WO2017206041A1

WO2017206041A1 - Method for determining endurance of interaction between ligand and target protein in cell

Info

Publication number: WO2017206041A1
Application number: PCT/CN2016/083998
Authority: WO
Inventors: 王帅
Original assignee: 王帅
Priority date: 2016-05-31
Filing date: 2016-05-31
Publication date: 2017-12-07
Also published as: CN109690316A

Abstract

Disclosed is a method for determining the endurance of the interaction between a ligand and a target protein in a cell, comprising: (1) treating the cell with the ligand; (2) removing the ligand outside the cell; (3) collecting the cell at different time points after the ligand is removed; (4) heating the cell; (5) lysing the cell; and (6) analysing the level of soluble or insoluble target protein in a cell lysis solution as a function of time. The method can be used to determine the dissociation rate constant, residence time and dissociation half-life of the interaction between the ligand and the target protein in the cell and the time range of therapeutic duration of the ligand so as to quantitatively describe the endurance of the interaction between the ligand and the target protein in the cell and to predict the duration of pharmacological action in the cell. The method can be used to develop new drugs and discover drugs with desired binding endurance. The method can be used to design a dosage regimen, especially a time interval of drug delivery.

Description

Method for determining the endurance of ligand interactions in intracellular ligands

Technical field

The invention belongs to the technical field of protein binding, and in particular relates to a method for determining the endurance of interaction of a target protein in a cell.

Background technique

Some drugs have surprisingly long-lasting effects even if they have been eliminated from the circulatory system for a long time. However, it is still not known what kind of drugs can have such long-lasting effects and how to design such drugs. To explain this contradictory pharmacological phenomenon, Robert A. Copeland et al., in Nat. Rev. Drug Discov., 5, 730-739, 2006, proposed a drug target protein retention time model, suggesting that the drug's efficacy mainly interacts with drug target proteins. The duration is related to the length of time. At present, drug target protein retention time is mainly measured by some biophysical and biochemical experimental techniques, such as surface plasmon resonance and fluorescence polarization, which are mainly related to the dissociation rate of drug target protein interaction. However, the residence time of the drug target protein in vitro is not equal to the retention time of the drug target protein in the cell or in vivo, and it is also uncertain whether the long-lasting drug target protein retention time in vitro can be converted into a sustained pharmacological action in the cell or in vivo, especially The long-term efficacy of quantification. In order to solve this problem, it is first necessary to accurately measure the retention time of drug target proteins in cells or in vivo, especially to determine the kinetics of drug target protein occupancy in cells or in vivo.

Since the interaction between the drug and the target protein usually occurs on the surface of the cell or inside the cell, the physiological environment inside the cell is much more complicated than the experimental conditions in vitro, so the retention time of the drug target protein in the cell is longer than the retention time of the drug target protein in vitro. Will be affected by more factors, including 1) the rate of dissociation of drug target protein interactions, 2) target proteins of different conformations, 3) recombination of drugs, 4) drugs that bind to other proteins, and 5) target proteins Natural substrates, post-translational modifications and binding partners, 6) active transport of drugs, 7) subcellular distribution of drugs and target proteins, 8) cell proliferation, 9) degradation and synthesis of target proteins. It can be seen that the retention time of the drug target protein in the cell is a new parameter different from the retention time of the drug target protein in vitro, and is used to describe the persistence of the drug target protein interaction at the cell level. For technical reasons, it is still difficult to measure the retention time of drug target proteins in the body. Therefore, it is more reasonable to predict the persistence of the drug in vivo by using the intracellular drug target protein retention time measured under physiological conditions. However, there is still a lack of methods for accurately assessing the persistence of intracellular drug target protein interactions and determining the retention time of intracellular drug target proteins.

Each method has its own advantages and disadvantages for the reported methods of assessing the binding endurance of drug target proteins in cells. Traditionally, radioisotope-labeled drug molecules have been used to measure their intracellular residence time (Expert Opin. Drug Discov., 7, 583-595, 2012). This method is simple but extremely expensive, and the experimental operation is complicated. More importantly, This method does not distinguish whether the detected radioactive signal is from a drug molecule that binds to the target protein or binds to the off-target protein.

The covalently bound fluorescently labeled probe molecule has the same binding site as the ligand to be detected and can be used in a competitive manner to study the persistence of interaction of the intracellular ligand with the target protein. Bradshaw et al., Nat. Chem. Biol., 11, 525-531, 2015, report the use of this method to study the intracellular binding endurance properties of BTK inhibitors. In addition, U.S. Patent Application Serial No. 14/104,860, the disclosure of which is incorporated herein by reference to the entire disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of The persistence of the interaction of the body with the target protein. The above two methods for applying probe molecules have the following problems: 1) it is very difficult or even impossible to design and synthesize a suitable fluorescently labeled probe molecule for each target protein; 2) it takes time for the probe molecule to enter the cell to reach equilibrium. About half an hour, so the above two methods can not accurately record the initial stage of the dissociation reaction; 3) especially because the fluorescent probe molecules can interfere with the interaction of the ligand with the target protein through competitive inhibition or allosteric regulation. . Therefore, the above two methods cannot accurately measure the endurance of intracellular ligand target protein interactions.

Protein thermal shift assays have been widely used in academia and industry to evaluate the interaction of ligand molecules with purified target proteins. Moreau et al., Mol. Biosyst., 6, 1285-1292, 2010, report a green fluorescent protein-based thermal transfer method that can be used to study ligands in cell lysates using non-purified GFP-fused target proteins. Interaction of GFP-fused target proteins. In addition, PCT/GB2012/050853 discloses a method for determining intracellular ligand binding to a target protein using a thermal transfer technique, which can be used to study natural unpurified target proteins in cells, including: 1) Whether the body can interact with intracellular target proteins, 2) the affinity of intracellular ligand target protein interactions. However, all the heat transfer tests mentioned above are The concentration of ligand and target protein remained unchanged under the equilibrium test conditions, ie throughout the test. In addition, all of the heat transfer test measurements mentioned above need to be completed in a short period of time, such as the recommended half hour, to prevent degradation or synthesis of the target protein (Nat. Protoc., 9, 2100-2122, 2014). ).

Summary of the invention

It is an object of the present invention to provide a method for determining the persistence of interactions of ligands in a cell with a target protein.

The technical solution adopted by the present invention to achieve the above object is as follows:

A method for determining the endurance of a ligand protein interaction in a cell, the method comprising the steps of:

Step 1, treating the cells with the ligand;

Step 2, removing the ligand outside the extracellular;

Step 3, collecting the cells at different time points after removing the ligand;

Step 4, heating the cells;

Step 5, lysing the cells;

In step 6, a function of the level of soluble or insoluble target protein in the cell lysate as a function of time is analyzed.

According to the method of the present invention, the ligand is added to the cell culture medium and enters the cell to interact with the target protein. The extracellular ligand is removed by replacing the fresh medium to break the balance between the extracellular and intracellular ligands, thereby triggering the dissociation reaction of the intracellular ligand. The cells at different time points after removal of extracellular ligands were collected, and the level of ligand-binding target protein in cells at different time points after removal of extracellular ligands was determined by cell thermal transfer technique to construct the dissociation of intracellular ligand target protein interactions. The curve calculates the dissociation rate constant, retention time and dissociation half-life of the intracellular ligand target protein interaction, and quantitatively describes the endurance of the intracellular ligand target protein interaction. In particular, the method of the present invention determines the kinetic profile of the intracellular ligand target protein occupancy, calculates the therapeutic duration of the ligand, and explains the persistence of the pharmacological effects of the intracellular ligand from the perspective of binding persistence.

