Detection method for promoting combination of SP1 and super enhancer by Niraparib based on combination of PLIP and ChIP-qPCR

Abstract

The invention discloses a protein-DNA (Deoxyribose Nucleic Acid) combination detection method integrating molecular docking, interaction analysis and micro experiment verification, which is used for analyzing a regulation mechanism of an SP1 transcription factor and an SPHK1 gene super enhancer in triple negative breast cancer. The method comprises the following steps: firstly, screening an SPHK1-SE core binding transcription factor SP1 through PROMO prediction, and further constructing an SP1-SE compound model by adopting a high-precision protein-nucleic acid docking technology; analyzing an atomic-scale interaction network based on a PLIP tool, and revealing a key action interface; the specific primer of the SE is further designed, a ChIP-qPCR system is established, and the SP1-SE binding activity (plt; 0.001) can be obviously enhanced by analyzing the PARP inhibitor Niraparib. The invention provides a methodological basis and an experimental basis for developing TNBC therapeutic drugs on a targeted SP1-SE interaction interface.

Description

Technical Field

[0001] The present invention belongs to the intersection of tumor molecular biology and precision medicine, and specifically relates to a method for discovering therapeutic targets for triple-negative breast cancer based on the interaction mechanism between SP1 protein and super enhancer. In particular, it involves analyzing the binding pattern of SP1 and SE by integrating molecular docking, interaction analysis and ChIP-qPCR verification technology, and revealing the molecular mechanism by which the PARP inhibitor Niraparib enhances this binding. Background Art

[0002] Triple-negative breast cancer (TNBC) lacks estrogen receptor (ER), progesterone receptor (PR), and HER2 expression, limiting clinical treatment options. Sphingosine kinase SPHK1, which metabolizes sphingosine to sphingosine-1-phosphate in the sphingolipid metabolism pathway, is abnormally overexpressed in TNBC. Its activity is dynamically regulated by superenhancers (SEs) and is closely associated with tumor proliferation, metastasis, and chemoresistance. However, the precise transcription factors through which SEs mediate SPHK1 regulation remain unclear, hindering the development of targeted intervention strategies. The inventors used the PROMO website to predict transcription factors that bind to SPHK1-SE. SPHK1, located in the forward strand, had the highest prediction score of 13.72. Although SP1 participates in tumorigenesis as a key transcription factor, its specific binding mode with SE (such as binding energy, key residues and dynamic regulatory mechanism) still lacks atomic-level analysis. Traditional technologies have significant limitations: conventional ChIP-qPCR technology usually requires a large number of cells. It is a complex multi-step technology, including cross-linking, chromatin fragmentation, immunoprecipitation, elution, reversal of cross-linking, DNA purification and quantification. Problems in any step may affect the final result; in addition, there is a gap in the research on the pharmacological regulation of SE and transcription factors, which restricts the development of targeted drugs to regulate SE.

[0003] PLIP (Protein-Ligand Interaction Profiler) is a tool for automatically detecting and analyzing protein-ligand interactions, commonly used in structural biology and drug discovery research. It can identify key interactions such as hydrogen bonds, hydrophobic interactions, salt bridges, π-stacking, halogen bonds, and generate visual reports. Chromatin immunoprecipitation (ChIP) is a key technology for studying the specific binding of proteins to DNA. It fixes protein-DNA complexes in living cells through cross-linking, uses specific antibodies to enrich DNA fragments bound to the target protein, and finally analyzes the bound DNA sequence. Quantitative polymerase chain reaction (ChIP-qPCR) is often used as a detection method downstream of ChIP and is widely used in the study of gene expression regulation, epigenetics, and disease mechanisms. Summary of the Invention

[0004] In response to the problems in the existing technology such as the unclear dynamic regulatory network of SPHK1-SE, the lack of atomic-level analysis of the SP1-SE interaction pattern, and the gap in pharmacological regulation research, the present invention innovatively integrates high-precision protein-nucleic acid docking (HDOCK), PLIP interaction analysis and ChIP-qPCR verification system, revealing for the first time the strong binding characteristics between SP1 and SE (binding energy -294.68 kcal / mol, confidence level 0.9475). Through PLIP, 11 pairs of hydrogen bonds, 2 pairs of salt bridges and π-π stacking and other key interactions were identified, and the ARG-13, TYR-9 and other residues of SP1 were locked in to form a stable interface with A-1067 and C-1069 of SE. ChIP-qPCR technology was used to verify that the PARP inhibitor Niraparib can significantly enhance SP1-SE binding (p<0.001), providing a new method for analyzing the interaction between proteins and DNA.

