v) as denaturing solvent combination. HEK293 cells were treated with 10 pM talmapimod for 15 min followed by treatment with of NMP:ethylene glyol: acetic acid or AEA at concentrations of 6 %, 9 %, 12 % or 15 % (v / v) for 20 min at 37 °C. Afterwards, cells were lysed, soluble proteins were separated via centrifugation and quantified by means of mass spectrometry. To obtain significantly (de-) stabilized proteins, protein intensities of Talmapimod treated samples were related to protein intensities of DMSO treated samples. A: Evaluation of assays reproducibility. B: Significant stabilization of the Talmapimod target MAPK14. C: Comparison of AEA and of NMP:ethylene glyokaa induced MAPK14 stabilization.

[0018] Figure 5: Evaluation of Panobinostat target identification using the SIP-MS technology with NMP:2-methylpropan-2-ol:acetic acid (volume ratio 50:50:0.1 v / v / v) as denaturing solvent combination. HEK293 cells were treated with 10 pM Panobinostat for 15 min followed by treatment with of NMP:2-methylpropan-2-ol:acetic acid or AEA at concentrations of 6 %, 9 %, 12 % or 15 % (v / v) for 20 min at 37 °C. Afterwards, cells were lysed, soluble proteins were separated via centrifugation and quantified by means of mass spectrometry. To obtain significantly (de-) stabilized proteins, protein intensities of Panobinostat treated samples were related to protein intensities of DMSO treated samples. A: Evaluation of assays reproducibility. B: Significant stabilization of the Panobinostat targets HDAC1 and HDAC8. C: Comparison of AEA and of NMP:2-methylpropan-2-ol:acetic acid induced HDAC stabilization.

[0019] Figure 6: Evaluation of Panobinostat target identification using the SIP-MS technology with NMP:ethylene glycokacetic acid (volume ratio 50:50:0.1 v / v / v) as denaturing solvent combination. HEK293 cells were treated with 10 pM Panobinostat for 15 min followed by treatment with of NMP:ethylene glycol: acetic acid or AEA at concentrations of 6 %, 9 %, 12 % or 15 % (v / v) for 20 min at 37 °C. Afterwards, cells were lysed, soluble proteins were separated via centrifugation and quantified by means of mass spectrometry. To obtain significantly (de-) stabilized proteins, protein intensities of Panobinostat treated samples were related to protein intensities of DMSO treated samples. A: Evaluation of assays reproducibility. B: Significant stabilization of the Panobinostat targets HDAC1 and HDAC8. C: Comparison of AEA and of N MP: ethylene glycokacetic acid induced HDAC stabilization.

[0020] Figure 7: Evaluation of Methotrexate target identification using the SIP-MS technology with NMP:2-methylpropan-2-ol:acetic acid (volume ratio 50:50:0.1 v / v / v) as denaturing solvent combination. HEK293 cells were treated with 10 pM Methotrexate for 15 min followed by treatment with of NMP:2-methylpropan-2-ol: acetic acid or AEA at concentrations of 6 %, 9 %, 12 % or 15 % (v / v) for 20 min at 37 °C. Afterwards, cells were lysed, soluble proteins were separated via centrifugation and quantified by means of mass spectrometry. To obtain significantly (de-) stabilized proteins, protein intensities of Panobinostat treated samples were related to protein intensities of DMSO treated samples. A: Evaluation of assays reproducibility. B: Significant stabilization of the Methotrexate target DHFR. C: Comparison of AEA and of NMP:2-methylpropan-2-ol:acetic acid induced DHFR stabilization.

[0021] Figure 8: Evaluation of Methotrexate target identification using the SIP-MS technology with NMP:ethylene glycokacetic acid (volume ratio 50:50:0.1 v / v / v) as denaturing solvent combination. HEK293 cells were treated with 10 pM Methotrexate for 15 min followed by treatment with of NMP:ethylene glycokacetic acid or AEA at concentrations of 6 %, 9 %, 12 % or 15 % (v / v) for 20 min at 37 °C. Afterwards, cells were lysed, soluble proteins were separated via centrifugation and quantified by means of mass spectrometry. To obtain significantly (de-) stabilized proteins, protein intensities of Panobinostat treated samples were related to protein intensities of DMSO treated samples. A: Evaluation of assays reproducibility. B: Significant stabilization of the Methotrexate target DHFR. C: Comparison of Methotrexate induced DHFR stabilization towards AEA or NMP:ethylene glycol: acetic acid.

[0022] Figure 9: Evaluation of Staurosporine target identification using the SIP-MS technology with NMP:2-methylpropan-2-ol:acetic acid (volume ratio 50:50:0.1 v / v / v) as denaturing solvent combination. HEK293 cells were treated with 10 pM Staurosporine for 15 min followed by treatment with of NMP:2-methylpropan-2-okacetic acid or AEA at concentrations of 6 %, 9 %, 12 % or 15 % (v / v) for 20 min at 37 °C. Afterwards, cells were lysed, soluble proteins were separated via centrifugation and quantified by means of mass spectrometry. To obtain significantly (de-) stabilized proteins, protein intensities of Panobinostat treated samples were related to protein intensities of DMSO treated samples. A: Evaluation of assays reproducibility. B: Significant stabilization of several kinases that are Staurosporine targets. C: Comparison of AEA and of NMP:2-methylpropan-2-ol:acetic acid induced kinase stabilization.

[0023] Figure 10: Evaluation of Staurosporine target identification using the SIP-MS technology with NMP:ethylene glycokacetic acid (volume ratio 50:50:0.1 v / v / v) as denaturing solvent combination. HEK293 cells were treated with 10 pM Staurosporine for 15 min followed by treatment with of N MP: ethylene glycokacetic acid or AEA at concentrations of 6 %, 9 %, 12 % or 15 % (v / v) for 20 min at 37 °C. Afterwards, cells were lysed, soluble proteins were separated via centrifugation and quantified by means of mass spectrometry. To obtain significantly (de-) stabilized proteins, protein intensities of Panobinostat treated samples were related to protein intensities of DMSO treated samples. A: Evaluation of assays reproducibility. B: Significant stabilization of several kinases that are Staurosporine targets. C: Comparison of Staurosporine induced kinase stabilization towards AEA or NMP:ethylene glycokacetic acid.

