Method for detecting ligand-target interactions using solvent- induced precipitation in intact cells

Abstract

The invention relates in one aspect to a method for detecting an interaction between a ligand and at least one target protein in in-tact cells, comprising inducing protein precipitation using one or more solvents in a first sample and in a second sample, both comprising intact cells and / or permeabilized cells, wherein said first sample is treated with said ligand, and said second sample is not treated with said ligand, wherein a difference in tolerance of the target protein to solvent-induced precipitation between the first and second samples indicates an interaction between a ligand and a target protein.

Description

[0001] METHOD FOR DETECTING LIGAND-TARGET INTERACTIONS USING SOLVENT- INDUCED PRECIPITATION IN INTACT CELLS

[0002] DESCRIPTION

[0003] The invention is in the field of biology, particularly in the field of biochemistry and cell biology, proteomics and drug discovery.

[0004] The invention relates in one aspect to a method for detecting an interaction between a ligand and at least one target protein in intact cells, comprising inducing protein precipitation using one or more solvents in a first sample and in a second sample, both comprising intact cells, wherein said first sample is treated with said ligand, and said second sample is not treated with said ligand, wherein a difference in tolerance of the target protein to solvent-induced precipitation between the first and second samples indicates an interaction between a ligand and a target protein.

[0005] BACKGROUND OF THE INVENTION

[0006] Among the chemical-proteomic approaches used in the field of target deconvolution, approaches analyzing the resistance of proteins to conditions causing protein denaturation and precipitation in combination with quantitative proteomics has become particularly popular in recent years.

[0007] One of said approaches is the SIPP approach ("Solvent-Induced Protein Precipitation for Drug Target Discovery on the Proteomic Scale") that was first described in WO2021098775, Zhang et al. 2019 (Anal Chem. 2020 Jan 7;92(1 ): 1363-1371 ) and Van Vranken et al., 2021 (eLife 10:e70784 https: / / doi.org / 10.7554 / eLife.70784). SIPP is a method for detecting interaction or affinity between a ligand and a protein on the basis of solvent-induced protein precipitation. The method employs the biophysical principle that once a protein binds a ligand, the protein’s tolerance against precipitation caused by solvent-induced denaturation is changed, such that e.g., the protein has a higher or lower tolerance against precipitation. WO2021098775 uses cell lysates or protein-comprising solutions to which a solvent is added to precipitate proteins. In WO2021098775 the differences between precipitated protein in the ligand-comprising sample is compared to a control sample without ligand to determine the binding of a target protein to the ligand. Using the SIPP approach, it was possible to generate protein-specific denaturation and aggregation curves (so-called "melting curves") for proteins by adding increasing relative amounts of a mixture of acetone, ethanol and acetic acid (AEA for short) to a complex lysate.

[0008] The SIPP approach according to WO2021098775 was, however, only applied to analyze lysates or solutions comprising a protein of interest. The analysis of living cells was not shown with the SIPP method of WO2021098775.

[0009] Among the chemical-proteomic approaches used in the field of target deconvolution, the CETSA® method (Cellular Thermal Shift Assay) in combination with quantitative proteomics has become particularly popular in recent years. The patent application WO2012143714A1 and Martinez Molina et al. 2013 (Science. 2013 Jul 5;341 (6141):84-7) describe the CETSA approach. The CETSA approach analyzes the binding of a protein to a ligand by application of a heat treatment that precipitates unbound target proteins faster than target proteins bound to a ligand. The method is also useful for analyzing whether drugs bind to their protein targets. The CETSA method is based on the long-known biophysical principle that proteins are stabilized against thermal denaturation by the binding of an active substance compared to the free protein. This principle is commonly used to determine thermodynamic parameters in drug discovery and protein research, e.g., in binding and protein folding measurements of purified proteins (classical thermal shift assays, TSA). If the target is known, CETSA can be used to measure whether the active substance actually binds the protein in the cell or cell extract (target engagement). In this process, aliquots of the sample are heated to different temperatures in the range between 37 °C and 65 °C, the less thermostable proteins denature and aggregate, and can be separated by centrifugation or filtration. The soluble proteins in the supernatant of the respective temperature can be detected and quantified, e.g., by Western blot, and melting curves and melting points of the protein with and without active substance can be derived from the data. With both classical TSAs and CETSA, the stabilization of the proteins is typically a few degrees Celsius (typical thermal shifts of 1-3 °C). Savitski et al. 2014 (Science, 2014 Oct 3;346(6205):1255784) were the first to describe a proteomics application of the CETSA (TPP) approach.

[0010] Mass spectrometry-based proteomics continues to gain importance in the pharmaceutical industry. A very important area of application here is above all the proteome-wide investigation and identification of interactions between pharmacological targets (proteins to which a pharmaceutical active ingredient can specifically bind and in this way influence a disease process) and active ingredients. The methods used for this have gained enormous importance with the increasing spread of phenotypic screening in the pharmaceutical drug search. Here, a relevant target is not known a priori, but substances are identified that show a certain desired effect in a cell or animal model, but whose site of action and mechanism of action are initially unknown and, like undesirable side effects, must only be identified subsequently at the molecular level.

[0011] Well-known methods for label-free target identification are, for example, SPROX (stability of proteins from rates of oxidation; West et al., Thermodynamic Analysis of Protein Stability and Ligand Binding Using a Chemical Modification- and Mass Spectrometry-Based Strategy Anal. Chem. 2008, 80, 4175-4185), DARTS (Lomenick et al. Target identification using drug affinity responsive target stability (DARTS) Proc Natl Acad Sci U S A. 2009;106:21984-9), LiP-MS (Limited proteolysis coupled with quantitative proteomics; Feng et al, Global analysis of protein structural changes in complex proteomes, Nature Biotechnology volume 32, pages 1036-1044, 2014), and CPP (Meng et al, Chemical Denaturation and Protein Precipitation Approach for Discovery and Quantitation of Protein-Drug Interactions Anal Chem. 2018, 90(15):9249-9255). These methods are referred to as "label-free" because neither labelling of the drug (or more generally: ligand) nor labelling of the (often unknown) protein is necessary for the analysis. Target identification methods (i.e., the elucidation of the target molecules of an active substance; often also referred to as target deconvolution) are important tools in drug research and in the elucidation of undesirable side effects. LiP (Limited Proteolysis) and CETSA or TPP (Cellular Therma Shift Assay or Thermal Proteome Profiling) are common implementations of physicochemical methods that do not require modification or derivatization of the drug. Both exploit the fact that a protein-drug interaction changes the physicochemical stability compared to the unbound protein. In LiP, the ligand-bound protein shows pronounced protection against digestion by a protease than the native protein, and in TPP, it is exploited that proteins stabilized by interactions with a ligand denature and precipitate only at higher temperatures than the respective free protein. Both procedures are read out with methods of quantitative proteomics and thus enable a comprehensive analysis of the stabilized proteins in the biological system under consideration.

[0012] In light of the prior art and the inherent strengths and weaknesses of each individual technology, there remains a significant need in the art to provide additional means for analyzing the binding of targets of interest to a ligand under the physiological conditions inside living cells.

[0013] SUMMARY OF THE INVENTION

[0014] In light of the prior art the technical problem underlying the present invention is to provide alternative and / or improved means for the analysis of ligand-target binding inside living cells.

[0015] This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

[0016] The invention therefore relates in one aspect to a method for detecting an interaction between a ligand and at least one target protein in intact cells, comprising inducing protein precipitation using one or more solvents in a first sample and in a second sample, both comprising intact cells (and / or (less preferably) permeabilized cells), wherein said first sample is treated with said ligand, and said second sample is not treated with said ligand, wherein a difference in tolerance of the target protein to solvent-induced precipitation between the first and second samples indicates an interaction between a ligand and a target protein.

[0017] In less preferred embodiments the method comprises inducing protein precipitation using one or more solvents in a first sample and in a second sample, both comprising intact cells and / or permeabilized (but intact / viable) cells, wherein said first sample is treated with said ligand, and said second sample is not treated with said ligand, wherein a difference in tolerance of the target protein to solvent-induced precipitation between the first and second samples indicates an interaction between a ligand and a target protein.

[0018] In embodiments the method comprises inducing protein precipitation using one or more solvents in a first sample and in a second sample (b), both comprising intact cells, wherein said first sample is treated with said ligand (a), and said second sample is not treated with said ligand, wherein a difference in tolerance of the target protein to solvent-induced precipitation between the first and second samples indicates an interaction between a ligand and a target protein (d-e). In embodiments of the present invention intact cells are or comprise viable or alive cells.

[0019] In embodiments the method comprises (a) treating the first sample with a ligand, (b) treating the first and second sample with one or more solvents to precipitate the proteins therein, (c) lysing the cells (after the addition of one or more solvents) preferably at mild conditions, (d) separating soluble protein (supernatant) from precipitated protein and cell debris, and (e) comparing an amount, quantity or level of un-precipitated target protein in the supernatant in the first and second samples, wherein a difference in tolerance of a protein to solvent-induced precipitation between the first and second samples indicates an interaction between the ligand and a protein.

[0020] In preferred embodiments the first and second sample both comprise intact cells or viable intact cells. In some preferred embodiments the first and second sample both don’t comprise permeabilized cells.

[0021] The method according to the invention provides a surprising and important advantage over the prior art (e.g., the SIPP method of Zhang et al. 2019 or CETSA / TPP of Molina et al. 2013 and Savitski et al. 2014) by facilitating the detection of protein-ligand interactions in live cell systems (living cells) and under physiological conditions. The methods of the prior art, that facilitate the observation of protein-ligand interactions in living cells or tissues require often conditions that differ significantly from physiological, intracellular conditions, e.g., regarding pH-value and / or temperature. For example, CETSA / TPP may be used with intact cells, but it requires non- physiologically high temperatures. In contrast to what has been shown for the SIPP method, the present method also allows to perform the present analysis in intact cells.

[0022] An important advantage and significant difference to the prior art CETSA / TPP approaches of Molina et al. 2013 and Savitski et al. 2014 is that in the present method the proteins are denatured isothermally, such that ultimately the stabilization of a protein of interest (through binding to a ligand of interest) can be measured at physiologically or pharmacologically relevant temperatures. This constitutes a significant difference and improvement over the prior art, especially over the TPP approach, wherein analysis is performed at unphysiological temperatures far above 40°C. Thus, the present method can provide more meaningful data on binding strengths in living cells than TPP / CETSA.

[0023] Another important advantage resulting from the inventive process compared to the prior art CETSA approach, which itself applies heat denaturation and precipitation, is that the present isothermal denaturation allows determination of EC / IC50-values at physiologically and pharmacologically relevant temperatures (e.g., around 37°C) within the cell. Further, the present method enables presumably a higher reproducibility of stabilization, as (solvent) concentrations can be very accurately and precisely set and maintained during an experiment, while it is technically much more challenging to maintain exact temperature values, which are applied in the CETSA approach. Moreover, the present method enables a simple and more straightforward implementation of automation compared to the CETSA approach. This is, for example, as the present method facilitates solvent-induced precipitation instead of heat precipitation. This has the advantage that it is easier to accurately and reproducibly pipet liquids in high throughput and thus precipitate proteins from many samples (e.g., with a pipetting robot) than to apply a certain temperature (heat) accurately and reproducibly to a plurality of samples. The approach used in the CETSA method requires an accurate heating of the samples to specific temperatures, which is more complicated to implement in high throughput and can constitute a hurdle to experimental performance, which significantly complicates the implementation of the CETSA method (higher costs, more time intensive). In comparison to CETSA the present method is preferably performed at physiological (isothermal) temperatures and denaturation is achieved by the application of a solvent instead of high (denaturation) temperatures.

[0024] The method according to the invention also provides an advantage over the prior art “SIPP” approach, which is only able to analyze interaction in cell lysates or extracts. This is because the present method enables the sustainment of physiological conditions during treatment (precipitation treatment), such that, e.g., the relevant cellular concentration of compound and targets, the relevant subcellular distribution of targets, compounds or molecular crowding and condensates, are sustained during the precipitation assay. Another advance over the SIPP method is that proteins are analyzed in their cellular state, e.g., with respect to other ligands and their activation or functional state. Further, the active metabolism of an added compound within a cell may be analyzed using the present method, as exemplified by the inventors in the Examples for the polyglutaminylation of Methotrexate (MTX), which binds to Thymidylate Synthase (TYMS) only after being metabolized. The present method further enables the analysis of secondary effects on targets and other proteins, such as modulation of post-translational modifications, of subcellular distributions, of activation and functional states, of their interactions with other biomolecules or ligands (such as e.g., other proteins, metabolites, DNA, RNA, etc.), of folding, biophysical stability and aggregation, of protein turnover, of substrate limitation or accumulation, etc. A substantial advantage of the present method is that none of the before enlisted exemplary effects and conditions can be analyzed using the SIPP method.

[0025] Another advantage of the present method over the SIPP approach is that the present method enables analysis of living cells and is therefore able to reproduce stabilization effects of experiments using cell lysates. Additionally, the present method is also able to detect stability changes that only arise from direct treatment of living cells.

[0026] When extensively validating and evaluating the present approach on living cells the inventors could surprisingly achieve a proteome coverage of >8,000 proteins and an unexpected high reproducibility of <5% median coefficient of variation of the protein intensities in mass spectrometry analysis of the soluble protein fraction (supernatant of precipitation reaction) and / or the precipitated protein fraction (obtained from precipitation reaction).

[0027] The present approach has been validated and evaluated on living cells using different implementations of the present approach with the chemotherapeutic and antifolate drug methotrexate, which binds to DHFR and TYMS (when polyglutaminylated), with the protein kinase inhibitors MK-2206, which targets the serine-threonine-protein kinases AKT1 and AKT2, and SCIO-469, which targets p38oc (MAPK14), the epigenetic drug Vorinostat, which targets histone deacetylases (HDACs), the covalent receptor tyrosine kinase inhibitor Ibrutinib, which targets BTK, and the natural product, molecular glue and immunosuppressive drug, Cyclosporin A, which targets peptidyl-prolyl cis-trans isomerases (cyclophilins).

