A method for quantitatively determining the concentration of protac drugs in cells with high specificity

Abstract

The application belongs to the technical field of pharmaceutical analysis, and provides a method for quantitatively determining the concentration of PROTAC drugs in cells with high specificity, comprising the following steps: (1) selecting a cell strain expressing a PROTAC drug target; (2) culturing the cell strain to the logarithmic growth phase; (3) inoculating the cell suspension on a cell culture plate for culture; (4) adding the PROTAC drug to the cell culture plate, mixing and continuing to incubate; (5) removing the cell culture medium in the cell culture plate; dispersing the cell culture medium into a single cell suspension, centrifuging to discard the supernatant and obtain a cell precipitate; (6) lysing the cell precipitate, collecting the supernatant by ultracentrifugation, and performing ultra-high performance liquid chromatography-tandem mass spectrometry analysis; (7) preparing a blank cell suspension and a series of standard solutions of PROTAC drug concentrations, performing ultra-high performance liquid chromatography-tandem mass spectrometry analysis, and constructing a standard curve; and (8) using the standard curve to obtain the concentration of the PROTAC drug in the cells.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical analysis technology. Specifically, this application provides a method for highly specific quantitative determination of intracellular PROTAC drug concentration. Background Technology

[0002] PROTAC technology is considered a revolutionary breakthrough in small molecule drug development. It degrades pathogenic proteins by recruiting the ubiquitin-proteasome system (UPS), rather than simply inhibiting their function. This mechanism dictates that PROTACs must be present at specific intracellular concentrations to form stable ternary complexes.

[0003] Intracellular drug concentration is a key indicator for assessing drug uptake efficiency, targeting, mechanism of action, and drug resistance within cells. Precise quantification of intracellular drug concentration is crucial for drug development and clinical application. It serves as a critical bridge connecting in vitro pharmacological activity with in vivo clinical efficacy. In the PROTAC drug development process, achieving precise quantification of intracellular concentration effectively assesses drug potential and permeation capacity, and provides essential references for the formulation and optimization of clinical dosing regimens.

[0004] Currently, commonly used methods for measuring intracellular drug concentrations mainly include the following categories: Cell lysis-solvent extraction: Cells are lysed through repeated freeze-thaw cycles, ultrasonic disruption, or enzymatic digestion to release intracellular drugs. The drugs are then extracted using organic solvents (such as methanol, acetonitrile, or ethyl acetate), and quantified using high-performance liquid chromatography (HPLC), gas chromatography (GC), or mass spectrometry (MS). This method is simple to operate and relatively inexpensive, making it a common approach for determining the intracellular concentration of traditional small molecule drugs. However, it suffers from drawbacks such as easy drug degradation and severe matrix interference.

[0005] Fluorescence detection methods include fluorescent labeling (drugs coupled with fluorescent groups) and autofluorescence (drugs themselves possess fluorescent properties, such as doxorubicin). These methods utilize fluorescence spectrophotometers, flow cytometry, or confocal microscopy to achieve quantitative or visual analysis of intracellular drug concentrations. While rapid and allowing for real-time monitoring, fluorescent labeling may alter the drug's physicochemical properties and biological activity, and is susceptible to interference from cellular autofluorescence. Therefore, these methods are suitable for qualitative or semi-quantitative analysis, or as an auxiliary verification method for mass spectrometry.

[0006] Immunoassays: Based on the principle of specific antigen-antibody binding, these methods include enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunofluorescence. They are suitable for determining the intracellular concentration of biopharmaceuticals (such as antibodies and protein drugs) or specific small molecule drugs. This method is highly specific and does not require complex separation equipment, but it relies on high-quality specific antibodies, is susceptible to cross-reaction interference, and is difficult to use for simultaneous quantification of multiple drugs. Its suitability is limited by the type of drug.

[0007] Chromatography-mass spectrometry (LC-MS / MS): Based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), combined with pretreatment techniques such as cell lysis, solid-phase extraction (SPE), or protein precipitation, this method achieves highly sensitive and specific drug quantification. For example, CN112505183A discloses an UPLC-MS / MS method for detecting intracellular tacrolimus concentration. However, this method is designed for small molecule drugs and does not consider the characteristics of PROTACs, such as large molecular weight, strong hydrophobicity, and easy binding to intracellular components, making it unsuitable for direct application. UPLC-MS / MS has advantages over other methods: it combines separation efficiency and detection accuracy, can tolerate complex intracellular matrices, and has detection limits as low as 8-10 ng / mL. It is suitable for the determination of trace intracellular concentrations of complex drugs such as PROTACs and is currently the most widely used standard method.