[0024] Figure 11 : Evaluation of the effect of different Talmapimod concentrations on solvent induced precipitation of the target protein MAPK14 using the SIP-MS technology. HEK293 cells were treated with different concentrations of Talmapimod (concentrations: 50 pM, 20 pM, 10 pM, 5 pM, 2 pM, 1 pM, 0.5 pM, 0.2 pM, 0.1 pM and 0.01 pM) or DMSO as a control for 15 min followed by treatment with NMP:2-methylpropan-2-ok acetic acid (volume ratio 50:50:0.1 v / v / v) or AEA at a concentration of 8 % (v / v) for 20 min at 37 °C. Afterwards, cells were lysed, soluble proteins were separated via centrifugation and quantified by means of capillary electrophoresis. To obtain dose-response curves, band intensities of all samples were related to band intensity of the respective DMSO control, which was set to 100 %.

[0025] DETAILED DESCRIPTION OF THE INVENTION

[0026] The present disclosure relates the use of N-methyl-2-pyrrolidone (NMP), dimethylacetamide (DMA) and / or ethylene glycol alone or in combination with each other, in combination with alcohols, diols, amides, and / or glycols, optionally with acetic acid as a protein precipitating agent during solvent-induced precipitation (SIP) assays for the determination of ligand-protein interactions.

[0027] As an embodiment there is provided a method for detecting the interaction between a ligand and one or more non-purified target protein(s) based on solvent-induced protein precipitation (SIP) using a denaturing solvent comprising N-methyl-2-pyrrolidone (NMP), Dimethylacetamide (DMA), and / or ethylene glycol.

[0028] In contrast to the state-of-the-art approaches, which use a mixture of acetone, ethanol and acetic acid (AEA, volume ratio 50:50:0.1), the herein described solvent mixtures are less volatile and therefore easier to handle and less prone to experimental errors. Besides being non-volatile and therefore less prone to experimental error, these solvent combinations surprisingly induce larger shifts for ligand-bound proteins and thereby allow identification of more interacting proteins with higher confidence.

[0029] N-Methyl-2-pyrrolidone (NMP) is an organic compound consisting of a 5-membered lactam. It is a colorless liquid with polar aprotic properties and a high boiling point at 202 °C and thus low volatility. NMP is miscible with water and most other organic solvents. (Ligocka, D. et al. Arch Toxicol 77, 261-266 (2003))

[0030] Dimethylacetamide (DMA, N,N-Dimethylacetamid) is a dipolar, aprotic oily solvent with a high boiling point of 165 °C. It is miscible with water and most other oxygen- or nitrogen-containing organic solvents.

[0031] Ethylene glycol (ethane-1 ,2-diol) is an organic compound with the formula (CH2OH)2 and a boiling point of 197 °C

[0032] 2-methylpropan-2-ol (aka tert-Butyl alcohol, tert-Butanol) is a tertiary alcohol with a boiling point of 83 °C and thus low volatility. It is miscible with water and various other organic solvents, including NMP, DMA and ethylene glycol.

[0033] Propan-2-ol (aka isopropanol, isopropyl alcohol) is an organic compound with the chemical formula (CHs CHOH) and it is the simplest example of a secondary alcohol, where the alcohol carbon atom is attached to two other carbon atoms. It has a boiling temperature of 82 °C.

[0034] Besides being less volatile and therefore easier to handle, the herein described solvent mixtures (in particular NMP:2-methylpropan-2-ol:acetic acid, NMP:ethylene glycokacetic acid, NMP:propan-2-ol:acetic acid, DMA:2-methylpropan-2-ol: acetic acid, and N-methyl-2- pyrrolidone : dimethylacetamide : acetic acid ) induce higher changes in the precipitation behavior of proteins and lead consequently to the identification of more interacting proteins and with higher significance.

[0035] There is provided an improved method for detecting the interaction between a ligand and one or more non-purified target protein(s) based on solvent-induced protein precipitation by using novel denaturing solvent compositions.

[0036] The term "target protein" as used herein, refers to a protein which is being assessed for ligand binding. The target protein can therefore be any protein which is present in a sample. The target protein may be naturally occurring e.g. in a cell or cell lysate or animal or patient sample, may be recombinantly expressed e.g. may be expressed from a plasmid which has been transformed into a cell, or may be added as a purified protein to the sample.

[0037] The term "non-purified target protein" refers to a target protein which is studied for interaction with the ligand. The “non-purified target protein” is not in an isolated form or alternatively viewed is present with other components e.g. proteins. The non-purified target protein to be used in the methods of the disclosure are in non-purified form before the addition of the ligand (test molecule) or in the absence of the ligand. Thus, the non-purified target protein is present with components other than the ligand. The samples comprising the non-purified target protein thus include cells, cell lysates and samples obtained directly from patients (human patients or animal patients or disease models e.g. dog, cat, monkey, rabbit, mouse, rat etc.) such as tissue samples, blood, serum, plasma, lymph etc. The non-purified target protein includes target protein when comprised in one or more cell colonies. The non-purified protein may also be comprised in a liquid culture of cells.

[0038] Key to the present disclosure is the finding that 'dirty' samples can yield reliable information when undergoing solvent-induced protein precipitation induced by a denaturing solvent composition. Thus, the sample at step a) is non-purified but there may be circumstances where a purified target protein has been added to a dirty starting sample. The sample is not purified and contains components such as other proteins, cell debris, nucleic acids etc., as described herein in the context of "non purified target protein".

[0039] It is also possible that the non-purified target protein could have been subjected to one or more steps e.g. the extraction step of a purification process, as long as the purification process was not completed and a purified protein was not isolated. The non-purified target protein is therefore typically present with other compounds or proteins and thus the target protein is not present in isolated form.

