Methods for determining drug-target residence times and selecting optimal drug-target candidates

Abstract

The present invention relates to a method for determining the residence time between at least one target and at least one ligand, optionally in their native biological context, using limited proteolysis in combination with data-independent acquisition (DIA), including, for example, selected reaction monitoring, parallel reaction monitoring, SWATH (Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra) techniques, and the like.

Description

[Technical Field]

[0001] The present invention relates to a method for determining the residence time between at least one target and at least one ligand, optionally in their native biological context, using limited proteolysis in combination with data-independent acquisition (DIA), including, for example, selected reaction monitoring, parallel reaction monitoring, SWATH (Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra) techniques, and the like. [Background technology]

[0002] Proteins are crucial effectors and regulators of a wide variety of cellular processes. Proteins can change their intracellular concentration and their structure in response to perturbations (e.g., in disease). Capturing such transitions is an essential challenge in life sciences for understanding the function of fundamental cellular processes in health and disease and for identifying new options for disease diagnosis and treatment. Changes in intracellular protein concentrations in response to perturbations can be routinely investigated using mass spectrometry (MS)-based proteomics techniques. However, little is known about intracellular protein conformational switches, primarily due to the lack of suitable approaches to study protein folding within cells. This poses a significant limitation for biological and clinical applications, as conformational changes can strongly affect protein activity and therefore cellular physiology.

[0003] Proteins can change their conformation in response to environmental changes such as binding to lipids, ions, small molecules, or nucleic acids, interactions with other proteins, chemical modifications (e.g., phosphorylation), or fluctuations in pH or temperature. The extent of conformational changes ranges from small local motions, such as allosteric rearrangements, through larger-scale fluctuations, such as domain motions, to dramatic switches between folded and unfolded states or between monomeric and polymeric states. In particular, the transition of monomeric proteins to higher-order aggregate structures has recently attracted increasing attention in both biology and biomedicine. Over the past two decades, various human diseases (more than 20 pathologies) known as protein aggregation diseases have been shown to be associated with the intracellular or extracellular accumulation of specific misfolded protein aggregates. Many neurodegenerative diseases with previously unknown causes, such as Parkinson's disease or Alzheimer's disease, are now classified in this category. Various diseases can also be classified according to the major protein components of their aggregates, which also distinguishes their clinical manifestations. For example, Lewy bodies containing α-synuclein (αSyn) are typically found in most types of Parkinson's disease (PD), whereas amyloid- β Peptide inclusions are produced in Alzheimer's disease (see, e.g., Non-Patent Document 1). The ability to monitor such protein conformational transitions in biological specimens would open new possibilities for the diagnosis and treatment of these protein-centric conditions and advance our understanding of their pathogenesis.

[0004] Many biophysical techniques have been applied to monitor protein conformational properties, including nuclear magnetic resonance (NMR), X-ray crystallography, infrared and Raman spectroscopy, circular dichroism, atomic force microscopy, and fluorescence spectroscopy. These techniques are primarily used to analyze (purified) proteins in vitro because they cannot address the complex biological context. This poses a significant limitation, as the conformation adopted by proteins is regulated in cells by multiple simultaneous events specific to the cellular context, such as environmental factors, binding events, or post-translational modifications, which cannot be reproduced in in vitro systems. Techniques based on Förster resonance energy transfer (FRET) offer the advantage of monitoring protein conformational changes in their native cellular environment, but require the introduction of fluorescent probes at appropriate sites in each target protein, making them inapplicable on a large scale or to clinical samples.

[0005] Gupta, Lapadula, and Abou-Donia reported the purification and characterization of pure cytochrome P450 isozymes from β-naphthoflavone-induced adult hen liver (Non-Patent Document 2). Characterization was performed by proteinase treatment using chymotrypsin under denaturing conditions.

[0006] In their paper entitled "Probing the solution structure of the DNA-binding protein Max by a combination of proteolysis and mass spectrometry" (Non-Patent Document 3), Cohen, Ferre-D'Amare, Burley, and Chait propose a simple biochemical method for investigating the solution structure of DNA-binding proteins by combining enzymatic proteolysis with matrix-assisted laser desorption / ionization mass spectrometry. This method is based on inferring structural information from determining protection against enzymatic proteolysis, which is governed by solvent accessibility and protein flexibility.

[0007] Patent document 1 discloses a limited proteolysis (LiP) protocol, i.e., a method for detecting the conformational state of a protein contained in a complex mixture of additional proteins and / or other biomolecules, in particular in a complex native biological matrix, as well as an assay for such a method. The method comprises, optionally after an extraction and / or lysis step, the following steps: 1. limited proteolysis of the complex mixture under conditions in which the protein is in the conformational state to be detected, to obtain a first fragment sample; 2. denaturing the first fragment sample to obtain a denatured first fragment sample; 3. completely fragmenting the denatured first fragment sample in a digestion step to obtain a fully fragmented sample; 4. analytically analyzing the fully fragmented sample to determine fragments having characteristics resulting from both the limited proteolysis of step 1 (corresponding to step D of the method according to the present invention) and the complete fragmentation in digestion step F to determine the conformational state.

[0008] In their paper titled "Measuring protein structural changes on a proteome-wide scale using limited proteolysis-coupled mass spectrometry" (Non-Patent Document 4), Schopper et al. reported on protein structural changes induced by external perturbations or internal factors that significantly affect protein activity and thus modulate cellular physiology. Limited proteolysis coupled with mass spectrometry (LiP-MS) is reported to be a recently developed proteomic approach that enables the direct identification of protein structural changes in complex biological contexts on a proteome-wide scale. After targeted perturbations, proteome extracts are subjected to a double protease digestion step, applying a nonspecific protease under native conditions, followed by complete digestion with the sequence-specific protease trypsin under denaturing conditions. This sequential process generates structure-specific peptides suitable for bottom-up MS analysis. Next, structure-dependent proteolytic patterns are measured directly in the proteome extracts using a proteomic workflow involving shotgun or targeted MS and label-free quantification. Potential applications of LiP-MS include probing perturbation-induced protein structural changes, identifying drug targets, detecting disease-related protein structural states, and directly analyzing protein aggregates in biological samples. This approach also allows for the identification of specific protein regions involved in structural transitions or affected by binding events.

[0009] In their paper titled "The cellular thermal shift assay for evaluating drug target interactions in cells" (Non-Patent Document 5), Jafari et al. report a thermal shift assay used to study protein thermal stabilization upon ligand binding. Such assays have been used on purified proteins to detect interactions in the drug discovery industry and academia. They have published a proof-of-principle study describing the performance of a thermal shift assay in a cellular format, which they call the cellular thermal shift assay (CETSA). This method enables the study of target engagement of drug candidates in a cellular context, as exemplified by experimental data on the human kinases p38a and ERK1 / 2. The assay involves treating cells with the compound of interest, heating to denature and precipitate proteins, cell lysis, and separation of cellular debris and aggregates from the soluble protein fraction. Unbound proteins denature and precipitate at elevated temperatures, while ligand-bound proteins remain in solution. They describe two procedures for detecting stabilized proteins in the soluble fraction of a sample. The first approach involves sample post-processing and detection using quantitative Western blotting, whereas the second is performed directly in solution and relies on the induced proximity of two targeting antibodies upon binding to soluble proteins. The latter protocol has been optimized for high throughput, as potential applications require large amounts of sample.

[0010] In their paper titled "Dynamic 3D proteomes reveal protein functional alterations at high resolution in situ" (Non-Patent Document 6), Cappelletti et al. reported that global protein structure readout based on limited proteolysis mass spectrometry (LiP-MS) is possible, detecting many functional changes simultaneously and in situ in bacteria during nutritional adaptation and yeast responding to acute stress. The structural readout, visualized as a structural barcode, captured changes in enzymatic activity, phosphorylation, protein aggregation, and complex formation, along with the elucidation of individual regulated functional sites, such as binding and active sites. Comparison with prior knowledge, including other omics data, showed that LiP-MS detects many known functional changes within well-studied pathways. This suggested distinct metabolite-protein interactions and enabled the identification of the regulatory mechanism for fructose-1,6-bisphosphate-based glucose uptake in Escherichia coli. Structural readouts dramatically increase the coverage of classical proteomics, generate mechanistic hypotheses, and pave the way for in situ structural systems biology.

[0011] In their paper titled "Tracking cancer drugs in living cells by thermal profiling of the proteome" (Non-Patent Document 7), Savitzki et al. reported performing thermal proteome profiling (TPP) on human K562 cells by heating either intact cells or cell extracts. They observed significant differences in melting characteristics between the two settings, with a trend toward increased protein stability in cell extracts. Thermal profiling of cellular proteomes has been reported to enable differential assessment of protein ligand binding and other protein modifications, provide an unbiased measure of drug target occupancy for multiple targets, and facilitate the identification of markers of drug efficacy and toxicity. [Prior art documents]

Patent Documents

[0012]

Patent Document 1

Non-Patent Documents

[0013]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Summary of the Invention

[0014] Concept of drug residence time There are fundamental differences between drug-target interactions in closed systems (in vitro) compared with open systems (in vivo). In in vitro systems, the target, its substrate, and drug are present at fixed concentrations, whereas in vivo, the concentrations of the drug, target, and its substrate can vary significantly in time and space. Therefore, although equilibrium binding measurements such as Kd or Ki / IC50 reflect the concentration and thus potency of the drug-target complex in vitro, they cannot accurately predict in vivo pharmacokinetics. Because a significant proportion of all approved drugs exhibit non-equilibrium properties, it has been proposed that drug residence time is more important for in vivo efficacy than in vitro equilibrium binding affinity. Accurate identification of drug-target interactions (DTIs) remains a critical turning point in discovering new insights and understanding the binding process. Therefore, DTI identification is essential for leveraging available information for new drug development, optimization of the entire process chain, and drug repositioning.