The present invention relates to the binding endurance of intracellular ligand target protein interactions, from the angle of time The degree of interaction of intracellular ligands with target proteins was investigated. The method of the present invention can be used to screen compounds, and find suitable binding endactive active molecules as seed molecules, which can be used to guide the optimization of lead compounds, and to design and synthesize drugs with desired binding endurance. The methods of the invention can be used to measure and compare the binding potency of a drug to a plurality of intracellular target proteins, such as target proteins and off-target proteins, wild-type and mutants, to determine the selectivity of the intracellular target protein of the drug from a time perspective. The method of the present invention can be used to measure and compare the binding persistence of drug and target protein interactions in different types of cells, such as different types of tumor cells, different tissue cells, etc., and determine the cell selectivity of the drug from a time perspective. . The methods of the present invention can be used to predict and design the most effective dosing regimen, particularly by setting a suitable dosing interval to reduce off-target related side effects caused by drug exposure depending on the duration of treatment of the drug.

Preferably, the ligand is a cell metabolite, a small molecule or a drug; the target protein is a protein molecule that interacts with a ligand in the cell; the cell is a mammalian cell, a cell strain, an engineered cell or a primary cell. .

The term "ligand" as used in this specification refers to a molecule or compound to be detected that is capable of interacting with a target protein in a cell. The ligand can be a polypeptide, a nucleic acid, a substrate for an enzyme, a cellular metabolite or a hormone. The ligand is preferably a pharmacologically active compound, a seed molecule, a lead compound, a drug candidate or a drug. The ligand may also be a prodrug that binds to a target protein after being converted into a drug by hydrolysis or an enzymatic reaction or the like. The ligand may be naturally occurring or unnaturally chemically synthesized. The ligand described in this specification is not limited to its size or structure.

The term "target protein" as used in this specification refers to a protein molecule that interacts with a ligand in a cell. The target protein can be any protein of the cell, including proteins of the cell membrane, cytoplasm, nucleus or other subcellular organelles. The interaction of the ligand with the target protein can occur within the cell, for example, the ligand interacts with the intracellular kinase; interaction of the ligand with the target protein can occur on the cell membrane, for example, the ligand interacts with the extramembranous portion of the membrane protein. The target protein may be naturally occurring or may be recombinantly expressed after transfection of a plasmid or vector encoding the target protein. The target protein can be expressed as a fusion protein with a tag molecule or polypeptide, such as a FLAG tag, a His tag, a GFP tag, a GST tag or a luciferase. Target proteins within cells include GPCR receptors, ion channels, proteases, kinases, nuclear receptors, transcription factors or enzymes. Target egg White can be wild type or mutant. The target protein in the present invention is not limited to its type or kind.

The term "cell" as used in this specification refers to a cell or similar structure containing a target protein that interacts with a ligand. Cells include prokaryotic cells such as bacteria, eukaryotic cells such as yeast, single cell eukaryotes such as Leishmania, insect cells, mammalian cells, engineered cells or cell lines. Purified subcellular organs such as mitochondria can also function as cells. Preferably, the cells are isolated from cultured primary cells of the animal or human, and more preferably cultured under physiological or pathological conditions close to the body. Ligand target protein interactions may have different binding endurance in different cells due to different cellular environments, so the method of the invention determines the cell selectivity of the ligand from a time perspective.

Preferably, the method for determining the endurance of the ligand interaction of the ligand in the cell, wherein the amount of the ligand added in the step 1 is determined according to one of the following:

Mode 1, determined according to the EC50 value of the effect of the ligand on the cells, wherein the concentration of the ligand in the medium in the step 1 is 0.5-20 times the EC50 value;

2, according to the ligand to the target protein occupancy rate in the cell, the concentration of the ligand in the medium in the step 1 is 70%-100% of the ligand in the cell to the target protein occupancy;

Mode 3, according to the metabolic kinetics of the ligand in the animal or human body, the concentration of the ligand in the medium in the step 1 is the maximum blood concentration and the minimum effective concentration of the ligand in the animal or human body. Range concentration range.

Preferably, the treatment time of the ligand to the cells in step 1 is determined according to the binding rate of the ligand to the intracellular target protein, and the treatment of the ligand is terminated when the intracellular ligand target protein interaction reaches equilibrium.

In the first step of the present invention, after the ligand is added to the cell culture medium, the ligand enters the cell by diffusion, active or passive transport, or endocytosis, disperses into different subcellular organelles, and finally interacts with the target protein to establish Ligand target protein complex. The level or amount of the intracellular ligand target protein complex is closely related to the concentration of the ligand (hereinafter referred to as the test concentration of the ligand) and the action time added to the medium. The test concentration of the ligand may be in any concentration range as long as sufficient ligand target protein complex can be produced. The test concentration of the ligand can be determined according to its cellular activity, and can produce at least 5%, 10%, 20%, 30%, 50%, 70%, 80%, 90% or 100% cell activity, Or the test concentration of the ligand is equal to at least 0.5, 1, 2, 4, 5, 10, 20 or more EC50 values. The test concentration of the ligand is preferably determined according to its intracellular target protein occupancy rate, and can produce at least 5%, 10%, 20%, 30%, 50%, 70%, 80%, 90% or 100% of the target protein. Occupancy, and more preferably at least 70%, 80%, 90% or 100% target protein occupancy. The test concentration of the ligand is preferably determined based on its metabolic kinetics in the animal or human, such as the maximum plasma concentration, or the lowest effective concentration in the therapeutic concentration range. The action time of the ligand is determined by the rate of binding of the ligand to the target protein in the cell, and the treatment of the ligand is terminated when the intracellular ligand target protein interaction reaches equilibrium. For example, it can be 10, 30, 60 minutes, 2, 4, 6, 12 hours or longer, usually 30 minutes to 1 hour. In addition, cell viability should be monitored throughout the dissociation test, especially for high activity compounds at high test concentrations and long dissociation times, which may result in cell death, correspondingly Adjust the dose of the ligand and the time of the dissociation reaction. For example, in the case of the present invention, the ligand molecule staurosporin at a test concentration of 10 μM can cause significant apoptosis 6 hours after removal of extracellular staurosporine.

Preferably, the method for determining the endurance of the ligand interaction of the ligand in the cell, the method of removing the ligand in the step 2 is: replacing the fresh medium or washing the cells with the solution and then replacing the fresh medium.

The removal of the ligand in the cell culture medium in step 2 of the present invention is to break the balance between the intracellular and extracellular ligands, thereby triggering the dissociation reaction of the intracellular ligand target protein. To remove the extracellular ligand, it is preferred to replace the fresh cell culture medium, and it is better to wash the cells with the solution for 1, 2 or 3 times before replacing the fresh medium. In addition, other methods include the addition of chemicals that specifically react or bind to the ligand, or dilution with a large amount of fresh medium. Upon removal of the extracellular ligand, the intracellular ligand will leave the cell and diffuse into the culture medium, referred to as the remaining ligand. In order to initiate an intracellular dissociation reaction, the target protein occupancy of the remaining ligand must be significantly less than the target protein occupancy of the starting ligand, and preferably the target protein occupancy of the remaining ligand is very small and negligible. In the case of a high activity ligand at high test concentrations, for example 10,000 times the equilibrium dissociation constant value (Kd), the remaining ligand may result in a false positive strong binding endurance test result. In this case, a more thorough extracellular ligand clearance should be used. Order, for example 3, 4, 5 or more cleanings.

