A SUMO-C7H 13 NO2 probes, their synthesis methods, and applications

Abstract

This invention discloses a SUMO-C7H 13 This invention relates to the field of biotechnology, specifically to NO2 probes, their synthesis methods, and applications. The molecular formula of the probe described in this invention is SUMO-C7H. 13 NO2, wherein the SUMO is the human SUMO-1 protein, the nucleotide sequence of which is shown in SEQ ID NO:1 and the amino acid sequence of which is shown in SEQ ID NO:2. This invention obtains the polypeptide sequence encoding the human SUMO-1 protein through gene cloning technology, and further prepares a structurally stable SUMO-C7H protein using chemical synthesis methods. 13 NO2, this probe has a stable molecular structure and is easy to synthesize and detect; SUMO-C7H 13 NO2 fluorescent probes possess high specificity and good detection sensitivity, providing an innovative research tool for exploring tumorigenesis mechanisms and assessing SUMOylation modification levels.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and relates to a SUMOylated trapping probe, its synthesis method, and its application. Specifically, it refers to a SUMOylated trapping probe (SUMO-2-(E)-5-(methylamino)pent-3-enoic acid methyl ester) (SUMO-C7H 13 NO2), its synthesis method and its application in tumor detection. Background Technology

[0002] Breast cancer is a malignant tumor originating from the epithelial tissue of the mammary glands and ducts, and its incidence rate ranks first among female malignant tumors. Currently, the pathogenesis of breast cancer is not fully understood, and effective preventive measures are lacking. It is well known that the occurrence and development of most cancers, including breast cancer, is a long and gradual pathological process. Existing diagnostic methods usually only identify the tumor when it has grown to a certain size, often delaying the optimal intervention time. Current treatments for breast cancer mainly include drug therapy, radiotherapy, and surgical resection; however, a highly efficient and universally applicable diagnostic and treatment strategy has not yet been established. Surgical resection, while removing tumor tissue, often inevitably damages or removes corresponding normal tissues and organs, affecting their physiological functions; radiotherapy, while killing tumor cells, also damages surrounding normal tissues, thus limiting its clinical application; and drug therapy, due to its limited efficacy and significant side effects, imposes a severe physiological and psychological burden on patients. Similar to other cancers, effective prevention and treatment of breast cancer still rely on a deeper understanding of its pathogenesis; therefore, gene therapy strategies are particularly important.

[0003] Small ubiquitin-associated modification (SUMOylation) is a crucial and dynamically reversible post-translational modification mechanism of proteins, studied for over two decades since its discovery. Currently, more than 3000 proteins have been identified as targets of SUMOylation, which plays a central role in regulating target protein function, involving multiple aspects such as cellular sublocalization, protein stability, signal transduction, enzyme activity, gene transcription regulation, cell cycle, and differentiation. In mammals, four SUMO protein isoforms have been identified: SUMO-1, SUMO-2, SUMO-3, and SUMO-4. Under the synergistic action of E1 activating enzymes, E2 conjugating enzymes, and E3 ligases, the C-terminal diglycine residue of the SUMO molecule covalently binds to the ε-amino group of the lysine side chain of the target protein via an isopeptide bond, thereby regulating the structure and function of the substrate protein. For example, SUMOylation can occur at lysine residue 386 of the p53 protein; PIAS family proteins can enhance p53 stability, and studies have shown that SUMOylation can increase the transcriptional activity of p53, thereby promoting apoptosis. Lysine residues at positions 254, 266, and 289 of the PTEN protein can be modified by SUMO-1 and SUMO-2, thereby downregulating the PI3K / AKT signaling pathway and inhibiting cell proliferation and tumor growth. Several proteins interacting with p53 can also undergo SUMOylation, and the roles of key signaling pathway factors such as NF-κB and PTEN in tumorigenesis and development are partly dependent on the activity of SUMO proteins. Therefore, SUMOylation systems are considered highly promising molecular targets for future cancer detection and treatment. Developing SUMO-specific fluorescent probes to reveal the dynamic changes in SUMOylation levels during tumor progression holds promise for providing new strategies for early tumor diagnosis and treatment.