[0040] In a first aspect there is provided a method for detecting the interaction between a ligand and one or more non-purified target protein(s) based on solvent-induced protein precipitation, comprising: a) Incubation of the one or more non-purified target protein(s) with the ligand dissolved in an appropriate dissolving solution forming the ligand-treated group or incubation with the dissolving solution alone forming the control group. b) Add the same amount of denaturing solvent to the ligand-treated group and to the control group to initiate protein denaturation and resulting in precipitation, wherein the denaturing solvent comprises: i. N-methyl-2-pyrrolidone (NMP) and a diol selected from the group consisting of ethylene glycol, polyethylen glycol, propylene glycol, 1,3-butanediol, and 1,4- butanediol, optionally with acetic acid, ii. N-methyl-2-pyrrolidone (NMP) and an alcohol selected from the group consisting of methanol, ethanol, propan-1-ol, propan-2-ol, 2-methylpropan-2-ol (tert-buthanol), n-butanol, isobutanol, ethanolamine, triflourethanol, and glycerol, optionally with acetic acid, iii. N-methyl-2-pyrrolidone (NMP) and an amide selected from the group consisting of dimethylacetamide (DMA), dimethylformamide (DMF), optionally with acetic acid, iv. Dimethylacetamide (DMA) and an alcohol selected from the group consisting of methanol, ethanol, propan-1 -ol, propan-2-ol, 2-methylpropan-2-ol (tert- buthanol), n-butanol, isobutanol, ethanolamine, triflourethanol, and glycerol, optionally with acetic acid, v. Dimethylacetamide (DMA) and a diol selected from the group consisting of ethylene glycol, polyethylen glycol, propylene glycol, 1,3-butanediol, and 1,4- butanediol, optionally with acetic acid, vi. Ethylene glycol and an alcohol selected from the group consisting of methanol, ethanol, propan-1-ol, propan-2-ol, 2-methylpropan-2-ol (tert-buthanol), n- butanol, isobutanol, ethanolamine, triflourethanol, and glycerol, optionally with acetic acid, or vii. Ethylene glycol and an amide selected from the group consisting of dimethylacetamide (DMA), dimethylformamide (DMF), optionally with acetic acid, c) Separation of the insoluble and soluble fractions of proteins in both, ligand-treated group and control group. d) Detection of the abundance of proteins in the soluble and / or insoluble fractions of the ligand-treated group and control group. e) Determining the one or more target protein(s) interacting with the ligand by comparison of the abundance of proteins measured in the ligand-treated group with the abundance of the proteins in the control group and revealing of differences of said abundance between these groups.

[0041] In step a) the one or more non-purified target protein(s) is (are) incubated with the ligand dissolved in an appropriate dissolving solution forming the ligand-treated group and the one or more non-purified target protein(s) is (are) incubated with the dissolving solution alone forming the control group.

[0042] The protein samples comprising the non-purified target protein(s) that can be studied range vastly. These include but are not limited to a protein mix of one or more initially purified proteins, enrichment of one or more protein samples from a complex mixture, complex mixtures of proteins from cell lysates or tissue lysates, living cells cultured in vitro, living cells cultured to form organoids, tissue extracts, blood, plasma or other body fluids. The above-mentioned samples can be derived from one or more of human, animal, plant or bacterial origin.

[0043] The ligands which are studied include any chemical compounds, as well as mixture of one or more chemical compounds, micro- or macro-molecules of biological origin (including but not limited to antibodies, DNA, RNA, amino acids, peptides, proteins, lipids, carbohydrates, metabolites) from animals, plants or bacteria, chemical agents of various use (including environmental agents, herbicides, pesticides), metal ions, nanoparticles and / or other agents that may interact with proteins in cellular or extracellular contexts. Ligands are applied in one or multiple concentrations over analogous analytes. Samples treated with one or more concentrations of a ligand are analyzed separately or combined after step b) or after step c) and measured as a pooled sample.

[0044] The ligand is dissolved in an appropriate dissolving solution. For small molecular compounds the dissolving solution may be DMSO. For macromolecules of biological origin, a buffered aqueous solution may be used as dissolving solution.

[0045] The protein solution incubated with a ligand is used as ligand group, the protein solution incubated with equivalent amount of dissolving solvent in the absence of ligand is used as the control group.

[0046] In step b) the same amount of denaturing solvent is added to the ligand-treated group and to the control group to initiate protein denaturation and resulting in precipitation.

[0047] In certain embodiments of the first aspect the denaturing solvent used in step b) comprises: i. N-methyl-2-pyrrolidone and ethylene glycol, optionally with acetic acid, ii. N-methyl-2-pyrrolidone and 2-methylpropan-2-ol, optionally with acetic acid, iii. N-methyl-2-pyrrolidone and propan-2-ol, optionally with acetic acid, iv. N-methyl-2-pyrrolidone and dimethylacetamide, optionally with acetic acid, or v. Dimethylacetamide and 2-methylpropan-2-ol, optionally with acetic acid.

[0048] In certain embodiments of the first aspect the denaturing solvent used in step b) consists of: i. N-methyl-2-pyrrolidone and ethylene glycol, optionally with acetic acid, ii. N-methyl-2-pyrrolidone and 2-methylpropan-2-ol, optionally with acetic acid, iii. N-methyl-2-pyrrolidone and propan-2-ol, optionally with acetic acid, iv. N-methyl-2-pyrrolidone and dimethylacetamide, optionally with acetic acid, or v. Dimethylacetamide and 2-methylpropan-2-ol, optionally with acetic acid.

[0049] In certain embodiments of the first aspect the denaturing solvent used in step b) has a volume ratio: i. N-methyl-2-pyrrolidone : ethylene glycol of 50:50, ii. N-methyl-2-pyrrolidone : 2-methylpropan-2-ol of 50:50, iii. N-methyl-2-pyrrolidone : propan-2-ol of 50:50, iv. N-methyl-2-pyrrolidone : dimethylacetamide of 50:50, or v. Dimethylacetamide : 2-methylpropan-2-ol of 50:50.

[0050] In certain embodiments of the first aspect the denaturing solvent used in step b) has a volume ratio: i. N-methyl-2-pyrrolidone : ethylene glycol : acetic acid of 50:50:0.1 , ii. N-methyl-2-pyrrolidone : 2-methylpropan-2-ol : acetic acid of 50:50:0.1 , iii. N-methyl-2-pyrrolidone : propan-2-ol : acetic acid of 50:50:0.1 , iv. N-methyl-2-pyrrolidone : dimethylacetamide : acetic acid of 50:50:0.1 , or v. Dimethylacetamide : 2-methylpropan-2-ol : acetic acid of 50:50:0.1.

[0051] The protein samples are treated with one or more concentrations of the denaturing solvent described herein. The protein samples treated with different concentrations can be analyzed separately or combined after step b) or c) and measured as a pooled sample. The choice of concentrations of the denaturing solvent combinations can be adjusted based on specific questions of the experimental set-up. Different proteins possess different degrees of resistance to chemical denaturation, as shown by Van Vranken et al (eLife, 10, e70784, 2021-12). As such, different concentrations of denaturing solvent combinations are better suited to analyze different kinds of proteins. The use of multiple concentrations can be used to probe a larger spectrum of proteins with different physico-chemical properties simultaneously and to evaluate doseresponse effects.

[0052] In certain embodiments the final concentration in step b) of the added denaturing solvent in the ligand-treated group and in the control group to initiate protein denaturation and resulting in precipitation is 1 to 20 % (v / v).

[0053] In certain embodiments the final concentration in step b) of denaturing solvent in the ligand- treated group and in the control group to initiate protein denaturation and resulting in precipitation is 6 % (v / v), 9 % (v / v), 12 % (v / v) or 15 % (v / v). In certain embodiments in step b) several different final concentrations of the denaturing solvent, preferentially 6% (v / v), 9% (v / v), 12% (v / v) and 15 % (v / v), in the ligand-treated group and the control group are used and these different samples of the ligand-treated group and the control group are pooled to form one sample of the ligand-treated group and one sample of the control group.

