A small molecule fluorescent probe, a screening method and application thereof

Abstract

This invention belongs to the field of biochemistry and drug screening technology, and relates to a small molecule fluorescent probe, its screening method, and its application. The small molecule fluorescent probe is obtained by the condensation of a pharmacophore and a fluorescent group; the pharmacophore is a compound, optical isomer, or pharmaceutically acceptable salt thereof represented by general formula II; wherein X is selected from: [missing information - likely a specific compound or ingredient]. This invention connects a high-affinity pharmacophore of the TYK2 pseudokinase domain to FITC, creating a small molecule fluorescent probe that can efficiently target target proteins, exhibits high specificity for the TYK2 pseudokinase domain, and can sensitively reflect the competitive effect of inhibitors, making it very suitable for the detection of weak interactions and the accurate evaluation of inhibitor efficacy.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry and drug screening technology, and relates to a small molecule fluorescent probe, its screening method and application. Background Technology

[0002] TYK2 is an important member of the Janus kinase family, playing a crucial role in mediating various cytokine signaling pathways and is a popular target for the treatment of autoimmune diseases. Compared with traditional inhibitors targeting the kinase domain, inhibitors targeting the TYK2 pseudokinase domain show promise for higher selectivity, thereby reducing side effects.

[0003] Although the TYK2 pseudokinase domain retains a kinase-like structure, it lacks ATP hydrolysis and phosphate transfer capabilities, making traditional kinase activity assays such as ADP-Glo ​​and HTRF unsuitable for direct application. Secondly, the pseudokinase domain has a poor affinity for ADP (Kd ~10 μM), far lower than the nanomolar binding of the kinase domain, resulting in weak signals and low signal-to-noise ratios in ADP-based detection methods. Currently, screening for small-molecule inhibitors targeting kinases often employs traditional biochemical kinase activity assays or surface plasmon resonance (SPR) techniques. The former struggles to directly reflect the conformational regulatory function of the target and is susceptible to false positives common in compound libraries; the latter suffers from low throughput and high cost.

[0004] TR-FRET is a highly sensitive analytical technique that combines time-resolved fluorescence detection with fluorescence resonance energy transfer. Its core principle involves using long-lived lanthanide fluorophores (such as europium, Eu). 3+ terbium (Tb) 3+ As an energy donor, the TYK2 pseudokinase domain molecule is detected with a delayed response after excitation, thus filtering out short-lived background fluorescence. When the donor and acceptor-labeled molecules approach each other due to interaction, energy transfer occurs, producing characteristic acceptor fluorescence. The key advantages of this technique are extremely low background interference, a homogeneous detection method requiring no washing, and high stability and anti-interference capability derived from fluorescence ratio calculations. Therefore, it is highly suitable for molecular interaction studies in complex samples, high-throughput drug screening, and biomarker detection. Although time-resolved fluorescence resonance energy transfer (TRFR) technology has been widely used in molecular interaction studies, its application in competitive screening of small molecules in the TYK2 pseudokinase domain is limited, highlighting the urgent need for a dedicated fluorescent probe with high affinity and a high signal-to-noise ratio. Summary of the Invention

[0005] The purpose of this invention is to provide a high-affinity small molecule fluorescent probe based on the TYK2 pseudokinase domain, its screening method, and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A small molecule fluorescent probe is obtained by the condensation of a pharmacophore and a fluorophore; wherein the pharmacophore is a compound, optical isomer, or pharmaceutically acceptable salt thereof represented by general formula II. ; Where X is selected from: , .

[0008] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows:

[0009] In one preferred embodiment, the fluorescent reporter group is fluorescein isothiocyanate FITC, carboxytetramethylrhodamine, boron dipyrrole fluorescein, carboxyfluorescein, cyanine dye 3, or cyanine dye 5.

[0010] In one preferred embodiment, the small molecule fluorescent probe is a compound of general formula I, an optical isomer, or a pharmaceutically acceptable salt thereof: ; Where R is selected from: , .