[0004] SUMOylation of proteins is a dynamic and reversible process, making deSUMOylating enzymes important drug targets. DeSUMOylating enzymes (DSPs) are mainly composed of the SENP family (Sentrin / SUMO-specific proteases). These enzymes regulate the SUMOylation level and biological activity of target proteins, and their expression and activity are also regulated by various factors. Sequence alignment shows that SENPs all contain an active site of approximately 200 amino acids, belonging to the C48 cysteine ​​protease family, and can specifically cleave the isopeptide bond between SUMO and the target protein. Based on this biochemical characteristic of DSPs, molecular probes targeting cysteine ​​residues in their active site can be designed. These probes can specifically recognize and covalently bind to this cysteine ​​residue, thereby blocking their activity in cleaving the SUMO-target protein isopeptide bond and inhibiting the deSUMOylation reaction.

[0005] Due to the highly dynamic nature of SUMOylation modification, its precise intracellular localization and real-time dynamic monitoring are extremely difficult. There is an urgent need to establish detection technologies capable of spatiotemporal resolution and dynamic tracking of SUMOylation modification. Especially in some tumor cells, the level of SUMOylation modification is closely related to tumor malignancy and prognosis. Therefore, cellular localization, quantitative analysis, and dynamic process monitoring of this modification will contribute to the accurate diagnosis and prognostic assessment of malignant tumors.

[0006] The information disclosed above in this background section is only for enhancing the understanding of the background technology of this invention, and therefore may include prior art that is not known to those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide a SUMO-C7H 13 NO2 probe, its synthesis method and application: This probe can rapidly detect the subcellular localization, degree of modification and dynamic modification changes of SUMOylated proteins in cells.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a SUMO-C7H 13 NO2 probe, the molecular formula of which is SUMO-C7H 13 NO2, wherein the SUMO is a human SUMO-1 protein, the nucleotide sequence of which is shown in SEQ ID NO: 1 and the amino acid sequence of which is shown in SEQ ID NO: 2.

[0009] The present invention also provides SUMO-C7H as described above. 13 The method for synthesizing the NO2 probe includes the following steps:

[0010] Step 1: Obtain the full-length cDNA sequence of human SUMO through gene cloning;

[0011] Step 2: The plasmid containing the full-length cDNA sequence of SUMO obtained in Step 1 is used to recombinantly express the SUMO protein polypeptide in E. coli, and then purified by affinity to obtain the pure SUMO protein polypeptide.

[0012] Step 3: The purified SUMO protein peptide obtained in Step 2 is synthesized into SUMO-C7H through a fully chemical synthesis method. 13 NO2 fluorescent probe.

[0013] Preferably, step 3 is specifically as follows:

[0014] Step 3.1: Synthesis of methyl (E)-5-(methylamino)pent-3-enoate, a fluorescent reporter group

[0015] Using (E)-pent-2-en-1,5-diacid monomethyl ester as a starting material, oxalyl chloride was reacted with DMF in anhydrous THF to generate acyl chloride. After concentration under reduced pressure, the acyl chloride was dissolved in DCM and added dropwise to an ice-bath cooled aqueous methylamine solution. The mixture was stirred overnight at room temperature, washed with water, dried, and purified by column chromatography with petroleum ether / ethyl acetate as the eluent, yielding a white solid product. The product was confirmed by ¹H NMR and ESI-MS.

[0016] Step 3.2: Fluorophore thioesterification

[0017] The product from step 3.1 was dissolved in anhydrous DMF, and 3-mercaptopropionic acid, HOBt and EDC·HCl were added. The reaction was carried out at room temperature under nitrogen protection. The reaction solution was diluted with ethyl acetate, washed successively with saturated NaHCO3, 1M HCl and brine, dried and concentrated to obtain fluorophore-thioester, which was used as the donor module for the NCL reaction.