Preferably, the method for determining the endurance of the ligand interaction of the ligand in the cell, wherein the time point of collecting the cells in the step 3 is at least 8 time points, and the interval between adjacent time points is based on the solution of the ligand target protein complex The rate of departure is determined.

In step 3 of the present invention, cells at different time points after removal of the extracellular ligand are collected, representing different stages of the dissociation reaction of the intracellular ligand target protein. The number of time points of the dissociation reaction may be, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 20 or more, and preferably includes at least 8 data points. The time interval between different time points is determined according to the dissociation rate of the ligand target protein complex, which may be 1, 2, 3, 5, 10, 20, 30, 60 minutes; or 2, 3, 4, 6 , 12, 24, 48 hours or more. In addition, the methods of the invention also include collecting cells prior to removal of the extracellular ligand, representing the maximum initial level of ligand target protein interaction in the dissociation reaction. For a ligand with a fast cell dissociation rate, the intracellular ligand target protein complex will be rapidly dissociated after removal of the extracellular ligand, in which case more data should be set in the early stages of the dissociation reaction. Point, especially the analysis of cells before removal of extracellular ligands. For a ligand with a slow cell dissociation rate, the ligand target protein complex will slowly disintegrate after removal of the extracellular ligand, in which case sufficient data points should be set to cover the entire dissociation at appropriate intervals. reaction process. In addition to the above cell samples, in some cases, to calculate the level of intracellular ligand-bound target protein and to determine the kinetics of intracellular ligand target protein occupancy, the methods of the invention may further comprise a negative control and a positive control. The positive control is preferably a cell treated with a ligand but not having a ligand removed, having a known level of cell activity or a known target protein occupancy, and more preferably treated with the same test concentration of the ligand but Unremoved cells represent the maximum level of ligand-bound target protein under this assay condition. The negative control reflects the level of noise under this test condition, which may be cells without ligand treatment, and is preferably cells treated with the remaining ligands described above. In addition, the negative control may also be a cell that is sufficiently long after removal of the extracellular ligand until the intracellular ligand target protein complex is completely dissociated.

Preferably, the method for determining the endurance of the interaction of the ligand protein in the cell, wherein the temperature at which the cells are heat treated in step 4 is higher than the initial melting temperature of the ligand not bound to the target protein and And below the final melting temperature of the ligand binding target protein.

In step 4 of the present invention, the level of the intracellular ligand-bound target protein at different time points after removal of the extracellular ligand is detected by a cell thermal transfer technique. Heating the cells to a certain temperature for a period of time, when the ligand-unbound target protein and the ligand-bound target protein have different thermostability, usually the ligand-bound target protein is more than the unbound target protein. High heat melting temperature. The heating temperature may be a temperature above the initial melting temperature of the unbound target protein of the ligand but below the final melting temperature interval of the ligand-bound target protein. The heating temperature is preferably the temperature at which the soluble target protein has the most significant difference in the negative control and sample. The heating time can be any time range, such as 0.5, 1, 2, 3 or 5 minutes, as long as the level of soluble target protein in the negative control and the sample is significantly different in this case, and it is better to have the largest The difference in degree. Heating instruments include PCR machines, incubators, water baths, etc., as long as the cells can be heated to a specific temperature.

The method of cell lysis in the step 5 of the present invention may be any method capable of lysing cells to release intracellular proteins. The cell lysis method may be a sonication method, preferably a freeze-thaw method comprising 1, 2 or 3 cycles, and more preferably a cell lysate containing an ionic or non-ionic denaturing agent or amphiphilic molecule. Cells are lysed, especially membrane proteins, such as 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40. Since the released protease can degrade the soluble target protein during cell lysis, the cell lysate further contains a protease inhibitor, an enzyme cofactor such as Mg ²⁺ , or a redox agent such as DTT for stabilization and Protect the target protein. To facilitate the detection of soluble and insoluble target proteins, soluble and insoluble protein components can be separated by some separation technique. The protein separation technique can be any method capable of separating soluble and insoluble protein components. The protein separation technique is preferably centrifugal separation, the supernatant is a soluble protein component, and the precipitate is an insoluble protein component. Filtration separation is also preferred, the filtrate is a soluble protein component and the residue is an insoluble protein component. In addition, the soluble target protein can also be isolated by affinity purification of the target protein-specific antibody.

Preferably, the method for determining the endurance of the interaction of the ligand protein in the cell, the method for quantitatively analyzing the target protein in the step 6 comprises antibody technology, mass spectrometry, biological activity of the target protein or enzymatic activity of the fusion tag.

In step 6 of the present invention, the soluble or insoluble protein component is analyzed to determine the amount of target protein in which it is soluble or insoluble. To measure an insoluble target protein, it is necessary to first dissolve the insoluble protein component. It is therefore preferred to measure soluble target proteins. Quantitative methods for target proteins include mass spectrometry, such as orbitrap or ion traps; application of target protein or fusion tag-specific antibody methods, such as immunoblotting, ELISA; and quantitative analysis using the biological activity of the target protein or the enzymatic activity of the fusion tag. The best method for quantifying target proteins is to use high-throughput methods for directly detecting the level of soluble target proteins in cell lysates, such as alpha scanning, fluorescence resonance energy transfer, and fluorescence polarization.

Preferably, the method for determining the endurance of the interaction of the ligand protein in the cell, the step 6 further comprises: analyzing the soluble target protein, determining the level of the target protein bound by the ligand in the cell, and constructing the cell as a function of the dissociation time. The dissociation curve of the internal ligand target protein interaction calculates the dissociation rate constant, residence time and dissociation half-life of the ligand ligand protein interaction in the cell.

According to the method of the present invention, the soluble protein component is analyzed, the target protein soluble therein is measured, the amount of the target protein bound by the intracellular ligand is determined, and the dissociation curve of the intracellular ligand target protein interaction is constructed. A higher level of soluble target protein was detected in the sample cells than in the negative control representing the noise level, meaning that the ligand still binds to the intracellular target protein at this time point after removal of the extracellular ligand. The level of ligand-bound target protein is equal to the difference between the sample and the soluble target protein in the negative control, initially reflecting the strength of the endurance of the intracellular ligand target protein interaction. In addition, the ligand-unbound target protein at some heating temperatures can be completely unfolded and precipitated. In this case, a negative control may not be necessary, and the level of soluble target protein directly reflects the intracellular ligand binding. The level of the target protein. The dissociation curve of intracellular ligand target protein interactions can be constructed as a function of the dissociation time as a function of the dissociation time by removing the amount of target protein bound by the intracellular ligand at different time points after removal of the extracellular ligand. The dissociation rate and retention time of intracellular ligand target protein interactions can be determined based on the dissociation curve of the intracellular ligand target protein, quantitatively describing the endurance of the intracellular ligand target protein interaction. However, the present inventors have found that the dissociation reaction of the intracellular ligand target protein may also include a plurality of dissociation phases having significantly different dissociation rates, so that it is difficult to accurately apply the conventional dissociation rate and residence time in this case. The persistence of intracellular ligand target protein interactions was compared (Examples 1 and 2). In order to solve this problem, the method of the present invention advances One step comprises determining the time required for dissociation of 50% of the ligand target protein complex in the cell according to the dissociation curve of the intracellular ligand target protein, which is called the dissociation half-life of the intracellular ligand target protein, and can be used to describe intracellular distribution. The persistence of body target protein interactions. Similarly, the time required for dissociation of different percentages of ligand target protein complexes can also be determined, for example 10%, 20%, 30%, 40%, for more precise description of the persistence of intracellular ligand target protein interactions. force.