[0011] Based on the same inventive concept, this invention also claims protection for a method for preparing the small molecule fluorescent probe, comprising the following steps: S1, methyl chloroformylbutyrate, reacts with starting material I in a nucleophilic substitution reaction to give intermediate 1; intermediate 1 is hydrolyzed under alkaline conditions to give intermediate 2; S2, intermediate 2, and diamine undergo an amide condensation reaction to give intermediate 3; intermediate 3 is deBoc under acidic conditions to give intermediate 4; S3 and intermediate 4 undergo a nucleophilic substitution reaction with FITC to obtain the small molecule fluorescent probe; The structure of raw material I is as follows: ; The structure of intermediate 1 is as follows: ; The structure of intermediate 2 is as follows: ; The structure of intermediate 3 is as follows: ; The structure of intermediate 4 is as follows: ; n is 3 or 4.

[0012] In one preferred embodiment, the synthetic route of the small molecule fluorescent probe is as follows: .

[0013] Based on the same inventive concept, the present invention also claims a TR-FRET detection system for a TYK2 pseudokinase domain inhibitor, wherein the TR-FRET detection system includes the small molecule fluorescent probe.

[0014] In one preferred embodiment, the TYK2 pseudokinase domain inhibitor includes Deucravacitinib, TAK-279, or ABBV712.

[0015] In one preferred embodiment, the TR-FRET detection system comprises the following components: a recombinant TYK2 pseudokinase domain protein with a specific tag, the small molecule fluorescent probe, a TR-FRET donor conjugate, and a tagged antibody with a lanthanide element (terbium).

[0016] In one preferred embodiment, the lanthanide element is terbium.

[0017] Based on the same inventive concept, the present invention also claims a kit comprising the above-described small molecule fluorescent probe.

[0018] Based on the same inventive concept, the present invention also claims protection for the use of the small molecule fluorescent probe, the TR-FRET detection system, or the kit in TYK2 pseudokinase domain inhibitors.

[0019] Based on the same inventive concept, this invention also claims a method for screening TYK2 pseudokinase domain inhibitors, the screening method being based on TR-FRET technology and using the small molecule fluorescent probe.

[0020] In one preferred embodiment, the FITC group in the small molecule fluorescent probe serves as a TR-FRET energy acceptor.

[0021] In one preferred embodiment, the screening method includes the following steps: (1) Different concentrations of the analyte compound were added to the TR-FRET detection system; 2) Using a multi-functional microplate reader, excitation was performed at 320 nm, and fluorescence intensities at 490 nm and 520 nm were detected, respectively. The TR-FRET ratio of each test well was calculated using the following formula, and the inhibitory activity of the test compound was analyzed accordingly: .

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] (1) Innovative probe design: This invention connects the high-affinity pharmacophore of the TYK2 pseudokinase domain to FITC, creating a novel signaling molecule. This probe can efficiently target the target protein, and its FITC group can also serve as a receptor for TR-FRET, achieving the integration of "targeting" and "detection".

[0024] (2) Simple and homogeneous method: Since FITC is directly used as the receptor, there is no need to use additional anti-FITC antibodies and their conjugates, which simplifies the detection system, reduces costs, and ensures true "mix and read" homogeneous detection, making it easy to achieve automated high-throughput screening.

[0025] (3) High signal-to-noise ratio and anti-interference: The time-resolved fluorescence and ratio method is used to read the data, which effectively eliminates the interference of compound self-fluorescence, light scattering and sample loading error, and significantly improves the accuracy and reliability of detection.

[0026] (4) High specificity and high sensitivity: This method directly detects the binding of small molecules to proteins and has high specificity for the TYK2 pseudokinase domain. It can sensitively reflect the competitive effect of inhibitors and is very suitable for the detection of weak interactions and the accurate evaluation of inhibitor efficacy. Attached Figure Description

[0027] Figure 1 The binding curve of Deu-2C-FITC and 10 nM TYK2 JH2 is shown.

[0028] Figure 2 The binding curve of Deu-3C-FITC and 10 nM TYK2 JH2 is shown.

[0029] Figure 3 The graph shows the combination curves of Deu-4C-FITC and 10 nM TYK2 JH2.

[0030] Figure 4 The binding curve of Deu-5C-FITC and 10 nM TYK2 JH2 is shown.

[0031] Figure 5 The binding curves of 9 nM Deu-2C-FITC with different concentrations of TYK2 JH2 are shown.

[0032] Figure 6 The binding curves of 2 nM Deu-3C-FITC with different concentrations of TYK2 JH2 are shown.

