CPT1A agonist as well as construction method and application thereof
By establishing an efficient expression and purification system and optimizing the detection method, combined with DEL technology screening, the problem of high expression and detection of CPT1A protein was solved, and the construction and screening of highly active CPT1A agonists were achieved, which has good application prospects.
Patent Information
- Application Number
- CN202510654065.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-21
- Publication Date
- 2025-09-16
AI Technical Summary
Existing technologies make it difficult to achieve high expression, activity, and purity of CPT1A protein. The detection methods are cumbersome and unsuitable for high-throughput screening. There is a lack of efficient screening optimization solutions for CPT1A.
The full-length CPT1A gene was labeled with a His tag, and a high-efficiency expression and purification system was established by combining a GST tag with molecular sieve purification. The DTNB detection system was optimized to achieve real-time kinetic monitoring under low substrate consumption. A sub-library was designed based on DEL technology for screening of CPT1A agonists.
The efficient construction of highly active CPT1A agonists was achieved, the detection method was simplified, the screening efficiency and accuracy were improved, and it has good application prospects.
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Figure CN120647577A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of CPT1A targeted drugs, and particularly relates to a CPT1A agonist and a construction method and application thereof. Background Art
[0002] Carnitine palmitoyltransferase 1A (CPT1A) is the rate-limiting enzyme in mitochondrial fatty acid β-oxidation, responsible for transporting long-chain fatty acids to mitochondria for oxidative decomposition. Regulating CPT1A activity can affect energy metabolism and may have potential applications in the treatment of related diseases. However, the development of CPT1A targeted drugs still faces the following challenges: (1) Difficulty in expressing and purifying CPT1A protein: CPT1A is a membrane-bound protein. Traditional expression systems (such as Escherichia coli) easily lead to protein aggregation or inactivation, making it difficult to obtain high-purity, high-activity recombinant proteins. Although mammalian cell and insect cell expression systems can retain protein activity, the expression level is low, the technology is difficult, and the production cost is high. Existing methods are difficult to achieve high expression level, high activity, and high purity, which restricts subsequent drug screening and structural studies. (2) Limitations of CPT1A activity detection methods: Traditional detection relies on radiolabeled substrates (such as 3H-carnitine) or complex mass spectrometry analysis, which is cumbersome and not suitable for high-throughput screening. Colorimetric methods based on DTNB require optimized reaction systems, otherwise they suffer from low sensitivity and are susceptible to interference from thiols. Some studies have used fluorescent probes, but these probes suffer from poor stability and high costs.
[0003] Traditional screening methods, such as high-throughput screening (HTS), are time-consuming, costly, and difficult to cover large chemical spaces. DEL technology has been used for a variety of protein kinase targets, but DEL screening for CPT1A has not yet been reported. Currently, there is a lack of optimized DEL screening protocols tailored to the structural characteristics of CPT1A, resulting in high false-positive rates or missed active molecules. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to provide a CPT1A agonist and its construction method and application, which has been verified to have functional activity against CPT1A and has good application prospects.
[0005] The present invention provides a CPT1A agonist, the structural formula of the CPT1A agonist is:
[0006]
[0007] The present invention also provides a method for constructing a CPT1A agonist, comprising the following steps:
[0008] (1) The full-length CPT1A gene was cloned into the pcDNA3.1 vector via the N-terminal GST-Factor Xa restriction site (IEGR)-His tag to obtain a GST-IEGR-His-CPT1A plasmid; the plasmid was transiently transfected into HEK293 cells, followed by lysis, capture, enzyme digestion, and purification to obtain the His-CPT1A protein;
[0009] (2) Using the DELopen library, the His-CPT1A protein was subjected to DEL screening to obtain the CPT1A agonist.
[0010] Preferably, the buffer composition used in the lysis in step (1) is: 50mM 4-hydroxyethylpiperazineethanesulfonic acid HEPES, 500mM NaCl, 1mM tris(2-carboxyethyl)phosphine TCEP, 5% glycerol, 0.05mg Benzonase nuclease, 0.5% ethylphenyl polyethylene glycol NP-40 and protease inhibitor mixture.
[0011] Preferably, the capture in step (1) is performed by glutathione agarose resin.
[0012] Preferably, the enzymatic cleavage in step (1) is performed by Factor Xa enzymatic cleavage.
[0013] Preferably, the purification in step (1) is performed using a Superdex S200 molecular sieve column.
[0014] The present invention also provides a use of a CPT1A agonist in the preparation of medicaments for metabolic diseases and immune diseases.
[0015] Preferably, the drug comprises a CPT1A agonist and a pharmaceutically acceptable carrier.
[0016] Preferably, the dosage form of the drug includes at least one of tablets, capsules, pills, powders, granules, suspensions, oral solutions, powder injections and injections.