[0028] In these experiments, cells in culture were resuspended and equal cell numbers were distributed in aliquots. Each aliquot of cells was treated with one of the drugs (or a vehicle control, usually DMSO) at a fixed concentration for a fixed amount of time, and the treatment was stopped by the addition of solvent. Cells were lysed after precipitation of proteins inside cells by addition of IGEPAL CA-630 to a final concentration of 0.4 % and three freeze-thaw cycles. Precipitated proteins and cell debris after lysis were removed by centrifugation and the supernatant was further prepared for LC-MS / MS-based protein identification and quantification.

[0029] To this end, proteins were digested, the resulting peptide mixtures were labelled with Tandem Mass Tags (TMT) for isobaric quantification, and the different TMT samples were mixed, desalted and concentrated via solid-phase-extraction, prior to off-line basic reversed-phase liquid chromatographic fractionation, and LC-MS / MS analysis on a Q Exactive Plus mass spectrometer. The resulting raw data was processed with the MaxQuant software and the resulting protein lists, and quantification data were analyzed in R.

[0030] Therefore, in comparison to CETSA the present method is preferably performed at physiological (isothermal) temperatures and denaturation is achieved by the application of a solvent instead of high (denaturation) temperatures.

[0031] In summary, the present method poses a significantly improved and expanded approach in comparison to the methods of the prior art, such as CETSA / TPP or SIPP.

[0032] In embodiments the method according to the invention comprises: a) treating the first sample with a ligand, b) treating the first and second samples with one or more solvents to precipitate the proteins therein, optionally lysing the cells (after protein precipitation), preferably at mild conditions, c) separating soluble protein (supernatant) from precipitated protein, and d) comparing an amount, quantity or level of un-precipitated target protein in the supernatant in the first and second samples, e) wherein a difference in tolerance of the target protein to solvent-induced precipitation between the first and second samples indicates an interaction between the ligand and the target protein.

[0033] In embodiments a sample, such as a first and second sample, comprises cells and / or constitutes a suspension of cells. In embodiments a sample, such as a first and second sample, comprises living cells and / or constitutes a suspension of living cells. In embodiments a sample, such as a first and second sample, comprises intact cells and / or constitutes a suspension of intact cells.

[0034] In embodiments the first sample comprises the sample conditions to be analyzed (of interest) and the second sample comprises - in embodiments where the first sample comprises a compound, substance or substance composition of interest - only the vehicle (vehicle compound / composition) used in the first sample for dilution, dissolving, storage and / or administering of the compound, substance or substance composition of interest.

[0035] In some embodiments step b) (also) comprises permeabilization of cells (cell permeabilization) before, after, or less preferably in addition to and / or at the same time as the precipitation of proteins. In some embodiments a first sample and a second sample comprise intact and / or alive, but permeabilized cells. The skilled person knows how to select appropriate agents and conditions to permeabilize cells in a sample without lysing cells or impairing their viability.

[0036] In some embodiments the step b) comprises precipitation of the proteins, and preferably no lysis of the cells.

[0037] In some embodiments the present method (also) comprises a cell lysis (or lysing of cells), preferably after the precipitation of proteins (after treating the first and second samples with one or more solvents to precipitate the proteins therein). In preferred embodiments, if a cell lysis is to be induced within the present method, said cell lysis is induced after the solvent-induced precipitation of the proteins in step b). In some embodiment the cell lysis is induced by one or more physical (mechanical) means. In some embodiment the cell lysis is induced chemically by one or more lysis buffers or agents. In some embodiment the cell lysis is induced by one or more physical (mechanical) means and / or chemically, e.g., preferably by one or more lysis buffers or agents. In some embodiments the use of a lysis agent or buffer can aid the physically (mechanically) induced cell lysis. In some embodiments the use of physical (mechanical) lysis means can aid the solvent-induced cell lysis.

[0038] In some embodiments step b) comprises an additional cell lysis-step after the step of treating the first and second samples with one or more solvents to precipitate the proteins therein. Namely, in embodiments step b) comprises (i) treating the first and second samples with one or more solvents to precipitate proteins in the cells, and afterwards (ii) treating the first and second samples with one or more agents to lyse the cells.

[0039] In other words, in some embodiments the method comprises inducing protein precipitation using one or more solvents in a first sample and in a second sample, both comprising intact cells, wherein said first sample is treated with said ligand, and said second sample is not treated with said ligand, subsequently treating the first and second samples with one or more agents to lyse the cells, and wherein a difference in tolerance of the target protein to solvent-induced precipitation between the first and second samples indicates an interaction between a ligand and a target protein. In embodiments the one or more agents (to lyse the cells) comprise or consist of a cell lysis buffer, a detergent, or any combination thereof. The skilled person knows how to select appropriate agents and conditions to lyse cells in a sample.

[0040] In other embodiments the lysis of cells can be achieved alternatively or additionally to an agent by physical lysis, e.g., via ultrasound, a douncer, syringes, freeze-thaw cycles or French press etc. The skilled person knowns different methods to induce cell lysis. Therefore, in embodiments step b) comprises (i) treating the first and second samples with one or more solvents to precipitate proteins in the cells, and afterwards (ii) treating the first and second samples with one or more physical (mechanical) means inducing lysis of the cells (physically). Preferably the one or more physical (mechanical) means comprise or constitute means selected from the group comprising, e.g., ultrasound, a douncer, syringes or French press. In some embodiments the cells may be lysed with a combination of physical (mechanical) means and chemical means (one or more agents).

[0041] In embodiments the method according to the invention comprises: a) treating the first sample with a ligand, b) first (i) treating the first and second samples with one or more solvents to precipitate the proteins therein, second (ii) lysing the cells, c) separating soluble protein (supernatant) from precipitated protein, and d) comparing an amount, quantity or level of un-precipitated target protein in the supernatant in the first and second samples, e) wherein a difference in tolerance of the target protein to solvent-induced precipitation between the first and second samples indicates an interaction between the ligand and the target protein.

[0042] In embodiments comparing an amount, quantity or level of un-precipitated target protein in the supernatant comprises quantifying the un-precipitated target protein in the supernatant. In embodiments comparing an amount, quantity or level of un-precipitated target protein in the supernatant comprises quantifying the un-precipitated target protein in the supernatant and / or quantifying and / or identifying / determining the precipitated protein(s).

[0043] In embodiments the tolerance of the target protein to solvent-induced precipitation may be indicated by, derived from or calculated on the basis of a higher amount, level, concentration or abundancy of a target protein in the supernatant of a sample (after precipitation, e.g., in step b) to which the ligand of interest was added. The tolerance to solvent-induced precipitation may in preferred embodiments be indicative for an interaction between the target protein and the ligand of interest.

[0044] Hence, in embodiments a difference in tolerance of the target protein to solvent-induced precipitation is measured by determining differences in target protein amount, quantity or level between the first and second samples, preferably between the supernatant of the first and second sample, respectively.

[0045] Herein, amount, quantity or level can in embodiments also comprise mass or intensity. Hence, in embodiments a difference in tolerance of the target protein to solvent-induced precipitation is measured by determining differences in target protein amount, quantity, level, mass or intensity between, preferably the supernatant, of the first and second samples respectively.

[0046] In embodiments a sample comprises intact cells, wherein the cells comprise or consist of a mixture of different cell types or cells from different origins, e.g., tissues.

[0047] In embodiments the first and second samples comprise intact cells selected from one or more of cultured living cells, organs, tissues, tissue samples, blood, serum, plasma, urine, lymph, cerebrospinal fluid, bone marrow or another patient or animal sample type.

[0048] In embodiments the cells are adherent, semi-adherent or suspended in cell culture.

[0049] In embodiments the cells are mammalian, plant, insect or bacterial cells.

[0050] In embodiments the cell number in the sample is in the range of 100,000 cells - 10 million cells. In embodiments the cell number in the sample is in the range of 100,000 cells to 10 million (mio.) cells, in embodiments the cell number in the sample is in the range of 500,000 cells to 1 .5 mio. cells, in some embodiments the cell number in the sample is around 1 mio. cells.

[0051] In embodiments of the method according to the invention the target protein in the cells or tissues remain in their initial conformation. In embodiments the initial conformation may be a natural, native, denatured, pathogenic or other conformation. Preferably the initial conformation is the conformation of a protein (inside a living and intact cell) before the addition of one or more solvents and cell lysis.

[0052] In embodiments the ligand is selected from the group consisting of small molecules, biologies, metabolites, plant extracts or natural products, food additives, environmental pollutants, agricultural pesticides or herbicides, environmental agents, metal ions, nanoparticles, peptides, proteins, nucleic acids, a drug or another chemical or any combination thereof.

[0053] In embodiments of the method according to the invention the ligand may be one or more ligands. In embodiments the ligand may be one or more ligands, wherein at least one ligand is selected from the group consisting of small molecules, biologies, metabolites, plant extracts or natural products, food additives, environmental pollutants, agricultural pesticides or herbicides, environmental agents, metal ions, nanoparticles, peptides, proteins, nucleic acids, toxins, a drug or another chemical.

[0054] In embodiments the present method is particularly advantageous in determining the interaction between an agent, chemical or drug and one or more target proteins / polypeptides in living cells and under physiological conditions, e.g., in alive and metabolizing cells. The present method is particularly useful regarding the analysis of interactions and / or effects of a compound under the conditions and processes that exist in a living cell. Many prior art methods, such as SIPP, so far failed to enable this analysis, as only cell lysates or extracts could be analyzed.

[0055] In embodiments the cells within a sample may be permeabilized with lipofectamine or electroporation, preferably with an agent or method not influencing the viability of the cells, such that cells are still considered intact and / or viable after permeabilization. Preferably the permeabilization does not lyse the cells or causes damage to the cell, but only allows agents to enter the cell more easily. Preferably the permeabilization allows agents to enter the cell mor easily but does not enable the leakage (outflow) of components out of the cell. In embodiments the cells comprised in the sample may be permeabilized before, during or after the addition of a ligand or the precipitation step, wherein the permeabilization does not lyse or damage the cells, nor negatively impacts on their viability. In some embodiments the cells comprised in the sample may be permeabilized during / togetherwith the addition of a ligand, wherein the permeabilization does not lyse or damage the cells, or negatively impacts on their viability. In some embodiments the cells comprised in the sample may be permeabilized after the addition of a ligand, wherein the permeabilization does not lyse or damage the cells, or negatively impacts on their viability. In embodiments the permeabilization of the cells improves or facilitates the entrance of a ligand into a cell within the sample, such that the ligand may interact with a target within the cell, but without lysing or damaging the cells, or negatively impacting on their viability (and without enabling the leakage (outflow) of components out of the cell). In embodiments the application of a permeabilization (step) of the cells (for the added ligand) may depend on the ligand and / or the cells used in the experiment. A skilled person knows, for which ligands and / or cells a permeabilization step may be advantageous or elemental for the entrance of the ligand into the cells. In some embodiments the (control) sample, in which no ligand is added to the sample, may also be subjected to permeabilization, such that, e.g., cells are subjected to similar (assay) conditions in the first and the second sample.

[0056] As a non-limiting example of such embodiments, the ligand is added to a sample together with a liposomal agent, e.g., a lipofectamine or lipid- / liposome-nanoparticles. In some embodiments the ligand is added to the cells before, during or after an electroporation treatment of the cells / sample.

[0057] In some embodiments the conditions for cell lysis, e.g., the added lysis agent, provides or facilitates mild lysis conditions, wherein exact buffer compositions may vary dependent on the target protein or cell / tissue type analyzed.

[0058] “Mild lysis conditions" in the context of the present invention are preferably those that avoid (re-) solubilization of precipitated proteins, unfolding and / or and denaturation of previously soluble proteins. Non-limiting examples of buffers enabling mild lysis conditions can be found e.g., in Reinhard et al. (Thermal proteome profiling monitors ligand interactions with cellular membrane proteins, Nat Methods, 2015), e.g., comprising 0.4% NP40, 1% DDM, 1 % CHAPSO, 1% CHAPS, 1% OTG, 1 % Brij 35, 1% Pluronic F-127, with DDM: n-dodecyl-P-D-maltopyranoside, OTG = n- octyl-p-d-thioglucopyranosides. In embodiments the strength (mild / moderate / strong) of the lysis conditions is influenced by the detergent and / or chaotrope used. Hence, in embodiments strong detergents and / or chaotrope, such as SDS, urea, GndHCI, would be avoided or used at low concentrations. Hence, in embodiments the lysis buffer consists of phosphate buffered saline (PBS) and / or comprises a lysis agent selected from the group of mild detergents such as IGEPAL CA-630, n- Dodecyl-P-D-Maltopyranoside, n-octyl-p-d-thioglucopyranoside, CHAPSO, CHAPS, Brij 35 and Pluronic F-127.

[0059] In embodiments in step b) the first and second samples are treated with multiple solvent conditions, for example increasing concentration of said one or more solvents, and the precipitation of the target protein in the first and second samples is compared for each of said multiple solvent conditions.

[0060] In embodiments multiple solvent conditions refers to varied, e.g., increasing or decreasing solvent concentrations within the sample. In embodiments the treatment with multiple solvent conditions, e.g., increasing solvent concentrations, is performed in the context of a melt-curve experiment.

[0061] As a non-limiting example, by adding increasing relative amounts of solvent or solvent mixture, such as a mixture of acetone, ethanol and acetic acid (AEA), to a sample comprising living cells, it is possible to generate protein-specific precipitation, denaturation and / or aggregation curves ("melting curves") for one or more target proteins. Thereby, a difference in tolerance of target protein(s) to solvent-induced precipitation between the first and second samples indicated in differences of the melt-curves of the target protein(s) under such conditions preferably indicates an interaction between the ligand and the target protein.

[0062] The present method may be used in embodiments for various experimental applications, for example for melt-curve experiments (with varied solvent concentrations), concentration-response curve experiments (with varied ligand concentration), 2D-experiments (varied solvent and ligand concentrations), and “Area-under-the-curve” experiments.