[0015] Retrospective studies have shown that drugs with longer residence times have increased efficacy and fewer side effects because they engage a higher proportion of their targets for a longer period of time, even after clearance from the systemic circulation. Residence time has become an important parameter for hit prioritization and lead optimization in many small molecule drug discovery programs. Residence times can vary from less than a minute to several days, with covalently bound inhibitors being an extreme example, and can affect efficacy in a variety of ways.

[0016] Despite efforts to optimize drug-target affinity and specificity during drug development, over 90% of small molecule drugs fail in the clinic. Therefore, efforts have been made to identify additional in vitro parameters that affect drug pharmacokinetics in vivo.

[0017] In addition to unexpected toxicity, a lack of in vivo efficacy is frequently observed for many compounds that appear promising in early drug discovery programs but subsequently fail in clinical trials. One reason for this is growing evidence that kinetic parameters appear to correlate much better with efficacy than affinity.

[0018] In traditional in vitro methods, drug-target interactions have mostly been addressed in terms of affinity measures or static crystal structures of bound complexes [Schuetz, DA, et al., Drug Discovery Today, 2017. 22(6): pp. 896-911]. However, the concept of residence time also takes into account protein conformational dynamics that affect drug binding and unbinding. Therefore, it is believed that the residence time of the drug-target complex, rather than binding affinity, is the primary contributor to in vivo pharmacological activity. In recent years, it has become clear that in vitro information on drug-target binding kinetics is paramount for candidate compound selection. However, in vitro information should be combined with information on the in vivo context in which the drug must exert its ultimate effect.

[0019] Drug binding kinetics has attracted interest among the drug discovery community due to reports that the efficacy of new drugs can be predicted by their binding and unbinding kinetics. Consequently, in vitro dissociation half-life has been proposed as an important parameter for compound optimization.

[0020] Binding affinity provides information about the strength of the interaction between a drug-target (DT) pair and is usually expressed in terms of measures such as the dissociation constant (Kd), inhibition constant (Ki), or 50% inhibitory concentration (IC50). IC50 depends on the target and ligand concentrations (Cer et al., 2009), and a low IC50 value signals strong binding. Similarly, a low Ki value indicates high binding affinity. Kd and Ki values ​​are usually expressed as pKd or pKi, which are the negative logarithms of the dissociation constant or inhibition constant.

[0021] The binding kinetics of a drug to its target protein is characterized by the bimolecular association rate constant (k), which is the rate at which the drug binds, and the dissociation rate constant (koff), which is the rate at which the drug dissociates.

[0022] koff is a key parameter that controls the time over which a drug is active. While binding affinity calculations are now routine, calculating koff has proven more challenging because the timescales involved far exceed the limits of standard molecular dynamics simulations.

[0023] The ability to predict the mechanism of protein-ligand unbinding and its associated rate constants is of great practical importance in drug design.

[0024] The drug-target residence time (τ) is the length of time that a drug remains bound to its target and is mathematically defined as the reciprocal of the dissociation rate constant (1 / koff), which can be experimentally estimated based on the half-life of the drug-target complex.

[0025] Traditionally, methods for measuring drug binding kinetics and their drug-retention times can be divided into three categories: labeled-ligand methods, label-free methods, and enzyme activity assays. However, these methods have a major drawback: they rely on a "biased" analysis of purified predicted drug-targets because they only use purified proteins or specific groups of proteins assumed to be the primary targets of the drug of interest. As a result, these approaches ignore the assessment of potential off-targets because, during protein purification, the protein of interest is extracted from a complex protein sample while the rest of the proteome is discarded. Therefore, other proteins that may bind to the drug are not included in the reaction mixture, even if they have long retention times, and therefore their potential interactions with the drug are not considered.

[0026] The method for measuring drug retention time is described below.

[0027] Labeled Ligand Method The approach of labeled-ligand-based methods is to incubate a target protein of interest with a test drug for a set period of time, followed by a filtration step to remove unbound drug. The drug-bound protein is then incubated with a radiolabeled version of the ligand, which binds to the protein as the unlabeled ligand is displaced. By measuring the association rate of the radioligand over time, it is possible to measure the dissociation rate and, therefore, the residence time of the unlabeled ligand. These methods require the production of radiolabeled drugs, which can be a costly process and is not easily feasible. Furthermore, regulations regarding the disposal of radioactively contaminated samples make this type of methodology difficult to implement.

[0028] Fluorescence-based methods An alternative to radioligand binding assays is to use fluorescence to measure binding kinetics. In this case, the ligand of interest is labeled with a fluorophore, which emits fluorescence at different wavelengths when the ligand is in solution or when the ligand binds to the target protein. Therefore, measuring the emitted fluorescence over time can provide insight into the binding-unbinding dynamics of the ligand and the protein of interest. While this methodology avoids the need for a radiolabeled ligand, adding a fluorophore to the ligand can potentially alter its binding properties.

[0029] Another alternative is to use fluorescent tracers that specifically bind to a set of proteins of interest (e.g., kinases). In either case, labeled-ligand methods require the development of different reagents and different ligands / engineered proteins for each assay, which can be costly and time-consuming (e.g., synthesis of radiolabeled drugs, conjugation with fluorophores, development of fluorescent ligands). Furthermore, these methods use pre-purified proteins that are assumed to be the primary targets of the ligands. This prevents the identification of potential off-targets, as the reaction occurs with a small number of proteins in solution.

[0030] Label-Free Law Several label-free methods are available for measuring drug binding kinetics, thereby avoiding the obstacles associated with label-based methods.

[0031] Surface plasmon resonance Surface plasmon resonance (SRP) is an analytical technique for studying molecular interactions and is the most widely used label-free method for determining small molecule binding kinetics. In SRP, a protein or proteins of interest are first immobilized on the surface of a biosensor chip. Next, a ligand is injected over the chip in a continuous flow, allowing the molecule to bind to the protein target. A buffer then flows over the chip, and dissociation of the protein-ligand complex is monitored by detecting changes in the refractive index of the chip's surface, which depend on the increase in surface mass due to ligand binding. SPR can be used to study interactions between two proteins, between proteins and antibodies, and between DNA and proteins, among other applications. Despite SPR's flexibility in studying interactions between various types of molecules, it is primarily suited to characterizing binding kinetics on purified, immobilized proteins, which hinders unbiased application of this method. Furthermore, protein immobilization conditions must be optimized to maintain the protein's native state and avoid compromising its structure, conformation, and accessibility to binding sites, which can be a tedious procedure.

[0032] Acoustic Biosensor Acoustic biosensors are based on quartz crystals, whose vibrational modes depend on the cut and shape of the crystal. When a mass is applied to the surface of the quartz crystal, the vibration frequency decreases. By measuring the change in frequency, it is possible to determine the change in mass, thereby enabling the detection of ligand binding events to target molecules immobilized on the crystal. As with previous methods, acoustic biosensors use purified proteins, which prevents unbiased analysis of the ligand target.

[0033] Biolayer Interferometry Biolayer interferometry (BLI) is an optical technique that measures macromolecular interactions by analyzing the interference pattern of white light reflected from the surface of a biosensor chip on which molecules of interest are immobilized. By detecting changes in the pattern of reflected white light, this method allows for monitoring the kinetics and affinity of molecular interactions between immobilized proteins and ligands. Consequently, this method focuses on the interaction of one pre-established protein of interest with another entity of interest. Therefore, when applied to complex proteomes, the method lacks protein-level resolution and cannot be applied in an unbiased exploration fashion.

[0034] Resonance Acoustic Profiling Acoustic biosensors enable label-free detection of molecules and analysis of binding events. Typically, acoustic biosensors are based on quartz crystals, whose vibrational modes depend on the cut and shape of the crystal. When a mass is applied to the surface of the quartz crystal, the vibration frequency decreases. By measuring the change in frequency, it is possible to determine the change in mass, and thus the binding of a ligand to a molecule immobilized on the quartz crystal. However, like the above methods, resonant acoustic profiling requires the immobilization of the target protein of interest on the quartz crystal surface, thus preventing unbiased identification of protein-ligand interactions.

[0035] Enzyme activity assay If the protein of interest is an enzyme and there is an appropriate assay available to monitor enzymatic activity, the enzymatic reaction can be used to monitor the binding kinetics between the enzyme and the ligand, as the fraction of enzyme-bound complex is proportional to the enzyme activity.

[0036] These methods monitor the production / consumption of reaction products, generally by spectroscopic / fluorimetric measurements, thus avoiding the drawbacks of label-based methods and the need for immobilized proteins and specialized spectroscopic equipment for the label-free methods described above.