[0008] While existing UPLC-MS / MS methods are widely used for intracellular concentration detection of small molecule drugs, their pretreatment procedures and chromatographic-mass spectrometry parameters are designed based on the characteristics of small molecules, failing to consider the large molecular size, strong hydrophobicity, and easy binding properties of PROTACs. There is a lack of readily available and mature solutions. PROTAC drugs possess a unique "heterobifunctional chimera" structure, with large molecular weights, both hydrophilic and hydrophobic properties, and readily form stable complexes with intracellular target proteins and ubiquitination-related proteins, exhibiting significantly different physicochemical properties from traditional small molecule drugs. Furthermore, the complex intracellular matrix (proteins, lipids, nucleic acids, etc.) easily contaminates the ion source of UPLC-MS / MS, causing severe ion inhibition effects. Consequently, the intracellular concentration detection of most drugs tends to favor semi-quantitative methods such as immunoblotting rather than the highly precise UPLC-MS / MS technology. This invention optimizes the entire process from scratch, including cell line screening, cell culture, protein precipitation, drug-specific enrichment, and UPLC-MS / MS quantitative analysis. This includes selecting a suitable chromatographic column, optimizing the mobile phase composition, and adjusting ultracentrifugation conditions (e.g., centrifugation at 100,000 × g to remove precipitated impurities). This invention aims to leverage the advantages of ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) to develop a simple, highly specific, and highly sensitive quantitative method that effectively eliminates matrix interference. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of existing intracellular drug concentration determination methods, specifically addressing a major challenge in PROTAC drug research—the difficulty of adapting UPLC-MS / MS to the intracellular concentration detection of complex PROTAC drugs. It also solves problems such as incomplete drug release and severe matrix interference caused by conventional cell processing methods. This invention provides an integrated quantitative determination method based on PROTAC drug action in cells, specific enrichment, and detection by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). This method achieves accurate quantification of intracellular PROTAC drugs, providing reliable data support for drug development and clinical applications.

[0010] This invention provides an efficient sample pretreatment method for intracellular analysis of PROTAC drugs, aiming to overcome the technical bottlenecks of existing technologies in drug release efficiency, structural stability, and matrix interference control. In the cell disruption stage, existing technologies commonly use ultrasonic disruption, which fails to disrupt the binding of PROTACs to intracellular proteins, resulting in low drug release efficiency. This invention employs acetonitrile incubation for 30 min, effectively rupturing the cell membrane while preventing drug degradation, thus enabling efficient drug release. Regarding impurity removal, conventional centrifugation parameters have low removal rates for intracellular proteins, lipids, and other matrix components, leading to severe ion inhibition effects from large amounts of residual matrix. This invention utilizes acetonitrile precipitation combined with ultracentrifugation at 4°C and 100,000 × g for 1 h to efficiently remove the matrix and avoid severe ion inhibition effects. This pretreatment method is highly compatible with UPLC-MS / MS detection technology. Through targeted cell pretreatment and impurity removal steps, it overcomes the dual detection challenges posed by the structural characteristics of PROTAC drugs and the complex intracellular matrix, achieving precise quantification of PROTAC drug intracellular concentration.

[0011] The present invention provides a method for quantitative determination of intracellular drug concentration. By optimizing cell processing conditions, specific enrichment techniques, and detection parameters, it eliminates matrix interference and improves detection sensitivity and specificity. The specific technical solution is as follows: It adopts an integrated technical route of "drug action in cells - specific enrichment - ultra-high performance liquid chromatography - tandem mass spectrometry (UPLC-MS / MS) detection" to develop a method for quantitatively determining the concentration of newly designed PROTAC drugs in cells. This method directly reflects the differences in the quantitative determination of different PROTAC drugs in cells and is an important analytical method in the research and development process of PROTAC drugs.

[0012] On the one hand, this application provides a method for highly specific quantitative determination of intracellular PROTAC drug concentration, the method comprising: (1) Select cell lines that express the target of PROTAC drugs; (2) Culture the cell line selected in step (1) to the logarithmic growth phase; (3) Inoculate the cell suspension onto the cell culture plate, so that the cells are evenly spread in the culture plate, and culture for 12-36 h.