[0063] A non-limiting example of an “Area-under-the-curve” experiment, or "PISA" experiment is a workflow wherein first (1) a melt-curve-experiment is performed, subsequently (2) the supernatant of the different solvent-precipitations is pooled, and (3) this pooled samples is used for LC- MS / MS analysis. This has the advantage that only 1 instead of 8-10 (or more) samples have to be measured per condition. In that pooled sample the intensity of each protein represents the "integral" (=AUC) of the melt curve. One can perform these experiments in the simple setup comparing 2 samples (drug vs vehicle) or multiple samples (different drug concentrations). In an exemplary "PISA" experiment, the inventors (1) performed a melt-curve-experiment and then (2) pooled the supernatant of the different solvent-precipitations, and (3) used said pooled samples for LC-MS / MS analysis. Figure 3 depicts an overview over potential experimental setups for AUC experiments.

[0064] In some embodiments it may be difficult or even not possible to distinguish in some area-under- the-curve and / or PISA experiments changes in protein stability (i.e. , stabilization or destabilization) from changes in protein concentration (i.e., through modulation of protein synthesis or degradation) upon compound treatment. In embodiments the present method is used for melt curve analysis comprising fixed ligand concentrations and varied denaturant concentration.

[0065] In embodiments the present method is used for a “PISA” -format analysis comprising melting curve analysis. Therein, all samples of a melt curve are combined into a single sample (“area under the curve” or “integral” (mathematical) approach). Such compressed format can save LC- MS / MS analysis time and allows for higher throughput (e.g., analysis of more replicates, ligands, concentrations, and / or cell lines, compare Gaetani et al. Proteome Integral Stability Alteration assay dramatically increases throughput and sensitivity in profiling factor-induced proteome changes, J. Proteome Res. 2019, 18, 11, 4027-4037).

[0066] In embodiments the present method is used for concentration range analysis comprising fixed concentrations of denaturants and varied ligand concentrations.

[0067] In embodiments the present method is used for two-dimensional (2D) analysis comprising varied concentrations of both, denaturants and ligand(s).

[0068] In embodiments a mixture of solvents is used for precipitation and comprises at least two or more solvents.

[0069] In embodiments the solvent and / or mixture of solvents used for precipitation comprises at least one organic and / or inorganic substance capable of denaturing and precipitating proteins.

[0070] In embodiments the solvent and / or mixture of solvents used for precipitation comprises at least one acidic reagent, alkaline reagent, metal ion and / or salt.

[0071] In embodiments the solvent and / or mixture of solvents used for precipitation is selected from the group consisting of acetone, methanol, ethanol, acetic acid, ascorbic acid, citric acid, trifluoroacetic acid, iso-propanol, butanol, phosphoric acid, or any combination thereof, but without being limited to these chemicals.

[0072] In embodiments the solvent mixture comprises a mixture of acetone, ethanol and acetic acid (abbreviated as AEA).

[0073] In embodiments the ratio of said solvents in said AEA is 50:50:0.1 ( lvlv) of acetone:ethanol:acetic acid (all units in volume, as the melting point of acetic acid is 16.6 °C). In embodiments a preferred solvent is AEA (acetone:ethanol:acetic acid), a mixture of acetone, ethanol and acetic acid. In embodiments the mixing ratio (% v / v / v) of AEA is 50:50.0.1 (acetone:ethanol:acetic acid).

[0074] In embodiments the concentration in the sample of AEA, which is used to treat the samples is 0- 50% (v / v), preferably 5-50% (v / v), more preferably 9%-22% (v / v). Preferably the concentration is the concentration in the sample, such as, e.g., the final concentration in a cell suspension or in the supernatant of adherent cells. In embodiments ascorbic acid is added to the sample at a final concentration of 1 mM-15 mM and / or wherein citric acid is present in the solvent at a final concentration of 1 mM-5mM. In embodiments ascorbic acid is added to the sample at a final concentration of 0.5 mM-25 mM , 1- 10mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12 mM, 13 mM, 14 mM, 15mM, 20 mM, 25 mM, 30 mM. In embodiments the citric acid is present in the solvent at a final concentration of 1 mM-5mM, 0.5-10 mM, 1-7 mM, 1-10mM, 0.5 mM, 1 mM, 2 mM, 3mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12 mM, 13 mM, 14 mM, 15mM. The final concentration is preferably the concentration in the sample.

[0075] In embodiments the solvent-treated samples are incubated with shaking at 20-40°C for 20-40 minutes, preferably at 20-30°C for 20-40 minutes shaking, preferably at 30-40°C for 5-30 minutes shaking, more preferably at 37°C or between 36.5 and 37.5 °C for 10-30 minutes. In embodiments the solvent-treated samples are incubated with shaking at 20-40°C, at 25-38°C, at 30-37 °C, at 37°C, at 36.5-37.5°C, at 30 °C or at 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 29.5, .30, 30.5, 31 , 32, 33, 34, 35, 36, 36.5, 37, 37.5, 38, 39, 40 °C. Any ranges between the enlisted temperatures are also envisaged, as well as an uncertainty or deviation therefrom of 1-5 °C.

[0076] In embodiments the solvent-treated samples are incubated, preferably with shaking, for 5-60 minutes, preferably for 20-40 minutes, or for at least or exactly 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes or longer.

[0077] In embodiments the incubation can be performed according to a specific temporal regimen, i.e., regarding the duration of the ligand-target incubation and / or the analysis of a sample. Different incubation conditions can also be used to learn more about the ligand and / or the biological system (e.g., cell) that is used.

[0078] Generally, and without being bound by theory, during incubation at constant ligand concentration it will take some time before a maximum target occupancy (by the ligand) can be expected to have occurred. For example, in wash-out experiments, it will again take time for the ligand to disappear from the target protein and the system, e.g., cell used. Generally, and without being bound by theory, during incubation secondary effects can also be expected - depending on the type of effect - to have different kinetics.

[0079] In embodiments the temporal regime, e.g., the treatment duration and sampling, can also be controlled to learn more about the drug or the biological system. In can be assumed that it would take some time for maximum target occupancy to occur at a constant concentration. In wash-out experiments, it likely would take time for the substance to disappear from the protein and from the system. Moreover, it can be assumed that the secondary effects would also - depending on the type of effect - have different kinetics.

[0080] In embodiment the intact cells of the first and second samples are lysed by the addition of a lysis buffer and / or at least two freeze-thaw cycles, preferably using liquid nitrogen and a water bath at about 37°C. In embodiments intact cells of the first and second samples are lysed by the addition of a lysis buffer and / or at least two freeze-thaw cycles, preferably using liquid nitrogen and a water bath at 10-40 °C, 20-40°C, at 25-38°C, at 30-37 °C, at 37°C, at 36.5-37.5°C, at 30 °C or at 10, 15, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 29.5, .30, 30.5, 31 , 32, 33, 34, 35, 36, 36.5, 37, 37.5, 38, 39, 40 °C. Any ranges between the enlisted temperatures are also envisaged, as well as a uncertainty or deviation therefrom of 1-5 °C.

[0081] In embodiment the lysis buffer consists of or comprises phosphate buffered saline (PBS) and / or comprises a lysis agent selected from the group of “mild” detergents such as IGEPAL CA-630, n- Dodecyl-P-D-Maltopyranoside, n-octyl-p-d-thioglucopyranoside, CHAPSO, CHAPS, Brij 35 and Pluronic F-127. In embodiments the lysis conditions, which are influenced by the strength (mild / moderate / strong) of the detergent used, are preferably “mild” lysis conditions.

[0082] In embodiments the lysis agent has a final concentration in the lysis buffer of 0-2% (% v / v), preferably 0.2-1% (% v / v), more preferably wherein the lysis agent is IGEPAL CA-630 which has a final concentration of 0.4% (% v / v) in the lysis buffer.

[0083] In embodiments the multiple solvent conditions induce precipitation of 80-90% of total protein in a sample. In embodiments the multiple solvent conditions induce precipitation of 80-90%, 70-100%, 70-99,9%, up to 100%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99,9%, 100% of total protein in a sample. In embodiments the solvent conditions induce precipitation of 70-100% of total protein in a sample. In embodiments the (multiple) solvent conditions induce precipitation of 70-100% of the target protein in a sample.

[0084] In embodiments soluble proteins are separated from precipitated proteins by centrifugation and / or filtration. In embodiments soluble proteins are separated from precipitated proteins by centrifugation. In embodiments soluble proteins are separated from precipitated proteins by filtration. In embodiments soluble proteins are separated from precipitated proteins by affinity capture methods.

[0085] In embodiments soluble proteins are separated from precipitated proteins by precipitating / capturing the denatured / precipitated proteins on a solid support, such as e.g., (magnetic) beads or any other suitable solid support, or means suitable for affinity or protein aggregation capture.

[0086] In embodiments soluble proteins are separated from precipitated proteins by protein aggregation capture or similar methods. Preferred but non-limiting examples of protein aggregation capture comprise the non-specific binding of proteins to microparticles (e.g., beads, magnetic beads etc. or as described in Batth et al., Mol Cell Proteomics. 2019 May;18(5):1027-1035, PMID: 30833379). Protein aggregation capture commonly takes advantage of the property of insoluble protein aggregates (e.g., herein precipitated proteins) to preferentially precipitate on microparticles. In embodiments soluble proteins are separated from precipitated proteins by protein aggregation capture comprising microparticles, such as particles, beads or magnetic beads. In embodiments soluble proteins are separated from the precipitated proteins by centrifugation and / or filtration and equal amounts of supernatant from each of said first and second samples are taken and dried by vacuum centrifugation. In embodiments soluble proteins (supernatant) are separated from the precipitated proteins in the first and second sample and equal amounts of the supernatant from each of the first and second samples are taken and dried, preferably by vacuum centrifugation / vacuum concentration (e.g., SpeedVac).

[0087] In embodiments, the supernatants from different solvent concentrations are used and treated with acetone to induce protein precipitation. In other embodiments, the supernatants from different solvent concentrations may be precipitated onto magnetic beads (e.g., via SP3 / PAC beads precipitation).

[0088] In embodiments soluble proteins are separated from the precipitated proteins by centrifugation and / or filtration. In embodiments the (soluble) proteins of the supernatant (the supernatant of the first precipitation reaction (e.g., of step b)) are subsequently precipitated with acetone. In embodiments in the initial precipitation step (e.g., step b)) proteins are precipitated “onto” magnetic beads (e.g., SP3 / PAC beads precipitation) and / or are captured by magnetic beads. In embodiments the separated proteins are subsequently analyzed in step d) and / or e). In embodiments the supernatants and / or precipitates of several samples (at least two samples) that were treated with different solvent concentrations and were processed according to the method of the present invention can be analyzed and optionally compared to each other.

[0089] In embodiments in steps d)-e), the method for detecting the amount, quantity or level of the target protein is selected from the group consisting of immunoblotting, one-dimensional electrophoresis, two-dimensional electrophoresis, quantitative proteomic techniques, such as mass spectrometry, preferably LC-MS / MS, data-dependent acquisition (DDA), data-independent acquisition (DIA), and combinations thereof and / or Multiple reaction monitoring mass spectrometry (MRM) or parallel reaction monitoring mass spectrometry (PRM).

[0090] In embodiments in steps d)-e), the method for detecting the amount, quantity or level of the target protein is (quantitative) mass spectrometry (MS, MS / MS) and the method further comprises the separation / enrichment and / or processing of the in step c) separated proteins by liquid chromatography (LC) before mass spectrometry (MS, MS / MS) measurement. In embodiments the method for detecting the amount, quantity or level of the target protein in steps d)-e) comprises liquid chromatography coupled to (quantitative) mass spectrometry (MS) analysis (LC- MS), preferably after proteolytic (on-bead) digestion of the separated protein(s).

[0091] In embodiments the (quantitative) mass spectrometry (MS, MS / MS) analysis comprises the steps of (i) digesting the in step c) separated proteins (precipitated or soluble) “on-bead”, and / or (ii) eluting the peptides originating from the separated proteins (that are still bound to the solid phase, e.g. bead), and / or (iii) separating peptides from the isolated proteins by liquid chromatography (LC), and analysing the LC-separated peptides from the separated proteins by mass spectrometry. In embodiments in steps d)-e), the method for detecting the amount, quantity or level of the target protein is selected from the group consisting of immunoblotting, one-dimensional electrophoresis, two-dimensional electrophoresis, quantitative proteomic techniques, such as mass spectrometry, preferably LC-MS / MS and / or Multiple reaction monitoring mass spectrometry (MRM) or parallel reaction monitoring mass spectrometry (PRM), Aptamers (such as SomaLogic, Nautilus Biotechnology) and (Nano-)pore-protein-sequencing.

[0092] In embodiments in steps d) and / or e), the method comprises a computer-based analysis. In embodiments the computer-based analysis comprises a statistical analysis. In embodiments wherein the data analysis in step d) comprises mass spectrometry analysis, the computer-based analysis comprises one or more of the following steps: determination of mass and intensity of peptide peaks in MS spectra, determination of mass and intensity of peptide fragment peaks in tandem mass spectra, comparison and scoring of similarity of tandem mass spectra (i.e., fragment spectra) to theoretical peptide fragment spectra (e.g. from protein sequence databases), quantification of reporter ion intensities, fragment intensities or peptide intensities and aggregation of these intensities on protein level via a protein grouping approach (e.g., with the MaxQuant software).

[0093] In embodiments the method comprises a computer-implemented analysis to identify the difference in tolerance of the target protein to solvent-induced precipitation.

[0094] The type of evaluation or data analysis performed in steps d) and / or e) preferably depends on the respective embodiment of the method according to the invention, hence on the format of the experiment and / or the analysis methods used. In embodiments a curve fitting (analysis) can be performed for melting curve experiments. In embodiments unfolding / aggregation curves can, for example, be fitted using (simple) sigmoidal equations based on chemical denaturation theory of proteins. In embodiments concentration response curves can be fitted using typical dose / concentration response curve approaches. In embodiments statistical analysis (e.g., employing ANOVA, t-test, Limma, etc.) is performed when samples are compared only pairwise (e.g., in a PISA experiment with 1 substance vs. control). In embodiments where a PISA approach is performed with multiple ligand concentrations the analysis can comprise the fitting of a dose-response curve.