[0033] Figure 7 The binding curves of 4 nM Deu-4C-FITC with different concentrations of TYK2 JH2 are shown.

[0034] Figure 8 The binding curves of 4 nM Deu-5C-FITC with different concentrations of TYK2 JH2 are shown.

[0035] Figure 9 IC50 assays were performed in a 4 nM Deu-5C-FITC and 5 nM TYK2 JH2 system with different concentrations of Deucravacitnib. 50 Line graph.

[0036] Figure 10 This is a schematic diagram of the experimental principle of TR-FRET in Embodiment 1 of the present invention. Detailed Implementation

[0037] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0038] The general synthetic route for the small molecule fluorescent probe of the present invention, the synthetic route of Formula I is shown below: .

[0039] Methyl chloroformylbutyrate undergoes a nucleophilic substitution reaction with starting material I to obtain intermediate 1. It is then hydrolyzed under alkaline conditions to obtain key intermediate 2. Intermediate 2 then undergoes an amide condensation reaction with mono-Boc diamines of different lengths to obtain intermediate 3-1 / 3-2 / 3-3 / 3-4. Under acidic conditions, it undergoes deBoc removal to obtain intermediate 4-1 / 4-2 / 4-3 / 4-4. Finally, it undergoes nucleophilic substitution with FITC to obtain the final product.

[0040] The core principle of the probe prepared by this invention is as follows: Figure 10 As shown.

[0041] The specific preparation process is illustrated below through specific examples.

[0042] Example 1

[0043] Synthesis of the final product Deu-2C / 3C / 4C / 5C-FITC

[0044] Synthesis of Intermediate 1: In a reaction flask, add starting material I (prepared according to this patent: WO2022233286 A1, 500 mg, 1.3990 mmol) and ultra-dry tetrahydrofuran (15 mL), then add triethylamine (283.0 mg, 2.7980 mmol). Add methyl chloroformylbutyrate (276.0 mg, 1.6790 mmol) dropwise at 0°C. Return to room temperature and react for 4-5 hours. After the reaction is complete, directly evaporate the reaction mixture to dryness and then purify by column chromatography to obtain Intermediate 1, a yellow solid, with a yield of 48%. Chromatography of Intermediate 1: 1 H NMR(500 MHz, DMSO-d6) δ 11.01 (s, 1H), 10.99 (s, 1H), 9.14 (s, 1H), 8.59 (s,1H), 8.17 (s, 1H), 7.67 (dd, J = 7.8, 1.4 Hz, 1H), 7.54 (dd, J = 7.9, 1.3 Hz,1H), 7.30 (t, J = 7.9 Hz, 1H), 3.96 (s, 3H), 3.74 (s, 3H), 3.58 (s, 3H), 2.49(t, J = 7.3 Hz, 2H), 2.34 (t, J = 7.4 Hz, 2H), 1.79 (m, J = 7.4 Hz, 2H). HRMS(ESI) m / z calcd for [M+H] + : 486.2293; found: 486.2298.

[0045] Synthesis of Intermediate 2: In a reaction flask, add Intermediate 1 (200 mg, 0.4061 mmol), methanol (8 mL), and water (2 mL), then add lithium hydroxide (11.0 mg, 0.4467 mmol). After reacting at room temperature for 6-7 hours, post-processing can be performed. Adjust the pH of the reaction solution to approximately 3-4 with 1 M HCl at 0°C. A white solid precipitates, yielding the key intermediate 2, with a yield of 42%. Spectroscopy of Intermediate 2: 1H NMR (600 MHz, DMSO-d6) δ 12.08 (s, 1H), 11.02 (s, 1H), 11.01(s, 1H), 9.15 (s, 1H), 8.58 (s, 1H), 8.21 (s, 1H), 7.68 (dd, J = 7.8, 1.3 Hz,1H), 7.61-7.49 (m, 1H), 7.31 (t, J = 7.9 Hz, 1H), 3.97 (s, 3H), 3.76 (s, 3H),2.50 (t, J = 7.4 Hz, 2H), 2.26 (t, J = 7.4 Hz, 2H), 1.79 (m, J = 7.4 Hz, 2H).HRMS (ESI) m / z calcd for [M+H] + : 472.2136; found: 472.2141.