[0013] (4) Add the PROTAC drug to the cell culture plate, mix it well, and continue incubation for 4-8 hours; (5) Remove the cell culture medium from the cell culture plate; disperse it into a single-cell suspension, centrifuge and discard the supernatant to obtain the cell precipitate; (6) After lysing the cell pellet, the supernatant was collected by ultracentrifugation and analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry. (7) Prepare blank cell suspension and PROTAC drug concentration standard solutions, perform ultra-high performance liquid chromatography-tandem mass spectrometry analysis, and construct standard curves; (8) Substitute the result of step (6) into the standard curve obtained in step (7) to obtain the intracellular PROTAC drug concentration.

[0014] Furthermore, in step (2), cell passage is performed when the cell fusion rate reaches 80-90%.

[0015] Furthermore, in step (3), the concentration is taken as 4×10 5 Cell suspensions of cells / mL were seeded onto cell culture plates, ensuring even distribution of cells, and cultured at 37°C in a 5% CO2 incubator for 24 h.

[0016] Further, in step (4), the PROTAC drug is added to the cell culture plate, mixed well, and then incubated for 6 hours.

[0017] Further, step (5) includes removing the cell culture medium from the cell culture plate; washing the cells in the cell culture plate with PBS; adding trypsin digestion solution to the cell culture plate; incubating at 37 °C until the adherent cells are completely detached and dispersed into a single-cell suspension; adding complete culture medium to terminate digestion; centrifuging and discarding the supernatant to obtain the cell pellet.

[0018] Further, step (6) includes adding acetonitrile to the cell pellet for incubation, and after 30 min, centrifuging at 4°C and 10000×g for 1 h to collect the supernatant for analysis by ultra-high performance liquid chromatography-tandem mass spectrometry.

[0019] Furthermore, the ultra-high performance liquid chromatography-tandem mass spectrometry analysis uses a 1.7 μm particle size ACQUITY UPLCBEH C18 column.

[0020] Furthermore, in the ultra-high performance liquid chromatography-tandem mass spectrometry analysis, mobile phase A is a 0.1% v / v formic acid aqueous solution, and mobile phase B is acetonitrile.

[0021] Furthermore, the mass spectrometry parameters in the ultra-high performance liquid chromatography-tandem mass spectrometry analysis are: ESI positive ion mode, 3.8 kV spray voltage, and 133.4-2000.0 m / z scan range.

[0022] Furthermore, the method includes: (1) Select cell lines that express the target of PROTAC drugs; (2) Culture the cell line selected in step (1) to the logarithmic growth phase, and passage the cells when the cell confluence reaches 80-90%. (3) Take a concentration of 4×105 Cell suspensions of cells / mL were seeded onto cell culture plates, ensuring even distribution of cells, and cultured at 37°C in a 5% CO2 incubator for 24 h.

[0023] (4) Add the PROTAC drug to the cell culture plate, mix it well, and continue incubation for 6 h; (5) Remove the cell culture medium from the cell culture plate; wash the cells in the cell culture plate with PBS; add trypsin digestion solution to the cell culture plate, incubate at 37 °C until the adherent cells are completely detached and dispersed into a single cell suspension, add complete culture medium to stop digestion, centrifuge and discard the supernatant to obtain cell pellet; (6) Add acetonitrile to the cell pellet and incubate for 30 min. After centrifugation at 4℃ and 10000×g for 1 h, collect the supernatant and perform ultra-high performance liquid chromatography-tandem mass spectrometry analysis. (7) Prepare blank cell suspension and PROTAC drug concentration standard solutions, perform ultra-high performance liquid chromatography-tandem mass spectrometry analysis, and construct standard curves; (8) Substitute the result of step (6) into the standard curve obtained in step (7) to obtain the intracellular PROTAC drug concentration.

[0024] The ultra-high performance liquid chromatography-tandem mass spectrometry analysis used a 1.7 μm particle size ACQUITY UPLC BEH C18 column; mobile phase A was 0.1% v / v formic acid aqueous solution, and mobile phase B was acetonitrile; the mass spectrometry parameters were: ESI positive ion mode, 3.8 kV spray voltage, and 133.4-2000.0 m / z scan range.

[0025] Furthermore, the cell culture plate has a diameter of 35 mm.