[0095] In embodiments the analysis may involve the “TPP” R-statistics software package, which provides a framework for analyzing TPP (and likewise SIPP) data as described in Franken et al. 2015 (“Thermal proteome profiling for unbiased identification of direct and indirect drug targets using multiplexed quantitative mass spectrometry”. Nat Protoc. 2015 Oct; 10(10): 1567-93). The TPP package may be applied to SIPP data according to Van Vranken et al., 2021 . The nonparametric analysis of TPP data regarding the melting curve data is described in Childs et al. (Mol Cell Proteomics. 2019 Dec;18(12):2506-2515). A non-parametric analysis of TPP data regarding the 2D (2D-experiment = varied solvent + varied ligand concentrations) and concentration response format is described in Kurzawa et al. (“A computational method for detection of ligand- binding proteins from dose range thermal proteome profiles”. Nat Commun. 2020 Nov 13;11 (1):5783).

[0096] In embodiments in steps d)-e), the method for detecting the amount, quantity or level of the target protein is performed by immunological detection methods, such as Western blot or ELISA. In embodiments in steps d)-e), the method for detecting the amount, quantity or level of the target protein is performed by electrophoresis, such as one-, two- or three-dimensional electrophoresis. In embodiments in steps d)-e), the method for detecting the amount, quantity or level of the target protein is performed by quantitative proteomic techniques, such as mass spectrometry, preferably LC-MS / MS and / or LC-MS in combination with MRM or PRM.

[0097] In embodiments methods suitable for detection or analysis of the sample are, without limitation thereto, e.g., binder-based or immunological detection methods (such as, e.g., antibodies, aptamers, immunoblotting, ELISA-assays, Western blot analysis, Olink, SomaLogic, etc.), methods in which a (purely) cloned protein is visualized (e.g., via GFP labelling or tagging or similar methods), mass spectrometry-based methods (e.g., targeted proteomics using MRM / PRM mass spectrometry, quantitative discovery proteomics using LC-MS / MS with different acquisition methods, such as data-dependent or data-independent acquisition, and different quantification methods, e.g., as disclosed herein).

[0098] Hence, in embodiments the method for detecting the amount, quantity or level of the target protein is selected from the group consisting of immunological detection methods, immunoblotting, antibodies, aptamers, immunoblotting, ELISA-assays, Western blot analysis, Olink, SomaLogic, affinity-based detection, electrophoresis (one, two or three dimensional), (transgenic or molecular biology-based) labelling of target protein(s), such as proteins fused to tags, fluorescence-tags, or other, quantitative proteomic techniques (e.g., mass spectrometry, preferably LC-MS / MS and / or MRM / PRM mass spectrometry).

[0099] Examples for (transgenic) labelling of target protein(s), such as tags or fluorescence-tags fused to proteins are FLAG-tag, GFP-tag, YFP-tag etc., GST-tag, HA-tag, HRP-tag, Myc-tag, LacZ-tag, SUMO- or Strep-tag or any other affinity or fluorescence tag.

[0100] In embodiments protein detection approaches suitable for analyzing single proteins via antibody- or aptamer-based detection systems, e.g., ELISA, Western Blot or similar, may be used in steps d)-e).

[0101] In embodiments during analysis, e.g., by mass spectrometry or Western Blot, also other (chemical) properties or states of (target) proteins may be analyzed, such as, e.g., post- translational modifications (e.g., phosphorylation, acetylation, methylation, SUMOylation etc.) or other types of chemical modifications (e.g., by covalent inhibitors), isoforms or any proteoforms of the analyzed protein. Hence, in some embodiments the analysis under steps d)-e) further comprises the analysis of post-translational modifications or isoforms of a target protein.

[0102] In embodiments in the final analysis any one or both of the fractions (derived from step c) comprising either soluble / non-precipitated proteins, or insoluble / precipitated proteins respectively can be analyzed. In other words, in embodiments after the precipitation of the sample (and optional separation of the two fractions) either one or both of the fractions comprising respectively (i) soluble / non-precipitated proteins or (ii) insoluble / precipitated proteins, are analyzed.

[0103] In embodiments soluble proteins may be isolated or separated after the prior removal of insoluble proteins. In embodiments (after the addition of a precipitation inducing agent, such as a solvent or solvent mixture) unfolded and / or aggregated proteins are removed, discarded or separated from the sample fraction comprising the soluble proteins. In embodiments insoluble proteins may be separated or extracted, for example, via centrifugation, incubation (gravity based), or on chromatographic material (e.g., suspension trapping, protein aggregation capture or via filters, chromatographic materials or affinity columns, e.g., under application of gravity, centrifugation or vacuum. In embodiments also filtration may be used, e.g., using 100 kDa MWCO filters or similar.

[0104] In embodiments the solvent(s) may be removed before analysis in steps d)-e), e.g., from the supernatant comprising the soluble protein fraction and / or from the precipitated fraction of a sample. In embodiments the solvent(s) may be removed before analysis, e.g., via precipitation, heating, suspension trapping or vacuum centrifugation (e.g., via SpeedVac: vacuum centrifugation with optional heating).

[0105] In embodiments analysis of the separated proteins (step c), e.g., the soluble proteins of the supernatant and / or the precipitated proteins, may be conducted by quantitative proteomics such as label-free shotgun proteomics, or isobaric labelling based proteomics.

[0106] In embodiments the quantitative proteomic technology comprises label-free quantification and / or label-based quantification.

[0107] In embodiments the method of labelled quantification comprises one or more isotope labeling methods, such as dimethyl labeling, SILAC, TMT, ICAT, MeCat or iTRAQ.

[0108] In embodiments the data analysis comprises approaches for curve fitting, e.g., comprising deriving thermodynamic parameters from the isothermal method according to the invention.

[0109] In embodiments the method comprises a computer-implemented analysis to identify the difference in tolerance of the target protein to solvent-induced precipitation.

[0110] In embodiments the present method may comprise varied denaturation and lysis conditions.

[0111] In embodiments the affinity of at least two or a multiplicity of different ligands for the target protein can be measured, wherein the protein is capable of unfolding due to a chemical change (due to a change of the solvent (or chemical) composition), wherein the method comprises

[0112] (i) contacting said target protein with a combination of at least two or more different ligands of said multiplicity in separated samples, respectively,

[0113] (ii) simultaneously heating said multiplicity of samples from step (a); (iii) measuring in each of said samples a physical change associated with the chemical unfolding of said target protein resulting from said denaturation;

[0114] (iv) generating a chemical unfolding curve for said target protein as a function of temperature for each of said samples;

[0115] (v) comparing each of said curves in step (d) with each of said other curves obtained for said target protein and to the chemical unfolding curve for said target protein in the absence of any of said ligands; and

[0116] (vi) determining the affinities of said combinations of different ligands for said target protein according to the change in each of said chemical unfolding curves.

[0117] Hence, in embodiments other solvents or mixtures than the herein described may be used. For example, in embodiments applying different types of solvents or denaturation mixtures may provide other or an improved resolution of isothermal protein stabilization. The skilled person knows how to determine or select suitable solvents to be used within the present method.

[0118] Also, in embodiments different pH conditions may be used within the method of the invention, e.g., comprising different denaturation strengths for different sample materials). The skilled person knows how to determine or select suitable pH conditions.

[0119] In embodiments other detergents, or even the omittance of detergent, may be equally suited to lyse cells and extract proteins mildly.

[0120] In embodiments of the invention unfolded and aggregated proteins may be removed or enriched e.g., by filtration, centrifugation and / or protein aggregation capture (e.g., as described in Batth et al, Protein Aggregation Capture on Microparticles Enables Multipurpose Proteomics Sample Preparation, Mol Cell Proteomics. 2019 May; 18(5): 1027-1035).

[0121] In embodiments the method according to the invention may be applied in the context of a high throughput screening. The present method enables in embodiments also a high degree of automatization. In embodiments the present method may also be combined with “PISA” analysis. In some embodiments the present method may be combined with a readout of selected target proteins using antibody-based approaches. The skilled person is familiar with suitable read out methods to be used in the context of the present method.

[0122] In embodiments the data analysis or readout of the present method may be achieved by a variety of quantitative proteomics approaches, such as, without limitation to, SILAC, TMT, ICAT, MeCat or iTRAQ dimethyl labelling, label-free quantification, DIA, targeted proteomics via PRM / MRM / SRM, or comparable methods. In embodiments the data analysis or readout of the present method may be achieved with antibody- or aptamer-based approaches, such as, without limitation to, ELISA, Western blot, dot blot or similar. The skilled person is familiar with suitable read out methods. In embodiments quantitative proteomics approaches comprising mass spectrometry comprise any state-of-the-art data acquisition and quantification methods, such as data-independent acquisition, Tandem Mass Tags, SILAC, Dimethyl-Labelling, and targeted proteomics (e.g., using parallel-reaction monitoring or TMT-based approaches, such as TOMAHAQ).

[0123] The present method may in embodiments be used for the analysis of sample materials and biological systems, such as primary cells, IPSCs, body fluids (such as in Perrin et al., “Identifying drug targets in tissues and whole blood with thermal-shift profiling”; Nat Biotechnol. 2020 Mar;38(3):303-308).

[0124] The present method may in embodiments also be used for ex vivo analysis of tissue samples, isolated organelles or subcellular structures (e.g., nuclei), bacteria, fungi, plants, or other organisms.

[0125] In embodiments the method according to the invention may be used as a generic assay for different steps of basic research and in preclinical drug development.

[0126] Non-limiting examples of such assays are label-free target class agnostic approaches to study protein igand interactions and for characterization or interrogation of cellular or physiological protein states, such as, e.g., different stability depending on activation and functional state, or sequence. Hence, in embodiments the present method may be used to analyze, for example, protein isoforms, mutations, SNPs, post-translational modifications of proteins, binding partners, and / or subcellular localization of molecules or proteins, or similar.

[0127] In embodiments the present method may be used to analyze, for example, proteinJigand interactions, wherein the ligand can be one or more protein, nucleic acid or (oligo)nucleotide (e.g., DNA, RNA), metabolite, lipid, glycan or small molecule compound etc.

[0128] In embodiments the present method may be used for high throughput screenings of pharmacologically active compounds .

[0129] In embodiments the present method may be used to analyze target deconvolution, such as ,e.g., for the identification of (drug dose resolved) target interactions, the identification of unexpected off-targets at different drug doses), and / or selectivity profiling of compounds.

[0130] In embodiments the present method may be used in the context of high-throughput screenings to identify proteins of interest. Non-limiting examples of high-throughput screenings are target validation (determination of the in vivo or in living cells potency of target engagement or downstream effects), target engagement, lead selection / optimization (use of target engagement information to optimize the on-target potency and concomitantly reduce the potency for undesired off-target hits), biomarker discovery for target engagement, residence time measurements in living cells (e.g., as described in Sabatier et al., System-Wide Profiling by Proteome Integral Solubility Alteration Assay of Drug Residence Times for Target Characterization, Analytical Chemistry, 2022) and mechanism of action analysis. In embodiments the present method may be used in the context of biomarker discoveries to identify proteins which exhibit differential stability against denaturation in different biological states or different biological systems (examples of biomarker discovery studies using state of the art approach are Mackmull, et al. Global, in situ analysis of the structural proteome in individuals with Parkinson's disease to identify a new class of biomarker. Nat Struct Mol Biol. 2022 Oct; 29(10):978-989)

[0131] DETAILED DESCRIPTION OF THE INVENTION

[0132] All cited documents of the patent and non-patent literature are hereby incorporated by reference in their entirety.

[0133] The present invention is directed in one aspect to a method for detecting an interaction between a ligand and at least one target protein in intact cells, comprising inducing protein precipitation using one or more solvents in a first sample and in a second sample, both comprising intact cells , wherein said first sample is treated with said ligand, and said second sample is not treated with said ligand, wherein a difference in tolerance of the target protein to solvent-induced precipitation between the first and second samples indicates an interaction between a ligand and a target protein.

[0134] A “sample” may comprise herein, without limitation thereto, a suspension of cells, living cells, a suspension of living cells, permeabilized but intact and / or viable cells, fixated cells, body fluids, organ samples, tissue samples, tissue samples of dosed animals or subcellular extracts (e.g., nuclei). In embodiments a sample may be taken from a subject, a patient, a cell culture of patient cells or cell lines, an animal, or a cell culture of animal cells or cell lines of a biopsy, a tissue sample, a blood sample, or an environmental sample. Basically, any kind of sample that is suspected to contain biochemical information of interest. As used herein, the term “sample” is a biological sample that is obtained or isolated from the subject, sample as used herein may, e.g., refer to a sample of bodily fluid, tissue or surface (e.g., mucosal swap sample) obtained for the purpose of diagnosis, prognosis, or evaluation of a subject of interest. In case of a liquid sample, or a liquid biopsy the sample may be in embodiments a sample of a bodily fluid, such as blood, plasma, serum, urine, cerebrospinal fluid, pleural effusions, saliva, sputum, a cellular extract, and the like. In further embodiment the sample may be a solid sample, such as a tissue sample, a biopsy, cells, a cell culture sample, a tissue sample, a tissue biopsy, a stool sample or a swap- derived sample.

[0135] Herein a “ligand” is a substance, molecule or agent binding to a target protein (protein of interest). In embodiments a “ligand” is selected from the group comprising small molecules, biologies, metabolites, plant extracts or natural products, food additives, environmental pollutants, agricultural pesticides or herbicides, environmental agents, metal ions, nanoparticles, peptides, proteins, nucleic acids, toxins, a drug or another chemical. In embodiments a ligand may be, without limitation, a substance, chemical or molecule, an ion, a particle, a binding agent or binding ligand, an antibody, a protein, a nucleic acid, a label, a chemical, agent or substance, a dye, a tag, an aptamer or any other type of ligand.

[0136] “Tolerance” herein preferably refers to the stability of molecules, such as, e.g., peptides, polypeptides, proteins, nucleic acids, ligand / agent and target complexes, antibody and antigen complexes, protein complexes, protein-nucleic acid complexes, ligand and (poly)peptide complexes, ligand and protein complexes, ligand and nucleic acid complexes, against solvent- induced denaturation and precipitation.