[0018] Step 3.3: Coupling of SUMO-1 protein with fluorophores

[0019] The chemically synthesized SUMO-1 protein was mixed with an excess of fluorophore-thioester in NCL buffer and reacted in the dark. The reaction specifically occurred at the α-amino group at the C-terminus or a specific Cys site of the SUMO-1 protein. The SUMO-1 fluorophore probe was purified by RP-HPLC and lyophilized, and confirmed by HRMS and fluorescence imaging.

[0020] The present invention also provides SUMO-C7H as described above. 13 Application of NO2 probes in the preparation of products for detecting SUMOylation modifications in cells, particularly for cell localization, quantitative analysis and / or dynamic modification change analysis of said modifications.

[0021] The present invention also provides SUMO-C7H as described above. 13 Application of NO2 probes in the preparation of reagents for cancer diagnosis and / or prognosis.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. This invention obtains the polypeptide sequence encoding the human SUMO-1 protein through gene cloning technology, and further prepares a structurally stable SUMO-C7H protein using chemical synthesis methods. 13 NO2, this probe has a stable molecular structure and is easy to synthesize and detect; SUMO-C7H 13 NO2 fluorescent probes possess high specificity and good detection sensitivity, providing an innovative research tool for exploring tumorigenesis mechanisms and assessing SUMOylation modification levels. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0025] Figure 1 This is a three-dimensional spatial structure diagram of the human SUMO-1 molecule of this invention;

[0026] Figure 2 This is the mass spectrum for identifying SUMO proteins in this invention;

[0027] Figure 3 The present invention is SUMO-1-C7H 13 NO2 fluorescent probe identification mass spectrum;

[0028] Figure 4 The present invention is SUMO-1-C7H 13 Schematic diagram of the imaging effect of NO2 fluorescent probe molecules in MCF7. Detailed Implementation

[0029] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0030] Example 1: Cloning and Expression of Human SUMO-1 Gene

[0031] Total RNA was extracted from human breast cancer MCF-7 cells, mRNA was purified, mRNA was reverse transcribed, and cDNA libraries were constructed. Primers were designed, and the human SUMO1 gene was screened using PCR. The 5' primer was 5'-ATGTCTGACCAGGAGGCAAAACCT-3' (SEQ ID NO: NM_003352.8), and the 3' primer was 5'-CTAAACTGTTGAATGACCCCCCGT-3' (SEQ ID NO: NM_003352.8). Positive clones were subjected to gene nucleotide sequencing. Gene sequencing results showed that the sequence encoding human SUMO-1 consists of 306 nucleotides, and the sequence from the 5' end to the 3' end is (SEQ ID NO: 1):

[0032] 1 ATGTCTGACC AGGAGGCAAA ACCTTCAACT GAGGACTTGG GGGATAAGAA

[0033] 51 GGAAGGTGAA TATATTAAAC TCAAAGTCAT TGGACAGGAT AGCAGTGAGA

[0034] 101 TTCACTTCAA AGTGAAAATG ACAACACATC TCAAGAAACT CAAAGAATCA

[0035] 151 TACTGTCAAA GACAGGGTGT TCCAATGAAT TCACTCAGGT TTCTCTTTGA

[0036] 201 GGGTCAGAGA ATTGCTGATA ATCATACTCC AAAAGAACTG GGAATGGAGG

[0037] 251 AAGAAGATGT GATTGAAGTT TATCAGGAAC AAACGGGGGG TCATTCAACA

[0038] 301 GTTTAG

[0039] The SUMO-1 sequence, the SUMO-1 sequence (SEQ ID NO:2;NP_001005781.1):Met-Ser-Asp-Gln-Glu-Ala-Lys-Pro-Ser-Thr-Glu-Asp-Leu-Gly-Asp -Lys-Lys-Glu-Gly-Glu-Tyr-Ile-Lys-Leu-Lys-Val-Ile-Gly-Gln-Asp-Ser-Ser-Glu-Ile-His- Phe-Lys-Val-Lys-Met-Thr-Thr-His-Leu-Lys-Lys-Leu-Lys-Glu-Ser-Yyr-Cys-Gln-Arg-Gln-G ly-Val-Pro-Met-Asn-Ser-Leu-Arg-Phe-Leu-Phe-Glu-Gly-Gln-Arg-Ile-Ala-Asp-Asn-His-Th r-Pro-Lys-Glu-Leu-Gly-Met-Glu-Glu-Glu-Asp-Val-Ile-Glu-Val-Tyr-Gln-Glu-Gln-Thr-Gly-Gly-His-Ser-Thr-Val