[0046] Synthesis of intermediate 3 (3-1 / 3-2 / 3-3 / 3-4): In a reaction flask, intermediate 2 (1.0 eq), HATU (1.5 eq), DMF, and DIPEA (2.0 eq) were added. After stirring at room temperature for 5-10 min, mono-Boc ethylenediamine / mono-Boc propylenediamine / mono-Boc butyladiamine / mono-Boc pentanediamine (1.1 eq each) were added. After reacting at room temperature for 6-7 hours, post-processing was performed. The reaction solution was extracted 2-3 times with DCM and water. The organic phases were separated and combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and finally purified by column chromatography. Intermediates 3-1 / 3-2 / 3-3 / 3-4 of different lengths were obtained as white solids with yields of 40-71%.

[0047] The graph of intermediate 3-1: 1H NMR (600 MHz, DMSO-d6) δ 11.00 (s, 1H), 10.98 (s,1H), 9.14 (s, 1H), 8.57 (s, 1H), 8.19 (s, 1H), 7.80 (t, J = 5.5 Hz, 1H), 7.67(dd, J = 7.8, 1.6 Hz, 1H), 7.54 (dd, J = 7.9, 1.5 Hz, 1H), 7.30 (t, J = 7.9Hz, 1H), 6.77 (t, J = 5.6 Hz, 1H), 3.96 (s, 3H), 3.74 (s, 3H), 3.05 (m, J =6.3 Hz, 2H), 2.96 (m, J = 6.2 Hz, 2H), 2.44 (t, J = 7.3 Hz, 2H), 2.08 (t, J =7.5 Hz, 2H), 1.77 (m, J = 7.4 Hz, 2H), 1.36 (s, 9H).

[0048] The graph of intermediate 3-2: 1 H NMR (400 MHz, DMSO-d6) δ 10.99 (s, 1H), 10.98 (s,1H), 9.14 (s, 1H), 8.57 (s, 1H), 8.19 (s, 1H), 7.76 (t, J = 5.7 Hz, 1H), 7.67(d, J = 7.8 Hz, 1H), 7.54 (d, J = 7.9 Hz, 1H), 7.30 (t, J = 7.9 Hz, 1H), 6.75(t, 1H), 3.95 (s, 3H), 3.73 (s, 3H), 3.01 (t, J = 6.9 Hz, 2H), 2.90 (t, J =6.8 Hz, 2H), 2.43 (t, J = 7.2 Hz, 2H), 2.08 (t, J = 7.4 Hz, 2H), 1.77 (q, J =7.3 Hz, 2H), 1.53-1.43 (m, 2H), 1.36 (s, 9H).

[0049] The graph of intermediate 3-3: 1H NMR (600 MHz, DMSO-d6) δ 10.99 (s, 1H), 10.98 (s,1H), 9.14 (s, 1H), 8.57 (s, 1H), 8.19 (s, 1H), 7.75 (t, J = 5.6 Hz, 1H), 7.67(dd, J = 7.8, 1.6 Hz, 1H), 7.54 (dd, J = 7.9, 1.5 Hz, 1H), 7.29 (t, J = 7.9Hz, 1H), 6.76 (t, J = 5.5 Hz, 1H), 3.95 (s, 3H), 3.74 (s, 3H), 3.00 (t, 2H),2.89 (t, 2H), 2.43 (t, 2H), 2.07 (t, 2H), 1.76 (m, 2H), 1.38 (m, 2H), 1.36(s, 9H), 1.34 (m, 2H).

[0050] Spectra of intermediates 3-4: 1 H NMR (600 MHz, DMSO-d6) δ 11.00 (s, 1H), 10.98 (s,1H), 9.14 (s, 1H), 8.57 (s, 1H), 8.19 (s, 1H), 7.73 (t, J = 5.4 Hz, 1H), 7.67(dd, J = 7.8, 1.3 Hz, 1H), 7.54 (dd, J = 8.0, 1.6 Hz, 1H), 7.29 (t, J = 7.9Hz, 1H), 6.74 (t, J = 5.1 Hz, 1H), 3.96 (s, 3H), 3.74 (s, 3H), 2.99 (m, 2H),2.89-2.84 (m, 2H), 2.43 (t, J = 7.3 Hz, 2H), 2.07 (t, J = 7.5 Hz, 2H), 1.76 (m, 2H), 1.36 (m, 2H), 1.36 (s, 9H), 1.34 (m, 2H), 1.20 (m, 2H).