[0037] Jump-dilution method The jump dilution method is the most widely used enzyme activity assay for measuring ligand binding kinetics. In this method, the target enzyme is first incubated with a saturating concentration of the drug of interest to allow the enzyme-ligand complex to form. The reaction volume is then increased to decrease the ligand concentration in solution, slowing the binding kinetics. Enzyme activity is then monitored over time, and the residence time is calculated by fitting an equation to the progress curve.

[0038] Despite its advantages of being simple to perform, the jump dilution method is only applicable to enzymes, which significantly narrows the scope of protein targets, and it relies on the activity of purified / predicted enzymes / enzyme sets, thus hindering unbiased off-target identification.

[0039] Jump-dilution limited proteolysis The jump dilution method concept relies on incremental reaction volumes to reduce the drug concentration in the reaction mixture as a means of slowing down binding kinetics, thereby preserving the native state of the proteome, a fundamental requirement for applying LiP-MS. Therefore, by combining the analytical potential of LiP-MS / DarkLip-MS technology with the jump dilution method, we describe a method for investigating drug-protein interactions at the proteome level. Thus, the method described here does not rely on purified proteins and thus simultaneously analyzes all potential binding partners for a test drug. It thereby simultaneously overcomes the limitations of the label-based and label-free methods mentioned above (particularly the need to immobilize / label a pre-purified, predicted drug target) and the limitations of the jump dilution method for enzymatically active proteins. Thus, the present invention describes a method for unbiased, proteome-wide measurement of drug retention times.

[0040] In a paper entitled "Kinetics for Drug Discovery: an industry-driven effort to target drug residence time" (Drug Discovery Today, 2017, pp. 896-911), the K4DD (Kinetics for Drug Discovery) consortium provided an updated report addressing drug binding kinetics following a multidisciplinary approach.

[0041] By reviewing existing methods for drug binding kinetics, particularly those based on energy transfer, such as surface plasmon resonance (SPR), confocal microscopy, bioluminescence resonance energy transfer (BRET), or time-resolved fluorescence resonance energy transfer (TR-FRET), the authors conclude that "for decades, drug discovery and development have focused on optimizing binding affinity, while drug binding kinetics has essentially been overlooked." In recent years, it has become clear that in vitro information about drug-target binding kinetics is paramount in the selection of candidate compounds. However, in vitro information must be coupled with information about the in vivo context in which the drug must exert its ultimate effect.

[0042] The method for determining the residence time between at least one target and at least one ligand according to the present invention addresses this long-desired and unmet need as there is a trend towards in vivo contextual analysis using targets contained in complex mixtures of additional proteins and / or other biomolecules and / or in complex native biological matrices. Drug target residence time is a key parameter for hit prioritization and lead optimization in many small molecule drug discovery programs.

[0043] The inventors have surprisingly found that quantitative mass spectrometry-based techniques such as LiP-MS™ or DarkLiP-MS™, which utilize structural proteomics for a variety of applications ranging from basic biology to target deconvolution and biomarker discovery, can be used to assess drug target residence time for candidate compound selection, hit prioritization and lead optimization in many small molecule drug discovery programs.

[0044] The method according to the invention makes it possible to: a) Prioritizing target candidates based on their dwell time. This is preferably achieved in the LiP-MS and DarkLiP-MS approaches by providing a list of potential drug-target candidates ranked by scores derived from computational analysis, thus allowing understanding which drug-targets are likely to have the longest interactions according to drug residence times. b) Selecting the best drug for a particular protein / set of proteins. Recent studies suggest that drugs with longer residence times at their intended targets are more efficient and lead to better results in vivo and during clinical trials. Drugs that exhibit long residence times at their expected target and minimal residence time at other proteins (off-targets) are considered more specific and less likely to cause severe side effects. A correlation can be seen between drug target residence time and drug selectivity and / or efficacy. Therefore, by measuring drug residence times for multiple drugs directed against the same protein / set of proteins, we can predict which drugs are likely to be successful in vivo. c) To predict the in-vivo off-target / side effects of different drugs based on their binding kinetics.

[0045] Due to the complexity of biological systems, most drugs bind to more than one target protein and often affect unpredictable off-target proteins. The effect of a drug on its target depends on the drug-protein interaction, and therefore, a longer interaction promotes a longer-lasting effect. Measuring the relative drug residence time across the entire proteome makes it possible to understand which proteins interact with a drug for a longer time and, therefore, to predict the drug's primary target / off-target. The greater the number of identified protein targets with longer residence times, the greater the likelihood of severe side effects. Drugs that have a longer residence time on their primary target and are associated with fewer off-targets that interact for longer periods of time are more specific and less likely to cause severe side effects. Thus, the method of the present invention can be used to determine medically relevant targets.

[0046] A primary use of the method according to the present invention relates to the in vitro comparison of drug specificities, which addresses a long-felt and unmet need for understanding and comparing the mechanisms of action of multiple drugs, for example, providing a valuable readout of early stage drug mechanisms of action.

[0047] This is achieved by estimating the drug residence time between the target and the drug of interest.

[0048] The method according to the invention allows for the identification of drugs that have relatively few off-targets relative to their primary target and are therefore most likely to be successful in in vivo testing.

[0049] In a first embodiment, the present invention provides a method for determining the residence time between at least one target and at least one ligand, said target being contained in a complex cellular mixture of further targets and / or other biomolecules, comprising the following steps: Step A: Incubating at least one target with at least one ligand for a suitable incubation period; Step B: Stopping the binding reaction by removing at least one ligand (unless it is bound to the target); or reducing the concentration of the ligand (unless it is bound to the target) to reduce the number of binding events (per unit time); Step C: Incubating at least one ligand bound to at least one target for different periods of time; Step D: limited proteolysis of the complex mixture under conditions in which at least one target is in the original conformational state to be detected to obtain a first fragment sample.

[0050] Step D is then followed (if using the Lip.MS protocol) by one of the following steps: Step E: Denaturing the sample into a denatured first fragment sample. Step F: Digesting the sample to obtain a fully fragmented sample Or (if using the DarkLiP-MS protocol) immediately after step D the following step is performed: Step G: Removing large peptides and proteins or other biomolecules from said first fragment sample to form an enriched fragment sample. In both cases (for both LiP-MS and DarkLiP-MS protocols), this is followed by the following: analytical analysis, in which quantitative mass spectrometry-based assays in the form of selected / parallel reaction monitoring (SRM / PRM) and / or data-independent acquisition of product ion spectra are used; For steps E and F (LiP-MS protocol), analytical analysis is performed on the fully fragmented sample to determine characteristic fragments that are the result of both the limited proteolysis step D and the full fragmentation step F. For step G (DarkLiP-MS protocol), analytical analysis is performed on the enriched fragment sample. Peptide abundance is measured over different incubation times after ligand removal to determine dissociation half-lives.

[0051] The residence time (τ) is calculated using Equation 1

[0052]

number

[0053] is calculated according to During the ceremony, τ=residence time k off = dissociation constant t 1 / 2 = dissociation half-life and For steps E and F (for LiP-MS protocols), the determination of the residence time between the target and the ligand is It is based on a quantitative comparison of analytical analysis of a fully fragmented sample with analytical analysis of a fully fragmented control sample. For step G (DarkLiP-MS protocol), the determination of the residence time between the target and the ligand is It is based on a quantitative comparison of analytical analysis of an enriched fragment sample with analytical analysis of an enriched fragmented control sample.

[0054] The complex cellular mixture of additional proteins and / or other biomolecules can be, for example, a native biological complex matrix or a biological probe such as a body fluid in general (plasma, cerebrospinal fluid, urine, etc.), can be based on tissue, environmental samples (e.g., seawater, etc.), biological secretions, etc.

[0055] In another embodiment of the method according to the invention, the limited proteolysis of the complex mixture (step D) is carried out over a time span of 1 minute to 60 minutes, preferably in the range of 2 minutes to 30 minutes, or 2 minutes to 10 minutes, or 2 minutes to 5 minutes, more preferably at a temperature in the range of 20°C to 40°C.

[0056] In another embodiment of the method according to the invention, in the case of the LiP-MS protocol, trypsin is used in the digestion step, preferably at a temperature between 15°C and 70°C, for a time span between 2 hours and 24 hours, and at an enzyme to substrate weight ratio in the range of 1 / 10.

[0057] In another embodiment of the method according to the invention, the target is at least a portion of a protein or proteome under native conditions and the ligand comprises one of the following entities: a small molecule drug compound, a naturally occurring protein cofactor, a coenzyme or cosubstrate (e.g., a metal ion, an organic molecule), an amino acid, a synthetic peptide drug, a naturally occurring peptide, a macromolecule (i.e., another protein, a nucleic acid, a lipid, or a macromolecule complex).

[0058] In another embodiment of the method according to the invention, at least one target is a protein based solely on proteinogenic amino acids or based on proteinogenic amino acids and carrying post-translational modifications, and the complex mixture of further proteins and / or other biomolecules is a native biological complex matrix.

[0059] In another embodiment of the method according to the invention, in the step of limited proteolysis the proteolytic system is selected from the group consisting of proteinase K, thermolysin, subtilisin, pepsin, papain, α-chymotrypsin, elastase, and mixtures thereof.

[0060] In a further embodiment of the method according to the invention, in the step of limited proteolysis, the proteolytic system is used at a weight concentration in the range of 1 / 25 to 1 / 10000, preferably in the range of 1 / 50 to 1 / 1000, given as the ratio of enzyme to biomolecule content with respect to the total biomolecule content in the sample.