[0026] Beneficial effects: This invention employs an acetonitrile-treated cell followed by ultracentrifugation, which disrupts cell membrane structure while precipitating interfering proteins and releasing the total intracellular drug. This avoids drug degradation problems associated with conventional cell disruption methods, providing more accurate data for drug activity assessment. Combining ultracentrifugation with the separation of precipitate and supernatant effectively removes intracellular matrix impurities, significantly reduces matrix effects, and improves detection specificity, making it particularly suitable for drugs with complex structures such as PROTAC that are susceptible to matrix interference.

[0027] The optimized UPLC-MS / MS detection system is adapted for the determination of intracellular concentrations of PROTAC drugs. Instead of simply using the existing UPLC-MS / MS equipment, the system optimizes the chromatographic column (selecting a 1.7 μm particle size ACQUITY UPLC BEH C18 column, adapted for macromolecular retention), mobile phase composition (0.1% formic acid aqueous solution-acetonitrile, improving PROTAC ionization efficiency), and mass spectrometry parameters (ESI positive ion mode, 3.8 kV spray voltage, 133.4-2000.0 m / z scan range) to effectively separate PROTAC from matrix impurities, improving ionization efficiency by more than 3 times. The UPLC-MS / MS detection conditions in this invention combine high separation efficiency with high sensitivity, with a detection limit as low as 8-10 ng / mL, enabling precise quantification of trace intracellular drugs and meeting the needs of low-concentration drug uptake assessment in drug development.

[0028] The pretreatment process is simple and time-efficient (≤4 hours in total), requiring no complex derivatization steps and avoiding drug degradation or loss. It is applicable to various cell types, including adherent and suspension cells, as well as drugs with complex PROTAC structures, demonstrating strong innovation. All indicators meet pharmaceutical analysis technical specifications, ensuring accurate and reliable results. It can be widely used in drug development for cell uptake efficiency screening, pharmacodynamic mechanism research, drug resistance analysis, and therapeutic drug monitoring in personalized clinical medication, possessing significant practical application value. Attached Figure Description

[0029] Figure 1 The UPLC-MS / MS detection DP1 linear representation plot provided by the present invention; Figure 2 The UPLC-MS / MS chromatogram for detecting DP1 in blank cells provided by this invention; Figure 3 The present invention provides a representative chromatogram of DP1 sample in cells for UPLC-MS / MS detection. Figure 4 To compare with the UPLC-MS / MS detection of DP1 sample in cells in Example 1; Figure 5 To compare the UPLC-MS / MS chromatogram of the DP1 sample in cells with that in Example 2; Figure 6 The UPLC-MS / MS detection TPD81020 linear representative plot provided by this invention; Figure 7 The UPLC-MS / MS chromatogram representing TPD81020 in blank cells is provided by the present invention; Figure 8The chromatogram of a representative TPD81020 sample in cells for UPLC-MS / MS detection provided by this invention. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0031] The experimental reagents and instruments used in this invention are shown in Tables 1 and 2: Table 1 Main Experimental Instruments

[0032] Table 2 Main Experimental Reagents

[0034] Example 1: Determination of intracellular drug DP1 concentration in T-47D cells The drug tested was a newly designed PROTAC drug targeting the DDR1 protein, DP1 (molecular weight: 851.8 Da), published in the European Journal of Medicinal Chemistry, Vol. 294, No. 15, entitled "Targeted degradation of DDR1 by proteolytic targeting chimera reverses immune exclusion for tumor immunotherapy".

[0035] The selected cell line was human breast cancer cell line T-47D (adherent cells); the expression level of DDR1 protein in T-47D cells was log2(TPM+1) = 8.50; The experimental steps are as follows: Step 1: Through database and literature searches, a human breast cancer cell line with high-level expression of DDR1 protein, namely T-47D cells, was selected.

[0036] Step 2: After reviving T-47D cells, culture them to the logarithmic growth phase. When the cell confluence is 80-90%, passage them for subsequent experiments.

[0037] Step 3: Take a concentration of 4×10 5 Cell suspensions of 10 cells / mL were seeded into cell culture plates, ensuring uniform cell spread, and incubated in a 37°C, 5% CO2 incubator for 24 h. The next day, 1 μM DP1 was added to the corresponding wells according to the experimental design, gently mixed, and incubated for another 6 h. After incubation, the culture plates were removed.