[0054] In certain embodiments step b) is performed at an isothermal temperature and shaking, to induce protein denaturation and precipitation. In specific embodiments the isothermal temperature is between 20 to 40° C, preferentially at 37° C. In specific embodiments the shaking is performed at 100 to 1000 rpm, preferentially at 1000 rpm.

[0055] In step c) after incubation with the denaturing solvent the insoluble and soluble fractions of proteins in both, ligand-treated group and control group are separated. The separation step can involve any separation method which is capable of separating soluble from insoluble protein. For example, a step of centrifugation can be used, or a step of filtration may be used. The soluble protein (supernatant) may be separated from the precipitate by centrifugation before the quantification steps. A filter can be used to separate soluble from insoluble proteins where soluble proteins will pass through a filter. Standard filter membranes can be used for filtering protein samples where the filters will typically have a pore size from 0.015 pm to 12 pm. When the target protein is produced or expressed in cells, such as bacteria e.g. E.coli, an optimal pore size may be 0.1-1.5 pm. Where target protein is from a eukaryotic cell or sample, preferred pore sizes may be larger.

[0056] In certain embodiments alternatively, or additionally, where the target protein is present in a cell (or on the cell surface), a step of lysis may be carried out before, simultaneously or after the ligand has been added. The lysis step is generally carried out before the separation step c) of the method (Separation of the insoluble and soluble fractions of proteins). It will be apparent that a step of lysis may only need to be carried out on samples where the target protein is comprised within a cell.

[0057] For cells subjected to lysis, the lysis step is preferentially non-denaturing, allowing target proteins to retain a native i.e. correctly folded or native-like conformation. This is referred to herein as native lysis. This can be carried out chemically or otherwise using reagents which are well known in the art e.g. urea, lysozyme containing buffers or detergents. The degree of lysis must be sufficient to allow proteins of the cell to pass freely out of the cell. Typically, when dealing with membrane bound proteins, lysis is performed in the presence of detergents or amphiphiles, for example NP-40, Triton X-100 or dodecylmaltoside, to release the protein from the membrane.

[0058] In certain embodiments the lysis step is carried out by freeze thawing the cells or colonies. In specific embodiments, lysis is carried out using both native lysis buffer and freeze thawing the cells. In a preferred embodiment for extraction of the proteins, mild lysis conditions are applied to maintain the native protein conformation prior treatment with the ligand of interest and / or treatment with the denaturing solvent . For this purpose, cells are harvested and washed several times with PBS prior to addition of lysis buffer containing PBS and 0.4 % NP-40. Cells are then subjected to three cycles of freezing in liquid nitrogen and subsequent thawing at 23 °C. Cell debris as well as any other insoluble components are removed via centrifugation. If the sample comprises living cells or tissues, or in general non-pre-extracted material, the mild extraction is performed after treatment with the ligand described in step a) and after solvent induced protein precipitation and sample pooling described in step b).

[0059] Differential protein abundance between ligand-treated and control samples are detected in the soluble and / or in the insoluble fractions. Protein quantification, as in step d) is achieved with one or more technologies, depending on the nature of the protein and availability of detection reagents. Recombinant proteins can be detected via specific methodologies, dependent on the specific activity and function of the modifications. These include but are not exclusive to fluorescence and chemiluminescence. Detection of recombinant, as well as non-recombinant proteins, can be done by immunoblotting or other immunoassays but is not limited to these. Quantification of one or more proteins from the same sample can also be achieved by quantitative proteomics technologies, which include but are not exclusive to mass spectrometrybased identification of proteins. Proteins in mass spectrometry are quantified in label-free or label-based quantifications. Label-based approaches include but are not exclusive to stable metabolic labeling during cell culture growth and / or labeling of proteins or peptides with stable isotopes after lysis and / or protein digestion. These latter methods include but are not exclusive to iTRAQ and TMT. In these methods, protein abundance is inferred by the abundance of peptides measured in the mass spectrometer. More precisely, the abundance of stable isotopes in the chemical moieties added by TMT or iTRAQ methods is measured. These measurements are then compared for all peptides, and then proteins, identified in the protein mixture from which the analyte was derived. Detection and quantification of proteins or peptides in the mass spectrometer is achieved using different methods of acquisition. These approaches include but are not exclusive to Data Dependent Acquisition (DDA), Data Independent Acquisition (DIA) or Parallel Reaction Monitoring (PRM). Here, protein abundances are inferred from the primary signal measured from the proteins that comprised the analyte.

[0060] As indicated in step e), analytical methodologies are required to determine if the difference in protein abundance upon ligand incubation and subsequent solvent precipitation herein described indicates a protein-ligand binding event. These methodologies require the quantification of protein abundance in the samples (step e)), achieved through one of the methods described above. Comparing the abundance difference of one or more proteins between ligand-treated and control condition constitutes the major read-out. If a single measurement of ligand-treated and control-treated analyte is made, this is a measure of sizeeffect of the compound-induced (de-)stabilization of the protein(s) measured. Multiple independent measurements of the analytes and controls treated in the same way (or replicates) constitute a way to gain statistical power in evaluating the likelihood that an observed shift in abundance of one or more proteins between ligand-treated and control treated samples is consistent, reproducible and can be confidently called a true ligand-protein interaction. The evaluation of how different distribution of protein(s) abundance values of replicates from ligand- treated and control are achieved with different statistical methods, such as Student t-tests. The use of cut-offs (as minimal or maximal values for specific numerical outcomes of the analysis) improves confidence in the identification of a true protein-ligand binding event. If the experimental set-up comprises the measurement of ligand-treated and control samples at multiple concentrations of the organic compound composition herein described, then the analysis can focus on the comparison of the melting profiles of protein(s). We define here “precipitation profiles” as the representations of amount, relative or absolute, of protein in the soluble and / or insoluble fraction, observed at different concentration of denaturant. In this setup, the analysis consists of first fitting the data to a sigmoid profile described by the equation: y = Bottom + g(xMog(e)) ■ Differences between curves originating from ligand-treated and control samples are achieved with different statistical methods. Similarly, if the experimental set-up comprises the measurement of multiple concentrations of ligand, then the analysis can focus on the comparison of dose-response profile of protein(s). We define here “dose-response profiles” as the representations of amount, relative or absolute, of protein in the soluble and / or insoluble fraction, observed at in samples treated with the organic compound composition herein described and at different concentrations of ligand. Analysis of this data can be achieved by fitting the data to the same equation described above and analyzed in an analogous way.