[0061] In yet a further embodiment of the method according to the invention, when the DarkLiP-MS protocol is used, the removal of large peptides and proteins (step G) is carried out by a filtration, separation or concentration step, including size filtering; chromatography, including size exclusion, hydrophobic or anion exchange chromatography; physical removal, including phase separation, absorption, precipitation; filtration, separation or concentration based on hydrophilic / hydrophobic properties; filtration, separation or concentration based on electric / magnetic fields; or a combination thereof.

[0062] In another embodiment of the method according to the invention, when using the DarkLiP-MS protocol, peptides, proteins or other biomolecules with a molecular weight greater than 20 kDa, preferably greater than 15 kDa, most preferably greater than 10 kDa are removed from the first fragment sample (step G).

[0063] In another embodiment of the method according to the invention, the analytical analysis step is preceded by a proteomics workflow, in particular a proteomics workflow involving denaturation, C18 cleanup, or a combination thereof.

[0064] In another embodiment of the method according to the invention, SWATH-MS and / or optionally data-dependent acquisition (shotgun) is used for the analytical analysis step.

[0065] In another embodiment of the method according to the invention, for quantitative mass spectrometry based assays, heavily labeled fragments bearing characteristics resulting from limited proteolysis or complete fragmentation are spiked into the original complex mixture or fully fragmented sample.

[0066] In a further embodiment of the method according to the invention, for quantitative mass spectrometry based assays, heavily labeled fragments having characteristics resulting from the limited proteolysis of step D and characteristics remaining after removal of large peptides and proteins or other biomolecules of step G are spiked into the original complex mixture and / or the first fragment sample and / or the enriched fragment sample.

[0067] In another embodiment, the method according to the invention is used for at least one of the following: a) prioritizing potential targets based on their residence time, or b) Selecting the best drug for a particular protein / set of proteins, or c) predicting the in vivo off-target / side effects of different drugs based on their binding kinetics, or d) Determining medically appropriate targets.

[0068] In another embodiment, the present invention provides a method for aiding in the prognosis or diagnosis of a disorder condition in a patient early in the progression of the disorder / disease, comprising: a) determining the presence of at least one selected target protein; b) determining the residence time between at least said selected target protein and at least one ligand in a biological sample of the patient, such as plasma, serum, cerebrospinal fluid or urine; c) comparing this concentration data with concentration data from a population affected by a disorder / disease to confirm or negate the presence of a given pathology; The method includes:

[0069] Possible experimental setups for the DarkLiP-MS approach: protein-drug incubation and proteolysis Protein extracts / purified proteins are incubated with the drug of interest for a defined period of time (eg, 10 minutes) in triplicate. Using jump dilution, increasing the volume of solution (eg, 100-fold) reduces binding of the drug to its target. After dilution, each sample is incubated for a specified time (for example, 0 minutes to 1 hour). A limited proteolysis step is performed on native cell extracts using the enzyme thermolysin and the DarkLiP-MS method. Thermolysin is inhibited with EDTA, and the resulting partially fragmented sample is purified on C18 resin, which removes large peptides and intact protein. The resulting fragmented sample is analyzed by mass spectrometry and compared to a drug-free sample (treated with DMSO).

[0070] Possible experimental setups for the LiP-MS approach: protein-drug incubation and proteolysis Protein extracts / purified proteins are incubated with the drug of interest in triplicate for a defined period of time (eg, 10 minutes). Using jump dilution, increasing the volume of solution (eg, 100-fold) reduces binding of the drug to its target. After dilution, each sample is incubated for a specified time (eg, 0 minutes to 1 hour). A limited proteolysis step is carried out on native cell extracts using the enzyme thermolysin and LiP-MS techniques. The thermolysin is inhibited with EDTA and the resulting partially fragmented sample is dried to reduce the volume. The dried, partially fragmented sample is redissolved and digested with LysC / trypsin to generate a fully fragmented sample. The fully fragmented peptides are purified on C18 resin, analyzed by mass spectrometry, and compared to a drug-free sample (treated with DMSO).

[0071] Performing LiP-MS / DarkLiP-MS at low protein concentrations To minimize the impact on maintaining the native state of the proteome, the jump dilution method discussed above is combined with LiP-MS to assess drug retention times.

[0072] To examine the effect of low protein concentration on limited proteolysis, cell extracts are incubated with a standard concentration of a drug (e.g., staurosporine) in the cell extract and the volume is increased by the addition of enzyme (the concentration of the drug is kept constant).

[0073] Evaluation of drug-target residence time by LiP-MS / DarkLiP-MS technology LiP MS and DarkLiP-MS technologies use LC-MS, DIA (Data Independent Acquisition of Product Ion Spectra) mass spectrometry, and non-specific database searching to enable MS-based identification of unique structural and / or conformational states of proteins and / or structural and / or conformational changes of proteins in complex biological or clinical specimens with high sensitivity, coverage, and throughput.

[0074] Identifying such unique protein conformational changes and states provides valuable information about protein structure and function, enables identification of drug or metabolite targets of interest, contributes to the characterization of biochemical and signaling pathways involved in the response to perturbations, and informs information about disease mechanisms.

[0075] To clearly demonstrate that the LiP-MS / DarkLiP-MS method can monitor the decay of drug-induced structural changes upon drug dilution, cell extracts were incubated with drugs whose retention times had been measured, and LiP-MS was performed at different time points after a 100-fold jump dilution.

[0076] A potential comparator of choice is the pan-CDK inhibitor loniclib, which shows kinetic specificity for CDK2 and CDK9 (albeit with IC50 values ​​in the nanomolar range for CDK1, CDK2, CDK4, CDK6, CDK7, and CDK9). The residence time of staurosporine will also be evaluated, as half-lives in the minute range for certain kinases have already been described.

[0077] Finally, irreversible inhibitors such as AEBSF and E64 (general inhibitors of serine and cysteine ​​proteases, respectively) should result in an "infinite" residence time.

[0078] Relative evaluation of drug residence times among multiple compounds To illustrate how LiP-MS / DarkLiP-MS can be used to predict the in vivo efficacy of different drugs based on their binding kinetics, we compare FDA-approved compounds with compounds known to induce strong side effects, thereby determining whether clinical or preclinical failure of a drug can be predicted by its residence time in off-target proteins.

[0079] LIP-MS technology The limited protein proteolysis mass spectrometry (LiP-MS) technique (designated as the LiP protocol) used in the present invention is described in WO201408273, the contents of which are incorporated herein by reference.

[0080] US Patent No. 5,999,623 discloses a limited proteolysis (LiP) protocol, i.e., a method for detecting the conformational state of proteins contained in complex mixtures of additional proteins and / or other biomolecules, in particular in complex native biological matrices, and an assay for such a method, which preferably comprises, optionally after an extraction and / or lysis step, the following steps: Step 1 (corresponding to step D of the method according to the present invention): subjecting the complex mixture to limited proteolysis under conditions in which the protein is in a conformational state to be detected, to obtain a first fragment sample; Step 2 (corresponding to step E of the method according to the present invention): denaturing the first fragment sample to form a denatured first fragment sample; Step 3 (corresponding to step F of the method according to the invention): completely fragmenting the denatured first fragment sample in a digestion step to obtain a completely fragmented sample; Step 4: Analytical analysis of the fully fragmented sample to determine the fragment characteristics resulting from both the limited proteolysis of step 1 and the complete fragmentation in digestion step 3 to determine conformational state.

[0081] Limited Protein Proteolysis (LiP) and Advanced Targeted Mass Spectrometry Workflows The proposed method is preferably based on the combination of a biochemical technique called limited proteolysis (LiP) with advanced targeted mass spectrometry workflows, including selected reaction monitoring (SRM) or SRM-like approaches (e.g., SWATH-MS), or other mass spectrometry approaches such as parallel reaction monitoring (PRM), data-dependent acquisition (DDA), isobaric label quantification, or untargeted analysis of DIA.

[0082] Liquid chromatography coupled with mass spectrometry (LC-MS) has been used for many years in the field of proteomics to identify and quantify peptides (and therefore proteins) from complex sample mixtures. The most commonly used approach is a variation of the so-called LC-MS / MS or "shotgun" MS approach, which relies on the generation of fragment ions from precursor ions automatically selected based on the precursor ion profile (data-dependent analysis, DDA). The most mature technique is called selected reaction monitoring (SRM), often also referred to as multiple reaction monitoring (MRM). Targets in an MRM experiment are defined rationally and depend on the hypotheses tested in the experiment. Selected combinations of precursor ions and fragment ions for these targets (so-called transitions; a set of transitions for one target precursor is called an MRM assay) are programmed into the mass spectrometer, which then generates measurement data only for the defined targets. Another variation of targeted proteomics is data-independent acquisition, a more recently described variation commonly referred to as the SWATH-MS approach. Here, the targeted aspect is introduced only at the data analysis level. In contrast to MRM, this approach does not require preliminary method design prior to sample injection. LC-MS acquisition encompasses the entire analyte content of a sample across the entire mass and retention time (RT) range, allowing post-hoc data mining for peptides / precursors of interest. Data is acquired in a data-independent manner across the entire mass range (e.g., 200 Thomsons to 2000 Thomsons) and the entire chromatography, regardless of sample content. This is typically achieved by stepping the mass spectrometer selection window across the entire mass range. In effect, this data acquisition method creates a complete fragment ion map for all analytes present in the sample and correlates fragment ion spectra back to the precursor ion selection window from which the fragment ion spectra were acquired.This is achieved by widening the mass spectrometer's precursor isolation window and thus taking into account multiple precursors that co-elute and simultaneously contribute to the fragmentation pattern recorded during the analysis. Such precursor windows are called swaths. As a result, complex fragment ion spectra result from the fragmentation of multiple precursors, which requires more challenging data analysis. Unlike shotgun proteomics, MRM and SWATH technologies repeatedly record spectra of the same analyte with high time resolution. This high time resolution compared to shotgun proteomics, combined with the limited fragment ion information in MRM and the limited association of fragment ions with precursor ions in SWATH, necessitates entirely new types of data analysis. Because only a limited, predefined number of analytes are monitored, shotgun proteomics-type database searches by comparing spectra to a complete theoretical proteome are not necessary. Instead, a number of scores based on signal characteristics such as shape, coelution of transitions, and similarity of transition intensity to an assay library are described.