[0051] Synthesis of intermediate 4 (4-1 / 4-2 / 4-3 / 4-4): Take a reaction flask, add intermediate 3-1 / 3-2 / 3-3 / 3-4 (1.0 eq) and methanol, then add 5 M HCl, react at room temperature for 4-5 hours, evaporate the reaction solution directly to dryness, and use the white solid crude product obtained after evaporating the solution with methanol three times directly for the next step of the reaction.

[0052] Synthesis of Deu-2C / 3C / 4C / 5C-FITC: In a reaction flask, intermediate 4 (1.0 eq) and DMF were added, and the mixture was stirred at -20°C for 5 min. DIPEA (10.0 eq) was added, and stirring continued for 10 min. Finally, FITC (1.0 eq) was added, and the mixture was allowed to react overnight at room temperature in the dark. Four different lengths of the final product, Deu-2C / 3C / 4C / 5C-FITC, were obtained as orange solids with yields ranging from 52% to 77%.

[0053] Deu-2C-FITC diagram: 1 H NMR (500 MHz, DMSO-d6) δ 11.01 (s, 1H), 11.00 (s, 1H), 10.66 (s, 1H), 9.14 (s, 1H), 8.59 (s, 1H), 8.57 (s, 1H), 8.33 (s, 1H), 8.20 (s, 1H), 8.04 (s, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.67 (dd, J = 7.8, 1.6Hz, 1H), 7.54 (dd, J = 8.0, 1.6 Hz, 1H), 7.29 (t, J = 7.9 Hz, 1H), 7.18 (d, J= 8.3 Hz, 1H), 6.70 (s, 1H), 6.70 (s, 1H), 6.61 (d, J = 8.7 Hz, 2H), 6.57(dd, J = 8.6, 2.3 Hz, 2H), 3.95 (s, 3H), 3.74 (s, 3H), 3.28 (m, 2H), 2.92 (m,2H), 2.46 (t, J = 7.2 Hz, 2H), 2.14 (t, J = 7.4 Hz, 2H), 1.80 (m, 2H). 1313C NMR (126 MHz, DMSO) δ 173.37, 172.96, 172.57, 169.05, 167.04, 160.18, 159.36, 156.33, 152.42, 150.99, 146.05, 145.59, 145.17, 141.94, 135.51, 132.99, 129.78, 129.49, 126.75, 126.54, 124.74, 124.57, 123.14, 113.17, 110.25, 102.72, 97.24, 84.31, 61.65, 52.10, 41.01, 38.21, 36.48, 35.97, 35.02, 18.79. HRMS (ESI) m / z calcd for [M+H] + : 903.3076; found: 903.3080.

[0054] The spectrum of Deu-3C-FITC: 1 1H NMR (400 MHz, DMSO-d6) δ 1 1H NMR (400 MHz, DMSO-d6) δ 11.00 (s, 1H), 9.14 (s, 1H), 8.57 (s, 1H), 8.24 (s, 1H), 8.22 (d, J = 1.6 Hz, 1H), 8.20 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.93 - 7.85 (m, 1H), 7.76 (s, 1H), 7.68 (dd, J = 7.8, 1.4 Hz, 1H), 7.58 - 7.49 (m, 1H), 7.31 (d, J = 7.9 Hz, 1H), 7.29 - 7.26 (m, 1H), 7.21 (d, J = 4.3 Hz, 1H), 7.19 (d, J = 4.4 Hz, 1H), 6.69 (s, 1H), 6.64 - 6.60 (m, 4H), 3.96 (s, 3H), 3.74 (s, 3H), 3.52 (t, 2H), 3.12 (t, 2H), 2.46 (t, J = 7.2 Hz, 2H), 2.13 (t, J = 7.4 Hz, 2H), 1.80 (m, 2H), 1.70 (m, 2H). 13¹³C NMR (101 MHz, DMSO) δ 173.36, 172.30, 169.00, 168.89, 167.04, 159.98, 159.93, 159.35, 156.32, 152.34, 151.01, 145.59, 145.19, 135.51, 132.97, 129.52, 127.06, 126.75, 126.59, 124.76, 123.22, 113.09, 113.04, 110.18, 110.05, 102.71, 97.23, 83.47, 61.66, 41.92, 36.57, 36.48, 35.97, 35.05, 29.21, 21.24. HRMS (ESI) m / z calcd for [M+H] + : 917.3232; found: 917.3225.