[0137] Herein the term “supernatant” preferably refers to the supernatant (fraction of soluble and / or nonprecipitated proteins) of a sample after the addition of one or more solvents or precipitationinducing substance(s), such as e.g., after step b) of the present method.

[0138] Herein the term "different implementations" refers to melting curve experiments in which different concentrations of solvents are used per condition (e.g., vehicle, ligand (e.g., drug), etc.) and each supernatant sample is analyzed individually after precipitation. Thus, for each condition melting curves for each protein measured are obtained and the melting curves of each protein can be compared for different conditions.

[0139] Herein "1 D experiments" describe the selection of a suitable solvent concentration at which the target engagement can be measured accurately in the presence of a ligand (e.g., a substance, drug etc.), such that also dose-response relationships can be derived when using multiple drug concentrations.

[0140] In “2D experiments”, melting curves are recorded for different ligand concentrations, not only + / - ligand (e.g., 1 substance, 1 control sample), such that EC50 values can be estimated / determined. Such experiments are commonly quite costly, especially in terms of measurement time to be invested.

[0141] The EC50 describes the half maximal effective concentration, which is the concentration of a ligand, drug, chemical, antibody or substance which induces a response halfway between the baseline and maximum after a specified exposure time. In other words, EC50 can be defined as the concentration required to obtain 50% of a ligands’ effect.

[0142] The PISA format refers to melting-curve formats, where all samples of a melt curve are combined into a single sample (a so-called “area under the curve” approach). For MS analysis this compressed format saves LC-MS / MS time and allows for higher throughput. PISA allows simplification of data analysis, since for PISA all samples from a denaturation curve are preferably pooled and subsequently analyzed as a single sample. The intensity of this sample is preferably proportional to the integral of or the area under the denaturation curve, with e.g., higher intensities reflecting curves that are shifted by protein stabilization. "PISA"-experiments, can either simply comprise or lack the addition of an active ingredient (+ / -), or are concentration resolved (e.g., consider / analyze multiple concentrations of an active ingredient). The conformation of a protein can be defined as the three-dimensional arrangement of atoms in an amino acid-chain molecule which determines the overall shape of the protein molecule. A protein’s conformation arises from the bonding arrangements within its structure and / or its interactions with non-covalent binding partners, in a dynamic fashion.

[0143] Protein denaturation describes the alteration of the secondary and tertiary structures of a protein, while the peptide bonds between the amino acids of the primary structure stay intact. All structural levels (primary, secondary and tertiary) of a protein determine its function. Accordingly, a denaturated protein is considered to be no longer able to perform its function.

[0144] Herein “initial conformation” refers to the conformation a respective protein in a sample has before being analyzed or processed according to the present method. The initial conformation may in embodiments be a natural conformation, a native confirmation, a pathogenic conformation, a denatured conformation or any other conformation a protein can adopt or fold into.

[0145] In embodiments a “cell lysis buffer” or “lysis buffer” may induce the lysis of cells (e.g., in a sample). In embodiments the “cell lysis step”, “cell lysis” or “lysis step” comprises the partial lysis of cells or only the lysis of a portion of the cells. In embodiments the terms the “cell lysis step”, “cell lysis” or “lysis step” may be used interchangeably or may refer to related processes. In other embodiments a cell lysis agent comprises or consists of a cell lysis buffer, a detergent, or any combination thereof. The skilled person knows how to select appropriate agents and conditions to lyse cells in a sample. In other embodiments the lysis of cells can be achieved alternatively or additionally to an agent by physical lysis, e.g., via ultrasound, a douncer, freeze-thaw cycles, syringes or French press etc. The skilled person knowns different methods to induce cell lysis.

[0146] “Precipitation” of a protein in general occurs by a change in the conditions of its aqueous environment, which through a thermal change or change in pH or change in the chemical composition of the aqueous buffer (by the addition of solvents, salts, metals, etc.) disrupt native intramolecular interactions, and the proteins denature, aggregate and / or precipitate out of solution. A thermal change or change in pH or change in the chemical composition of the aqueous buffer (by the addition of solvents, salts, metals, etc.) can lead to an unfolding of the protein, thereby exposing hydrophobic structures of the protein, which in turn aggregates with hydrophobic structures of other similarly unfolded protein molecules through hydrophobic interactions.

[0147] A “soluble protein” may be a protein, which is dissolved or solubilized within a solution, suspension or any solvent, such as aqueous solvents. A soluble protein or solubilized protein may be present in a supernatant of a solution, suspension, solvent or sample even after centrifugation of said solution, suspension, solvent or sample. The centrifugation may herein be performed at between 1 g and 25,000g, wherein g is the relative centrifugal force or gravitational force. In biochemistry and pharmacology in general, a ligand is commonly a substance, chemical or molecule that forms a complex with a biomolecule.

[0148] In embodiments a “ligand” is selected from the group comprising small molecules, biologies, metabolites, plant extracts or natural products, food additives, environmental pollutants, agricultural pesticides or herbicides, environmental agents, metal ions, nanoparticles, peptides, proteins, nucleic acids, toxins, a drug or another chemical. In embodiments a ligand may be, without limitation, a substance, chemical or molecule, an ion, a particle, a binding agent or binding ligand, an antibody, a protein, a nucleic acid, a label, a chemical, agent or substance, a dye, a tag, an aptamer or any other type of ligand.

[0149] In general cell permeabilization is considered to differ from cell lysis. Permeabilized cells differ significantly from lysed cells and cell extracts, whereby permeabilized cells comprise properties of intact cells, such as, e.g., (mostly) intact organelles, sustained metabolic activity, molecular crowding and / or condensates. Permeabilized cells herein refer to cells that are still viable and / or may be considered intact. In the context of the invention permeabilized cells comprise preferably also membrane permeability for solvents and other reagents, but no lysis of the cells and does not facilitate the leakage (outflow) of components out of the cell. In summary, permeabilized cells are in the context of the present method not to be equated with cell lysates or lysed cells, as they still comprise various properties of intact and viable cells and preferably no internal components of the cells are able to exit the cell.

[0150] Hence, in embodiments cells may be permeabilized with mild reagents not having a negative effect on the viability and / or intactness of the cell as a whole, such as lipofectamine or electroporation. In embodiments permeabilization of cells allows larger molecules, such as antibodies or probes, to access the inside of cell, thereby enabling the targeting of proteins or conditions of interest inside the cell without lysing or damaging the cell. Permeabilization in general provides access to intracellular or intraorganellar antigens without lysing or damaging the cell. Common types of permeabilizing techniques and agents are, without being limited thereto: electrophoresis, or liposome-forming agents such as, e.g., lipofectamine,.

[0151] Adherent cells, also termed anchorage-dependent cells require surface fixation / adherence to grow in vitro. In vivo anchorage-dependent cells are residing embedded within the (connective) tissue of an organ or tissue.

[0152] Suspension cells are not dependent on surface connection to grow in vitro and remain suspended in vitro in a tissue culture vessel, e.g., dish or flask. In vivo suspension cells form commonly part of the circulatory system.

[0153] Some cultures grow as semi-adherent cells, namely as a mixed population with a proportion of cells not attaching in vitro to a tissue culture vessel, e.g., dish or flask, and remaining in suspension. Cells can also be provided as functionally cryopreserved cells or “assay ready” cells, which may be obtained as aliquots of functionally cryopreserved cells that can be used immediately after thawing without prior cultivation (“assay ready”).

[0154] Methotrexate (MTX) is a chemotherapy agent and immune-system suppressant, which is commonly used in the treatment of cancer or autoimmune diseases. Methotrexate is known to target DHFR and TYMS (when polyglutaminylated).

[0155] MK-2206 is a protein kinase inhibitor, which targets the serine-threonine-protein kinases AKT1 and AKT2.

[0156] Talmapimod (SCIO-469) is an orally active, selective, and ATP-competitive p38a (MAPK14) inhibitor with an IC50 of 9 nM.

[0157] Vorinostat (SAHA) is a potent and orally active pan-inhibitor of histone deacetylases HDAC1 , HDAC2 and HDAC3 (Class I), HDAC7 (Class II) and HDAC11 (Class IV), an “epigenetic drug”, with IC50 values of 10 nM and 20 nM for HDAC1 and HDAC3, respectively.

[0158] Ibrutinib is a covalent receptor tyrosine kinase inhibitor, which targets BTK.

[0159] Cyclosporin A is a natural product, molecular glue and immunosuppressive drug, which targets peptidyl-prolyl cis-trans isomerases (cyclophilins).

[0160] Analysis methods

[0161] In embodiments before the mass spectrometry measurement the isolated proteins are subjected to preparation of the proteins for mass spectrometric analysis. The preparation of proteins for mass spectrometric analysis preferably contains one or more of the followings steps: removal or dilution of agents, which may interfere with subsequent steps of the procedure (e.g., chaotropic reagents, detergents, solvents, salts), the proteolytic digestion of proteins into peptides preferably with sequence-specific proteases trypsin or LysC, the desalting and concentration of peptides, and the elution of peptides from a solid phase prior to the analysis by mass spectrometry, preferably combined with liquid chromatography (LC) previous to mass spectrometry (LC-MS). Elution from a solid support can occur, e.g., in embodiments by acetonitrile (ACN) and / or trifluoroacetic acid (TFA).

[0162] The proteolytic digestion is preferably performed before the mass spectrometry measurement of enriched / isolated proteins. The digestion can in embodiments be performed in solution or “on- bead”. “On bead” digestion refers to the digestion of proteins while still bound to a solid enrichment-phase or solid support, such as stationary solid phases, for example, a column, a surface, a resin or (magnetic) beads.

[0163] The term "mass spectrometry" (MS) generally refers to a technology for the separation of electrically charged molecules (ions) in the gas phase. The ions are preferably generated in an ion source, e.g., electrospray ionisation (ESI) or nano ESI (for higher ionization frequency and analytic sensitivity), which allows the transfer and ionisation of analytes from a solid or liquid phase (e.g., from liquid chromatography; LC) into the gas phase. These gas-phase ions are then analysed by a mass analyser, which sorts the ions in space or time according to their mass-to- charge ratio (m / z). Finally, the separated ions are detected as electrical signals by an ion detector in the space or time domain and translated into mass spectra (showing the number of ions at different m / z values). Mass spectrometry thus facilitates the identification of molecules based on their characteristic mass-to-charge ratio and fragmentation patterns.

[0164] Tandem mass spectrometry, also known as MS / MS, involves the coupling of two or more mass analyser steps in time or space and the use of an additional gas-phase reaction step to increase the resolution of sample analysis. In MS / MS, the molecules of a sample are ionised in a first step and the first spectrometer (called MS1) separates these ions according to their mass-to-charge ratio (m / z). In a second step, ions of a specific m / z ratio are isolated in the same or a different mass analyser and then fragmented, e.g., by collision-induced dissociation, higher-energy collision dissociation (HCD), electron transfer dissociation (ETD), electron capture dissociation (ECD), ion-molecule reaction or (ultraviolet) photodissociation. The fragments are then analysed in a mass analyser to separate and detect the fragments according to their m / z ratio (MS2). The separation and fragmentation increases the resolution of the detection by enabling the separation and identification of ions with very similar m / z ratios in single mass spectrometry (MS).

[0165] Quantitative mass spectrometry can be used for quantitative proteomics, i.e., determining the amount of proteins in a sample. For MS, there are relative and absolute quantification methods. An example of relative quantification is the labelling of samples with stable isotope labels, which allow identical proteins in different samples to be distinguished. Examples of such relative quantification methods are isotope-coded affinity tags (ICAT), isobaric labelling (tandem mass tags (TMT), dimethyl labelling, stable isotope labelling with amino acids in cell culture (SILAC), and isobaric tags for relative and absolute quantification (iTRAQ), N-terminal labelling, terminal amine isotope labelling of substrates (TAILS) and metal-coded tags for label-free quantification (MeCAT). Label-free approaches involve analysing different samples separately and then comparing their mass spectra to determine the relative abundance of peptides in each sample. Label-free quantification is usually based on either precursor signal intensity or spectral counting. In area under the curve (AUC) methods, the area under the spectral peak is calculated for each peptide spectrum of an LC-MS / MS run, which is linearly proportional to the concentration of protein in the analysed sample. In spectral counting, the spectra of an identified protein are counted and standardised using an applicable normalisation.

[0166] Tandem Mass Tags (TMT, tandem atomic mass tags) are isobaric chemical tags that enable multiplexed functions for relative quantitative proteomic analysis. In relative quantification experiments, different isobaric tags are used to label different systemic conditions. Without being bound by theory, after tagging, all samples are mixed and analyzed in a single experiment using liquid chromatography-mass spectrometry (LC-MS). Preferably, after fragmentation, the tags generate a unique signature reporter from each individual systemic state in the lower m / z region of the MS / MS spectrum, wherein peptide identification is achieved by matching the resulting ion peaks to values provided in fragment databases. Peptide quantification is performed by comparing the intensities of the reporter ions.

[0167] Stable Isotope Labelling with Amino Acids in Cell Culture (SILAC) involves labelling protein samples in living cells and / or in vivo by replacing an isotopically heavy amino acid form with a naturally occurring light form. SILAC allows the combination of labelled and unlabelled samples during sample preparation so that SILAC can both minimize quantitative error and allow mixing of samples to enable a variety of enrichment procedures. Without being bound by theory, these procedures can improve the detection of abundance changes in both low abundance proteins and post-translational modifications.

[0168] Label-free quantitation (LFQ) enables cost-effective relative quantitation of protein samples. Samples are individually tested using advanced software with chromatographic capabilities prior to measurement. Advantageously, the number of sample comparisons is not limited. In addition, peptide identification can be performed using any fragmentation method (CID, ETD, EThcD and / or HCD). Targeted mass spectrometry methods are, for example, multiple reaction monitoring (MRM), also termed selected reaction monitoring (SRM), and parallel reaction monitoring (PRM).