[0038] Step 4: First, collect the cell culture medium from each well into a 2 mL centrifuge tube for later use. Add 1 mL of phosphate-buffered saline (PBS) to each well of the culture plate, gently wash the cell surface, and collect the washing solution and combine it with the aforementioned culture medium. Then, add 200 μL of trypsin digestion solution to the 35 mm diameter culture plate, and incubate at 37°C, 5% CO2 for 5 min until the adherent cells are completely detached and dispersed into a single-cell suspension. Immediately add an equal volume of complete culture medium to stop digestion, gently agitate the bottom of the well with a sterile pipette tip, transfer the cell suspension to a 1.5 mL centrifuge tube, and centrifuge at 4°C, 4000 rpm for 3 min. Discard the supernatant in the centrifuge tube. Add 300 μL of acetonitrile to the cell pellet, vortex thoroughly on an adjustable vortex mixer, and lyse at room temperature for 30 min. After lysis, transfer the sample to an ultracentrifuge tube and centrifuge at 4°C, 10000×g for 1 h. After centrifugation, the supernatant was transferred to an injection tube for analysis by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS).

[0039] Step 5: Select quantitative ions. Use Q-Exactive Plus liquid chromatography-mass spectrometry to detect DP1 solution, identify characteristic quantitative ions, and select the quantitative ions with higher sensitivity as target ions for quantitative analysis. Step 6: Preparation of standard solutions. The concentration of DP1 standard stock solution is 6 mM. Accurately transfer a certain amount of DP1 standard stock solution and dilute it with dimethyl sulfoxide (DMSO) to prepare linear working solutions of 1 μM, 0.5 μM, 0.2 μM, 0.1 μM, 0.05 μM, 0.02 μM, and 0.01 μM. Prepare the required amount as needed.

[0040] Step 7: After obtaining the standard solution for quantitative analysis, use a Q-Exactive Plus liquid chromatography-mass spectrometry system to perform liquid chromatography and mass spectrometry quantitative detection on the sample from Step 4; In this invention, the DP1 standard solution has a linear concentration range of 0.01 to 1 μM, which provides high sensitivity and improves selectivity, precision and accuracy.

[0041] In this invention, the quantitative detection conditions for the chromatography include: Chromatographic column: ACQUITY UPLC BEH C18 column, specifications: column length 100 mm, inner diameter 2.1 mm, particle size 1.7 μm, column temperature: 40℃ Mobile phase: Mobile phase A is a 0.1% formic acid aqueous solution, wherein the formic acid accounts for 0.1% of the volume of the formic acid aqueous solution; Mobile phase B is pure acetonitrile.

[0042] Gradient elution conditions: 0–0.5 min, mobile phase A: 95%, mobile phase B: 5%; 0.5–8.3 min, mobile phase A: 95%→5%, mobile phase B: 5%→95%; 8.3–9.5 min, mobile phase A: 5%, mobile phase B: 95%; 9.5–9.51 min, mobile phase A: 5%→95%, mobile phase B: 95%→5%; 9.51–11 min, mobile phase A: 95%, mobile phase B: 5%; flow rate: 0.3 mL / min. Injection volume: 5 μL. Under these chromatographic conditions, the drug and impurities in the cells were well separated, with good resolution.

[0043] In this invention, the quantitative detection conditions for the chromatography include: The ion source used was an electrospray ionization (ESI) source, and the scanning mode was Full MS. 2 Positive ion mode scanning; spray voltage: 3.8 kV; capillary temperature: 320℃; sheath gas flow rate: 35 mL / min; auxiliary gas flow rate: 10 mL / min; scanning range: 133.4000~2000.0000.

[0044] The molecular weight of the quantitative ion selected in this invention is 852.30334, which has the highest quantitative ion response value and no matrix interference.

[0045] The detection method in this invention has a wide linear range and can be applied to the detection of biological samples in clinical research and the monitoring of clinical drug concentrations.

[0046] The experimental results of this invention are as follows: Depend on Figure 1 Representative chromatograms of the standard curve (UPCC-MS / MS linear representation of DP1 concentration in cells, linear range 0.01–1 μM) show that the target ion molecular weight is 852.3, and the linearity r is... 2 Greater than 0.99, indicating good linearity.

[0047] Based on the linear regression equation, the intracellular drug concentration was calculated to be 0.42 μM.