[0061] ADVANCES OF THE INVENTION

[0062] (1) The herein described solvent combinations lead to greater alterations of compoundbound proteins compared to control samples (greater Iog2(fold changes)) than the state-of-the-art solvent combination AEA. Consequently, the use of these solvents in SIP-MS experiments leads to:

[0063] 1 . More robust identification of target proteins with higher statistical confidence

[0064] 2. Identification of more relevant compound-target interactions and thus more target proteins

[0065] (2) The herein described solvent combinations are characterized by a lower volatility than the state-of-the-art solvent combination AEA. Consequently, the use of these organic solvents in solvent-induced precipitation (SIP) assays facilitates the lab-work to less evaporation and leads to lower assay variability and higher confidence on identified target proteins.

[0066] (3) The physico-chemical properties of the herein describes solvent compositions, especially the surface-tension and lower volatility clearly facilitate the experimental work during SIP-MS sample preparation compared to the state-of-the-art solvent combination AEA. Due to the higher surface-tension, the new solvent combinations can be pipette with much higher accuracy, which allows for more streamlined automation of pipetting steps. Finally, this leads to higher assay reproducibility and consequently to higher confidence into identified target proteins.

[0067] EXAMPLES

[0068] Experiment 1 : Evaluation of Talmapimod induced MAPK14 stabilization towards different solvent combinations

[0069] To obtain dose-response SIP curves, Talmapimod at a concentration of 100 pM or an equivalent amount of DMSO was diluted in PBS and 20 pL of the solutions were distributed into 12 wells of a 96-well multitier plate. Afterwards, 20 pL of HEK293 cells at a density of 1 E7 cells / mL were added to each well to reach a final compound concentration of 50 pM. Cells were incubated with the compound or DMSO for 60 min at 37 °C and 300 rpm. A dilution series of the different solvent combinations in PBS was prepared with concentrations of 0, 2, 4, 8, 12, 16, 20, 24, 28, 32, 36 and 40 % (v / v). Afterwards, 10 pL of these solvent dilutions were added to each well containing the cells to reach final solvent concentrations of O, 1 , 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 % (v / v). For protein precipitation, cells were incubated with the solvent dilutions for 20 min at 1000 rpm. For cell lysis, NP40 solution (at a final concentration of 0.4 % (v / v)) containing proteasome inhibitor was added and cells were snap frozen in liquid nitrogen and thawed at 23 °C for 5 min in three repetitive cycles. Samples were then centrifuged at 20000 xg for 20 min at 4 °C and supernatants were collected and analyzed via capillary-electrophoresis using the Jess™ system (Bio-Techne) and the 12-230 kDa Separation Module (#SM-W004, Bio Techne). For this purpose, equal volumes of 3 pL of the supernatants were prepared using the EP-Z Standard Pack l (PS-ST01 EZ-8, Bio Techne) according to the instructions of the manufacturer. For protein detection, an antibody against MAPK14 (#9212S, Cell Signaling Technology) was used, along with the Anti-Rabbit Detection Module (#DM-001 , Bio Techne) containing the secondary antibody as well as the corresponding detection reagents. Detected MAPK14 protein bands were quantified using the Compass for SW software (Bio Techne) via the band area. Signals obtained for compounds or DMSO and solvent-treated samples were related to the signal of the respective non-solvent treated control sample, which was set to 100 %.

[0070] The immuno-detection of MAPK14 (Figure 2) show a marked stabilization of the target protein after treatment of cells with 50 pM Talmapimod compared to DMSO for each of the solvent combinations tested. This stabilization is also clearly confirmed after quantification of the detected bands, which are visualized in the corresponding dose-response curves. However, the size effect of the detected MAPK14 stabilization differs significantly depending on the solvent concentration used. Compared to the solvent combinations evaluated in this disclosure, the state-of-the-art solvent combination AEA detects the smallest stabilization of MAPK14 after T almapimod binding. Consequently, the use of other solvent combinations than AEA, specifically the use of a combination of 2-methylpropan-2-ol : N-Methyl-2-pyrrolidone (volume ratio 50:50), ethylene glycol : N-Methyl-2-pyrrolidone (volume ratio 50:50), dimethylacetamide : 2- methylpropan-2-ol (volume ratio 50:50) or N-Methyl-2-pyrrolidone : Propan-2-ol (volume ratio 50:50) in presence or absence of acetic acid (with a volume ratio 50:50:0.1) can be highly beneficial to gain a more significant and more robust results.

[0071] Experiment 2: Evaluation of solvent-induced precipitation method using a combination 2-methylpropan-2-ol: N-Methyl-2-pyrrolidone: acetic acid (volume ratio 50:50:0.1) and N- Methyl-2-pyrrolidone: ethylene glycol: acetic acid (volume ratio 50:50:0.1) for in cell target identification of different small molecules

[0072] To evaluate the effects of the 2-methylpropan-2-ol: N-Methyl-2-pyrrolidone: acetic acid (volume ratio 50:50:0.1) (2-methylpropan-2-ol:NMP:aa) and N-Methyl-2-pyrrolidone: ethylene glycol: acetic acid (volume ratio 50:50:0.1) (N-Methyl-2-pyrrolidone: ethylene glycokaa) solvent combinations on several compound-target interactions, a mass-spectrometry based solvent- induced shift assay (SIP-MS) was performed. For this purpose, HEK293 cells at a density of 1 E7 cells / mL were treated with 10 pM of the respective compound or DMSO as a control and incubated at 37 °C and 300 rpm. Meanwhile, 4X concentrated solvent dilutions in PBS were prepared (24 %, 36 %, 48 % and 60 % (v / v)). After 15 min, the samples were divided into four parts with a volume of 30 pL per sub-samples and 10 pL of the 4X solvent dilutions were added to the respective samples to final solvent concentrations of 6 %, 9 %, 12 % and 15 % (v / v). Samples were incubated for 20 min at 37 °C and 1000 rpm for protein precipitation. Afterwards, sub-samples were pooled back together, and 0.4 % (v / v) NP-40 and protease inhibitor were added to each sample. For lysis, cells were then snap frozen in liquid nitrogen three times and thawed for 3 min at 23 °C and 300 rpm. To separate cell debris and precipitated protein, samples were centrifuged for 30 min at 20000 xg and the supernatants containing the soluble proteins were collected in fresh vials. Protein concentrations were detected by means of a BCA assay (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, #23225). Afterwards, equal volumes of the samples (~10 pg protein) were used for mass spectrometry sample preparation. For reduction and alkylation (RA), 25 pL of the samples were treated with 5 pL RA buffer (50 mM HEPES, 10 mM TCEP, 20 mM CAA and 1% (v / v) SDS) and incubated for 30 min at 23 °C and 300 rpm. Afterwards 150 pg magnetic beads (Sera-Mag beads hydrophobic (#44152105050250, GE Healthcare) and Sera-Mag beads hydrophilic (#24152105050250, GE Healthcare), ratio 1 :1) were added to each sample and samples were processed using the single-pot, solid-phase- enhanced sample-preparation (SP3) technology. Subsequently, 0.5 pg Trypsin / LysC solution were added to each sample, and samples were incubated for 5 h at 37 °C and 1000 rpm for protein digestion. Finally, samples were acidified with 0.1 % (v / v) Trifuoric acid (TFA) and beads were removed, prior to submission to mass spectrometry. The acquired mass spectra were analyzed to identify the proteins present in the samples and their respective abundances. Significant solvent-induced shifts were evaluated by comparing compound-treated against DMSO-treated cells via a Student t-test statistic. Example 1 : Validation of different solvents for SIP-MS for Talmapimod target identification