[0169] The acquisition of mass spectrometry raw data can be performed in embodiments using Data Dependent Acquisition (DDA) or Data Independent Acquisition (DIA) or hybrid approaches thereof. DIA fragments all precursors within defined mass ranges (precursor isolation windows), which is in contrast to data-dependent acquisition (DDA), where precursors are selected for fragmentation based on their intensity. In general, MS1 acquisition scans the entire mass range, usually between 350-1 ,650 m / z. In a DIA method, precursor isolation windows are set across the entire mass range for MS2 acquisition. The number and width (fixed or variable) of the windows depend on the LC gradient and instrument. All peptides within an isolation window are simultaneously fragmented and their fragments then detected by MS / MS. The identity of the peptides can be obtained by matching the ion peaks in a mass spectrum to a spectral library containing information on the pattern of peptide fragment ions and their elution time from LC. The method can be further improved by coupling ion mobility spectrometry (high-field asymmetric waveform or trapped) prior to MS analysis.

[0170] Tandem mass spectrometers can in embodiments be coupled with additional analytical online or offline separation techniques to achieve a multi-dimensional separation and increase analytical resolution. The coupling with liquid chromatography is referred to as LC-MS / MS, and the coupling with ion mobility spectrometry (IMS) and its variety of concepts (e.g., trapped ion mobility spectrometry; TIMS; or field asymmetric ion mobility spectrometry; FAIMS) can add further to the analytical resolution of such instruments.

[0171] Liquid chromatography (LC) is a method of physical separation wherein the components of a liquid mixture are distributed between two immiscible phases, for example, a stationary and a mobile phase. The practice of LC can be divided into five categories, such as adsorption chromatography, partition chromatography, ion-exchange chromatography, size-exclusion chromatography, and affinity chromatography. The most commonly employed method is the reverse-phase (RP) mode of the partition chromatography technique, which uses a nonpolar (hydrophobic) stationary phase and a polar mobile phase. High-performance liquid chromatography (HPLC), is a method applied to separate, identify, and quantify multiple components of a sample. HPLC employs pumps to pass a pressurized liquid solvent containing the sample through a column filled with a solid adsorbent material, wherein each component in the sample interacts slightly differently with the adsorbent material, resulting in different flow rates for the different components and leading to the separation of the components as they flow out of the column.

[0172] In embodiments of the present invention a substance or drug of interest or candidate substance may be any substance of interest. In embodiments the present method is used to analyse the ability of a substance of interest to interact with a certain target molecule or to analyse its effect on the interactions, e.g., protein-protein interactions, of a target molecule. Accordingly, in some embodiments the substance of interest / candidate substance is a chemical compound, a pharmaceutical compound or a drug. In some embodiments the substance or compound is a pollutant or toxin. Herein the terms “substance” or “compound” or even “drug” may be used interchangeably.

[0173] The term "at least one" may herein refer to at least one, more than one, at least two at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 500, 1000, 10.000.

[0174] The instant disclosure comprises methods and / or means for performing said methods. Each feature of the invention that is disclosed in the context of one aspect of the invention is herewith also disclosed in the context of the other inventive aspects disclosed herein. Accordingly, embodiments and features of the invention described with respect to one embodiments of the method disclosed herein, are considered to be disclosed with respect to each and every other aspect and embodiment of the disclosure.

[0175] The various aspects of the invention are unified by, benefit from, are based on and / or are linked by the common and surprising finding of the unexpected advantageous effects of the present method to identify and analyse ligand-target interaction in living cells and tissues, such as, e.g., substance-target interactions in living and metabolizing cells after substance treatment or exposure.

[0176] FIGURES

[0177] The invention is further described by the following figures. These are not intended to limit the scope of the invention but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

[0178] Brief description of the figures:

[0179] Figure 1 : Overview of one embodiment of the present method. Figure 2: Solvent Induced Protein Precipitation in live cells.

[0180] Figure 3: Overview of AUC experiments.

[0181] Figure 4: Comparison of MTX targets in lysate and in living cells (Fig. 3 cont.).

[0182] Figure 5: Extension of SIPP to adherent cell lines.

[0183] Figure 6: Generation of dose response curves and ECso-values by application of PISA.

[0184] Figure 7: LC-MS / MS analysis of SIPP melting curve experiments with MTX on cells and dose response curve experiments with MTX on cells (Fig. 6 cont.).

[0185] Figure 8: LC-MS / MS analysis of SIPP experiment with SCIO-469 on cells.

[0186] Figure 9: LC-MS / MS analysis of SIPP experiment with MK-2206 on cells.

[0187] Figure 10: LC-MS / MS analysis of SIPP experiment with Vorinostat on cells.

[0188] Figure 11 : LC-MS / MS analysis of SIPP experiment with Ibrutinib on cells.

[0189] Figure 12: LC-MS / MS analysis of SIPP experiment with Cyclosporine A in lysate or on cells (Fig. 11 cont.).

[0190] Figure 13: LC-MS / MS analysis of SIPP experiment with tissue.

[0191] Figure 14: Direct SP3-digestion of SIPP supernatants.

[0192] Figure 15: Cell lysis during the denaturation procedure.

[0193] Figure 16: Comparison of protein denaturation between SIPP on lysate and on cells.

[0194] Detailed description of the figures:

[0195] Figure 1 : Overview of embodiments of the present method. The key difference to the prior art method is that the present method allows detecting protein-ligand interactions in alive cell systems, and not only in cell or tissue extracts.

[0196] Figure 2: Solvent Induced Protein Precipitation in live cells. First, cells (for instance K-562, others possible) are seeded in respective growth medium 24 hours (h) before start of each experiment. (1) For subsequent treatment of live cells with chemical compounds, seeded cells are washed twice with prewarmed PBS and resuspended in growth medium containing the chemical compound. Incubation for 1 h at 37 °C and 5 % CO2 (the incubation is in general not limited to 1 h. but depends on experimental design / compound). (2) For following protein precipitation inside intact cells, treated cells are harvested by centrifugation, briefly washed with PBS and resuspended in PBS. 1e6cells per denaturing condition were treated with increasing concentrations of AEA (acetone:ethanol:acetic acid; 50:50.0.1) ranging from 0 to 20 %. The samples were incubated at 37 °C with vigorous shaking (800 rpm) for 20 min, to induce protein denaturation and aggregation inside intact cells. (3) For protein extraction, cells are lysed by addition of IGEPAL CA-630 to a final concentration of 0.4 % and three freeze-thaw cycles using liquid nitrogen and a water bath at 37 °C. (4) Insoluble and precipitated proteins were separated by centrifugation at 4 °C and 3000 x g for 45 min and equal amounts of supernatant from each sample were removed and dried by vacuum centrifugation. (5) Samples are resuspended in LDS- buffer for analysis by Western Blot or in buffer suitable for MS-sample preparation method of choice (S-Trap, SP3, others possible).

[0197] Figure 3: Solvent Induced Protein Precipitation can be performed as Area-under-the-Curve- Experiment. Equal volumes of the supernatant of a denaturation curve are pooled. The differences in the amount of each protein between pooled samples reflect a shift of the underlying denaturation curve and therefore (de-)stabilization. Increasing stabilization of a protein by increasing compound concentration can be exploited to extract EC50 values from this dosedependent effect.

[0198] Figure 4: The Figure shows the experimental results of Example 1 regarding the comparison of MTX targets in lysate (first three graphs) and in living cells (graph four to six). Treatment of living cells for 1 h with 10 pM of the ligand Methotrexate (MTX) and analysis according to the present method was compared with treatment of cell extract for 20 minutes with 100 pM MTX, followed by state of the art SIPP-analysis. DMSO treatment solved as control (vehicle) treatment (indicated as curves with circles in graphs). MTX treatment is indicated in the graphs as curves with boxes. The Western blot analysis (panels next to graph) showed, that DHFR is stabilized in both experiments of cell lysate (Figure 4; MTX in lysate) and living cells (Figure 4; MTX on living cells) against solvent-induced denaturation, while TYMS is only stabilized after metabolization of MTX in living cells (Figure 4; MTX on living cells). p-Tubulin serves as a negative control, showing denaturation in general, but no (de-)stabilization by MTX. The graphs show a quantification of the relative band intensity (y-axis) of the bands in the respective western blots depicted below the graphs. The X-axis of the graphs shows the solvent concentration in the sample (increasing from 0-20% v / v).

[0199] Figure 5: The Figure shows the experimental results of Example 2. The present method was performed with the treatment of living, previously detached adherent cells inside cell culture dishes designed for suspension cells and with slow agitation to prevent re-attachment of cells. The cells were either treated with 10 pM Methotrexate (MTX) as ligand or with vehicle (DMSO). Western Blot analysis of stability changes for DHFR and TYMS after ligand-treatment of living, adherent cells (HCT-116) shows stabilization of both proteins, similar to living suspension cells (see Fig. 3). The graphs show a quantification of the relative band intensity (y-axis) of the bands in the respective western blots depicted below the graphs. The X-axis of the graphs shows the solvent concentration in the sample (increasing from 0-20% v / v). In the graph, DMSO (vehicle) treatment is indicated as curves with circles, Methotrexate (ligand) treatment is indicated as curves with boxes.

[0200] Figure 6: The Figure depicts the results of Example 3 considering the generation of dose response curves and ECso-values from 2D-experiments (2D = varied solvent + varied ligand concentrations) by application of PISA. The PISA format refers to melting-curve formats, where all samples of a melt curve are combined into a single sample (“area under the curve” approach); this compressed format saves LC-MS / MS time and allows for higher throughput. The PISA approach was utilized to determine ECso-values for MTX from a two-dimensional experiment, with 12 denaturant concentrations (pooled to PISA samples) and treatment of cells for 1 h with 5 MTX concentrations. DHFR and TYMS were increasingly stabilized with increasing concentrations of ligand (MTX), as shown in the denaturation curve analyses. Fitting of a dose response curve to the intensities of PISA samples (one for each compound concentration) allowed determination of ECso-values, which are similar to previously published data (Seeger et al., 1947, Allegra et al., 1985).

[0201] Figure 7: The Figure depicts the results of the LC-MS / MS analysis (based on Tandem Mass Tags (TMT) quantification) of Example 4 as denaturation curves and dose response curves. Experiments similar to Expl. 1 and 3 were analyzed by LC-MS / MS and the results were comparable to Western Blot analysis. The denaturation curves of DHFR and TYMS are both shifted to higher denaturant concentrations upon treatment of cells with ligand (MTX). The dose response curves of DHFR and TYMS allow determination of ECso-values of MTX in the expected range. The Y-axis of the denaturation curve plots depicts the relative residual reporter intensity determined by LC-MS / MS, the X-axis depicts the concentration (%v / v) of denaturating agent (denaturant). In the denaturation curve plots, DMSO (vehicle) treatment is indicated ascurve with circles, Methotrexate (MTX) treatment is indicated as curve with boxes. The Y-axis of the dose response curve plots depicts the relative intensity determined by LC-MS / MS, the X-axis depicts the concentration (nM) of MTX.

[0202] Figure 8: The Figure depicts the results of the LC-MS / MS analysis (based on Tandem Mass Tags (TMT) quantification) of Example 5 as volcano plot. K562 cells were treated for 1 h with 100 pM of the selective serine-threonine protein kinase inhibitor SCIO-469 and the designated target protein, p38oc (MAPK14), is stabilized against denaturation and aggregation / precipitation in treated samples. The volcano plot depicts the effect size of the stabilization or destabilization as Iog2 fold change reporter intensity values of the treated and the untreated sample per protein on the X-axis and the statistical significance as negative log-io p value on the Y-axis. The expected target protein p38 a is labelled.

[0203] Figure 9: The Figure depicts the results of the LC-MS / MS analysis (based on Tandem Mass Tags (TMT) quantification) of Example 6 as volcano plot. K562 cells were treated for 1 h with 25 pM of the allosteric serine-threonine protein kinase inhibitor MK-2206. The designated target proteins, AKT1 and AKT2 are stabilized against denaturation and aggregation / precipitation in treated samples. The volcano plot depicts the effect size of the stabilization or destabilization as Iog2 fold change reporter intensity values of the treated and the untreated sample per protein on the X-axis and the statistical significance as negative log-io p value on the Y-axis. Expected target proteins are labelled. Figure 10: The Figure depicts the results of the LC-MS / MS analysis (based on Tandem Mass Tags (TMT) quantification) of Example 7 as volcano plot. K562 cells were treated for 1 h with 25 pM of the epigenetic drug Vorinostat. The designated target proteins, HDAC1 and HDAC2 are stabilized against denaturation and aggregation / precipitation in treated samples. The volcano plot depicts the effect size of the stabilization or destabilization as Iog2 fold change reporter intensity values of the treated and the untreated sample per protein on the X-axis and the statistical significance as negative log-io p value on the Y-axis. The significantly stabilized and expected target HDAC1 is labelled.

[0204] Figure 11 : The Figure depicts the results of the LC-MS / MS analysis (based on Tandem Mass Tags (TMT) quantification) of Example 8 as volcano plot. K565 cells were treated for 1 h with 25 pM of the selective covalent receptor-tyrosine-kinase inhibitor Ibrutinib. The designated target protein, BTK, and known kinase off-targets (YES1 , LYN, RIPK2, FYN) are stabilized against denaturation and aggregation / precipitation in treated samples. The volcano plot depicts the effect size of the stabilization or destabilization as Iog2 fold change reporter intensity values of the treated and the untreated sample per protein on the X-axis and the statistical significance as negative log-io p value on the Y-axis. Expected target proteins are labelled.

[0205] Figure 12: The Figure depicts the results of the LC-MS / MS analysis (comprising Tandem Mass Tags (TMT) quantification) of Example 9 as volcano plot and Venn diagrams. HeLa cells were treated for 1 h with 120 pM of the natural substance and molecular glue Cyclosporine A. The designated direct target proteins, Peptidyl-prolyl cis-trans isomerases B and F, are stabilized against denaturation and aggregation / precipitation both in cell extract experiments and live cell experiments in treated samples. Peptidyl-prolyl cis-trans isomerase A is only stabilized in lysate. The known mode of action for Cyclosporine A includes induced interaction of the direct target proteins with calcineurin / Protein Phosphatase 2 B. Three out of five calcineurin subunits were detected in this experiment and all of them were significantly destabilized in live cell experiments. In addition, there are many other proteins stabilized and destabilized in live cell experiments of Cyclosporine A, which are not affected in experiments with cell extracts. Many of the stabilized proteins are ribosomal proteins, including the translation initiation factor EIF2A, which is crucial for regulating translation under stress conditions. One may deduce that the stabilization of ribosomal proteins occurs due to stalled ribosomes caused by unfolded protein stress and integrated stress response. The volcano plots depict the effect size of the stabilization or destabilization as Iog2 fold change reporter intensity values of the treated and the untreated sample per protein on the X-axis and the statistical significance as negative log-io p value on the Y-axis. The Venn diagrams depict which proteins are significantly (de-)stabilized in both of the two compared experiments or only in one of the two experiments.