[0048] The linearity results of the standard solution are as follows: Figure 1 As shown In the blank cells, DP1 represents the chromatogram as follows: Figure 2 As shown In this invention, the intracellular DP1 representative chromatogram is as follows: Figure 3 As shown Comparative Example 1 The tested drugs, cell lines, T-47D cells, DP1 drugs, reagents and instruments were exactly the same as those in Example 1.

[0049] Step 1: Perform steps 1 to 4 in Example 1 to obtain T-47D cell pellets incubated with 1 μM DP1 for 24 h.

[0050] Step 2: Add 300 μL of phosphate-buffered saline (PBS) to the cell pellet for resuspending, transfer to a centrifuge tube for ultrasonic disruption, and place in an ultrasonic disruptor for ultrasonic disruption. Set the parameters to 200W, run for 3 seconds, with a 5-second interval, for a total disruption time of 5 minutes.

[0051] Step 3: After the protein fragmentation is complete, add an equal volume of methanol to the system for protein precipitation, vortex for 5 min, and centrifuge at 4℃ and 10000×g for 1 h. After centrifugation, transfer the supernatant to a sample injection tube for analysis by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS).

[0052] Step 4: Perform steps 5 to 7 in Example 1, using the same UPLC-MS / MS detection conditions for quantitative analysis.

[0053] The test results are as follows: Based on the linear regression equation, the intracellular drug concentration was calculated to be 0.0096 μM. Experimental data showed that the drug concentration detection result of this method was only 1 / 44 of the result obtained by the detection method of this invention. Furthermore, peaks of the drug's specific molecular weight appeared in the chromatogram at different retention times, indicating the presence of various impurities in the sample obtained by this method. These impurities may compete with the target drug for ionization (e.g., competing for charge in ESI mass spectrometry), leading to reduced ionization efficiency, weakened signal intensity, and ultimately, a lower quantitative result, thus affecting the quantitative detection of the drug.

[0054] In comparison to Example 1, the intracellular DP1 chromatogram is as follows: Figure 4 As shown.

[0055] Comparative Example 2 The tested drugs, cell lines, T-47D cells, DP1 drugs, reagents and instruments were exactly the same as those in Example 1.

[0056] Step 1: Perform steps 1 to 3 in Example 1 to obtain T-47D cell pellets incubated with 1 μM DP1 for 24 h.

[0057] Step 2: Add 300 μL of acetonitrile to the cell pellet, place it on an adjustable vortex mixer and vortex thoroughly, then lyse at room temperature for 30 min.

[0058] Step 3: After lysis, centrifuge at 4℃ and 20000×g for 30 min using standard centrifugation parameters. Transfer the supernatant to the injection tube for analysis by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS).

[0059] Step 4: Perform steps 5 to 7 in Example 1, using the same UPLC-MS / MS detection conditions for quantitative analysis.

[0060] The test results are as follows: DP1 cannot be detected In comparison to Example 2, the intracellular DP1 representative chromatogram is as follows: Figure 5 As shown Example 2: Determination of intracellular drug concentration in Raji cells The drug tested was a newly designed PROTAC drug targeting the BRD4 protein, based on a long flexible chain linker: TPD81020 (molecular weight 1424.22 Da).

[0061] The selected cell line was human Burkitt lymphoma cancer cell line Raji (suspension cells); the expression level of BRD4 protein in Raji cells was log2(TPM+1) = 5.126; The experimental steps are as follows: Step 1: Through database and literature searches, the human Burkitt lymphoma cell line Raji cells, which express BRD4 protein at a high level, was selected.

[0062] Step 2: After reviving Raji cells, culture them to the logarithmic growth phase. When the cell confluence is 80-90%, passage them and conduct subsequent experiments.

[0063] Step 3: Take a concentration of 4×10 5 Cell suspensions of cells / mL were seeded into cell culture plates, ensuring uniform cell spread, and incubated in a 37°C, 5% CO2 incubator for 24 h. The next day, 1 μM TPD81020 was added to the corresponding wells according to the experimental design, gently mixed, and incubated for another 6 h. After incubation, the culture plates were removed.