[0005] The present invention discloses a method for detecting the binding of SP1 protein to SPHK1 super enhancer based on molecular docking-PLIP interaction analysis-micro-quantitative ChIP-qPCR. The three-level technical system breaks through the bottleneck of traditional research: first, the HDOCK platform is used to construct a high-precision SP1-SE binding model (binding energy -294.68kcal / mol, confidence level 0.9475), and the surface mode simulation shows that the two form a stable complex through surface complementarity; secondly, the atomic-level interaction network is analyzed by the PLIP tool, and 11 pairs of hydrogen bonds, 2 pairs of salt bridges and 1 pair of π-π stacking are identified. Specifically, a salt bridge is formed between A-1067 of the SE-A ​​chain and ARG-13 of SP1, a hydrogen bond is formed between A-1067 of the SE-A ​​chain and SER-8 of SP1, and a hydrogen bond is formed between TYR-9 of SP1 and C-1069 of the SE-A ​​chain. In addition, there are multiple groups of hydrophobic interactions, which together maintain the stable binding of proteins and nucleic acids. SE-specific primers (F-SE: 5′-CCTGTGTGACAGCCTTGGTTGAAACCC-3′; R-SE: 5′-GAGTGTGGACATTAGGGTCGAGAAACTC-3′) were further designed and a ChIP-qPCR system was established to verify that the PARP inhibitor Niraparib can significantly enhance SP1-SE binding activity (p<0.001), providing a new method for analyzing the binding between proteins and DNA, and providing a patentable methodological basis for analyzing the binding between proteins and DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 HDOCK server docking results: SP1 (green); SE-A ​​chain (yellow); SE-B chain (purple).

[0007] Figure 2 :Surface visualization of docking results (A) SP1 (green); (B) SE-A ​​chain (yellow) and SE-B chain (purple).

[0008] Figure 3 : PLIP analysis and demonstration of the interaction between SP1 and SE.

[0009] Figure 4 : pyMOL shows the interaction details between SP1 and SE, where A / B is Figure 3 Detailed diagram of the binding mode in .

[0010] Figure 5 : ChIP-qPCR verified that Niraparib promoted the binding of SP1 and SPHK1-SE. DETAILED DESCRIPTION

[0011] Experimental materials involved in this invention:

[0012] 1. Cell lines: TNBC cell lines, including MDA-MB-231 and MDA-MB-468, were purchased from the cell bank of Shanghai Institute of Biochemistry and Cell Biology (SIBCB, Shanghai, China).

[0013] 2. Reagents: Anti-SP1 (1:1000, proteintech, Lot No: 21962-1-AP); IgG isotype control antibody (Cell Signaling Technology, #2729); Niraparib (Pubchem CID: 24958200, GlpBio, GC17802)

[0014] Example 1 Obtaining protein structure:

[0015] Uniprot (Universal Protein) is a protein database that contains protein sequences, functional information, and research article indexes. It integrates resources from three major databases: the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB), and the Protein Information Resource (PIR). Using SP1 as the keyword, we searched for protein structures from Uniprot and selected the SP1_HUMAN (Uniprot ID: P08047) NMR structure of 6UCP as the receptor protein. We then used AlphaFold3 to build a model based on the DNA sequence. The model with the highest confidence, as ranked by the software plddt, was selected as the experimental model.

[0016] The PROMO website was used to predict the transcription factors that bind to SPHK1-SE, with an error tolerance of 1%. The JASPAR website was used to predict the transcription factor binding sites that bind to SPHK1-SE. The top four transcription factors and their binding sites were listed according to the score ranking (Table 1).

[0017] Table 1 Prediction of transcription factor binding sites for SPHK1-SE by combining PROMO and JASPAR websites

[0018]

[0019] Example 2 Protein (SP1)-Nucleic Acid (SPHK1-SE) Docking:

[0020] Protein-nucleic acid docking uses molecular simulation methods to predict near-native structures of complexes based on known three-dimensional protein and nucleic acid structures. Docking is a sampling and scoring process. This paper uses the HDOCKlite v1.1 local server for protein-nucleic acid docking. HDOCK combines physics-based and bioinformatics approaches to develop efficient molecular docking algorithms and accurate scoring functions for biomolecular interactions. HDOCK attempts to sample all binding modes for protein and nucleic acid structures. Scoring functions are then used to rank the sampling process and the resulting binding modes. Due to the lack of information about the binding site, ab initio global docking is often required to sample six degrees of proposed binding modes (three rotations plus three translations). This scoring function uses two metrics: the docking score, with more negative docking scores indicating a more likely binding model; and the confidence score. When the confidence score is above 0.7, the probability of binding between the two molecules is high; when the confidence score is between 0.5 and 0.7, the two molecules are considered likely to bind; and when the confidence score is below 0.5, the probability of binding between the two molecules is low.