[0073] For validation of improved target identification using different solvent combinations beyond AEA, SIP-MS was performed after treatment of living cells with the MAPK14 inhibitor Talmapimod, as described above. The results obtained for the NMP:2-methylpropan-2-ol:aa solvent combination are shown in figure 3.

[0074] The data in figure 3A and 3B show that talmapimod-bound MAPK14 is significantly and reproducibly stabilized against NMP:2-methylpropan-2-ol:aa treatment with a Log2(fold change) > 2. Consequently, the NMP:2-methylpropan-2-ol:aa solvent combination can be used for successful identification of talmapimod target. Figure 3C shows a comparison of Talmapimod-induced MAPK14 stabilization towards AEA compared to NMP:2-methylpropan-2- ol:aa. The data clearly show that NMP:2-methylpropan-2-ol:aa leads to a greater Log2(fold change) of MAPK14 compared to AEA, underlining the great advantage of the use of this new solvent combination compared to the state-of the art.

[0075] Similarly, the influence on the stability of MAPK14 towards NMP:ethylene glyokaa treatment after binding talmapimod was tested in comparison to the unbound state (Figure 4).

[0076] The data in figure 4A and 4B clearly show that talmapimod-bound MAPK14 is significantly and reproducibly stabilized towards NMP:ethylene glyokaa treatment compared to unbound MAPK14, with a Log2(fold change) > 2. Conclusively, this solvent combination can be used as an alternative to AEA, for identification of successful target identification using the SIP-MS method. The Log2(fold change) induced by NMP:ethylene glyokaa is significantly higher than the Log2(fold change) induced by the standard solvent solution AEA (Figure 4C). Consequently, also this solvent combination holds great advantage over the state-of-the-art solvent combination AEA, that is reported for solvent-induced precipitation approaches for target identification in complex protein mixtures (e.g., lysates or living cells).

[0077] Example 2: Validation of different solvents for SIP-MS for Panobinostat target identification

[0078] To confirm the suitability of other solvent combinations than state-of-the-art (AEA) as precipitating agents in SIP MS assays for target identification after compound treatment, especially the use of NMP:2-methylpropan-2-ol:aa and NMP:ethylene glyokaa, the HDAC inhibitor Panobinostat was evaluated as a second example. For this purpose, samples were prepared and analyzed as described above. The results obtained for the NMP:2-methylpropan- 2-ol:aa solvent combination are shown in figure 5. The data shown in figure 5 show a clear stabilization and thus successful identification of the Panobinostat target proteins HDAC1 and HDAC8 in samples treated with 10 pM Panobinostat compared to DMSO treated samples. This effect is reproducible (figure 5A) and significant (figure 5B). Consequently, the NMP:2-methylpropan-2-ol:aa can be used as a solvent combination in SIP-MS assays in living cells. The Iog2(fold change) values obtained for HDAC target proteins using NMP:2-methylpropan-2-ol:aa is markedly higher compared to the state-of-the-art combination AEA. This confirms that the usage of NMP:2-methylpropan-2-ol:aa in is beneficial compared to the state-of-the-art.

[0079] Figure 6 shows the results obtained for Panobinostat in a SIP-MS experiment using NMP:ethylene glyokaa as solvent combination for protein precipitation.

[0080] The data in Figure 6A show a clear and reproducible stabilization of HDAC1 towards NMP:ethylene glyol after treatment of cells with 10 pM Panobinostat. Although the shift for HDAC8 is smaller compared to the SIP-MS using NMP:2-methylpropan-2-ol:aa as solvent combination, Figure 6B shows that both proteins shifted with a -loglO(p-value) > 2 and were consequently both identified as Panobinostat target proteins. Comparison of the Log2(fold changes) obtained by usage of NMP:ethylene glyokaa and the state-of-the-art solvent combination AEA (Figure 6C) again shows, that both target proteins show greater shifts in case of the herein described new solvent combination. Consequently, our newly identified solvent combination outperforms the state-of-the-art solution.

[0081] Example 3: Validation of different solvents for SIP-MS for Methotrexate target identification

[0082] The suitability of the herein described solvent combinations, especially the use of NMP:2- methylpropan-2-okaa and NMP:ethylene glyokaa, compared to state-of-the-art combination AEA as precipitating agents in SIP MS assays for target identification after compound treatment, was additionally evaluated for the DHFR inhibitor Methotrexate. Samples were prepared and analyzed as described above. The results obtained for the NMP:2-methylpropan-2-okaa solvent combination are shown in Figure 7.

[0083] Figure 7A shows a clear and reproducible stabilization of the target protein DHFR towards NMP:2-methylpropan-2-okaa, with a Log2(fold change) > 2 after treatment of cells with 10 pM Methotrexate. As visualized in Figure 7B, this great shift is also highly significant with a -Iog10(p- value) > 4. Comparison of the herein reached Methotrexate induced DHFR stabilization to the state-of-the-art solvent combination AEA shows much greater Log2(fold change) in case of NMP:2-methylpropan-2-okaa usage (Figure 7C). In line with previous results discussed above, these data strongly underline the benefit of this solvent combination over the state-of-the-art. In analogy to other compounds described above, target identification using NMP:ethylene glyokaa as protein precipitating solvent combination in SIP-MS experiments was explored. The obtained data are represented in Figure 8.