Preferably, the method for determining the endurance of the interaction of the ligand protein in the cell, the step 6 further comprises determining the target protein occupancy rate at different time points after removing the extracellular ligand, and constructing the kinetics of the ligand protein occupancy rate in the cell. The curve calculates the duration of treatment of the ligand above the lowest effective target protein occupancy.

Ligand has strong binding endurance with intracellular target proteins, meaning that the ligand has the potential to produce long-lasting intracellular pharmacological effects, however in some cases, for example, different doses of administration, even the same ligand target protein binding Persistence can still lead to different intracellular pharmacological effects over time. In order to determine the duration of intracellular pharmacological activity after removal of the extracellular ligand under different conditions, the method of the invention further comprises determining the occupancy of the ligand target protein and the level of the target protein bound by the intracellular ligand in the positive control. Calculate the level of intracellular ligand target protein occupancy at different time points after removal of the extracellular ligand, thereby constructing a kinetic curve of the intracellular ligand target protein occupancy after removal of the extracellular ligand, reflecting this In the case of removal of extracellular ligands, the relationship between intracellular ligand target protein occupancy and dissociation time. The lowest effective target protein occupancy that produces intracellular pharmacology can be determined based on the biological properties of the target protein and the pathogenic mechanism. The time range in which the intracellular ligand target protein occupancy kinetic curve is higher than the lowest effective ligand target protein occupancy rate is the time range in which the intracellular pharmacological action can be sustained after the extracellular ligand is eliminated. The invention is referred to as the "Therapeutic duration time range" of the ligand. In different cases, there may be different kinetic curves of the intracellular ligand target protein occupancy, and there may be different durations of intracellular pharmacological action of the ligand. When the target protein occupancy rate of the dissociation reaction is 100%, the dissociation curve of the intracellular ligand target protein is directly equal to the kinetic curve of the intracellular ligand target protein occupancy rate, in which case the treatment of the ligand The duration range can also be determined from the dissociation curve of the intracellular ligand target protein.

Preferably, the method for determining the persistence of interaction of a ligand protein in a cell, and the dissociation reaction of an intracellular ligand target protein interaction comprises dissociation reaction during removal of the extracellular ligand and removal of the extracellular ligand Dissociation reaction.

According to the method of the present invention, it is found that the dissociation reaction of the intracellular ligand target protein can be apparently occurred in the process of removing the extracellular ligand, in which case it is possible to ignore the dissociation reaction during removal of the extracellular ligand, possibly This leads to erroneous conclusions about the strength of the binding endurance of the ligand target protein in the cell. To determine the dissociation reaction during removal of the extracellular ligand, the method of the invention further comprises analyzing the cells prior to removal of the extracellular ligand as a positive control representing the ligand-bound target protein in the dissociation assay. The maximum initial level, as compared to the first time point sample of the dissociation reaction, determines the extent to which the intracellular ligand target protein complex dissociates during removal of the extracellular ligand. As shown in Example 1, if the cells before removal of the extracellular ligand are not analyzed and the dissociation reaction during removal of the extracellular ligand is neglected, the second stage of the dissociation reaction may be mistaken for the entire intracellular ligand target. The dissociation reaction of the protein results in a strong binding endurance of the false positive. However, after considering the dissociation reaction during removal of the extracellular ligand, it was found that most of the ligands in the cell had dissociated from the target protein during removal of the extracellular ligand, demonstrating a fast dissociation rate, and conversely only A small amount of the ligand slowly dissociates from the target protein. Considering the entire dissociation reaction during removal of the extracellular ligand, it can be determined that the test ligand molecule actually has a weak binding endurance with the intracellular target protein. In order to accurately assess the binding endurance of intracellular ligand target proteins, consideration should be given to the application of a complete intracellular ligand target protein dissociation reaction, including removal of extracellular ligands during dissociation and removal of extracellular ligands. Dissociation reaction.

As described above, unlike recombinantly expressed and purified target proteins, intracellular target proteins are not completely homogeneous components, and actually contain target proteins of different conformations, different post-translationally modified target proteins, different subcellular organelle target proteins, and Target proteins that interact with different binding partners may have different fast or slow dissociation rates for target proteins and ligand molecules in different states. In addition, during the dissociation reaction, intracellular ligand-bound or unbound target proteins may be degraded, new unbound target proteins may be synthesized, and degradation and synthesis of intracellular target proteins may attenuate intracellular distribution. The binding endurance of the body target protein. Similar to cell proliferation can also lead to reduction The persistence of ligand-protein interactions in weak cells. These mechanisms may be helpful in explaining the multi-stage dissociation reaction of the intracellular ligand target protein found in the present invention.

Dissociation assays due to intracellular ligand target proteins may persist for a long period of time, especially for ligands with slow dissociation rates, such as 48 or 96 hours. The cells continue to grow during the dissociation reaction, so in order to accurately compare the levels of intracellular ligand-bound target proteins at different time points, the method of the present invention further includes normalization. The standard for normalization can be based on the protein concentration of the soluble cell lysate or on the number of cells in the sample. Proliferation of cells reduces the average level of ligand-target protein complexes in cells, which in turn leads to a decrease in the persistence of ligand-protein interactions in cells. However, the rate of cell proliferation in vivo is usually significantly lower than the rate of cell proliferation in vitro. In this case, the survival of the ligand target protein interaction is underestimated due to the rapid cell proliferation rate in vitro, so in order to evaluate the endurance of the ligand interaction of the intracellular ligand in the case of proximity to the body, it is necessary to exclude the in vitro For high cell proliferation rates, cells of the same sample volume, such as 1 ml or 1 well, can be used as normalization criteria. In this case, samples at different time points may contain different numbers of cells but possess the same initial level of ligand target protein complex. To some extent, it is also understood that the cell proliferation rate is 0 in this case. Example 3). This normalization standard is especially suitable for cells with slow proliferation rate in vivo, which can avoid the insufficiency of ligand cell target interaction due to in vitro cell proliferation, and better reflect the endurance of ligand target protein interaction in vivo.

The method of the invention can be used for the development of new drugs, determining the binding endurance of intracellular target proteins of different compounds, selecting suitable binding endurance compounds as seed molecules, lead compounds, drug candidates according to the biological characteristics and pathological characteristics of the target protein. Or drugs. Strongly potent compounds have higher levels of soluble target protein at the same dissociation time point than weakly durable compounds, as well as slower intracellular dissociation rates and longer intracellular residence times. In particular, it has a longer duration of treatment. The binding endurance parameter of the compound can be used to guide the optimization of the lead compound and to design and synthesize a drug molecule having the desired binding endurance. In addition, when faced with multiple compounds with the same cellular activity, the persistence of intracellular compound target protein interactions can be used as a new parameter to further sort and distinguish compounds from a time perspective, which is helpful for selection. The most active molecule.