[0040] (MSDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKESYCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGGHSTV)

[0041] The three-dimensional structure of the human SUMO-1 molecule is as follows: Figure 1 As shown.

[0042] Specifically, the cloning of the SUMO-1 molecule gene includes:

[0043] 1) Total RNA extraction from human MCF7 cells:

[0044] ① Take MCF7 cells cultured in a 35mm culture dish, add 1ml of RNAiso Plus (Trizol, TAKARA, Japan), place on a decolorizing shaker, and lyse at room temperature for 5min.

[0045] ② Transfer the cell lysis buffer to a 1.5 mL centrifuge tube, add 200 μL of chloroform to the tube, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes.

[0046] ③ Place the centrifuge tube from ② into a low-temperature refrigerated centrifuge and centrifuge at 12,000 rpm for 15 minutes at 4°C.

[0047] ④ Transfer the supernatant to a 1.5 mL RNase-Free centrifuge tube, add 500 μL of isopropanol, gently invert the centrifuge tube to mix the liquid, and let it stand at room temperature for 10 min.

[0048] ⑤ Place the centrifuge tube from ④ into a low-temperature refrigerated centrifuge and centrifuge at 12,000 rpm for 10 minutes at 4°C.

[0049] ⑥ Remove the supernatant, add 1 mL of 75% ethanol to the centrifuge tube, and gently pipette to precipitate the RNA.

[0050] ⑦ Place the centrifuge tube from ⑥ into a low-temperature refrigerated centrifuge and centrifuge at 5000 rpm for 3 minutes at 4°C.

[0051] ⑧ Carefully remove the supernatant with a pipette, and let it dry at room temperature for several minutes.

[0052] ⑨ Add 30 μL of RNase-free water to the centrifuge tube in ⑧ to dissolve the RNA.

[0053] 2) Construction of human cDNA library:

[0054] I. cDNA first-strand synthesis (mRNA reverse transcription):

[0055] ① Add 1.0 μL of total RNA from human MCF7 cells, 1.0 μL of LOligo(dT) primer, and 5.0 μL of RNase-free ddH2O to a PCR tube that has had RNase removed, bringing the total volume to 7 μL. Mix well and centrifuge briefly (2000 rpm, 30 s). After centrifugation, incubate at 72°C for 10 minutes. After incubation, incubate the centrifuge tube at 4°C for 2 minutes.

[0056] ② Add the following reagents to the centrifuge tubes: 2.0 μL 5×M-MLV Buffer, 0.5 μL 10 mM dNTPMix, 0.5 μL 40 U / μL RNase Inhibitor, and 0.25 μL RTase M-MLV. Mix the reagents in the centrifuge tubes and centrifuge briefly (2000 rpm, 30 s). Incubate at 42 °C for 60 min, then at 70 °C for 15 s. After the incubation period, place the centrifuge tubes on ice to stop the synthesis and set aside for later use.

[0057] II. Amplification of the SUMO1 gene using polymerase chain reaction (PCR):

[0058] ① Mix 1 μL cDNA template, 0.25 μL Taq enzyme, 10 μL 5×PCR buffer, 1 μL 10 mM dNTP, 1.0 μL 5' PCR primer, 1.0 μL 3' PCR primer and 35.75 μL ddH2O in a PCR tube, and centrifuge to allow the mixture to collect at the bottom of the tube.

[0059] ② Amplify in a PCR instrument according to the following program: 95℃, 5 min; 35 cycles: 94℃, 30 sec, 56℃, 30 s, 72℃, 1 min. After the cycles are completed, incubate at 72℃ for 10 min. After PCR, store in a -80℃ freezer.