[0064] Step 4: Gently aspirate the cells from the bottom of the well using a pipette tip to prepare a single-cell suspension. Collect the suspension in a 2 mL centrifuge tube and centrifuge at 4°C and 4000 rpm for 3 min. Carefully collect the supernatant into a new 2 mL centrifuge tube. Add 1 mL of phosphate-buffered saline (PBS) to the cell pellet, gently resuspend, and centrifuge again under the same conditions. Carefully collect the supernatant into a new 2 mL centrifuge tube. Then add 300 μL of acetonitrile to the cell pellet and vortex thoroughly on an adjustable vortex mixer for lysis at room temperature for 30 min. After lysis, transfer the sample to an ultracentrifuge tube and centrifuge at 4°C and 10000×g for 1 h. After centrifugation, transfer the supernatant to a sample injection tube for analysis by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS).

[0065] Step 5: Select quantitative ions. Use Q-Exactive Plus liquid chromatography-mass spectrometry to detect DP1 solution, identify characteristic quantitative ions, and select the quantitative ions with higher sensitivity as target ions for quantitative analysis. Step 6: Preparation of standard solutions. The TPD81020 standard stock solution has a concentration of 6 mM. Accurately transfer a certain amount of TPD81020 standard stock solution and dilute it with dimethyl sulfoxide (DMSO) to prepare linear working solutions of 10 μM, 0.5 μM, 2 μM, 1 μM, 0.5 μM, 0.2 μM, and 0.1 μM. Prepare the required amount as needed.

[0066] Step 7: After obtaining the standard solution for quantitative analysis, use a Q-Exactive Plus liquid chromatography-mass spectrometry system to perform liquid chromatography and mass spectrometry quantitative detection on the sample from Step 4; In this invention, the TPD81020 standard solution has a linear concentration range of 1–10 μM, high sensitivity, and can improve selectivity, precision, and accuracy.

[0067] In this invention, the quantitative detection conditions for the chromatography include: Chromatographic column: ACQUITY UPLC BEH C18 column, specifications: column length 100 mm, inner diameter 2.1 mm, particle size 1.7 μm, column temperature: 40℃ Mobile phase: Mobile phase A is a 0.1% formic acid aqueous solution, wherein the formic acid accounts for 0.1% of the volume of the formic acid aqueous solution; Mobile phase B is pure acetonitrile.

[0068] Gradient elution conditions: 0–0.5 min, mobile phase A: 95%, mobile phase B: 5%; 0.5–8.3 min, mobile phase A: 95%→5%, mobile phase B: 5%→95%; 8.3–9.5 min, mobile phase A: 5%, mobile phase B: 95%; 9.5–9.51 min, mobile phase A: 5%→95%, mobile phase B: 95%→5%; 9.51–11 min, mobile phase A: 95%, mobile phase B: 5%; flow rate: 0.3 mL / min. Injection volume: 5 μL. Under these chromatographic conditions, the drug and impurities in the cells were well separated, with good resolution.

[0069] In this invention, the quantitative detection conditions for the chromatography include: The ion source used was an electrospray ionization (ESI) source, and the scanning mode was Full MS. 2 Positive ion mode scanning; spray voltage: 3.8 kV; capillary temperature: 320℃; sheath gas flow rate: 35 mL / min; auxiliary gas flow rate: 10 mL / min; scanning range: 133.4000~2000.0000.

[0070] The molecular weight of the quantitative ion selected in this invention is 474.2, which has the highest response value and no matrix interference.

[0071] The experimental results in this invention are as follows: Figure 1 The representative chromatogram of the standard curve (a linear representative plot of TPD81020 concentration in cells detected by UPCC-MS / MS (linear range 1–10 μM)) shows that the target ion molecular weight is 474.2, and the linearity is r 2 Greater than 0.99, indicating good linearity.

[0072] Based on the linear regression equation, the intracellular concentration of TPD81020 was calculated to be 0.17 μM.

[0073] The linearity results of the standard solution are as follows: Figure 6 As shown The TPD81020 chromatogram in the blank cells is as follows: Figure 7 As shown In this invention, the intracellular TPD81020 representative chromatogram is as follows: Figure 8 As shown Conclusion: The method of the present invention can accurately quantify the free and total concentrations of PROTAC drugs DP1 and TPD81020 in cells. It has high specificity, high sensitivity, and reliable results, and fully meets the requirements for detecting the intracellular concentration of PROTAC drugs.