The method of the present invention can be used to compare the binding endurance of a compound to a different target protein in a cell, and to evaluate the selectivity of a target protein in a compound cell from a time perspective, which is called the selectivity of a target protein in time. Active molecules usually interact with multiple proteins in the cell, of which the target protein is responsible for pharmacological activity, and the other is called off-target protein. The binding of active molecules to off-target proteins can sometimes lead to deleterious side effects, so the target protein selectivity of the drug is an important goal in the optimization phase of the lead compound. The traditional target protein selectivity is mainly to compare the affinity of the active molecule to the target protein and the off-target protein from a thermodynamic point of view. Complementing the traditional target protein selectivity, the intracellular target protein selectivity of the present invention refers to the kinetics of target protein occupancy and off-target protein occupancy in the cell, comparing target protein occupancy and off-target protein at the same time point. The occupancy rate, or the retention time of the target protein in the cell and the retention time of the off-target protein, determine the intracellular target protein selectivity of the active molecule from the kinetic point of view. In the presence of an active molecule having the same affinity as the target protein and the off-target protein, the target protein selectivity of the active molecule can be further evaluated from the kinetic point of view using the intracellular target protein of the present invention. Similarly, the methods of the invention can also be used to compare the binding endurance of an active molecule to a wild-type and mutant target protein, determining the selectivity of the active molecule for wild-type and mutant from a time perspective.

The method of the present invention can be used to compare the persistence of an active compound interacting with a target protein in different cells, and to evaluate the cell selectivity of the active molecule from a time perspective. The target protein can be distributed in multiple tissues and organs in the body, such as heart, lung, kidney, liver, brain, etc., wherein the tissue related to the pharmacological activity of the active molecule is called a target tissue, and the active molecule interacts with a target protein in other tissues. May cause undesirable side effects. Although target proteins in different tissues have the same amino acid sequence, different cellular environments may result in the persistence of interactions between different active molecular target proteins. The present invention evaluates the selectivity of active molecules in different tissue cells from a time perspective to help explain the mechanism of action of the drug, design a rational dosing regimen, and reduce the side effects associated with off-target tissue. Similarly, the method of the present invention can be used to compare the persistence of active compounds interacting with target proteins in different tumor cells, and to evaluate the selectivity of tumor cells of active molecules from a time perspective, and to help predict drugs in different tumors. The efficacy of the design of personalized dosing regimens.

The methods of the present invention can be used to predict the most effective dosing regimen for design in an animal or human, particularly in predicting the time interval of administration based on the duration of treatment of the drug. Dosing regimens, including dosing and dosing intervals, have a significant impact on the efficacy and toxicity of bioactive molecules in animals and humans. The time interval for administration is usually determined based on the pharmacokinetics of the compound, and it is considered that once the blood concentration of the drug is below the therapeutic concentration range, the pharmacological activity of the drug will be immediately terminated. However, the present inventors have demonstrated that certain compounds can interact with intracellular target proteins at about 50% target protein occupancy even after removal of extracellular compounds (Examples 2 & 3), meaning that the cellular activity of the compound is even if the cells are removed. The compound is still present 24 hours after the compound. It is more scientific and rational to predict the time interval of administration in combination with the pharmacological kinetic properties of the drug in combination with the therapeutic duration of the drug of the present invention, and thus the method of the present invention facilitates the design of an optimal dosing regimen, The rate of cell dissociation maintains a sustained target protein occupancy and achieves therapeutic efficacy, and the toxic side effects associated with the off-target protein are attenuated by appropriate dosing intervals, thereby obtaining the largest therapeutic window of the drug. For example, a compound can be completely eliminated within 2 hours in the body, however in this case there is a 24 hour treatment duration range. Depending on the pharmacokinetic properties, the compound requires multiple administrations per day, however, considering the therapeutic duration of the compound, the once-daily dosing regimen can be reduced.

Compared with the prior art, the beneficial effects of the present invention are as follows:

The method of the present invention does not require the use of fluorescent probe molecules, so the method of the present invention can accurately measure the endurance of intracellular ligand target protein interaction without the interference effect brought by the probe molecule; the method of the present invention has no probe molecule The delayed effect can completely record the dissociation reaction of the ligand target protein in the cell. Secondly, the method of the invention does not require the construction of a fusion protein, which can detect native proteins, especially directly from the patient. Finally, the method of the invention is a versatile method for the analysis of the vast majority of ligands and target proteins. Using this approach, it was found that the intracellular ligand target protein dissociation reaction is much more complex than the reported data and can contain multiple dissociation phases with significantly different dissociation rates. Most importantly, the intracellular ligand target protein occupancy was maintained at about 60% 24 hours after removal of the extracellular ligand. Thus the method of the invention represents an effective method for determining the persistence of interactions of target protein proteins within a cell.

DRAWINGS

Figure 1 is a graph of the thermal melting of Aurora A in Jurkat cells in the absence (•) and presence (▲) staurosporine.

Figure 2 is a graph showing the dose response of Aurora A and staurosporine in Jurkat cells.

Figure 3 is a dissociation plot of the interaction of staurosporin Aurora A in Jurkat cells, normalized to the same number of cells at each time point.

Figure 4 is a kinetic plot of the occupancy of staurosporin Aurora A in Jurkat cells, normalized to the same number of cells at each time point.

Figure 5 is a graph of the thermal melting of PARP-1 in Jurkat cells in the absence (•) and presence (▲) Talazoparib.

Figure 6 is a graph showing the dose response of Talazoparib and PARP-1 in Jurkat cells.

Figure 7 is a dissociation plot of Talazoparib interaction with PARP-1 in Jurkat cells, normalized to the same number of cells at each time point.

Figure 8 is a graph showing the kinetics of Talazoparib and PARP-1 occupancy in Jurka cells, the normalization criterion being the same number of cells at each time point.

Figure 9 is a dissociation plot of Talazoparib interaction with PARP-1 in Jurkat cells, normalized to the same volume of cells at each time point, simulating a cell proliferation rate of zero.

Figure 10 is a kinetic curve of Talazoparib and PARP-1 occupancy in Jurkat cells. The normalization criterion is the same volume of cells at each time point, simulating a cell growth rate of zero.

detailed description

The technical solution of the present invention will be described in detail below with reference to the embodiments.

Example 1 Determination of the persistence of intracellular staurosporin Aurora A interaction

The purpose of this experiment was to determine the persistence of staurosporine interaction with Aurora A in Jurkat cells and found that most of the staurosporins can be removed from cells during the removal of extracellular staurosporine. Dissociation on Aurora A demonstrates that intracellular staurosporine has a weak binding endurance with Aurora A.

Thermal melting test: Jurkat cells were cultured in a RPMI 1640 medium at 37 ° C in a 5% carbon dioxide incubator. Jurkat cells were treated with 10 μM staurosporin (#19-123MG, Merck KGaA, Germany) for half an hour in serum-free RPMI1640 medium, and Jurkat cells treated with 0.1% DMSO in parallel were used as negative controls. 1x10 ⁶ cells were collected from each data point and resuspended in 20 μL of PBS, heated by PCR instrument (Eppendorf, Germany) at different temperatures in the temperature range of 37-64 ° C for three minutes, and then containing protease inhibitors (Roche, Switzerland) The cell lysate (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 7.5) was lysed on ice for half an hour. After centrifugation, the supernatant was collected and Aurora A was detected by SDS-PAGE and immunoblotting. The thermal melting curve of Aurora A in Jurkat cells under conditions of no treatment with staurosporine was constructed relative to the level of Aurora A at the lowest heating temperature.