[0060] 3) Screening of human SUMO-1 gene clones:

[0061] After the PCR reaction, the PCR products were electrophoresed on a 1% agarose gel at a constant voltage of 120V for 30 min. The gel was then observed and photographed using a gel imaging system. The gel containing the SUMO-1 target fragment was excised and recovered using an agarose gel DNA recovery kit. The recovered target fragment was ligated into the pGEX-4T-3 vector and transformed into DH5α competent cells. The cells were plated and subjected to ampicillin and blue-white screening. Single colonies were picked and the insert size was detected by PCR using M13 primers. Positive colonies were picked, and plasmids were extracted by shaking. Nucleotide sequencing was then performed, and the resulting amino acid sequences were translated.

[0062] 4) SUMO protein expression and in vitro purification

[0063] pGEX-4T-3-SUMO1 was transformed into E. coli BL21. After screening for positive clones, the cells were cultured in shake flasks until they reached the logarithmic growth phase. After induction with IPTG for 4 hours, the cells were cultured for another 16 hours, followed by sonication and protein purification using an affinity purification column. The protein was then identified by MS (e.g., ...). Figure 2 (As shown).

[0064] Example 2: SUMO-C7H 13 NO2 probe molecule synthesis

[0065] 1) Synthesis and activation of fluorescent reporter groups

[0066] ① Synthesis of (E)-5-(methylamino)pent-3-enoic acid methyl ester

[0067] Starting with (E)-pent-2-en-1,5-dicarboxylic acid monomethyl ester, 1.2 equivalents of oxalyl chloride and a catalytic amount of DMF were added to anhydrous THF, and the mixture was stirred at 0°C for 1 hour to generate the corresponding acyl chloride. The solvent was removed under reduced pressure, and the residue was dissolved in anhydrous DCM and slowly added dropwise to a reaction system cooled in an ice bath containing excess methylamine (40% aqueous solution). The mixture was stirred overnight at room temperature, washed with water, dried, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 3:1) to give a white solid (E)-5-(methylamino)pent-3-enoic acid methyl ester. The structure was confirmed by ¹H NMR and ESI-MS.

[0068] ② Fluorophore thioesterification

[0069] The product from step ① was dissolved in anhydrous DMF, and 1.5 equivalents of 3-mercaptopropionic acid, 1.5 equivalents of HOBt, and 1.5 equivalents of EDC·HCl were added. The reaction mixture was reacted at room temperature for 12 hours under nitrogen protection. The reaction solution was diluted with ethyl acetate and washed successively with saturated NaHCO3 solution, 1M HCl, and brine. The organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain the key intermediate fluorophore-thioester. This compound serves as a donor module for NCL reactions.

[0070] 2) Coupling of SUMO-1 protein with fluorophores

[0071] ① NCL reaction of SUMO-1 with fluorophore-thioester

[0072] The chemically synthesized SUMO-1 protein (whose native sequence contains a reactive inert cysteine ​​residue, or a site-directed mutagenesis introducing a cysteine ​​residue specifically for linking, replacing Val at position 101 with Cys) was mixed with an excess of the fluorophore-thioester (step 1) product. The reaction was carried out in NCL buffer (as above), pH adjusted to 7.2, with the addition of 2% thiophenol. The reaction was incubated at 25°C in the dark for 4–8 hours. This reaction is highly specific, occurring only between the α-amino group at the C-terminus of the SUMO-1 protein (or the introduced specific Cys site) and the fluorophore-thioester. After the reaction was complete, the protein was finely purified using RP-HPLC, and the main peak was separated and collected.

[0073] ② After lyophilization, the final product—SUMO-1-(E)-5-(methylamino)pent-3-enoic acid methyl ester probe—was obtained. Final confirmation was performed using high-resolution mass spectrometry (HRMS) and fluorescence imaging. After trypsin digestion, LC-MS was used for identification (e.g., ...). Figure 3 (As shown).