[0055] The spectrum of Deu-4C-FITC: 1 ¹H NMR (400 MHz, DMSO-d6) δ 10.99 (s, 1H), 10.14 (s, 1H), 9.89 (s, 1H), 9.12 (s, 1H), 8.56 (s, 1H), 8.25 (d, J = 7.5 Hz, 1H), 8.18 (d, J = 5.1 Hz, 1H), 8.09 (s, 1H), 7.83 (t, J = 5.5 Hz, 1H), 7.73 (d, J = 7.2 Hz, 1H), 7.66 (dd, J = 7.8, 1.5 Hz, 1H), 7.54 (dd, J = 8.0, 1.6 Hz, 1H), 7.29 (t, J = 7.9 Hz, 1H), 7.17 (d, J = 8.3 Hz, 1H), 6.68 (s, 1H), 6.68 (s, 1H), 6.61 (d, J = 8.7 Hz, 2H), 6.56 (dd, J = 8.7, 2.3 Hz, 2H), 3.9 (s, 3H), 3.73 (s, 3H), 3.50 (t, 2H), 3.06 (t, 2H), 2.44 (t, 2H), 2.09 (t, 2H), 1.77 (m, 2H), 1.56 (m, 2H), 1.45 (m, 2H). 1313C NMR (101 MHz, DMSO) δ 173.30, 171.86, 169.01, 166.89, 159.71, 159.33, 156.19, 152.32, 150.95, 145.60, 145.02, 144.61, 135.53, 135.51, 132.85, 129.51, 126.74, 126.61, 126.59, 124.76, 123.23, 123.12, 116.81, 112.96, 110.23, 102.64, 97.17, 83.64, 61.66, 40.63, 38.52, 36.49, 35.95, 34.99, 27.17, 25.60, 21.26. HRMS (ESI) m / z calcd for [M+H] + : 931.3389; found: 931.3399.

[0056] Spectrum of Deu-5C-FITC: 1 1H NMR (500 MHz, DMSO-d6) δ 11.00 (s, 1H), 10.55 (s, 1H), 9.14 (s, 1H), 8.57 (s, 1H), 8.52 (s, 1H), 8.38 (s, 1H), 8.20 (s, 1H), 7.82 (t, J = 5.7 Hz, 2H), 7.67 (d, J = 6.9 Hz, 1H), 7.55 (d, J = 7.5 Hz, 1H), 7.29 (t, J = 7.9 Hz, 1H), 7.17 (d, J = 8.3 Hz, 1H), 6.70 (d, J = 1.9 Hz, 2H), 6.64 - 6.51 (m, 5H), 3.95 (s, 3H), 3.74 (s, 3H), 3.04 (q, J = 6.4 Hz, 2H), 2.96 (q, J = 7.0 Hz, 2H), 2.44 (t, J = 7.2 Hz, 2H), 2.10 (t, J = 7.4 Hz, 2H), 1.77 (m, 2H), 1.56 (m, 2H), 1.42 (m, 2H), 1.37 - 1.28 (m, 2H). 13C NMR (126 MHz, DMSO) δ 173.39, 172.70, 171.85, 169.09, 167.05, 160.06, 159.36, 156.34,152.38, 151.00, 145.59, 145.17, 142.20, 139.52, 138.35, 135.51, 132.99,129.48, 126.76, 126.55, 124.74, 124.37, 123.15, 113.11, 110.26, 102.71,97.25, 77.51, 61.29, 52.50, 38.83, 36.48, 36.02, 35.02, 24.36, 21.67, 21.28,18.58. HRMS (ESI) m / z calcd for [M+H] + : 945.3545; found: 945.3552.

[0057] Example 2: Establishment and optimization of the TR-FRET competitive binding experimental system

[0058] The consumables required for the experiment are shown in Table 1.

[0059] The establishment and optimization of the TR-FRET competitive binding experimental system includes the following experimental steps:

[0060] 1: Binding experiment of four probes of different concentrations with TYK2 and JH2 (probe titration experiment)

[0061] (1) Buffer formulation: Hepes pH 7.5 (20 mM), MgCl2 (10 mM), Brij-35 (0.015%), DTT (2 mM), BSA (50 ug / mL).