[0021] The results of HDOCK docking are shown in Table 1. It can be seen that the docking score of model_1 with the lowest binding energy is -294.68 and the confidence score is 0.9475 ( Figure 1 ). This shows that the binding between protein and nucleic acid is stronger in this form. The binding process between protein and nucleic acid is displayed in the form of Surface ( Figure 2 ).

[0022] Example 3 Analysis of protein (SP1)-nucleic acid (SPHK1-SE) interaction:

[0023] Protein-nucleic acid complexes are formed through non-covalent interactions between proteins, primarily consisting of hydrophobic, hydrogen-bonding, and electrostatic interactions. The PLIP interaction analysis platform was used to comprehensively characterize and systematically analyze the binding interfaces of protein-nucleic acid complexes. pyMOL was then used to further refine the interaction details.

[0024] PLIP is an analytical tool for protein-ligand non-covalent interactions. In addition to considering important influencing factors such as size and shape, residue surface complementarity, residue interface tendency, etc., it can also analyze the non-covalent interactions of protein-ligand complexes at the atomic level, including hydrophobic interactions, hydrogen bonds, water-mediated interactions, salt bridges, π stacking, cation interactions, and halogen bonds. Its detection mechanism is mainly based on the spatial position and geometric relationship between atoms. The specified distance is There are hydrophobic interactions between all hydrophobic atom pairs within the When the minimum angle at the hydrogen bond donor is greater than 100°, a hydrogen bond is considered to be formed between the acceptor and donor groups. When , a salt bridge is considered to be formed between the acceptor and donor groups.

[0025] ① Hydrophobic interaction

[0026] The aggregation of hydrophobic molecules or chemical groups in aqueous solution reduces their contact surface area with water, leading to the release of water molecules into the body. With the increase in entropy, the coordination of hydrophobic amino acids with the corresponding ligand groups plays a dominant role in protein-ligand binding.

[0027] ② Hydrogen bond

[0028] Hydrogen bonding is considered the most important of all directional covalent interactions, and it is hypothesized that each additional hydrogen bond increases the binding affinity of a ligand by an order of magnitude. Polar covalent bonds form between a donor group (DH), which provides the positive end in the form of a hydrogen atom, and an acceptor group with high electron density. Typical values ​​of hydrogen bond energy range from 10 to 40 kJ / mol.

[0029] ③ Salt bridge

[0030] Salt bridges play a key role in molecular recognition. They are formed by ligand atoms of opposite charge being more than The binding energy it contributes is around 3-13 kJ / mol.

[0031] ④Interactions between aromatic rings (π stacking)

[0032] π stacking is controlled by electrostatic interactions, van der Waals forces, and hydrophobic interactions. to The angles range from 30° to 90+°. The interaction energies range from 8 kJ / mol for sandwich structures to 11 kJ / mol for π stacking.

[0033] The interaction between SP1 and SE analyzed by PLIP ( Figure 3 ), and then use pyMOL to fill in the details of the interaction ( Figure 4 ). SP1 is used as the reference chain for interaction analysis. Green represents SP1, yellow represents SE-A ​​chain, and purple represents SE-B chain. Eleven pairs of hydrogen bonds are formed between proteins ( inside), 2 pairs of salt bridges, 1 pair of Pi-Pi stacking interactions such as Figure 4As shown in A, a salt bridge is formed between A-1067 of SE-A ​​chain and ARG-13 of SP1 (yellow dashed line), a hydrogen bond is formed between A-1067 of SE-A ​​chain and SER-8 of SP1 (blue solid line), a hydrogen bond is formed between TYR-9 of SP1 and C-1069 of SE-A ​​chain, a salt bridge is formed between LYS-31 of SP1 and G-1075 of SE-A ​​chain, a Pi-Pi stacking interaction is formed between SP1SER-8 and G-732 of SE-B chain, and A- Hydrogen bonds are formed between 1064 and C-1065 of SP1 and SER-8; between THR-26 of SP1 and G-764 of SE-B chain; between ARG-25 of SP1 and T-763 of SE-B chain; between ARG-22 of SP1 and G-1035 of SE-A ​​chain; between ARG-16 of SP1 and G-1035 of SE-A ​​chain; and between T-791 of SE-B chain and THR-15 of SP1. In addition, multiple hydrophobic interactions exist (gray dashed lines).