[0074] Example 3: SUMO-C7H 13 Application of NO2 probe molecules in MCF7 cells

[0075] MCF7 cells were seeded in 35 mm cell culture plates, and fusion experiments were performed when the cell confluence reached approximately 40%. SUMO-C7H 13 NO2 probe molecules were co-incubated with MCF7 cells, and fluorescence microscopy was performed after 36 hours. The results are as follows: Figure 4 As shown.

[0076] The probe detection method is as follows:

[0077] Because SUMO proteins can specifically recognize lysine residues of substrate proteins, the modified protein undergoes SUMOylation through the action of enzymes involved in the process, converting SUMO-C7H... 13 NO2 probe molecules bind directly to the substrate. Since the probe molecules are autofluorescent, with an excitation wavelength of 485 nm and an emission wavelength of 515 nm, the intensity and position of the autofluorescence emission light of the probe molecules can be detected to locate, quantify, and monitor the dynamic changes of intracellular SUMOylation modification.

[0078] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A SUMO-C7H 13 NO2 probe, characterized in that: The molecular formula of the probe is SUMO-C7H. 13 NO2, wherein the SUMO is a human SUMO-1 protein, the nucleotide sequence of which is shown in SEQ ID NO: 1 and the amino acid sequence of which is shown in SEQ ID NO:

2.

2. A SUMO-C7H as described in claim 1 13 The method for synthesizing NO2 probes is characterized by: Includes the following steps: Step 1: Obtain the full-length cDNA sequence of human SUMO through gene cloning; Step 2: The plasmid containing the full-length cDNA sequence of SUMO obtained in Step 1 is used to recombinantly express the SUMO protein polypeptide in E. coli, and then purified by affinity to obtain the pure SUMO protein polypeptide. Step 3: The purified SUMO protein peptide obtained in Step 2 is synthesized into SUMO-C7H through a fully chemical synthesis method. 13 NO2 fluorescent probe.

3. A SUMO-C7H according to claim 2 13 The method for synthesizing NO2 probes is characterized by: The specific steps of step 3 are as follows: Step 3.1: Synthesis of methyl (E)-5-(methylamino)pent-3-enoate, a fluorescent reporter group Using (E)-pent-2-en-1,5-diacid monomethyl ester as a starting material, oxalyl chloride was reacted with DMF in anhydrous THF to generate acyl chloride. After concentration under reduced pressure, the acyl chloride was dissolved in DCM and added dropwise to an ice-bath cooled aqueous methylamine solution. The mixture was stirred overnight at room temperature, washed with water, dried, and purified by column chromatography with petroleum ether / ethyl acetate as the eluent, yielding a white solid product. The product was confirmed by ¹H NMR and ESI-MS. Step 3.2: Fluorophore thioesterification The product from step 3.1 was dissolved in anhydrous DMF, and 3-mercaptopropionic acid, HOBt and EDC·HCl were added. The reaction was carried out at room temperature under nitrogen protection. The reaction solution was diluted with ethyl acetate, washed successively with saturated NaHCO3, 1M HCl and brine, dried and concentrated to obtain fluorophore-thioester, which was used as the donor module for the NCL reaction. Step 3.3: Coupling of SUMO-1 protein with fluorophores The chemically synthesized SUMO-1 protein was mixed with an excess of fluorophore-thioester in NCL buffer and reacted in the dark. The reaction specifically occurred at the α-amino group at the C-terminus or a specific Cys site of the SUMO-1 protein. The SUMO-1 fluorophore probe was purified by RP-HPLC and lyophilized, and confirmed by HRMS and fluorescence imaging.

4. The SUMO-C7H as described in claim 1 13 The application of the NO2 probe in the preparation of products for detecting SUMOylation modification in cells is characterized by: Specifically, it is used for cell localization, quantitative analysis, and / or dynamic modification change analysis of the modifications.

5. The SUMO-C7H as described in claim 1 13 Application of NO2 probes in the preparation of reagents for cancer diagnosis and / or prognosis.

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