Dose-effect assay: Jurkat cells were treated with different concentrations of staurosporine from 0.1 nM to 10 μM in serum-free RPMI 1640 medium for 1 hour; DMSO concentration was 0.1%. 1×10 ⁶ cells were resuspended in 20 μL of PBS at each concentration point, and heated by a PCR machine at 58 ° C for three minutes. The cells were then lysed on ice for half an hour with a cell lysate containing protease inhibitor (Roche, Switzerland) (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 7.5). After centrifugation, the supernatant was collected and Aurora A was detected by SDS-PAGE and immunoblotting. The level of Aurora A in the negative control was subtracted from the sample as the level of staurosporine-conjugated Aurora A, and a dose-response curve of intracellular staurosporine and Aurora A was constructed.

Cell dissociation assay: Jurkat cells were treated with 100 nM staurosporine in serum-free RPMI 1640 medium for half an hour with a DMSO concentration of 0.1%. Extracellular staurosporine was removed by replacing fresh RPMI1640 medium containing 10% serum. Then collected at various time points Jurkat cells, 1x10 ⁶ per time point were collected cells were resuspended in 20μL PBS and heated at 58 deg.] C using a PCR machine for three minutes. The cells were then lysed on ice for half an hour with a cell lysate containing protease inhibitor (Roche, Switzerland) (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 7.5). After centrifugation, the supernatant was collected and Aurora A was detected by SDS-PAGE and immunoblotting. Parallel, using Jurkat cells prior to removal of extracellular staurosporine as a positive control, represents the maximum initial signal level for the dissociation test and the 76% Aurora A occupancy measured (based on the above dose-response curve). Jurkat cells treated with 0.1% DMSO as a negative control represent the noise levels under the conditions of this test. A higher level of Aurora A was detected in the sample than in the negative control, meaning that staurosporin still interacted with intracellular Aurora A at this time point after removal of extracellular staurosporine. Dissociation of intracellular staurosporin Aurora A interaction by time as a function of the level of Aurora A in the negative control from the sample as the level of staurosporine-conjugated Aurora A in the sample The curve determines the dissociation rate, retention time and dissociation half-life of intracellular staurosporin Aurora A interaction. Further, based on the known occupancy rate of staurosporin Aurora A in the positive control, the kinetic curve of intracellular staurosporin Aurora A occupancy in this test was determined, and the staurosporin was determined under the test conditions. Duration of treatment.

SDS-PAG & Immunoblotting: Aurora A protein was separated by SDS-PAGE and transferred to PVDF membrane using Trans-Blot Turbo (Bio-Rad, USA), blocked and tested with anti-Aurora A antibody (#14475, Cell Signaling Technology) , USA), the chemiluminescence signal was recorded using a CCD camera (GE Healthcare, USA).

See Figure 1, which is a graph of the thermal melting of Aurora A in Jurkat cells in the absence (•) and presence (▲) staurosporin. Among them, (●) indicates a thermal melting curve of Aurora A in the case of no treatment with staurosporine; (▲) indicates a thermal melting curve of Aurora A in the case of treatment with staurosporine. Here, the ordinate "relative strip intensity" refers to the ratio of the Aurora A luminescence intensity to the strongest Aurora A luminescence intensity of each data result measured by immunoblotting in Example 1. According to the Aurora A thermal melting curve shown in Fig. 1, the staurosporin treatment group exhibited a higher level of Aurora A than the control group at a temperature interval of 55-61 ° C, so that 58 ° C can be selected as the heating temperature for the cells. Dissociation test of endosporin and Aurora A.

See Figure 2, which is a graph of the dose response of intracellular Aurora A and staurosporine. As shown in Figure 2, the level of intracellular staurosporin-conjugated Aurora A increases with the concentration of staurosporine (in the range of 0.1-10000 nM), so a concentration of 100 nM staurosporine can be selected. Corresponding to the 76% Aurora A occupancy in Jurkat cells as the test concentration of staurosporin in the dissociation test.

Referring to Figures 3 and 4, Figure 3 is a dissociation curve of the interaction of staurosporin Aurora A in Jurkat cells; Figure 4 is a kinetic plot of the occupancy of staurosporin Aurora A in Jurkat cells. The normalization criterion is the same number of cells at each time point. As shown in Figure 3, the level of staurosporin-conjugated Aurora A decreased non-linearly with increasing dissociation time. A two-phase dissociation model was used to analyze the dissociation curve of staurosporin Aurora A in Jurkat cells (shown in Figure 3). The first phase is a very fast dissociation phase that occurs during the removal of extracellular staurosporine, comparing cell samples prior to removal of extracellular staurosporine and removal of extracellular staurosporin A dissociation time point sample found that about 70% of the staurosporin Aurora A complex dissociated during about 5 minutes, and it was difficult to accurately fit the phase solution due to the very fast dissociation rate and limited data points. Rate constant. The second phase is a slow dissociation phase, and approximately 26% of the staurosporin Aurora A complex gradually dissociates in the next 36 hours. The dissociation rate constant of this phase is 0.042 h ^-1 , presumably Jurkat The retention time of intracellular staurosporin Aurora A interaction is about 36 hours, or longer, but its dissociation half-life is only about 0.1 hour. According to the 76% Aurora A occupancy rate in the positive control, the intracellular staurosporin dissociation curve (shown in Figure 3) can be converted to the occupancy of staurosporin Aurora A in Jurkat cells under the test conditions. A kinetic curve (shown in Figure 4) from which the time range over which a certain Aurora A occupancy is exceeded under the test conditions can be determined.

Example 2 Determination of the persistence of Talazoparib and PARP-1 interactions in Jurkat cells

The purpose of this experiment was to determine the persistence of the Talazoparib PARP-1 interaction in Jurkat cells and found that intracellular Talazoparib has a strong binding endurance with PARP-1.

Thermal Melting Test: Jurkat cells were cultured in a RPMI 1640 medium at 37 ° C in a 5% carbon dioxide incubator. Jurkat cells were treated with 1 μM Talazoparib (Selleckchem, USA) in serum-free RPMI 1640 medium for half an hour, and cells treated with 0.1% DMSO in parallel served as a negative control. 1x10 ⁶ cells were collected from each data point and resuspended in 20 μL of PBS, heated with a PCR instrument (Eppendorf, Germany) at different temperatures ranging from 37-55 ° C for three minutes, and then containing a protease inhibitor (Roche, Switzerland). Cell lysate (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 7.5) was lysed on ice for half an hour. After centrifugation, the supernatant was collected, and PARP-1 was detected by SDS-PAGE and immunoblotting. The thermal melting curve of intracellular PARP-1 was constructed in the presence and absence of Talazoparib treatment relative to the level of PARP-1 at the lowest heating temperature.

Dose-effect test: Jurkat cells were treated with Talazoparib at different concentrations from 0.001 nM to 10 μM in serum-free RPMI 1640 medium for 0.5 hour at a DMSO concentration of 0.1%. 1×10 ⁶ cells were collected from each concentration point and resuspended in 20 μL of PBS, and heated at 49 ° C for three minutes using a PCR machine. The cells were then lysed on ice for half an hour with a cell lysate containing protease inhibitor (Roche, Switzerland) (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 7.5). After centrifugation, the supernatant was collected, and PARP-1 was detected by SDS-PAGE and immunoblotting. The level of PARP-1 in the negative control was subtracted from the sample as the level of intracellular Talazoparib-bound PARP-1, and a dose-response curve of Talazoparib and PARP-1 in Jurkat cells was constructed.