[0206] Figure 13: The Figure depicts the results of the LC-MS / MS analysis of Example 10 as melt curves of example proteins. The denaturation curves of the example proteins are different. The protein HSPD1 has the lowest melting point (approximately 8% (v / v)), the highest melting point has protein PAFAH1 B1 (approximately 16% (v / v)). The eight members of the CCT complex, TCP1 , CCT2, CCT3, CCT4, CCT5, CCT6A, CCT7, and CCT8, show congruent melting curves. The Y-axis of the denaturation curve plots depicts the relative residual intensity determined by LC-MS / MS, the X-axis depicts the concentration (%v / v) of denaturating agent (denaturant).

[0207] Figure 14: The figure depicts a comparison of SIPP supernatants, that have been pooled according to the PISA principle and have either been dried before LC-MS / MS sample preparation, or directly used for LC-MS / MS sample preparation, using an SP3 workflow. The dried samples were resuspended in PBS with 5 % SDS, before addition of methanol to a final concentration of 70 % (v / v), and continuation of the sample preparation workflow. The supernatants of the samples that have been directly used for MS sample preparation have been brought directly to final concentration of 70 % (v / v) methanol. The correlation heatmap depicts the correlation between the protein intensities within and between sample types. The upset plot depicts the overlap in identified proteins between the sample types and replicates.

[0208] Figure 15: The figure depicts cell lysis during the denaturation procedure. Live K562 cells were subjected to the denaturation procedure described above, which consists of addition of different concentrations of AEA and incubation at 37 °C and 800 rpm on a thermo shaker. Cells were then either lysed by addition of IGEPAL CA-630 to a final concentration of 0.4 % and three freezethaw cycles (shown as black data points), or this lysis step was omitted (shown as white data points). Insoluble proteins and cells were in both samples subsequently separated from the soluble proteins by centrifugation. Amounts of total soluble proteins were determined by BCA and are plotted against the respective AEA concentrations.

[0209] Figure 16: The figure depicts the differences of overall protein denaturation between SIPP on lysate and SIPP on living cells. The average denaturation curves (left panel) were filtered as described earlier for TPP experiments (Savitski et al. 2014, Science, 2014 Oct 3;346(6205): 1255784) for a bottom plateau of 0.3 relative to the 0 % sample and a R-squared value regarding the curve fit of 0.8 or higher. Histograms and boxplots (right panel) depict the distribution of C1 / 2 values across all denaturation curves after filtering.

[0210] EXAMPLES

[0211] The invention is further described by the following examples. These are not intended to limit the scope of the invention but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

[0212] Methods

[0213] Quantitative proteomics

[0214] Protein digestion, TMT labelling, and basic reversed phase chromatographic fractionation.

[0215] Proteins were digested into peptides using the suspension trapping approach (Zougman et al., Proteomics. 2014; 14(9): 1006-0) with 1 :20 trypsimprotein overnight or using the R2P1 approach (Leutert et al, Mol Syst Biol 2019; 15(12):e9021). Peptides were desalted by C18 solid-phase extraction cartridges (BRAVO C18 cartridges, Agilent) and dried in vacuum.

[0216] TMT11 labelling was performed according to (Zecha et al, Mol Cell Proteomics. 2019; 18(7): 1468- 1478), equal amounts combined per TMT plex, and desalted by C18 solid-phase extraction cartridges (Sep-Pak® C18 Cartridges, Waters).

[0217] Desalted peptides were chromatographically separated via basic reversed phase chromatography into 24 fractions, dried down, and stored at -20°C until further analysis (Bian et al, Nat Commun. 2020;11 (1):157).

[0218] LC-MS / MS analysis and data processing

[0219] The samples were measured by nLC-MS / MS system (NanoLC: UltiMate3000; MS: Orbitrap Eclipse Tribrid; both Thermo Fisher Scientific) with the following settings: 120 min nLC gradient, data (in-)dependent acquisition, fragmentation method: MS2 (HCD).

[0220] Peptides and proteins were identified and quantified using MaxQuant (Version: 2.0.3.1), with the following settings: protease: Trypsin; label: TMT11-plex; fixed modifications: Carbamidomethyl I; variable modifications: Oxidation (M); Acetyl (Protein N-term); Phospho(STY); protein sequence database: human SwissP rot database; PSM / Protein FDR: 1%.

[0221] Data analysis

[0222] Depending on the experimental format and experimental design, different data analysis approaches have been developed and can be used. Unfolding / aggregation curves can, for instance, be fitted using simple sigmoidal equations based on chemical denaturation theory of proteins; concentration response curves can be fitted using typical dose / concentration response curve approaches. Other experimental formats can also be analyzed, e.g., using statistical hypothesis testing approaches (ANOVA, t-test, Limma, or similar).

[0223] In the analysis may involve the TPP R package, which provides a framework for analyzing TPP (and likewise SIPP) data as described in Franken et al. (“Thermal proteome profiling for unbiased identification of direct and indirect drug targets using multiplexed quantitative mass spectrometry”. Nat Protoc. 2015 Oct; 10(10): 1567-93). The TPP package may be applied to SIPP data according to Van Vranken et al., 2021 . The non-parametric analysis of TPP data regarding the melting curve data is described in Childs et al. (Mol Cell Proteomics. 2019 Dec;18(12):2506-2515).

[0224] The non-parametric analysis of TPP data regarding the 2D (2D-experiment = varied solvent + varied ligand concentrations) and concentration response format is described in Kurzawa et al. (“A computational method for detection of ligand-binding proteins from dose range thermal proteome profiles”. Nat Commun. 2020 Nov 13; 11 (1 ):5783).

[0225] Example 1 : Comparison of MTX targets in lysate and in living cells. Treatment of living cells with Methotrexate (MTX) as ligand and analysis according to the present method was compared with treatment of cell extract with MTX, followed by state of the art SIPP- analysis. DMSO treatment solved as control (vehicle) treatment. DHFR (Dihydrofolate Reductase) is expected to be stabilized in both cases, since MTX interacts directly with this protein. In living and therefore metabolically active cells, MTX gets polyglutaminylated (M. Huang et al., J. Pharmacol. Exp. Ther. 304, 753 (2003)) and can additionally interact with TYMS. In this experiment p-Tubulin served as a negative control, showing denaturation in general, but no stabilization by MTX.

[0226] The Western blot analysis showed, that DHFR is stabilized in both experiments of cell lysate (Figure 4a; MTX in lysate) and living cells (Figure 4b; MTX on living cells) against solvent-induced denaturation if MTX (ligand) is added, while TYMS is only stabilized after metabolization of MTX in living cells (Figure 4b; MTX on living cells).

[0227] Although the same experiment has been done before, during for the establishment of the CETSA method (Molina et al., Science, 2013), the present examples show that the method according to the invention surprisingly enables the analysis of target-ligand interaction in living cells - contrary to CETSA - at isothermal conditions. The approach of using the present method with living cells can therefore reproduce stabilization effects of experiments with lysate but can additionally detect stability changes that only arise from direct treatment of living cells. Hence, the present experiment shows, that the present method can be used in common drug testing experiments, but now - surprisingly - also at physiological and isothermal conditions in living cells. Therefore, the present method constitutes a significant improvement over the methods of the prior art.

[0228] Example 2: Extension of SIPP to adherent cell lines.

[0229] This example shows that the present method is not limited to suspension cells. To avoid prolonged exposure to medium / reagents without compound during detachment of adherent cells, which might dilute the final compound concentration before the protein precipitation step, the present method was performed with the treatment of living, previously detached adherent cells. The cells were kept inside cell culture dishes designed for suspension cells and with slow agitation to prevent re-attachment of cells. Alternatively, it is also possible to incubate the cells in 10 mL medium in a 15 mL Falcon with intermittent shaking in a thermoblock. For volumes <1 mL is also possible to use 2mL deep-well plates. The method was performed analogous to Example 1 with MTX serving as ligand (curve with boxes in Figure 5) and DMSO as vehicle control (curve with circles in Figure 5). Western Blot analysis of stability changes for DHFR and TYMS after treatment of living, adherent cells (HCT-116) with increasing solvent concentrations shows stabilization of both proteins, similar to suspension cells (Figure 5) in the presence of MTX as ligand. Accordingly, an interaction between MTX and DHFR, and MTX and TYMS can be assumed. This example shows that the method according to the invention is not limited to suspension cell lines.

[0230] Example 3: Generation of dose response curves and ECso-values from 2D-format by application of PISA. The PISA approach was described first by Gaetani et al. for TPP / CETSA (Gaetani et al. Proteome Integral Stability Alteration assay dramatically increases throughput and sensitivity in profiling factor-induced proteome changes, J. Proteome Res. 2019, 18, 11 , 4027-4037), but also applied to the SIPP method by Van Vranken et al. (Van Vranken et al., Assessing target engagement using proteome-wide solvent shift assay, eLife, 2021). PISA allows simplification of data analysis, since for PISA all samples from a denaturation curve are pooled and subsequently analyzed as a single sample. The intensity of this sample is proportional to the integral of or the area under the denaturation curve, with e.g., higher intensities reflecting curves that are shifted by protein stabilization. The PISA format refers to melting-curve formats, where all samples of a melt curve are combined into a single sample (“area under the curve” approach); this compressed format saves LC-MS / MS time and allows for higher throughput.

[0231] Here a two-dimensional experiment was carried out, comprising both, varied solvent and varied ligand concentrations. In the present example the PISA approach was utilized to determine EC50- values from a 2D-experiment, with 12 different denaturant concentrations (pooled to PISA samples) and 5 different MTX concentrations. DHFR and TYMS were stabilized by MTX in a dose dependent manner, as shown in the dose response curve analyses (Figure 6). Therefrom, an interaction between MTX and DHFR, and MTX and TYMS can be assumed. Fitting of a dose response curve to the intensities of PISA samples (one for each compound concentration) allowed determination of ECso-values for MTX, which are similar to previously published data.

[0232] Therefore, the present method allows for reliable determination of dose-response data, and therefrom EC50 values, for agents of interest in living cells.

[0233] Example 4: LC-MS / MS analysis of SIPP experiments on cells.

[0234] Experiments similar to Expl. 1 and 3 were analyzed by LC-MS / MS and produced results comparable to Western Blot analysis. In this example melt-curve experiments (with varied solvent concentrations) and concentration-response curve experiments (with varied ligand concentration) were performed.

[0235] The denaturation curves of DHFR and TYMS (Figure 7) are both shifted to higher denaturant concentrations upon treatment of cells with the ligand MTX. An interaction between MTX and DHFR and MTX and TYMS is thereby indicated. The dose response curves of DHFR and TYMS (Figure 7 cont.) allow determination of ECso-values for MTX in the expected range.

[0236] Since LC-MS / MS analysis is not limited to observation of a few proteins, additional proteins that are affected by the compound treatment can be detected. A dose-dependent destabilization of MTR could be detected in this experiment. This might be explained by decrease of its substrate’s concentration inside cells, due to the inhibition of DHFR, which produces a precursor of this substrate. The method according to the invention is therefore able to detect secondary effects that depend on an active metabolism of living cells and cannot be detected in lysate.

[0237] Example 5: Identification of targets of a selective serine-threonine protein kinase inhibitor using Solvent Induced Protein Precipitation in live cells K562 cells were treated with the serine-threonine protein kinase inhibitor SCIO-469 at 100 pM for one hour or vehicle control, washed with PBS and subjected to PISA-format SIPP using eight different solvent concentrations of AEA (0, 8, 10, 12, 14, 16, 18, 20% v / v), which were combined prior to sample preparation for LC-MS / MS based analysis. The experiment was performed in triplicates.

[0238] The volcano plot (Figure 8) shows the results of the statistical analysis. The designated target of SCIO-469, p38oc (MAPK14), was clearly identified as being stabilized upon treatment. The other significant proteins may represent proteins affected by secondary effects or unknown / uncharacterized off-targets of SCIO-469.

[0239] This example confirms that SIPP with live cells is capable of identifying ligand-protein interactions of selective serine-threonine protein kinase inhibitors in live cells.

[0240] Example 6: Identification of targets of an allosteric inhibitor using Solvent Induced Protein Precipitation in live cells

[0241] Experimental setup, format, and design as in Example 5.

[0242] K562 cells were treated with the allosteric serine-threonine protein kinase inhibitor MK-2206 at 25 pM for one hour or vehicle control, washed with PBS and subjected PISA-format SIPP using eight different solvent concentrations of AEA (0, 8, 10, 12, 14, 16, 18, 20% v / v), which were combined prior to sample preparation for LC-MS / MS based analysis. The experiment was performed in triplicates.

[0243] The volcano plot (Figure 9) shows the results of the statistical analysis. The designated targets of MK-2206, the serine-threonine protein kinases AKT1 and AKT2, were clearly identified as being stabilized upon treatment. The other significant proteins may represent proteins affected by secondary effects or unknown / uncharacterized off-targets of MK-2206.

[0244] This example confirms that SIPP with live cells is capable of identifying ligand-protein interactions of allosteric inhibitors in live cells.

[0245] Example 7: Identification of targets of an epigenetic drug using Solvent Induced Protein Precipitation in living cells

[0246] Experimental setup, format, and design as in Example 5.

[0247] K562 cells were treated with the histone deacetylase (HDAC) inhibitor Vorinostat at 25 pM for 1 hour or vehicle control, washed with PBS and subjected to PISA-format SIPP using eight different solvent concentrations of AEA (0, 8, 10, 12, 14, 16, 18, 20% v / v), which were combined prior to sample preparation for LC-MS / MS based analysis. The experiment was performed in triplicates.