[0083] Furthermore, MRM does not allow for the estimation of identification confidence using the false discovery rate (FDR) as in classical shotgun proteomics. Therefore, a novel approach has been developed that relies on measuring non-existent peptide transitions (decoy transitions) (Reiter L, Rinner O, Picotti P, Huttenhain R, Beck M, Brusniak MY, Hengartner MO, Aebersold R: mProphet: automated data processing and statistical validation for large-scale SRM experiments. Nature methods 2011, 8(5):430-435). Data from these decoy transitions can be used to derive FDRs, similar to those used in shotgun proteomics. This FDR confidence estimation is necessary to determine the significance level of the data and enable user-defined data quality filtering. SWATH data differs from MRM data. In contrast to MRM, full fragment ion spectra are recorded using the SWATH method. The time resolution is typically selected similarly to MRM. Compared to shotgun proteomics, SWATH differs in that the precursor selection window in shotgun proteomics is typically around 1 Th, whereas in SWATH, the selection is typically higher, typically 25 Th, resulting in fragment ion spectra derived from a much larger number of precursors. This high complexity of fragment ion spectra makes it impractical to analyze the data as in shotgun proteomics using database searches. However, with the added advantage of high time resolution in the data, the data can be analyzed similarly to MRM data. This can be done by extracting the ion currents corresponding to the MRM transitions. The resulting data can be analyzed very similarly to MRM. In all variations of LC-coupled mass spectrometry, proteins in samples for MRM experiments are digested into smaller peptides before analysis.The resulting peptide mixture is typically separated chromatographically to reduce sample complexity. Chromatographic separation adds a temporal dimension, called retention time (RT), to the data recorded by the mass spectrometer. Data-independently acquired data can also be analyzed in a spectrally centric or untargeted manner, where a search space is queried based on the data, for example, on precursor ion (MS1) signals within the data (different from SWATH). This is the same type of analysis typically used for DDA. Additionally, various quantification techniques can be used to measure absolute quantities of peptides and proteins, such as isobaric labeling (TMT) or iTRAQ, stable isotope labeling with amino acids in cell culture (SILAC), or isotope-heavy-labeled peptides. Parallel reaction monitoring (PRM), similar to MRM, can also be used, but performed on high-resolution instruments, where fragment ion scans (MS2 scans) are acquired across the entire range of the analyte(s) targeted in the analysis.

[0084] SRM assays are quantitative mass spectrometry-based assays specific for proteins of interest, similar to antibodies for Western blotting, but with higher multiplexing capabilities and shorter development times (assays for 100 peptides can be developed in 1 hour). Previously, SRM has been demonstrated to be capable of quantifying proteins with a wide range of cellular abundances, down to less than 50 copies per cell, in whole cell lysates (see P. Picotti et al., Cell 138 (4), 795 (2009), and Picotti et al. Nature Methods, VOL. 9 NO. 6, JUNE 2012; these references relate to SRM technology and are specifically included in this disclosure), to resolve proteins with high (>95%) sequence overlap, and to measure target peptides across multiple samples. Thus, this technology enables quantitative measurement of specific peptides in highly complex samples. In recent years, further developments in the SRM approach have included SRM-like approaches based on data-independent acquisition of product ion spectra and their targeted analysis (SWATH method, see LC Gillet et al., Mol Cell Proteomics 11 (6), O111 016717 (2012), which includes disclosures on SWATH method and data extraction).

[0085] DarkLiP-MS technology The DarkLiP-MS technique (also referred to as the DarkLiP protocol) used in the present invention is described in European Patent Application Publication No. 21212313.7, entitled "Method and tools for the determination of conformations and conformational changes of proteins and of derivatives thereof," filed by Biognoys AG on December 3, 2021, the contents of which are incorporated herein by reference.

[0086] The DarkLiP-MS approach is a novel, complementary approach to LiP-MS, focusing on the generation and analysis of unique peptide sets, which can provide information distinct from traditional LiP-MS experiments. By digesting only native proteins for a limited time, followed by a filtration / concentration step, the proposed technique reduces the number of uninformative peptides and / or protein fragments that can be problematic during sample preparation, data acquisition, and analysis. This increased signal-to-noise ratio can significantly improve data quality and subsequent biological insights.

[0087] More generally, the DarkLiP-MS approach relates to a method for detecting the conformational state of at least one protein, said at least one protein being contained in a complex mixture of further proteins and / or other biomolecules (such a complex mixture can be, for example, a complex cell extract mixture or a biological probe such as a body fluid in general (plasma, cerebrospinal fluid, urine, etc.)), wherein said at least one protein in said complex (cell extract) mixture has been subjected to conditions that induce a conformational change in said at least one protein.

[0088] The DarkLiP-MS approach also relates to a method for detecting the conformational state of at least one protein contained in a tissue, an environmental sample (e.g., seawater, etc.), or a biological secretion obtained by lysing cells according to a LiP-MS protocol, wherein the at least one protein has been subjected to conditions that induce a conformational change in the at least one protein.

[0089] Protein concentration can be determined using an assay kit.

[0090] The method preferably comprises, optionally after an extraction and / or dissolution step, the following sequence of steps: Step 1 (corresponding to step D of the method according to the invention): limited proteolysis of a complex mixture (e.g. a cell extract mixture) under conditions in which at least one protein to be detected is in its original conformational state to obtain a first fragment sample, immediately followed by Step 2 (corresponding to step G of the method according to the present invention): removing large peptides and proteins or other biomolecules from said first fragment sample to form an enriched fragment sample; Analyzing the enriched fragment sample to determine fragments having characteristics resulting from the limited proteolysis of step 1, as well as characteristics remaining after removal step 2 to determine the conformational state of said at least one protein.

[0091] The reference to "immediately following" at the end of step D excludes further denaturation and / or proteolysis steps, but does not exclude further steps involved in terminating the limited proteolysis of step D, i.e., steps of quenching the limited proteolysis by adding a corresponding reagent (e.g., sodium deoxycholate solution), increasing the temperature, washing, filtration, precipitation, adjusting the solvent and / or pH, or a combination thereof. In other words, step 1 is "complete" from the perspective of digestion, since there are no other steps during the limited proteolytic digestion that contribute to peptide production (e.g., denaturation or addition of other proteases to increase access to cleavage sites). However, it is possible to add deoxycholate and increase the temperature, e.g., to 98°C, prior to step 2, thereby terminating the protease activity of step D.

[0092] The DarkLiP-MS technique is a novel variation of the LiP-MS approach that focuses on increasing the relative ability to identify peptides that convey structural / conformational information from proteins, at the expense of identifying peptides (and therefore proteins) that do not necessarily report structural information. By focusing on enrichment of structurally informative peptides, the proposed technique increases the signal-to-noise ratio and therefore makes the identification of protein structural changes more robust.

[0093] A key feature of the proposed technique is that it significantly alleviates the dynamic range challenge inherent in proteomics samples by increasing the number and abundance of truly informative peptides relative to the total number of peptides contained in the sample. This problem is most pronounced in human body fluids such as plasma, but it also exists in samples where the protein(s) of interest are low in abundance, as is common in drug studies targeting a single (or a few) proteins. Among other factors, this wide dynamic range results from the protein size distribution combined with the distribution of peptide responses in mass spectrometry. In classical proteomics approaches, including LiP-MS approaches, larger proteins generate more tryptic peptides. Thus, there is a strong correlation between the size and / or abundance of a protein and the likelihood that such a protein will generate at least some peptides that show a strong response in mass spectrometry. In contrast, the proposed technique generates peptides primarily from protease-accessible regions of proteins in their native or near-native conformation (i.e., undenatured). These peptides tend to be located on the solvent-exposed surface of the protein. Because the relationship between protein surface area and volume is not a constant ratio but rather decreases on average as protein size increases, the proportional number of peptides derived from large proteins is significantly reduced. By exploiting this bias in the surface area-to-size ratio, the proposed technique aims to reduce the dynamic range inherent in proteomics samples, which is at least in part due to the natural size distribution of proteins. Furthermore, the proposed technique focuses on accessible, informative peptides that report on protein structure and protein structural changes.