Cell dissociation assay: Jurkat cells were treated with 100 nM Talazoparib in serum-free RPMI 1640 medium for 0.5 hour with a DMSO concentration of 0.1%. Replace the extracellular Talazoparib with fresh RPMI1640 medium containing 10% serum. Then collected at various time points Jurkat cells, 1x10 ⁶ per time point were collected cells were resuspended in 20μL PBS heated at 48.5 deg.] C using a PCR machine for three minutes. The cells were lysed on ice for half an hour with a cell lysate containing protease inhibitor (Roche, Switzerland) (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 7.5). After centrifugation, the supernatant was collected, and PARP-1 was detected by SDS-PAGE and immunoblotting. Parallel, using Jurkat cells prior to removal of extracellular Talazoparib as a positive control, represents the maximum initial signal level for the dissociation test and the 93% PARP-1 occupancy measured (based on the measured dose effect curve). Jurkat cells treated with 0.1% DMSO as a negative control represent the noise level under the test conditions. A higher level of PARP-1 was detected in the sample than in the negative control, meaning that Talazoparib still interacted with intracellular PARP-1 at this time point after removal of extracellular Talazoparib. The level of PARP-1 in the sample was subtracted from the level of PARP-1 in the negative control as the level of Talazoparib-bound PARP-1 in the sample, and the dissociation curve of the Talazoparib PARP-1 interaction in Jurkat cells was constructed as a function of time to determine the cell. Dissociation rate, retention time and dissociation half-life of the internal Talazoparib PARP-1 interaction. Based on the known Talazoparib PARP-1 occupancy rate in the positive control, the dissociation curve of intracellular Talazoparib PARP-1 interaction was converted to the kinetic curve of intracellular Talazoparib PARP-1 occupancy under the test conditions, and the experimental conditions were determined. The duration of treatment of the lower Talazoparib.

SDS-PAG & Immunoblotting: Separation of PARP-1 protein by SDS-PAGE Trans-Blot Turbo (Bio-Rad, USA) was transferred onto a PVDF membrane, blocked, and tested with anti-PARP-1 antibody (#sc-8007, SANTA CRUZ BIOTECHNOLOGY, USA) with a CCD camera (GE Healthcare, USA) Record the chemiluminescent signal.

See Figure 5, which is a graph of the thermal melting of PARP-1 in Jurkat cells in the absence (•) and in the presence of (▲) Talazoparib; wherein (●) indicates the heat of PARP-1 in the absence of treatment with Talazoparib Melt curve; (▲) shows the thermal melting curve of PARP-1 in the case of Talazoparib treatment; wherein, the ordinate "relative band intensity" refers to the PARP-1 luminescence intensity of each data result measured by immunoblotting in Example 2. The ratio of the intensity of the strongest PARP-1 luminescence. From the thermal melting curve shown in Figure 5, the Talazoparib treatment group exhibited a higher level of PARP-1 than the control group at a temperature range of 46-49 ° C, so 48.5 or 49 ° C could be selected as the heating temperature for intracellular determination. The persistence of Talazoparib PARP-1 interaction.

See Figure 6, which is a graph of the dose response of Talazoparib and PARP-1 in Jurkat cells. As shown in Figure 6, the level of soluble PARP-1 and Talazoparib (in the range of 0.001-10000 nM) showed a concentration-dependent increase, so 100 nM Talazoparib was selected, corresponding to 93% PARP-1 occupancy in cells, as dissociation Test concentration of Talazoparib in the test.

Referring to Figures 7 and 8, Figure 7 is a dissociation plot of the interaction of Talazoparib with PARP-1 in Jurkat cells. Figure 8 is a graph showing the kinetics of Talazoparib and PARP-1 occupancy in Jurka cells. The normalization criteria of Figures 7 and 8 are the same number of cells at each time point. As shown in Figure 7, the level of Talazoparib-bound PARP-1 decreased non-linearly with increasing dissociation time. A three-phase dissociation model was used to analyze the dissociation curve of Talazoparib interaction with PARP-1 in Jurkat cells (as shown in Figure 7). The first phase is a fast dissociation phase that occurs during the first hour after removal of extracellular Talazoparib, during which about 50% of the Talazoparib PARP-1 complex dissociates with a dissociation rate constant of 0.69 h ^-1 . And during the removal of extracellular Talazoparib, no dissociation of intracellular Talazoparib PARP-1 complex was observed. The second phase is a very slow dissociation phase that occurs during the next 23 hours during which there is little change in the level of intracellular Talazoparib PARP-1 complex, and its dissociation rate is very slow and difficult to fit accurately. The third phase is a slightly faster dissociation phase, and the remaining Talazoparib PARP-1 complex in the cell will gradually dissociate. About 25% of the Talazoparib PARP-1 complex is still present 36 hours after removal of extracellular Talazoparib, so it is speculated that the retention time of Talazoparib PARP-1 interaction in Jurkat cells will be more than 36 hours, much higher than the reported SPR technique. The measured Talazoparib PARP-1 retention time was 2.6 hours (Clin. Cancer Res., 19, 5003-5015, 2013). It was also determined that the intracellular Talazoparib PARP-1 dissociation half-life was 24 hours, which was much higher than the reported Talazoparib PARP-1 dissociation half-life determined by the SPR technique for 1.8 hours (Clin. Cancer Res., 19, 5003-5015, 2013). ). Based on the 93% PARP-1 occupancy rate in the positive control, the intracellular dissociation curve of Talazoparib (shown in Figure 7) was converted to the kinetic curve of Talazoparib PARP-1 occupancy in Jurkat cells under the experimental conditions (Fig. 8). As shown, the duration of treatment of Talazoparib under the test conditions can be determined from the PARP-1 occupancy kinetics curve. For example, assuming that the lowest effective PARP-1 occupancy in the cell is 50%, in this case the treatment duration of Talazoparib is in the range of 24 hours, meaning that the cell activity of Talazoparib can continue to be maintained for 24 hours after the removal of extracellular Talazoparib. .

Furthermore, in Comparative Example 1 and Example 2, it was found that the Talazoparib PARP-1 interaction in Jurkat cells has stronger binding endurance than the staurosporin Aurora A interaction, meaning different ligands and targets in the same cell. Protein combinations can produce different binding endurance.

Example 3

The purpose of this example was to demonstrate that cell proliferation can attenuate the persistence of intracellular ligand target protein interactions and the duration of intracellular pharmacological effects. In order to verify the effects of cell proliferation, this experiment uses a fixed cell sampling volume as a normalization standard to simulate a cell proliferation rate of 0. In this case, the sample may contain a different number of cells at each time point, but Ligand target protein complexes with the same starting level.

Jurkat cells were treated with 50 nM Talazoparib serum-free RPMI 1640 medium for 0.5 hour with a DMSO concentration of 0.1%. Extracellular Talazoparib was removed by replacing fresh RPMI1640 medium containing 10% serum. Then, 1 ml of cells were collected at each detection time point, counted, resuspended in 20 μL of PBS, and heated by a PCR instrument at 48.5 ° C for three minutes. Cell lysate (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 7.5) containing protease inhibitor (Roche, Switzerland) was lysed on ice for half an hour. After centrifugation, the supernatant was collected, and PARP-1 was detected by SDS-PAGE and immunoblotting. Parallel, using cells prior to removal of extracellular Talazoparib as a positive control, represents the maximum initial signal level for the dissociation test and the calculated 85% PARP-1 occupancy (based on the above dose-response curve). 1x10 ⁶ cells treated with 0.1% DMSO served as a negative control, representing the noise level in this case. The level of PARP-1 in the sample was subtracted from the level of PARP-1 in the negative control multiplied by the background noise of the proliferation rate as the level of Talazoparib-bound PARP-1 in the sample, constructed as a function of the dissociation time. The dissociation curve of the Talazoparib PARP-1 interaction in the Jurkat cells determines the dissociation rate, retention time and dissociation half-life of the intracellular Talazoparib PARP-1 interaction. Based on the known Talazoparib PARP-1 occupancy rate in the positive control, the dissociation curve of intracellular Talazoparib PARP-1 interaction was converted to the kinetic curve of intracellular Talazoparib PARP-1 occupancy under the test conditions, and the experimental conditions were determined. The duration of treatment of the lower Talazoparib.