[0084] Treatment of cell with 10 pM Methotrexate reproducibly stabilized the target protein DHFR towards NMP:ethylene glycokaa induced protein precipitation with a Iog2(fold change) > 1 (Figure 8A). This stabilization is highly significant with a -loglO(p-value) > 5 (Figure 8B). Consequently, these data again proof suitability of NMP:ethylene glycokaa as solvent for SIP- MS based target identification approaches. Moreover, fold changes obtained using this solvent combination are clearly greater than shifts that are obtained through usage of the state-of-the- art solvent combination AEA (Figure 8C), again underscoring the huge advantage of the herein described method.

[0085] Example 4: Validation of different solvents for SIP-MS for Staurosporine target identification

[0086] The pan kinase inhibitor Staurosporine was used as a fourth tool compound to evaluate the suitability of new solvent combinations, especially NMP:2-methylpropan-2-okaa and NMP:ethylene glyokaa as precipitating agent during SIP-MS experiments for target identification. The experiments were performed as described above. The respective results obtained for NMP:2-methylpropan-2-okaa as protein precipitating solvent combination are shown in Figure 9.

[0087] In line with data discussed before, several kinases that are targets of the pan kinase inhibitor Staurosporine show a reproducible stabilization or destabilization towards NMP:2- methylpropan-2-okaa, with Log2(fold changes) > 1 or < -1 after treatment of cells with 10 pM Staurosporine (Figure 9A). With loglO(p-values) > 4, the alterations in protein stability are highly significant (Figure 9B). The comparison of the new solvent combination NMP:2-methylpropan- 2-okaa to the state-of-the-art solvent combinate AEA (Figure 9C) clearly shows greater changes, especially higher stabilizing effects when using NMP:2-methylpropan-2-okaa as precipitating agent. In line with data discussed above, also in this case NMP:2-methylpropan-2-okaa clearly out-performs the current state-of-the-art.

[0088] Figure 10 shows the results obtained for SIP-MS after treatment of cells with 10 pM Staurosporine when using NMP:ethylene glycokaa solvent combination for protein precipitation.

[0089] Similar to the results discussed above, several kinases were reproducibly stabilized or destabilized towards NMP:ethylene glycokaa treatment after incubation of cells with the pan kinase inhibitor Staurosporine at a concentration of 10 pM (Figure 10A). As shown in Figure 10B, these effects were highly significant with Iog2(p-values) > 4. Moreover, the alterations were comparable, or even higher when using NMP:ethylene glycokaa as protein precipitating solvent SIP-MS, compared to the state-of-the-art solvent combination AEA (Figure 10 C).

[0090] Experiment 3: Evaluation of Talmapimod dose-response effects on in cell solvent- induced precipitation method using a combination 2-methylpropan-2-ol: N-Methyl-2- pyrrolidone: acetic acid (volume ratio 50:50:0.1) or AEA (volume ratio 50:50:0.1)

[0091] To evaluate compound induced dose-response effects on solvent induced precipitation assays in intact cells using the herein described solvent combinations compared to the state-of-the art solvent combination AEA (volume ratio 50:50:0.1), stabilization of MAPK14 was evaluated at different Talmapimod concentrations, but at one single concentration of 8 % (v / v) of each solvent. For this purpose, Talmapimod was diluted in PBS at a concentration of 100 pM, 40 pM, 20 pM, 10 pM, 4 pM, 2 pM, 1 pM, 0.4 pM, 0.2 pM and 0.02 pM. Afterwards, 20 pL of the dilutions were distributed into 12 wells of a 96-well multitier plate and 20 pL of HEK293 cells at a density of 1 E7 cells / mL were added to each well to reach final Talmapimod concentrations of 50 pM, 20 pM, 10 pM, 5 pM, 2 pM, 1 pM, 0.5 pM, 0.2 pM, 0.1 pM and 0.01 pM. As a control, samples were treated with DMSO, which was constantly set to a final concentration of 0.5 % (v / v) in all samples. Cells were incubated with the compound or DMSO for 60 min at 37 °C and 300 rpm. Afterwards, 10 pL of a 40 % (v / v) solution of AEA or NMP:2-methylpropan-2-ol:aa was added to each well containing the cells to reach final solvent concentrations of 8 % (v / v). For protein precipitation, cells were incubated with the solvent dilutions for 20 min at 1000 rpm. For cell lysis, NP40 solution (at a final concentration of 0.4 % (v / v)) containing proteasome inhibitor was added and cells were snap frozen in liquid nitrogen and thawed at 23 °C for 5 min in three repetitive cycles. Samples were then centrifuged at 20000 xg for 20 min at 4 °C and supernatants were collected and analyzed via capillary-electrophoresis using the Jess™ system (Bio-Techne) and the 12- 230 kDa Separation Module (#SM-W004, Bio Techne). For this purpose, equal volumes of 3 pL of the supernatants were prepared using the EP-Z Standard Pack 1 (PS-ST01 EZ-8, Bio Techne) according to the instructions of the manufacturer. For protein detection, an antibody against MAPK14 (#9212S, Cell Signaling Technology) was used, along with the Anti-Rabbit Detection Module (#DM-001 , Bio Techne) containing the secondary antibody as well as the corresponding detection reagents. Detected MAPK14 protein bands were quantified using the Compass for SW software (Bio Techne) via the band area. Signals obtained for Talmapimod, or samples were related to the signal of the respective DMSO treated control sample, which was set to 100 %. The obtained results are shown in Figure 11.

[0092] The immuno-detection in Figure 11 shows increasing MAPK14 band intensities at increasing concentrations of Talmapimod with both solvent combinations, reflecting a stabilizing effect of the compound on the target. Notably, 8 % (v / v) NMP:2-methylpropan-2-ol:aa demonstrates a more distinct manifestation of the dose-responsive effects than the state-of-the-art solvent combination AEA at a concentration of 8 % (v / v), as indicated by the stronger immuno-detection signal. This distinction is further accentuated upon the relative quantification of band intensities against the DMSO control. The use of NMP:2-methylpropan-2-ol:aa thus facilitates a highly refined discernment of the interactions between compound and protein. Besides qualitative evaluation of a binding-event, the substantial assay window is advantageous for detection of binding affinities. These observations strongly support the superiority of other solvent combinations, especially the implementation NMP:2-methylpropan-2-ol:aa over the state-of-the- art.