[0094] The proposed technique utilizes two key processes: limited digestion combined with enrichment of short MS-compatible peptides. The overall intent of this procedure is to increase the number of peptides that carry conformational / structural information (signal) in mass spectrometer-ready samples, while reducing the amount of peptides that do not contain such information (noise).

[0095] This is achieved in a surprisingly simple and efficient approach by employing a limited digestion step under non-denaturing (i.e., protein structure-preserving) conditions using specific or non-specific proteases. The limited digestion can be varied by changing the enzyme-to-substrate ratio, performing the digestion at lower temperatures, or performing the digestion on a relatively short timescale. In contrast to LiP-MS protocols, the resulting peptide mixture is not fully denatured and fully fragmented; instead, it is filtered or otherwise processed (see below) to remove large peptide / protein fragments, which make up the majority of the mixture and contain little information about protein structure / conformation and / or structural / conformational changes. By removing large peptide / protein fragments from the digest prior to mass analysis, the proposed technique also reduces the number of non-informative peptides that could introduce artifacts and / or noise into the experiment, both in terms of peptide identification on the mass spectrometer and during downstream data analysis. This removal can be achieved by processes that separate large peptide / protein fragments and enrich for suitable peptides, including size filtering (e.g., using 10k MWCO filtration devices), chromatography (e.g., size exclusion, hydrophobic or anion exchange), or physical processes (e.g., phase separation, absorption or precipitation). Compared to classical LiP-MS, the proposed technique omits the full trypsinization step under denaturing conditions after the limited proteolysis step.

[0096] Physical removal, including phase separation, absorption, precipitation; filtration, separation, or concentration based on hydrophilic / hydrophobic properties; filtration, separation, or concentration based on electric / magnetic fields; or a combination thereof. Filtration can also be performed using two or more filters to enrich for a specific peptide size range, i.e., to remove not only large peptides but also very small peptides whose very short amino acid sequences do not provide sufficient specificity or are not suitable for mass spectrometry analysis.

[0097] In addition to using a single protease for the limited digestion step, the proposed technique can be supplemented by the use of a protease mixture and / or a sensitizer (e.g., heat, urea). Instead of a protease mixture, two different proteases (or sets of proteases) can be used on aliquots of the sample, followed by pooling. Pooling can be performed after digestion or after separating peptides from the rest of the sample. Instead of pooling, samples can also be processed and measured completely separately. The protease mixture and sensitizer act in two ways to improve the identification of proteins of interest according to the proposed technique. The protease mixture contains proteases that target different amino acids for cleavage, thus allowing for the easy generation of more and more unique peptides during the digestion step. The sensitizer works by slightly disrupting the native state of the protein, allowing for novel protease cleavage. Sensitizers are particularly useful for the proposed technique when investigating specific conditions (e.g., the presence or absence of a drug or metabolite) for changes in protein structure. In such cases, when an event such as drug binding alters the structure (i.e., stabilizes or destabilizes), the protein of interest becomes more or less sensitive to a particular sensitizer, thereby amplifying the effect of the sensitizer.

[0098] Thus, the proposed technique can identify specific protein conformations or structures by utilizing previously overlooked peptides (peptides with two nontryptic ends located on the surface of proteins). The steps (e.g., filtration) included in the workflow of the proposed technique to remove large peptides and proteins alter the overall number of peptide / protein identifications, but lead to a relative enrichment of structurally informative peptides.

[0099] The workflow of the proposed technology includes or consists of the following steps: Step A: Protein- or proteome-containing samples from cells, tissues, or body fluids are interrogated for the structural and / or conformational state of the proteins. This includes, but is not limited to, incubation with a ligand (e.g., small molecule, metabolite, etc.) at a defined concentration (treatment mixture), including a control (vehicle) sample, or subjecting the lysate to conditions that induce structural state changes, e.g., temperature, metabolic stimulants, etc., including an unstimulated sample. Whatever conditions are utilized, native protein structure (primary, secondary, tertiary, and preferably (optionally) quaternary) is maintained throughout. Step B: Stopping the binding reaction by removing at least one ligand insofar as it is not bound to the target; or reducing the concentration of the ligand insofar as it is not bound to the target, thereby reducing the number of binding events (per unit time). Step C: Incubating the at least one ligand bound to the at least one target for different periods of time. Step D: Each sample is subjected to a limited digestion step using a specific or nonspecific protease (or a combination thereof). This digestion should be relatively short, typically 1-5 minutes, and quickly quenched. Step G: After this digestion step, larger peptides and protein fragments are removed, for example, via filtration, separation or concentration devices (see methods above and below).

[0100] The remaining peptides are then processed using a standard proteomics workflow (e.g., denaturation, C18 cleanup, etc.) and analyzed via LC-MS / MS so that peptides can be identified and quantified.

[0101] Further embodiments of the invention are also set forth in the dependent claims and the following description.

[0102] Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. However, the drawings are intended to explain the preferred embodiments of the present invention and are not intended to limit the present invention. [Brief explanation of the drawings]

[0103] [Figure 1] Figure 1 shows a schematic of the proposed approach. Native proteins are incubated with defined concentrations of ligand, including a control (vehicle) sample. Each sample undergoes a short, rapid-quenched, limited digestion step, after which larger peptides and protein fragments are removed, yielding a unique peptide population that depends on the protein conformation during the limited digestion. The remaining peptides can be used to perform a standard proteomics workflow. [Figure 2] FIG. 2 is a diagram showing the concept of measuring drug retention time by LiP-MS / DarkLiP-MS. [Figure 3] FIG. 3 shows the experimental workflow used to measure drug retention times using the jump dilution method in combination with LiP-MS. [Figure 4] FIG. 4 shows the workflow of sample processing for analysis by mass spectrometry. [Figure 5] FIG. 5 shows the expected results of measuring drug retention time by LiP-MS. [Figure 6] FIG. 6 shows proof of concept: ligand staurosporine and target bispecific mitogen-activated protein kinase kinase 3 (MP2K3). [Figure 7]FIG. 7 shows proof of concept: ligand calyculin A and target phosphatase PP2A-α (PPP2CA). [Figure 8] FIG. 8 shows proof of concept: ligand calyculin A and non-target protein kynureninase (KYNU). DETAILED DESCRIPTION OF THE INVENTION

[0104] A schematic showing an example of how the present technology supports the detection of structural / conformational changes is shown in Figure 1 , using the investigation of ligand binding events as an example.

[0105] Figure 1 shows a schematic of the DarkLiP-MS approach, where the condition that distinguishes conformational differences between a reference sample and an altered sample is the addition of a ligand. Native protein 100 is incubated with ligand 101a at a defined concentration in the altered sample (top path), while no ligand is added in the control (vehicle) sample 101b (bottom path). Each sample is typically subjected to a short (1-5 min) rapid-quenched limited digestion step 102 using a nonspecific protease. Next, in step 103, larger peptides and protein fragments are removed (e.g., via filtration), yielding a unique peptide population 104a / 104b that depends on the protein conformation during limited digestion 102. The remaining peptides can then be used for standard proteomics workflows (e.g., denaturation, C18 cleanup, LC-MS, and analysis).

[0106] The DarkLiP-MS approach is based on the combination of a biochemical technique called limited proteolysis (LiP) with an advanced targeted mass spectrometry workflow, including selected reaction monitoring (SRM) or SRM-like approaches (e.g., SWATH-MS), or other mass spectrometry approaches such as parallel reaction monitoring (PRM), data-dependent acquisition (DDA), isobaric label quantification, or untargeted analysis (DIA). The analytical analysis techniques used in this study, based on quantitative mass spectrometry, enable the MS-based identification of unique structural / conformational states and / or structural / conformational changes of proteins in complex biological or clinical specimens with high sensitivity, coverage, and throughput, using LC-MS, DIA (data-independent acquisition of product ion spectra) mass spectrometry, and nonspecific database searching. The structural / conformational states of proteins sampled by the proposed approach can be native, non-native, or a mixture of both, depending on the application. Implementation of this technique can be used to investigate protein structure under standard conditions, during protein binding to drugs / small molecules or metabolites, upon binding to other proteins (protein-protein interactions, i.e., protein complexes), upon chemical modifications (e.g., PTMs such as protein phosphorylation), or upon binding to various other molecules (e.g., lipids or DNA) as a result of changes in the local environment (e.g., elevated temperature, changes in ionic strength, or the presence of chaotropes). The proposed technique allows for the detection of proteins that undergo structural changes without hypothesis when perturbations are induced in the investigated system (e.g., immune signaling or disease triggers). Identifying such unique protein conformational changes and states provides valuable information about protein structure and function, enables the identification of drug or metabolite targets of interest, characterizes biochemical and signaling pathways involved in the response to perturbations, and informs information about disease mechanisms. Furthermore, altered protein structures can be used as surrogates (i.e., structural biomarkers) for disease detection.Insights into the structural states and dynamics of the structural proteome provide a deeper understanding of both physiological and non-physiological mechanisms of action, both of which can be major obstacles to better understanding disease and supporting drug design and improvement. Thus, the proposed technology enables the use of structural proteomics for a variety of applications, ranging from basic biology to target deconvolution and biomarker discovery.