[0062] (2) Protein working solution concentration: 3× protein TYK2 JH2 (30 nM).

[0063] (3) Tag antibody working solution concentration: 0.75×Tb tag antibody.

[0064] (4) Probe working solution concentration: 3×Tracer (Deu-2C / 3C / 4C / 5C-FITC, 125×3 nM, 62.5×3 nM, 31.25×3 nM, 15.625×3 nM, 7.8125×3 nM, 3.90625×3 nM, 1.953125×3 nM, 0.9765625×3 nM, 0 nM);

[0065] (5) Operation steps: First, add 5 µL of Buffer (no protein group) / TYK2 JH2 working solution (protein group) to the plate, centrifuge at 1000 rpm for 1 min; then add 5 µL of Tracer working solution, centrifuge at 1000 rpm for 1 min; finally add 5 µL of Tb antibody working solution, centrifuge at 1000 rpm for 1 min; total 15 µL, seal the plate, and incubate at room temperature for 30 min;

[0066] (6) Measure the readings of 520 nm / 490 nm.

[0067] The results are as follows Figures 1-4 As shown, the results indicate that after increasing the concentration, the Ratio values ​​of Deu-2C-FITC, Deu-3C-FITC, Deu-4C-FITC, and Deu-5C-FITC increased significantly compared with the protein-free group. According to the binding curve fitting, the Kd values ​​of these four probes were all less than 10 nM.

[0068] 2: Binding experiments of four different probes with different concentrations of TYK2 and JH2 (protein titration experiments)

[0069] (1) Protein working solution concentration: 3× protein JH2 (100 nM, 80 nM, 60 nM, 40 nM, 20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, 0.3125, 0 nM).

[0070] (2) Tag antibody working solution concentration: 0.75×Tb tag antibody.

[0071] (3) Probe concentration: After determining the final concentration based on the Kd values ​​of the four probes, the working solution concentration is then determined;

[0072] (4) Operation steps: First, add 5 µL of Buffer (protein-free group) / TYK2 JH2 working solution (protein group) to the plate, centrifuge at 1000 rpm for 1 min; then add 5 µL of Tracer working solution, centrifuge at 1000 rpm for 1 min; finally add 5 µL of Tb antibody working solution, centrifuge at 1000 rpm for 1 min; total 15 µL, seal the plate, and incubate at room temperature for 30 min;

[0073] It should be noted that the TYK2 JH2 binding experiment was performed on raw material I and FITC using a biolayer interferometer. The results showed that the affinity of the final product Deu-2C / 3C / 4C / 5C-FITC for the TYK2 pseudokinase domain was at least 20% stronger than that of raw material I. In particular, the Kd value of Deu-5C-FITC was increased by more than 30% compared with that of raw material I.

[0074] Under the same conditions, intermediates 1 / 2 / 3 / 4 also have at least 20% stronger affinity than raw material I. FITC cannot bind to proteins.

[0075] Therefore, in the probe structure, both the inhibitory portion of the TYK2 pseudokinase domain and the FITC labeling are indispensable.

[0076] (5) Measure the readings of 520 nm / 490 nm.

[0077] The results are as follows Figures 5-8 As shown, the results indicate that when the concentrations of the four probes were fixed (around the Kd value), increasing the protein concentration revealed that the ratio gradually reached saturation after the protein concentration increased to 10 nM. However, the signal / background (S / B) window curves of the four probes were not identical. This invention requires selecting probes with higher binding rates and larger S / B values ​​at low protein concentrations for subsequent high-throughput screening. The results show that Deu-2C-FITC has a high Kd value, but its overall window S / B is small within the protein concentration range, making it unsuitable for subsequent screening experiments. Although Deu-3C-FITC has a smaller Kd value, its overall window S / B is even lower than that of Deu-2C-FITC within the protein concentration range, also making it unsuitable. Deu-4C-FITC has a large window across the overall protein concentration range, but its window value is lower than that of Deu-5C-FITC at 5 nM. Therefore, Deu-5C-FITC is currently the probe with the highest binding rate, largest window, and highest ratio at 5 nM. Ultimately, Deu-5C-FITC was selected for subsequent Deucravacitinib positive drug validation and high-throughput screening experiments, with the protein concentration fixed at 5 nM and the probe concentration fixed at 4 nM.