[0248] The volcano plot (Figure 10) shows the results of the statistical analysis. One designated target of Vorinostat, HDAC1 , was clearly identified as being stabilized upon treatment. The other significant proteins may represent proteins affected by secondary effects or unknown / uncharacterized off-targets of Vorinostat.

[0249] This example confirms that SIPP with live cells is capable of identifying ligand-protein interactions of HDAC inhibitors in live cells.

[0250] Example 8: Identification of targets of a selective covalent inhibitor using Solvent Induced Protein Precipitation in live cells

[0251] Experimental setup, format, and design as in Example 5.

[0252] Adherent cells were detached, treated with the covalent receptor-tyrosine kinase inhibitor Ibrutinib at 25 pM for one hour or vehicle control with intermittent shaking to avoid reattachment, washed with PBS and subjected PISA-format SIPP using eight different solvent concentrations of AEA (0, 8, 10, 12, 14, 16, 18, 20% v / v), which were combined prior to sample preparation for LC-MS / MS based analysis. The experiment was performed in triplicates.

[0253] The volcano plot (Figure 11) shows the results of the statistical analysis. The designated target of Ibrutinib, BTK, was identified as being stabilized upon treatment. The other significant proteins include other known targets of Ibrutinib (YES1 , LYN, RIPK2, FYN) but also other proteins, which may represent proteins affected by secondary effects or unknown / uncharacterized off-targets of Ibrutinib.

[0254] This example confirms that SIPP with live cells is capable of identifying ligand-protein interactions of covalent inhibitors in live cells.

[0255] Example 9: Identification of targets of a natural substance and molecular glue using Solvent Induced Protein Precipitation in cell extracts and live cells

[0256] Experimental setup, format, and design of the in-cell experiment as in Example 5.

[0257] Adherent HeLa cells were detached, treated with the natural substance and molecular glue Cyclosporine A at 120 pM for 1 hour or vehicle control with intermittent shaking to avoid reattachment, washed with PBS and subjected to PISA-format SIPP using eight different solvent concentrations of AEA (0, 8, 10, 12, 14, 16, 18, 20% v / v), which were combined prior to sample preparation for LC-MS / MS based analysis. The experiment was performed in triplicates.

[0258] The SIPP experiment in the cell extract was performed by incubation of triplicate samples with DMSO as control or 120 pM Cyclosporine A for 20 minutes rotating at room temperature. Aliquots of 200 pg lysate in 50 pL volume were subjected to PISA-format SIPP like live cells.

[0259] The volcano plots (Figure 12), which displays the results of the statistical analysis of the experiment in the cell extract, shows clear stabilization of designated targets of Cyclosporine A, such as TESC, PPIA, PPIB, and PPIF.

[0260] The volcano plot (Figure 12 cont.), which displays the results of the statistical analysis of the experiment in live cells, shows a large number of stabilized and destabilized proteins upon treatment with 120 pM Cyclosporine A for 1 hour, including designated targets, such as PPIB and PPIF. Additionally, the three subunits of calcineurin that were detected in this experiment (PPP3CA, PPP3CC, PPP3R1) were all significantly destabilized. This is consistent with the known mode of action for Cyclosporine A, which includes induced interaction of the direct targets with calcineurin. The large number of significantly changed proteins indicate a significant modulation of intracellular events, which is not surprising in light of the fact that Cyclosporine A induces unfolded protein stress, leading to the integrated stress response.

[0261] This example confirms that SIPP with live cells is capable of identifying ligand-protein interactions of natural substances and molecular glues in live cells, as well as induced protein-protein interactions, and that the combination of cell extract and live cell experiments can lead to further insights on secondary effects of ligand-protein interactions.

[0262] Example 10: Denaturation and aggregation curves of proteins in tissue

[0263] Rodent brain tissue was aliquoted into pieces of similar size and each piece was incubated in 50 pL PBS at 37 °C for one hour, to simulate potential compound treatment conditions. Tissue pieces were then added directly to 50 pL of AEA-dilutions ranging from 0 to 24 % (v / v), incubated for 20 minutes at 37 °C and 800 rpm on a thermos shaker. For lysis, IGEPAL CA-630 was added to a final concentration of 0.4 % and tissues were mechanically lysed using a Precellys 24 at 5000 rpm, with two times 30 s lysis, and a 10 s break in between. Soluble proteins were retained after centrifugation of the lysed samples for 10 minutes at 17.500 g and 4 °C. 5274 Proteins were identified in total.

[0264] Examples of proteins which denature and aggregate / precipitate are shown in Figure 12. The denaturation curves of the example proteins are different. The protein HSPD1 has the lowest melting point (approximately 8% (v / v)), the highest melting point has protein PAFAH1 B1 (approximately 16% (v / v)). The eight members of the CCT complex, TCP1 , CCT2, CCT3, CCT4, CCT5, CCT6A, CCT7, and CCT8, show congruent melting curves.

[0265] This example confirms that SIPP is capable of denaturing and aggregating / precipitating proteins in intact tissues, and indicates that SIPP can be used to identify ligand-protein interactions ex vivo using intact tissue samples.

[0266] Example 11 : Direct SP3-digestion of SIPP supernatants

[0267] HeLa lysates were used in triplicate for a SIPP assay in PISA-format. Three replicates each were either dried after pooling of the supernatant, resuspended in PBS with 5 % SDS and then used for LC-MS / MS sample preparation, or directly used for LC-MS / MS sample preparation.

[0268] An upset plot (Figure 14) shows, that both approaches identify almost the same proteins. The correlation heatmap, further depicts high correlation between the intensities of all samples (>0.96). This example confirms, that SIPP-samples can be directly used for LC-MS / MS sample preparation, facilitating higher sample throughput. Results and discussion

[0269] An advantage resulting from the inventive process compared to the prior art SIPP approach, which only uses cell lysate, is that the present method enables the sustainment of physiological conditions during both ligand and precipitation treatment such that, e.g., the relevant cellular concentration of compound and targets, the relevant subcellular distribution of compounds or molecular crowding and condensates are sustained during the precipitation assay. Another advance over the SIPP method is that proteins are analyzed in their cellular state, e.g., with respect to other ligands and their activation or functional state. Further, the active metabolism of an added compound within a cell can be analyzed, as exemplified by the inventors for the polyglutaminylation of MTX, which binds to TYMS only after being metabolized. The present method further surprisingly enables the analysis of secondary effects on targets and other proteins, such as modulation of post-translational modifications, of subcellular distributions, of activation and functional states, of their interactions with other biomolecules or ligands (other proteins, metabolites, DNA, RNA, etc.) of folding, biophysical stability and aggregation, of protein turnover, of substrate limitation or accumulation, all of which is not possible using the SIPP method.

[0270] An advantage resulting from the inventive process compared to the CETSA approach, which itself applies heat denaturation and precipitation, is that the present isothermal denaturation allows determination of EC / IC50- or EC50 values at physiologically and pharmacologically relevant temperatures (e.g. 37°C) within the cell. Further, the present method enables presumably a higher reproducibility of stabilization, as (solvent) concentrations can be more accurately and precisely set and maintained during an experiment than temperature values. Moreover, the present method enables a simple and more straightforward implementation of automation compared to the CESTA approach.

[0271] Example 12: Cell lysis during the denaturation procedure

[0272] The aim of the present experiment was to provide evidence that AEA does not lyse cells treated or exposed to AEA..

[0273] K562 cells were subjected to the assay as described above and the total amount of soluble protein in the supernatant after centrifugation was quantified.

[0274] In detail, first, samples comprising intact and viable K562 cells were exposed to different concentrations of AEA at 37 °C.

[0275] In a second step, samples were divided into two groups, for each AEA concentration respectively. The cells in the samples of the first group were lysed completely using IGEPAL CA-630 and by applying a freeze-thaw approach.

[0276] Afterwards the samples of the first (“lysis”) and second (“no lysis”) group were subjected to centrifugation to separate the insoluble fraction of each sample from the supernatant. Subsequently, protein amounts were determined in the separated supernatant using BCA assay. The results of the BCA measurement are shown in Figure 14. The comparison of the BCA analysis of the supernatants from cells subjected either to AEA treatment only (“no lysis”), or to AEA treatment followed by cell lysis treatment (“lysis”) clearly evidenced that AEA treatment itself did not induce cell lysis at commonly used concentrations (as can be seen by the low protein concentration in the analyzed supernatant). Even at high AEA concentrations cell lysis occurred only to a minor degree. The decline in protein amount in the supernatant of lysed cells, which can be seen at high AEA concentrations over 20 % can presumably be attributed to the strong precipitation of proteins at high AEA concentrations before centrifugation. Further, the slight increase of protein amount in the supernatant of the non-lysed sample at AEA concentrations between 8 to 17 % were still more than 50 % lower than in the supernatant of samples subjected to additional cell lysis conditions.

[0277] In comparison, total amount of soluble protein in the supernatant from samples, which underwent the same assay without having been lysed after precipitation was consistently lower (Figure 15). This indicates, that solvents, such as AEA, did not lyse intact cells during the precipitation step, since most of these samples’ protein was trapped in the intact cells and was therefore not detectable in the supernatant after centrifugation. Though, protein amounts and therefore the amount of lysed cells are distinctly smaller than protein amounts after complete lysis and the present experiment evidences that solvent induced precipitation according to an embodiment of the present invention, e.g., using AEA, did not lead to relevant (significant) lysis of intact and viable cells within the samples.

[0278] Example 13: Comparison of protein denaturation between SIPP on lysate and on cells.

[0279] K562 cells were subjected to the assay as described above and the total amount of soluble protein in the supernatant after centrifugation was quantified by LC-MS / MS. Comparison of the relative average curves between both experiments shows that the average C1 / 2 value for experiments on live cells is 1.6 % (v / v) higher than on lysates (Figure 16, left panel). Also, the slope of the curve from an experiment on cells is notably shallower, reflected by a broader distribution of C1 / 2 values for the single proteins (Figure 16, right panel).

Claims

CLAIMS1 . A method for detecting an interaction between a ligand and at least one target protein in intact cells, comprising inducing protein precipitation using one or more solvents in a first sample and in a second sample, both comprising intact cells and / or permeabilized cells, wherein said first sample is treated with said ligand, and said second sample is not treated with said ligand, wherein a difference in tolerance of the target protein to solvent-induced precipitation between the first and second samples indicates an interaction between a ligand and a target protein.

2. The method according to claim 1 , wherein the method comprises: a) treating the first sample with a ligand, b) treating the first and second samples with one or more solvents to precipitate the proteins therein, c) separating soluble protein (supernatant) from precipitated protein, and d) comparing an amount, quantity or level of un-precipitated target protein in the supernatant in the first and second samples, e) wherein a difference in tolerance of the target protein to solvent-induced precipitation between the first and second samples indicates an interaction between the ligand and the target protein.

3. The method according to any of the preceding claims, wherein a difference in tolerance of the target protein to solvent-induced precipitation is measured by determining differences in target protein amount, quantity or level between the first and second samples.

4. The method according to any of the preceding claims, wherein in step b) the first and second samples are treated with multiple solvent conditions, for example increasing concentration of said one or more solvents, and the precipitation of the target protein in the first and second samples is compared for each of said multiple solvent conditions.

5. The method according to any of the preceding claims, wherein the ligand is selected from the group consisting of small molecules, biologies, metabolites, plant extracts or natural products, food additives, environmental pollutants, agricultural pesticides or herbicides, environmental agents, metal ions, nanoparticles, peptides, proteins, nucleic acids, toxins, a drug or another chemical, or any combination thereof.

6. The method according to any of the preceding claims, wherein a mixture of solvents is used for precipitation and comprises at least two or more solvents.

7. The method according to any of the preceding claims, wherein the solvent and / or mixture of solvents used for precipitation comprises at least one organic and / or inorganic substance capable of denaturing and precipitating proteins.

8. The method according to any of the preceding claims, wherein the solvent and / or mixture of solvents used for precipitation comprises at least one acidic reagent, alkaline reagent, metal ion and / or salt.

9. The method according to any of the preceding claims, wherein the solvent and / or mixture of solvents used for precipitation is selected from the group consisting of acetone, methanol, ethanol, acetic acid, ascorbic acid, citric acid, trifluoroacetic acid, iso-propanol, butanol, phosphoric acid, or any combination thereof, but without being limited to these chemicals.

10. The method according to any of the preceding claims, wherein the solvent mixture comprises a mixture of acetone, ethanol and acetic acid (abbreviated as AEA).11 . The method according to any of the preceding claim, wherein the ratio of said solvents in said AEA is 50:50:0.1 (v / v / v) of acetone:ethanol:acetic acid.

12. The method according to any of the preceding claims, wherein the final concentration in the sample of AEA used to treat the samples is 0-50% (% v / v), preferably 5-50% (% v / v), more preferably 9%-22% (% v / v).

13. The method according to any of the preceding claims, wherein ascorbic acid is added to the sample at a final concentration of 1 mM-15 mM and / or wherein citric acid is present in the solvent at a final concentration of 1 mM-5mM.

14. The method according to any of the preceding claims, wherein the solvent-treated samples are incubated with shaking at 20-40°C for 20-40 minutes, preferably at 20-30°C for 20-40 minutes shaking, preferably at 30-40°C for 10-20 minutes shaking, more preferably 37°C for 10-30 minutes shaking.

15. The method according to any of the preceding claims, wherein soluble proteins are separated from precipitated proteins by centrifugation and / or filtration.

16. The method according to any of the preceding claims, wherein in steps d)-e), the method for detecting the amount, quantity or level of the target protein is selected from the groupconsisting of immunological detection, aptamers, transgenic protein labelling, electrophoresis, quantitative proteomic techniques, such as mass spectrometry, preferably LC-MS / MS and / or Multiple reaction monitoring mass spectrometry (MRM) or parallel reaction monitoring mass spectrometry (PRM).

17. The method according to any of the preceding claims, wherein the method comprises a computer-implemented analysis to identify the difference in tolerance of the target protein to solvent-induced precipitation.

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