[0107] Thus, the proposed approach is a novel, complementary approach to LiP-MS, which, due to its focus on generating and analyzing unique peptide sets, can provide information that is entirely unique from conventional LiP-MS experiments. By digesting only native proteins in a limited time and introducing subsequent filtration / concentration steps, the proposed technique reduces the number of uninformative peptides and / or protein fragments that are problematic during sample preparation, data acquisition, and analysis. This increased signal-to-noise ratio can significantly improve data quality and subsequent biological insights.

[0108] Preferred embodiment of the limited proteolysis / removal-filtration process / analytical analysis According to a preferred protocol, in the limited proteolysis step D, a proteolytic system selected from the group consisting of proteinase K, thermolysin, subtilisin, pepsin, papain, α-chymotrypsin, elastase, and mixtures thereof is used.

[0109] In step D, the proteolytic system is preferably used at a weight concentration in the range of 1 / 50 to 1 / 10000, preferably 1 / 100 to 1 / 1000, given as the ratio of enzyme to biomolecule content with respect to the total biomolecule content in the sample.

[0110] Step D can be carried out over a time span of 1 minute to 60 minutes, preferably in the range of 2 minutes to 30 minutes, or 2 minutes to 10 minutes, or 2 minutes to 5 minutes, and more preferably at a temperature in the range of 20°C to 40°C.

[0111] Preferably, the temperature in the limited proteolysis step D is in the range of 20°C to 40°C or 4°C to 90°C. This temperature range is usually around room temperature (20°C to 25°C) or 37°C, while thermolysin is active up to 80°C. 4°C is also applicable to slow down the proteolysis reaction.

[0112] The properties of the non-specific proteases used are summarized in Table 1 below:

[0113] [Table 1]

[0114] Preferably, in step G, peptides and proteins are removed by a filtration, separation or concentration step. Preferred methods include size filtering (e.g., using a 10k MWCO filtration device); chromatography, including size exclusion, hydrophobic or anion exchange chromatography.

[0115] Preferably, particularly good results can be obtained if in step G peptides, proteins or other biomolecules, or both, having a molecular weight greater than 20 kDa, preferably greater than 15 kDa, most preferably greater than 10 kDa, are removed from the first fragment sample.

[0116] In line with the above, good results can be obtained if, preferably, in step G, peptides having a molecular weight of less than 0.1 kDa, or less than 0.2 kDa, or less than 0.4 kDa are also removed from the first fragment sample.

[0117] According to another preferred embodiment, step G comprises a proteomics workflow including, inter alia, denaturation, C18 cleanup, or a combination thereof, prior to the actual analysis.

[0118] For quantitative determination, heavily labeled fragments bearing the characteristics resulting from the limited proteolysis in step D and the characteristics remaining after removal step G can be spiked into the original complex mixture and / or the first fragment sample and / or the enriched fragment sample. Therefore, if necessary, absolute quantification can be achieved using heavily labeled synthetic internal standard peptides. This approach can be applied directly to unfractionated proteome extracts, or it can be combined with various isotope labeling and sample fractionation techniques previously used in proteomic experiments (e.g., iTRAQ labeling and the TAILS workflow, O. Kleifeld et al., Nature Biotechnology 28 (3), 281 (2010)).

[0119] For the analytical analysis step, specific, quantitative mass spectrometry-based assays in the form of selected reaction monitoring (SRM) and / or data-independent acquisition of product ion spectra are preferably used.

[0120] The present invention also relates to the use of methods as detailed above in combination with peptide fragment enrichment techniques such as TAILS on the peptides generated in step D.

[0121] The method according to the invention opens up many possibilities for biomedical, biotechnological and pharmaceutical applications as well as biological research.

[0122] Figure 2 shows a schematic diagram of the retention time measured by LiP-MS / DarkLiP-MS. In this approach, a native protein is incubated with at least one ligand. Interaction between the ligand and the target protein induces a conformational change in the target. The ligand is removed from the solution medium, and the ligand-bound target is incubated for a defined period and then subjected to a limited proteolysis step using LiP-MS or DarkLiP-MS. Finally, the generated peptides are analyzed by mass spectrometry. The peptide abundance measured by mass spectrometry depends on the amount of ligand still bound to the protein target at the time of limited proteolysis by LiP-MS or DarkLiP-MS.

[0123] Figure 3 illustrates the experimental workflow used to generate the data presented in the Examples section below. Native proteomes were incubated with the ligand of interest (staurosporine or calyculin A) (bottom panel) or solvent (DMSO) (top panel). DMSO-treated samples served as negative controls and were used to confirm that the observed effects were due to the interaction of the ligand with its target. The proteomes were then diluted 100-fold (jump dilution method), and aliquots containing samples of the ligand-bound proteome were extracted at different time points after dilution. Because the ligand concentration in solution decreases (as a result of the jump dilution), new binding events between the ligand and target are highly unlikely. Consequently, the proportion of ligand-bound target protein is expected to decrease over time as the ligand-target complex dissociates. Samples incubated for different times were then subjected to limited proteolysis using LiP-MS or DarkLiP-MS and measured by mass spectrometry. The intensity (I) as a function of retention time (RT) is shown on the right.

[0124] Figure 4 shows the experimental workflow used to process LiP-MS or DarkLiP-MS samples for analysis by mass spectrometry. In both LiP-MS and DarkLiP-MS, limited proteolysis is performed using the nonspecific protease thermolysin. After a short incubation, the reaction is stopped by adding EDTA.

[0125] In the LiP-MS experiment (1), samples are dried after quenching. After resolubilization with urea, the partially fragmented proteome is reduced and alkylated, diluted with ammonium bicarbonate, and treated with LysC and trypsin to generate a fully fragmented peptide sample. This peptide sample is acidified with TFA and washed with a C18 resin before analysis by mass spectrometry.

[0126] For processing in a DarkLiP-MS experiment (2), the partially fragmented proteome is denatured with guanidinium hydrochloride, reduced and alkylated, acidified with TFA, and washed with C18 resin. Without fragmenting the proteins with LysC and trypsin, the sample is washed directly with C18, removing proteins and large peptides still present in solution and enriching for fragmented peptides generated during the limited proteolysis step. This enrichment step is a hallmark of the DarkLiP-MS approach.

[0127] Figure 5 shows the expected pattern of recorded peptide abundance relative to vehicle for proteins with different drug residence times. The y-axis of the graph corresponds to the measured intensity for a particular peptide, while the x-axis corresponds to the time after jump dilution. Because peptide intensity depends on the proportion of ligand-bound target protein, the graph shows the lifetime of the protein-ligand complex. 1 - Peptide abundance measured in vehicle-treated proteome. It is assumed that the target protein does not exhibit significant structural changes during the experimental procedure. Therefore, after dilution of the proteome, no change in peptide abundance over time is expected. 2. Proteins with Short Drug Retention Times. For proteins with short drug retention times, the abundance of the target peptide is expected to differ from the abundance of the same peptide measured in the vehicle-treated proteome. This is due to the structural changes caused by the ligand binding to the target protein. Because the interaction between the ligand and the target is short-lived, the difference in intensity between the ligand-treated and vehicle-treated samples is expected to decrease immediately after jump dilution. Eventually, the peptide intensity may reach the value of the vehicle-treated sample. 3 - Proteins with intermediate drug retention times. For proteins with intermediate drug retention times, the abundance of the target peptide may differ from the abundance of the same peptide measured in the vehicle-treated proteome. However, in contrast to the proteins with short drug retention times described above, the peptide intensity is maintained for a longer period of time. Eventually, the peptide intensity may reach the value of the vehicle-treated sample. 4—Proteins with long drug residence times or irreversible modifications. As in Cases 2 and 3, it is assumed that the abundance of the target peptide differs from the abundance of the same peptide measured in the vehicle-treated proteome. However, because the structural modification is long or irreversible, it is assumed that the abundance of the peptide does not change during the measurement period. In this case, the difference in peptide abundance between the ligand-treated and vehicle-treated proteomes will be constant.

[0128] Specific examples Example 1: Determination of the residence time of staurosporine to target dual specificity mitogen-activated protein kinase kinase 3 (MP2K3) Staurosporine is a natural compound that mimics the biological molecule ATP and acts as a promiscuous kinase inhibitor. Due to its similarity to ATP, staurosporine exhibits a short retention time at its target proteins. To demonstrate the application of LiP-MS technology to measure short drug retention times, the experimental workflow described in Figures 3, 4, and 5 was applied to the promiscuous kinase inhibitor staurosporine.

[0129] The native cellular proteome was treated twice with staurosporine or DMSO (vehicle), diluted 100-fold, and analyzed by LiP-MS. The results of Example 1 are shown in Figure 6.

[0130] The graph in Figure 6 shows the average abundance (y-axis) of four different peptides, peptide 1 through peptide 4, of the kinase MP2K3, measured between 0 and 60 minutes (x-axis) after jump dilution. Peptides 1 through 4 are listed in the database PeptideAtlas under accession numbers PAp01457867, Pap00639637, PAp02016489, and PAp00503812. The graph shows the difference in peptide abundance between staurosporine and DMSO, which is greater at early time points after dilution (between 0 and 30 minutes). The difference in abundance between the staurosporine-treated samples and DMSO decreases as time progresses. This observation indicates that the drug staurosporine binds to its putative target, MP2K3, leading to a conformational change in MP2K3 via the formation of an MP2K3-staurosporine complex. The decrease in peptide abundance over time indicates that the conformational change disappeared over time (i.e., dissociation of the MP2K3-staurosporine complex) and the conformational state became similar to that of the DMSO-treated protein, demonstrating the pattern expected for drugs with short residence times, as described in Figure 5.