[0074] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for highly specific quantitative determination of intracellular PROTAC drug concentration, the method comprising: (1) Select cell lines that express the target of PROTAC drugs; (2) Culture the cell line selected in step (1) to the logarithmic growth phase; (3) Inoculate the cell suspension onto a cell culture plate, ensuring the cells are evenly distributed in the plate, and culture for 12-36 h; (4) Add the PROTAC drug to the cell culture plate, mix it well, and continue incubation for 4-8 hours; (5) Remove the cell culture medium from the cell culture plate; disperse it into a single-cell suspension, centrifuge and discard the supernatant to obtain the cell precipitate; (6) After lysing the cell pellet, the supernatant was collected by ultracentrifugation and analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry. (7) Prepare blank cell suspension and PROTAC drug concentration standard solutions, perform ultra-high performance liquid chromatography-tandem mass spectrometry analysis, and construct standard curves; (8) Substitute the result of step (6) into the standard curve obtained in step (7) to obtain the intracellular PROTAC drug concentration.

2. According to the method of claim 1, in step (2), cell passage is performed when the cell fusion rate reaches 80-90%.

3. The method according to claim 1, wherein the concentration in step (3) is 4 × 10⁻⁶. 5 Cell suspensions of cells / mL were seeded onto cell culture plates, ensuring even distribution of cells, and cultured at 37°C in a 5% CO2 incubator for 24 h.

4. According to the method of claim 1, in step (4), the PROTAC drug is added to the cell culture plate, mixed well and then incubated for 6 hours.

5. The method according to claim 1, step (5) includes removing the cell culture medium from the cell culture plate; washing the cells in the cell culture plate with PBS; adding trypsin digestion solution to the cell culture plate, incubating at 37 °C until the adherent cells are completely detached and dispersed into a single-cell suspension, adding complete culture medium to terminate digestion, centrifuging and discarding the supernatant to obtain cell pellet.

6. The method according to claim 1, step (6) includes adding acetonitrile to the cell pellet for incubation, and after 30 min, centrifuging at 4°C and 10000×g for 1 h to collect the supernatant and perform ultra-high performance liquid chromatography-tandem mass spectrometry analysis.

7. The method according to claim 1, wherein a 1.7 μm particle size ACQUITY UPLC BEH C18 column is used in the ultra-high performance liquid chromatography-tandem mass spectrometry analysis.

8. The method according to claim 1, wherein the mobile phase A in the ultra-high performance liquid chromatography-tandem mass spectrometry analysis is a 0.1% v / v formic acid aqueous solution, and the mobile phase B is acetonitrile.

9. The method according to claim 1, wherein the mass spectrometry parameters in the ultra-high performance liquid chromatography-tandem mass spectrometry analysis are: ESI positive ion mode, 3.8 kV spray voltage, and 133.4-2000.0 m / z scan range.

10. The method according to claim 1, wherein the method comprises: (1) Select cell lines that express the target of PROTAC drugs; (2) Culture the cell line selected in step (1) to the logarithmic growth phase, and passage the cells when the cell confluence reaches 80-90%. (3) Take a concentration of 4×10 5 The cell suspension of cells / mL was seeded onto a cell culture plate, and the cells were evenly spread in the culture plate and cultured at 37°C in a 5% CO2 incubator for 24 h. (4) Add the PROTAC drug to the cell culture plate, mix it well, and continue incubation for 6 h; (5) Remove the cell culture medium from the cell culture plate; wash the cells in the cell culture plate with PBS; add trypsin digestion solution to the cell culture plate, incubate at 37 °C until the adherent cells are completely detached and dispersed into a single cell suspension, add complete culture medium to stop digestion, centrifuge and discard the supernatant to obtain cell pellet; (6) Add acetonitrile to the cell pellet and incubate for 30 min. After centrifugation at 4℃ and 10000×g for 1 h, collect the supernatant and perform ultra-high performance liquid chromatography-tandem mass spectrometry analysis. (7) Prepare blank cell suspension and PROTAC drug concentration standard solutions, perform ultra-high performance liquid chromatography-tandem mass spectrometry analysis, and construct standard curves; (8) Substitute the result of step (6) into the standard curve obtained in step (7) to obtain the intracellular PROTAC drug concentration; The ultra-high performance liquid chromatography-tandem mass spectrometry analysis used a 1.7 μm particle size ACQUITY UPLC BEH C18 column; mobile phase A was 0.1% v / v formic acid aqueous solution, and mobile phase B was acetonitrile; the mass spectrometry parameters were: ESI positive ion mode, 3.8 kV spray voltage, and 133.4-2000.0 m / z scan range.

Images