Claims

CLAIMS1) A method for detecting the interaction between a ligand and one or more non-purified target protein(s) based on solvent-induced protein precipitation, comprising: a) Incubation of the one or more non-purified target protein(s) with the ligand dissolved in an appropriate dissolving solution forming the ligand-treated group and incubation of the one or more non-purified target protein(s) with the dissolving solution alone forming the control group. b) Add the same amount of denaturing solvent to the ligand-treated group and to the control group to initiate protein denaturation and resulting in precipitation, wherein the denaturing solvent comprises: i. N-methyl-2-pyrrolidone (NMP) and a diol selected from the group consisting of ethylene glycol, polyethylen glycol, propylene glycol, 1,3-butanediol, and 1,4- butanediol, optionally with acetic acid, ii. N-methyl-2-pyrrolidone (NMP) and an alcohol selected from the group consisting of methanol, ethanol, propan-1-ol, propan-2-ol, 2-methylpropan-2-ol (tert-buthanol), n-butanol, isobutanol, ethanolamine, triflourethanol, and glycerol, optionally with acetic acid, iii. N-methyl-2-pyrrolidone (NMP) and an amide selected from the group consisting of dimethylacetamide (DMA), dimethylformamide (DMF), optionally with acetic acid, iv. Dimethylacetamide (DMA) and an alcohol selected from the group consisting of methanol, ethanol, propan-1 -ol, propan-2-ol, 2-methylpropan-2-ol (tert- buthanol), n-butanol, isobutanol, ethanolamine, triflourethanol, and glycerol, optionally with acetic acid, v. Dimethylacetamide (DMA) and a diol selected from the group consisting of ethylene glycol, polyethylen glycol, propylene glycol, 1,3-butanediol, and 1,4- butanediol, optionally with acetic acid, vi. Ethylene glycol and an alcohol selected from the group consisting of methanol, ethanol, propan-1-ol, propan-2-ol, 2-methylpropan-2-ol (tert-buthanol), n- butanol, isobutanol, ethanolamine, triflourethanol, and glycerol, optionally with acetic acid, or vii. Ethylene glycol and an amide selected from the group consisting of dimethylacetamide (DMA), dimethylformamide (DMF), optionally with acetic acid,c) Separation of the insoluble and soluble fractions of proteins in both, ligand-treated group and control group. d) Detection of the abundance of proteins in the soluble and / or insoluble fractions of the ligand-treated group and control group. e) Determining the one or more target protein(s) interacting with the ligand by comparison of the abundance of proteins measured in the ligand-treated group with the abundance of the proteins in the control group and revealing of differences of said abundance between these groups.2) The method according to claim 1 wherein the denaturing solvent used in step b) comprises: i. N-methyl-2-pyrrolidone and ethylene glycol, optionally with acetic acid, ii. N-methyl-2-pyrrolidone and 2-methylpropan-2-ol, optionally with acetic acid, iii. N-methyl-2-pyrrolidone and propan-2-ol, optionally with acetic acid, iv. N-methyl-2-pyrrolidone and dimethylacetamide, optionally with acetic acid, or v. Dimethylacetamide and 2-methylpropan-2-ol, optionally with acetic acid.3) The method according to claim 1 wherein the denaturing solvent used in step b) consists of: i. N-methyl-2-pyrrolidone and ethylene glycol, optionally with acetic acid, ii. N-methyl-2-pyrrolidone and 2-methylpropan-2-ol, optionally with acetic acid, iii. N-methyl-2-pyrrolidone and propan-2-ol, optionally with acetic acid, iv. N-methyl-2-pyrrolidone and dimethylacetamide, optionally with acetic acid, or v. Dimethylacetamide and 2-methylpropan-2-ol, optionally with acetic acid.4) The method according to claim 2 or 3 wherein the denaturing solvent used in step b) has a volume ratio: i. N-methyl-2-pyrrolidone : ethylene glycol of 50:50, ii. N-methyl-2-pyrrolidone : 2-methylpropan-2-ol of 50:50, iii. N-methyl-2-pyrrolidone : propan-2-ol of 50:50, iv. N-methyl-2-pyrrolidone : dimethylacetamide of 50:50, or v. Dimethylacetamide : 2-methylpropan-2-ol of 50:50.5) The method according to claim 2 or 3 wherein the denaturing solvent used in step b) has a volume ratio: i. N-methyl-2-pyrrolidone : ethylene glycol : acetic acid of 50:50:0.1, ii. N-methyl-2-pyrrolidone : 2-methylpropan-2-ol : acetic acid of 50:50:0.1, iii. N-methyl-2-pyrrolidone : propan-2-ol : acetic acid of 50:50:0.1 , iv. N-methyl-2-pyrrolidone : dimethylacetamide : acetic acid of 50:50:0.1, or v. Dimethylacetamide : 2-methylpropan-2-ol : acetic acid of 50:50:0.1.6) The method according to any of claims 1 to 5 wherein in step b) the final concentration of the added denaturing solvent in the ligand-treated group and in the control group to initiate protein denaturation and resulting in precipitation is 1 to 20 % (v / v).7) The method according to any of claims 1 to 5 wherein in step b) the concentration of the added amount of denaturing solvent in the ligand-treated group and in the control group to initiate protein denaturation and resulting in precipitation is 6 % (v / v), 9 % (v / v), 12 % (v / v) or 15 % (v / v).8) The method according to any of claims 1 to 7 wherein in step d) the soluble fraction is analyzed.9) The method according to any of claims 1 to 8 wherein in step e) the one or more target protein(s) interacting with the ligand are determined via a greater abundance in the ligand- treated group compared the to the abundance in in the control group.10) The method according to any of claims 1 to 9 wherein step b) is performed at an isothermal temperature, preferentially at 37 °C, and shaking, preferentially at 1000 rpm, to induce protein denaturation and precipitation.11) The method according to any of claims 1 to 10 wherein in step b) several different final concentrations of the denaturing solvent, preferentially 6 % (v / v), 9 % (v / v), 12 % (v / v) and 15 % (v / v), in the ligand-treated group and the control group are used and pooling of thesedifferent samples of the ligand-treated group and the control group to form one sample of the ligand-treated group and one sample of the control group.12) The method according to any of claims 1 to 11 wherein in step c) the separation of the insoluble and soluble fractions of proteins in both, ligand-treated group and control group is performed by filtration, or centrifugation.13) The method according to any of claims 1 to 12, wherein if the method is performed with cells comprising the one or more non-purified target protein(s), a lysis step of the cells is performed after step b) and before step c).14) The method according to claim 13, wherein the lysis of the cells is performed by three cycles of freezing in liquid nitrogen and thawing, preferentially at 23 °C.15) The method according to any of claims 1 to 14 wherein in step d) the detection of the abundance of one or more proteins in the soluble and / or insoluble fractions of the ligand- treated group and control group is performed by fluorescence, chemiluminescence, affinity binding, immunoblotting or other immunoassays, or quantitative proteomics technologies.16) The method according to any of claims 1 to 15 wherein in step d) the detection of the abundance of one or more proteins in the soluble and / or insoluble fractions of the ligand- treated group and control group is performed by mass spectrometry.

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