[0017] The innovative features of the present invention include:
[0018] (1) Highly efficient and soluble CPT1A expression and purification system: The full-length CPT1A gene is tagged with a His tag, the HEK293 expression vector is optimized, and the GST tag is combined with molecular sieve purification to obtain a highly active protein.
[0019] (2) Highly sensitive DTNB detection system: Optimize reaction conditions to eliminate free thiol groups and achieve real-time kinetic monitoring under low substrate consumption.
[0020] (3) DEL-based agonist screening platform: DEL technology was applied to CPT1A for the first time, and sub-libraries were designed in combination with allosteric site information to improve the hit rate.
[0021] (4) Structure-guided compound optimization: After DEL screening, the DNA tags of positive compounds are removed, and key intermediates / pharmacophores (such as quinoline-7-carboxamide) are retained to further optimize the compound structure and properties and construct an agonist library.
[0022] Beneficial effects
[0023] The present invention proposes a strategy for developing highly efficient and controllable CPT1A agonists, with the aim of applying CPT1A agonists to the treatment of metabolic diseases, immune diseases and other diseases. The obtained CPT1A agonists have been verified to have functional activity against CPT1A and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 Schematic diagram of the synthesis of the CPT1A agonist of the present invention.
[0025] Figure 2 This is the result of the CPT1A agonist of the present invention acting on macrophages induced by mouse bone marrow cells. DETAILED DESCRIPTION
[0026] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.
[0027] Example 1
[0028] I. CPT1A protein expression and purification:
[0029] The full-length CPT1A gene was cloned into the pcDNA3.1 vector via an N-terminal GST-Factor Xa restriction site (IEGR)-His tag to generate the GST-IEGR-His-CPT1A plasmid. This plasmid was transiently transfected into HEK293 cells, which were lysed using a lysis buffer containing 50 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM TCEP, 5% glycerol, 0.05 mg Benzonase, 0.5% NP-40, and a protease inhibitor cocktail. The GST-IEGR-His-CPT1A protein was captured on glutathione agarose resin and then digested with Factor Xa to obtain the His-CPT1A protein. The collected fractions were further purified using a Superdex S200 molecular sieve column (equilibration buffer: 50 mM HEPES, pH 7.5, 300 mM NaCl, 10% glycerol).
[0030] II.DEL screening method:
[0031] The screening work was completed by WuXi AppTec according to standard procedures using the DELopen library (containing 27 sub-libraries with a chemical diversity of over 4 billion).
[0032] Brief protocol: After equilibration of HisPur Ni-NTA magnetic beads (20 μL) with elution buffer (50 mM HEPES (pH 7.5), 300 mM NaCl, 10 mM imidazole, 0.05% Tween 20), four screening conditions were set up (C1-C3 used CPT1A protein, and C4 was a no-target control). His-tagged CPT1A protein (5 μg) was incubated with pre-washed magnetic beads in screening buffer (50 mM HEPES (pH 7.5), 300 mM NaCl, 10 mM imidazole, 0.05% Tween 2, 0.1 mg / mL salmon sperm DNA) at 25°C for 30 minutes with rotation. The protein-bead complex was incubated with DEL molecules in 100 μL of screening buffer at room temperature for 1 hour. After three washes, bound compounds were released by heating at 95°C for 10 minutes. The collected eluate served as the input library for the next round of screening, and two rounds of screening were performed. Each round of elution samples was detected by the QuantStudio 7Flex real-time quantitative PCR system, and the final round of samples was used for PCR amplification, sequencing, and data analysis. The final compound 10531-10115-136-14-334-0 was obtained with the structural formula:
[0033]
[0034] Example 2
[0035] The synthetic route of compound 10531-10115-136-14-334-0 is as follows Figure 1 As shown:
[0036] (1) Preparation of (R)-3-((tert-butyloxycarbonyl)amino)-5-phenylpentanoic acid carboxamide (1-2):
[0037] (R)-3-((tert-Butoxycarbonyl)amino)-5-phenylpentanoic acid (1-1) (0.230 g, 0.78 mmol) was dissolved in dichloromethane (5 mL). 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (0.358 g, 0.94 mmol), N,N-diisopropylethylamine (DIPEA) (0.304 g, 2.35 mmol), and methylamine hydrochloride (0.064 g, 0.94 mmol) were added sequentially. The mixture was reacted at 25°C for 1 hour. After concentration, the product was purified by column chromatography to afford the product 1-2 as a white solid (0.220 g, 91.58% yield). LCMS: Rt = 0.516 min, [M-99]+ = 207.1.
[0038] (2) Preparation of (R)-3-amino-N-methyl-5-phenylpentanamide (1-3):
[0039] Compound 1-2 (0.220 g, 0.72 mmol) was dissolved in dichloromethane (5 mL), and a 2M HCl / dioxane solution (5 mL) was added. The mixture was reacted at 25°C for 1 hour. After concentration, the solution was used directly in the next step to obtain the product 1-3 as a colorless oil (0.160 g, 91.80% yield). LCMS: Rt = 0.363 min, [M+1]+ = 207.0.