[0078] 3: Deucravacitinib Validation Experiment

[0079] (1) Protein working solution concentration: Protein JH2 (3X, 15 nM).

[0080] (2) Tag antibody working solution concentration: 3X, 0.75×Tb tag antibody.

[0081] (3) Deucravacitinib working solution concentration: 6X, 300 nM, and then 3 times diluted for a total of 11 concentrations.

[0082] (4) Probe working solution concentration (Deu-5C-FITC): 6X, 24 nM.

[0083] (5) Procedure: First, add 5 µL of Buffer (protein-free) / TYK2 JH2 working solution (protein-free) to the plate and centrifuge at 1000 rpm for 1 min; then add 2.5 µL of Tracer working solution and centrifuge at 1000 rpm for 1 min; then add 2.5 µL of Deucravacitinib working solution and centrifuge at 1000 rpm for 1 min; finally add 5 µL of Tb antibody working solution and centrifuge at 1000 rpm for 1 min; total 15 µL, seal the plate and incubate at room temperature for 30 min;

[0084] (6) Measure the readings of 520 nm / 490 nm.

[0085] The results are as follows Figure 9 As shown, the positive drug test results indicate that in the presence of Deu-5C-FITC, Deucravacitinib exhibits a concentration-dependent trend in competing with the probe, and the fitted IC50 value is [missing information]. 50 The values ​​are basically consistent with those reported in the literature and can be used for subsequent target activity screening experiments.

[0086] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A small molecule fluorescent probe, characterized in that, It is obtained by the condensation of a pharmacophore and a fluorophore; the pharmacophore is a compound of general formula II, an optical isomer, or a pharmaceutically acceptable salt thereof. ； Where X is selected from: , .

2. The small molecule fluorescent probe according to claim 1, characterized in that, The fluorescent reporter group is fluorescein isothiocyanate FITC, carboxytetramethylrhodamine, boron dipyrrole fluorescein, carboxyfluorescein, cyanine dye 3, or cyanine dye.

3. The small molecule fluorescent probe according to claim 1, characterized in that, The small molecule fluorescent probe is a compound, optical isomer, or pharmaceutically acceptable salt thereof represented by general formula I. ； Where R is selected from: , .

4. The method for preparing the small molecule fluorescent probe according to any one of claims 1-3, characterized in that, Includes the following steps: S1, methyl chloroformylbutyrate, reacts with starting material I in a nucleophilic substitution reaction to give intermediate 1; intermediate 1 is hydrolyzed under alkaline conditions to give intermediate 2; S2, intermediate 2, and diamine undergo an amide condensation reaction to give intermediate 3; intermediate 3 is deBoc under acidic conditions to give intermediate 4; S3 and intermediate 4 undergo a nucleophilic substitution reaction with FITC to obtain the small molecule fluorescent probe; The structure of raw material I is as follows: ; The structure of intermediate 1 is as follows: ; The structure of intermediate 2 is as follows: ; The structure of intermediate 3 is as follows: ; The structure of intermediate 4 is as follows: ; n is 3 or 4.

5. The method for preparing the small molecule fluorescent probe according to claim 1, characterized in that, The synthetic route of the small molecule fluorescent probe is as follows: 。 6. A TR-FRET detection system for a TYK2 pseudokinase domain inhibitor, characterized in that, The TR-FRET detection system includes the small molecule fluorescent probe.

7. The TR-FRET detection system according to claim 6, characterized in that, The TR-FRET detection system comprises the following components: a recombinant TYK2 pseudokinase domain protein with a specific tag, the small molecule fluorescent probe, the TR-FRET donor conjugate, and a tagged antibody with lanthanides (terbium).

8. A reagent kit, characterized in that, The kit includes the small molecule fluorescent probe according to any one of claims 1-3 or the TR-FRET detection system according to claim 6 or 7.

9. The use of the small molecule fluorescent probe according to any one of claims 1-3, the TR-FRET detection system according to claim 6 or 7, or the kit according to claim 8 in TYK2 pseudokinase domain inhibitors.

10. A method for screening TYK2 pseudokinase domain inhibitors, characterized in that, The screening method is based on TR-FRET technology and uses the small molecule fluorescent probe described in any one of claims 1-3.

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