[0131] According to Equation 1, the residence time of staurosporine relative to MP2K3 can be determined based on the half-life of the MP2K3-staurosporine complex. Because the half-life of the MP2K3-staurosporine complex corresponds to the time interval for a 50% decrease in peptide abundance, the peptide abundance values ​​were fitted to a four-parameter logistic curve. The time for a 50% decrease in relative peptide abundance was then calculated (Table 2). Because experimental variability resulted in slightly different values ​​for the complex half-life for each peptide, the median of the four values ​​obtained based on the four graphs in Figure 6 was calculated. According to Equation 1, dividing the median half-life by 0.693 (logarithm of 2) yielded a residence time of approximately 65 minutes for the MP2K3-staurosporine complex.

[0132] [Table 2]

[0133] Example 2: Residence time patterns obtained for the irreversible drug calyculin A Calyculin A is a natural compound that acts as a potent and irreversible inhibitor of serine / threonine protein phosphatases. To demonstrate the application of LiP-MS technology to irreversible drug retention time measurements, the experimental workflow described in Figures 3, 4, and 5 was applied to the phosphatase inhibitor calyculin A. Native cellular proteomes were treated twice with calyculin A or DMSO (vehicle), diluted 100-fold, and analyzed by LiP-MS. The results of Example 2 are shown in Figures 7 and 8.

[0134] The graph in Figure 7 shows the abundance (y-axis) of peptide 5 from protein phosphatase PP2A-α (PPP2CA) measured between 0 and 60 minutes after jump dilution (x-axis). This peptide is listed in the database PeptideAtlas under accession number PAp00524112. The graph shows that the difference in peptide intensity between calyculin A and DMSO remained constant throughout the entire length of the experiment. This indicates that the drug calyculin A bound to its putative target, PPP2CA, but the conformational change resulting from the formation of the PPP2CA-calyculin A complex did not disappear over time. This result illustrates the expected pattern for an irreversible drug, as described in Figure 5.

[0135] The graph in Figure 8 shows the abundance (y-axis) of peptide 6 of the protein kynureninase (KYNU), a non-target of calyculin A. This peptide is listed in the database PeptideAtlas under accession number PAp04420703. The graph shows that calyculin A did not bind to KYNU and therefore did not induce a conformational change in KYNU, as the peptide intensities were similar in calyculin A- and DMSO-treated samples throughout the entire length of the experiment. The discrepancy between the calyculin A and DMSO lines can be explained by experimental variability, as the scale of the y-axis represents much smaller fluctuations than the graphs in Figures 6 and 7 (where actual conformational changes are assumed). [Explanation of symbols]

[0136] 100 native proteins 101a Ligand 101b Control (vehicle) sample 102 Limited digestion process 103 Filtration 104a / 104b Unique peptide population I strength RT retention time

Claims

1. A method for determining the residence time between at least one target and at least one ligand, wherein the target is contained in a complex cell mixture of at least one further target and other biomolecules, and the steps are as follows: Step A: A step of incubating the at least one target with the at least one ligand over an incubation period involving a binding reaction between the ligand and the target, Step B: Stopping the binding reaction by removing at least one ligand insofar as it is not bound to the target; or reducing the number of binding events by decreasing the concentration of the ligand, Step C: A step of incubating the at least one ligand bound to the at least one target over different periods of time to obtain an incubated ligand-bound mixture, Step D: A step of obtaining a first fragment sample by selectively grading the incubated ligand-bound mixture under conditions in which at least one target is in its original conformational state. Includes, Here, after step D, one of the following sequences: Step E: A step of modifying the sample to obtain a modified first fragment sample. Step F: A step of digesting the sample to obtain a completely fragmented sample. Will it be done? Alternatively, after step D, the following steps: Step G: A step of removing large peptides and proteins or other biomolecules from the first fragment sample to form a concentrated fragment sample. The event took place. In either case, the next step is: An analytical analysis step in which an assay based on quantitative mass spectrometry is used in at least one form of selected / parallel reaction monitoring (SRM / PRM) and data-independent acquisition of product ion spectra, Here, in the case of steps E and F, the analytical analysis is performed on the completely fragmented sample to determine the fragments that have characteristics that are the result of both the limited protein degradation step D and the complete fragmentation step F. In the case of process G, the analytical analysis is performed on the concentrated fragment sample, A step to determine the dissociation half-life by measuring the amount of peptide present over different incubation times after ligand removal. This continued, The residence time (τ) is given by the following formula: [Math 1] It is calculated from there according to, During the ceremony, τ = residence time koff = dissociation constant t1 / 2 = Dissociation half-life And, Here, in steps E and F, the determination of the residence time between the target and the ligand is based on a quantitative comparison of the analytical analysis of the completely fragmented sample and the analytical analysis of the completely fragmented control sample. The method, wherein, in step G, the determination of the residence time between the target and the ligand is based on a quantitative comparison between the analytical analysis of the concentrated fragment sample and the analytical analysis of the concentrated fragmented control sample.

2. The method according to claim 1, wherein the protein-limited degradation of the composite mixture (step D) is carried out over a time span of 1 to 60 minutes, preferably in the range of 2 to 30 minutes, 2 to 10 minutes, or 2 to 5 minutes, at a temperature preferably in the range of 20°C to 40°C.

3. The method according to claim 2, wherein in the digestion step F, trypsin is used in an enzyme-to-substrate weight ratio in the range of 1 / 10 at a temperature of preferably 15°C to 70°C for a time span of 2 hours to 24 hours.

4. The method according to any one of claims 1 to 3, wherein the target is at least a portion of a protein or proteome under native conditions, and the ligand comprises one of the following entities: small molecule drug compounds, metal ions or organic molecules, naturally occurring protein cofactors, coenzymes or comatrixes, amino acids, synthetic peptide drugs, naturally occurring peptides, another protein, nucleic acids, lipids or polymeric complexes.

5. The method according to any one of claims 1 to 3, wherein the at least one target is a protein based solely on amino acids that constitute a protein, or is based on amino acids that constitute a protein and is responsible for post-translational modifications, and the complex mixture of further proteins and / or other biomolecules is a native biological complex matrix.

6. The method according to any one of claims 1 to 3, wherein in the protein-limited degradation step D, the protein degradation system is selected from the group consisting of proteinase K, thermolysin, subtilisin, pepsin, papain, α-chymotrypsin, elastase, and mixtures thereof.

7. The method according to any one of claims 1 to 3, wherein in the protein-limited degradation step D, the protein degradation system is used at a weight concentration in the range of 1 / 25 to 1 / 10000, preferably in the range of 1 / 50 to 1 / 1000, given as the ratio of enzyme to biomolecular content with respect to the total biomolecular content in the sample.

8. The method according to any one of claims 1 to 3, wherein, when the DarkLiP-MS protocol is used, the removal of large peptides and proteins (step G) is carried out by a filtration, separation or concentration step including size filtering; chromatography including size exclusion, hydrophobicity or anion exchange chromatography; physical removal including phase separation, absorption or precipitation; filtration, separation or concentration based on hydrophilic / hydrophobic properties; filtration, separation or concentration based on electric / magnetic fields; or a combination thereof.

9. The method according to any one of claims 1 to 3, wherein in step G, peptides, proteins, or other biomolecules having a molar weight greater than 20 kDa, preferably greater than 15 kDa, and most preferably greater than 10 kDa, are removed from the first fragment sample (step G).

10. The method according to any one of claims 1 to 3, wherein a proteomics workflow, in particular a proteomics workflow involving denaturation, C18 cleanup, or a combination thereof, is performed prior to the analytical analysis step.

11. The method according to any one of claims 1 to 3, wherein SWATH-MS and / or optionally data-dependent acquisition (shotgun) are used for the analytical analysis.

12. The method according to any one of claims 1 to 3, comprising spiking the original composite mixture or the completely fragmented sample with a heavily labeled fragment having characteristics that result from the limited degradation or complete fragmentation of the protein for an assay based on quantitative mass spectrometry.

13. The method according to any one of claims 1 to 3, wherein, for an assay based on quantitative mass spectrometry, a heavily labeled fragment having characteristics resulting from the limited protein degradation of step D, and characteristics remaining after the removal of large peptides and proteins or other biomolecules in step G, is spiked into the original composite mixture and / or the first fragment sample and / or the concentrated fragment sample.

14. The following objectives: Prioritizing target candidates based on their dwell time, Selecting the optimal drug-target candidate, Determining a medically appropriate target, Predicting in vivo off-target / side effects of different drugs based on their binding kinetics, Controlling the quality of protein-based pharmaceutical formulations Use of the method according to any one of claims 1 to 3 for at least one of the following.

15. A method for supporting the prognosis or diagnosis of a disorder in a patient in the early stages of functional impairment / disease progression, comprising: determining the presence of at least one selected target protein; determining the residence time between at least the selected target protein and at least one ligand as described in any one of claims 1 to 3 in a biological sample of the patient, e.g., plasma, serum, cerebrospinal fluid, or urine; and comparing the concentration data with concentration data from a population affected by the disorder / disease to confirm or cancel the presence of a given disorder.

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