[0040] (3) Preparation of (R)-4-chloro-2-methyl-N-(1-(methylamino)-1-oxo-5-phenylpentane-3-yl)quinoline-7-carboxamide (1-4):
[0041] Compound 1-3 (0.100 g, 0.41 mmol) and 4-chloro-2-methylquinoline-7-carboxylic acid (1-3A, 0.091 g, 0.41 mmol) were dissolved in dichloromethane (3 mL). HATU (0.188 g, 0.50 mmol) and DIPEA (0.160 g, 1.24 mmol) were added and reacted at 25°C for 1 hour. Purification on a silica gel column afforded the product 1-4 as a white solid (0.160 g, 94.8% yield). LCMS: Rt = 0.499 min, [M+1]+ = 410.1.
[0042] (4) Preparation of the final product 10531-10115-136-14-334-0:
[0043] Compound 1-4 (0.140 g, 0.34 mmol) and (4-((4-(2-methoxyethyl)phenoxy)methyl)phenylboronic acid (1-4A, 0.098 g, 0.34 mmol) were dissolved in dioxane (5 mL), and methanesulfonic acid (2-dicyclohexylphosphino-2',4',6'-tri-isopropyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl) palladium Pd-XPhos G3 (0.027 g, 0.03 mmol) and 2M potassium carbonate solution (0.51 mL) were reacted at 100°C under nitrogen for 1 hour. Purification by semi-preparative HPLC and lyophilization afforded the final product as a white solid (0.015 g, 7.13% yield, 99.9% purity). LCMS: Rt = 0.543 min, [M+1]+ = 616.3. 1H NMR (400 MHz, MeOD-d4) spectral data confirmed the structure.
[0044] Example 3
[0045] Compound 10531-10115-136-14-334-0 was dissolved in DMSO and stored at 4°C. Mouse bone marrow-derived macrophages (BMDM) were treated with the DTNB assay for 24 hours at a final concentration of 25 μM. CPT1A activity was then determined using the DTNB assay. This assay involves the reaction of carnitine and acyl-CoA with CPT1A to form acylcarnitine, which releases thiol-CoA (COA-SH). This thiol-CoA reacts with DNTB to produce yellow TNB. CPT1A activity is quantified by measuring the absorbance change at 412 nm. Equal amounts of cell lysate were mixed with DTNB reaction buffer (116 mM Tris, 2.5 mM EDTA, 2 mM DTNB, 0.2% Triton X-100, pH 8.0) and incubated at room temperature for 30 minutes to eliminate free thiol groups. The reaction was initiated by adding the substrates palmitoyl-CoA (100 μM) and L-carnitine (5 mM), and the reading was monitored for 1 hour. The final activity data was obtained by subtracting the absorbance value of the no-substrate control and correcting for total protein.
[0046] like Figure 2 As shown, the results show that the compound is functionally active against CPT1A.
Claims
1. A CPT1A agonist, characterized in that: The structural formula of the CPT1A agonist is:
2. A method for constructing the CPT1A agonist according to claim 1, comprising the following steps: (1) The full-length CPT1A gene was cloned into the pcDNA3.1 vector via the N-terminal GST-Factor Xa restriction site (IEGR)-His tag to obtain a GST-IEGR-His-CPT1A plasmid; the plasmid was transiently transfected into HEK293 cells, followed by lysis, capture, enzyme digestion, and purification to obtain the His-CPT1A protein; (2) Using the DELopen library, the His-CPT1A protein was subjected to DEL screening to obtain the CPT1A agonist.
3. The construction method according to claim 2, wherein: The components of the buffer used for the lysis in step (1) are: 50 mM 4-hydroxyethylpiperazineethanesulfonic acid HEPES, 500 mM NaCl, 1 mM tris(2-carboxyethyl)phosphine TCEP, 5% glycerol, 0.05 mg Benzonase nuclease, 0.5% ethylphenyl polyethylene glycol NP-40 and a protease inhibitor mixture.
4. The construction method according to claim 2, wherein: The capture in step (1) is performed by glutathione agarose resin.
5. The construction method according to claim 2, wherein: The enzymatic cleavage in step (1) is performed by FactorXa enzymatic cleavage.
6. The construction method according to claim 2, wherein: The purification in step (1) is performed using a Superdex S200 molecular sieve column.
7. Use of the CPT1A agonist according to claim 1 in the preparation of drugs for metabolic diseases and immune diseases.
8. The use according to claim 7, characterized in that: The drug includes a CPT1A agonist and a pharmaceutically acceptable carrier.
9. The use according to claim 7, characterized in that: The dosage form of the drug includes at least one of tablets, capsules, pills, powders, granules, suspensions, oral solutions, powder injections and injections.