[0034] Example 4 Cultivation of cell lines:

[0035] MDA-MB-231 (ER- / PR- / HER2-, BRCA1 wild-type) and MDA-MB-468 (ER- / PR- / HER2-, BRCA1 wild-type) cells were cultured in RPMI-1640 medium (Sigma-Aldrich, R8758) supplemented with 10% fetal bovine serum (FBS; Gibco, 10099141) and 1% penicillin-streptomycin (HyClone TM All cell lines were authenticated by short tandem repeat (STR) typing within six months before the experiment and were incubated at 37°C in 5% CO2.

[0036] Example 5: Confirmation of the Binding of SP1 and SPHK1-SE by ChIP-qPCR:

[0037] To further investigate whether niraparib promotes the binding of SP1 transcription factors to SE sequences, the inventors used SnapGene software to design specific primers targeting the blue SE sequence (Forward-SE-(homo) / Reverse-SE-(homo)). Chromatin immunoprecipitation (ChIP) protocols were as follows: cells were cross-linked with 1% formaldehyde (Sigma, #F8775) at 25°C for 10 minutes, followed by quenching with 125 mM glycine. Nuclei were isolated using NP-40 lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 0.5% NP-40), and chromatin was fragmented into 200-500 bp fragments by sonication (6 cycles of 30 seconds on / 30 seconds off). Chromatin was then incubated at 4°C with 5 μg of target-specific SP1 antibody or IgG isotype control antibody (Cell Signaling). Technology, #2729) for immunoprecipitation overnight, followed by incubation with ProteinA / G magnetic beads (ThermoFisher, #88802) for 2 hours. The beads were washed sequentially with low salt buffer (20mM Tris-HCl, pH 8.0, 150mM NaCl, 0.1% SDS), high salt buffer (500mM NaCl), LiCl buffer (250mM LiCl, 1% NP-40) and TE buffer. Cross-linking was reversed by incubation with 200mM NaCl at 65°C for 6 hours, followed by proteinase K digestion (ThermoFisher, #AM2548). SYBR Green Master Mix and primers covering the SE region, and purified DNA was analyzed by qPCR. Input DNA was used as a normalization control. Enrichment was calculated by the ΔΔCt method and expressed as the fold change of the IgG control. Statistical significance was determined by a two-tailed Student's t-test (n = 3 biological replicates; *p < 0.05, **p < 0.01, ***p < 0.001). ChIP-qPCR experiments showed that Niraparib promoted the binding of SP1 transcription factor to SE sequences in TNBC cells ( Figure 5 ).

[0038] The primers used for real-time quantitative PCR experiments are as follows:

[0039]

[0040] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A protein-DNA binding detection method integrating molecular docking, interaction analysis and micro-quantification experimental verification, characterized in that: The above method is used to analyze the binding mechanism of SP1 transcription factor and SPHK1 gene super enhancer SPHK1-SE in triple-negative breast cancer, and specifically includes the following steps: (1) Obtain the SP1 protein structure and use the PROMO website to predict the transcription factors that bind to SPHK1-SE; (2) Use HDOCK to dock protein SP1 and nucleic acid SPHK1-SE; (3) The interaction between SP1 and SPHK1-SE was analyzed using the PLIP interaction analysis platform, and the interaction details were supplemented by pyMOL; (4) ChIP-qPCR technology was used to verify that the PARP inhibitor Niraparib can enhance the binding between SP1 and SPHK1-SE.

2. The method according to claim 1, characterized in that In step (1), the transcription factors binding to SPHK1-SE were predicted using the PROMO website, with the error tolerance set to 1%. The transcription factor binding sites binding to SPHK1-SE were predicted using the JASPAR website and listed according to the score ranking.

3. The method according to claim 1, characterized in that The docking in step (2) includes two indicators: one is the docking score, where a more negative docking score indicates a more likely binding model; the other is the confidence score. When the confidence score is higher than 0.7, the possibility of the two molecules binding is very high; when the confidence score is between 0.5 and 0.7, it is considered that the two molecules can bind; when the confidence score is lower than 0.5, it is considered that the possibility of the two molecules binding is low.

4. The method according to claim 1, wherein The analysis in step (3) is as follows: when the maximum distance between the hydrogen bond acceptor and the donor is less than When the minimum angle at the hydrogen bond donor is greater than 100°, a hydrogen bond is considered to be formed between the acceptor and the donor group; when the distance between the two oppositely charged centers is within When , a salt bridge is considered to be formed between the acceptor and donor groups.

5. The method according to claim 1, wherein The verification using ChIP-qPCR technology in step (4) is specifically as follows: cell lines MDA-MB-231 and MDA-MB-468 are cultivated and verified by designing specific primers for SE sequences.

6. Use of the method according to any one of claims 1 to 5 in preparing a drug for treating triple-negative breast cancer.

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