The PARP-1 protein was separated by SDS-PAGE and transferred to a PVDF membrane using Trans-Blot Turbo (Bio-Rad, USA). After blocking, the chemiluminescent signal was recorded with a CCD camera (GE Healthcare, USA) using an anti-PARP-1 antibody assay (#sc-8007, SANTA CRUZ BIOTECHNOLOGY, USA).

Referring to Figures 9 and 10, Figure 9 is a dissociation plot of Talazoparib interaction with PARP-1 in Jurkat cells. The normalization criterion is the same volume of cells at each time point, simulating a cell growth rate of zero. Figure 10 is a graph showing the kinetics of Talazoparib and PARP-1 occupancy in Jurkat cells. The normalization standard is the same volume of cells at each time point, and the simulated cell growth rate is zero. As shown in the dissociation plot of Figure 9, the level of Talazoparib-bound PARP-1 decreases non-linearly with increasing dissociation time. A dissociation curve of the interaction between Talazoparib and PARP-1 in Jurkat cells under the experimental conditions of this example was analyzed using a two-phase dissociation model. Similarly, the first phase is a fast dissociation phase that occurs during the first hour after removal of extracellular Talazoparib, during which about 50% of the Talazoparib PARP-1 complex dissociates with a dissociation rate constant of 0.62 h ^-1 . It was confirmed again that no dissociation of the intracellular Talazoparib PARP-1 complex was observed during the removal of extracellular Talazoparib. The second phase is a very slow dissociation phase that occurs during the next 35 hours during which there is little change in the level of intracellular Talazoparib PARP-1 complex, and the dissociation rate is very slow and difficult to fit accurately. In particular, about 60% of the Talazoparib PARP-1 complex was still present at the 36-hour time point, so the retention time of Talazoparib interacting with PARP-1 in Jurkat cells was much longer than 36 hours under this test condition. Intracellular Talazoparib and PARP The -1 dissociation half-life is also more than 36 hours. Based on the 85% PARP-1 occupancy rate in the positive control, the intracellular dissociation curve of Talazoparib (shown in Figure 9) was converted to the kinetic curve of Talazoparib PARP-1 occupancy in Jurkat cells under the experimental conditions (Fig. 10). As shown), the duration of treatment of Talazoparib in this case can be determined from the PARP-1 occupancy kinetic curve. A similar hypothesis is that the least potent PARP-1 occupancy rate in the cell is 50%, so the duration of Talazoparib treatment is more than 36 hours under this test condition, meaning that the cell viability of Talazoparib can continue to be maintained after removal of extracellular Talazoparib. hour.

Example 2 uses the normalization method of the same cell number to represent the normal cell proliferation rate, and Example 3 uses the normalization method of the fixed cell sampling volume to simulate the case where the cell proliferation rate is zero. Comparing the dissociation curves of Talazoparib PARP-1 in both cases (as shown in Figures 7 and 9), especially at the 36 hour time point, and the dissociation half-life, Example 3 was found to be stronger than Example 2. The persistence of intracellular Talazoparib PARP-1 interactions demonstrates that cell proliferation can attenuate the persistence of ligand-target protein interactions in cells. Comparing the kinetic curves of Talazoparib PARP-1 occupancy (as shown in Figures 8 and 10) and the duration of treatment in both cases, it can be found that Example 3 is still more effective than Example 2 even if a slightly lower Talazoparib test concentration is applied. With a longer duration of treatment, it is demonstrated that cell proliferation can attenuate the duration of cellular pharmacological effects.

The above is only some of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. It will be apparent to those skilled in the art that various changes and modifications may be made without departing from the scope of the invention.

Claims

A method for determining the endurance of a ligand protein interaction in a cell, the method comprising the steps of:

Step 1, treating the cells with the ligand;

Step 2, removing the ligand outside the extracellular;

Step 3, collecting the cells at different time points after removing the ligand;

Step 4, heating the cells;

Step 5, lysing the cells;

In step 6, a function of the level of soluble or insoluble target protein in the cell lysate as a function of time is analyzed.
The method for determining the endurance of an intracellular ligand target protein interaction according to claim 1, wherein the ligand is a cell metabolite, a small molecule or a drug; and the target protein is an intracellular and ligand interaction with each other. A protein molecule that acts; the cell is a mammalian cell, a cell strain, an engineered cell, or a primary cell.
The method for determining the endurance of an intracellular ligand target protein interaction according to claim 1, wherein in the step 1, the ligand is added to the cell culture medium and enters the cell to interact with the target protein.
The method for determining the endurance of an intracellular ligand target protein interaction according to claim 1, wherein the method for removing the extracellular ligand in the step 2 comprises replacing the fresh medium or replacing the cells with the solution and then replacing the cells. Fresh medium.
The method for determining the endurance of an intracellular ligand target protein interaction according to claim 1, wherein said step 3 collects a cell sample at one or more time points after removal of the extracellular ligand.
The method for determining the endurance of an intracellular ligand target protein interaction according to claim 1, wherein the step of heating the cells to a ligand-bound and unbound target protein has different thermal stability. temperature.
A method for determining the endurance of interaction of a target ligand protein in a cell according to claim 1, wherein The method further comprises: after lysing the cells in the step 5, further comprising extracting intracellular proteins, and separating the soluble and insoluble cell lysate fractions by centrifugation or filtration.
The method for determining the endurance of an intracellular ligand target protein interaction according to claim 1, wherein the level of the target protein in the step 6 is determined by quantitative use of a target protein, and the quantitative method of the target protein includes antibody technology. , mass spectrometry, biological activity of the target protein or enzymatic activity of the fusion tag.
The method for determining the endurance of an intracellular ligand target protein interaction according to claim 1, wherein the step 6 specifically comprises measuring a soluble target protein and determining the level of a target protein bound by the ligand in the cell, The dissociation curve of intracellular ligand target protein interaction was constructed by dissociation time, and the dissociation rate constant, retention time and dissociation half-life of intracellular ligand target protein interaction were calculated.
The method for determining the endurance of an intracellular ligand target protein interaction according to claim 1, further comprising collecting a cell sample before removing the extracellular ligand.
The method for determining the endurance of an intracellular ligand target protein interaction according to claim 10, wherein the dissociation reaction of the intracellular ligand target protein interaction comprises dissociation reaction and removal during removal of the extracellular ligand. Dissociation reaction after extracellular ligand.
A method of determining the persistence of interaction of a target ligand protein in an intracellular ligand according to claim 1, further comprising a positive control after ligand treatment but not removed, having a known target protein occupancy level.
The method for determining the endurance of interaction of a target ligand protein in an intracellular ligand according to claim 12, wherein the target protein occupancy rate at different time points after removal of the extracellular ligand is determined, and the occupancy rate of the ligand protein in the cell is constructed. The kinetic curve calculates the duration of treatment of the ligand above the lowest effective target protein occupancy.