A rapid design method for fluorescent probes to discover and detect preliminary screening indicators of tumors

Abstract

The present invention discloses a method for rapid design of fluorescent probes for preliminary screening indicators of tumors. First, zinc finger proteins, biomarkers related to tumor invasion and migration, are selected. Quinoline is selected as the parent structure based on its structural characteristics. Then, the zinc finger proteins are transformed using the concept of combinatorial chemistry to obtain a reasonable probe candidate library. Then, a gradient optimized conformation search algorithm is used to specify a zinc finger protein, and the binding effect between the zinc finger protein and various candidates is scored and evaluated to find the structure with the highest score. The spectral properties are preliminarily verified. Finally, by comparing the serum test results of breast cancer patients and healthy people, it is proved that the fluorescent probe has a good signal response to the sample, and the application potential of the total amount of zinc finger proteins as a preliminary screening indicator for tumors is explored. The method in the present invention can accurately and efficiently design fluorescent probes for exploring preliminary screening indicators for tumors, providing a new idea for realizing rapid screening of tumors.

Description

Technical Field

[0001] The invention relates to a rapid design method of a fluorescent probe for exploring and detecting preliminary screening indicators of tumors. Background Art

[0002] Tumor invasion and metastasis are the main causes of poor prognosis and death in patients with malignant tumors. How to quickly diagnose the invasion and migration of patients' tumors has always received widespread attention. At present, pathological biopsy is the gold standard for clinical judgment of whether a tumor will metastasize, but it takes more than a week and is not a rapid screening method. In addition, while waiting for the biopsy results, the patient's condition is likely to change, which will hinder the doctor from determining the most accurate treatment plan and even miss the best treatment time. At the same time, this process brings great psychological pressure to patients. At the same time, the process of discovering new tumor indicators in clinical body fluid samples itself takes a long time and consumes a lot of manpower and material resources.

[0003] Small molecule fluorescent probes are one of the reliable tools for exploring life mechanisms and pathological mechanisms. They are highly sensitive, highly selective, and stable in nature, and are one of the important methods for assisting disease diagnosis. Similar to drug design, the design steps of probes are cumbersome, costly, and time-consuming, and require a large number of experiments to verify. Therefore, there is an urgent need for a rapid design and screening system for fluorescent probes that can overcome the above shortcomings, and through this system, new tumor indicators can be discovered in a time-saving and labor-saving manner.

[0004] In recent years, the update and iteration of computational chemistry software has made it possible to predict the properties of chemical structures. Researchers use software to predict structural properties, but there is still a lack of a complete and effective system for how to quickly design and screen suitable fluorescent probe structures for a certain disease biomarker. Therefore, the present invention uses computational chemistry software as a tool, takes zinc finger protein, a disease biomarker closely related to tumor metastasis, as an example, and takes the serum of breast cancer patients as the test sample to set up a complete process system, which can effectively and quickly screen out effective fluorescent probe structures, discover new tumor indicators, and achieve auxiliary diagnosis of diseases. It can also be used as a disease risk indicator for routine health screening. Summary of the invention

[0005] In the design and optimization process of existing fluorescent probes for diagnosing the malignancy of cancer, a large number of experiments are often required to synthesize and verify suitable fluorescent structures. The present invention combines simulation calculation with experimental verification, considers zinc finger proteins as new tumor indicators for clinical data verification, and provides a fluorescent probe design system for discovering and detecting preliminary screening indicators of tumors. According to the structural characteristics of zinc finger proteins, quinoline is selected as the fluorescent parent structure, and the parent structure is derivatized in a targeted manner within a small range to obtain a potential fluorescent probe candidate structure. This method is both economical and convenient and can achieve disease screening.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] Step 1: Select zinc finger proteins closely related to tumor invasion and migration through existing databases and literature, and select quinoline as the fluorescent parent structure based on its structural characteristics;

[0008] Step 2: Using the concept of combinatorial chemistry to modify quinoline, that is, attaching specific auxiliary groups to the structure of quinoline to obtain a batch of probe candidates;

[0009] Step 3: Use ChemDraw chemical software to construct the molecular structure of the probe candidate, and use the AutodockVina program under Autodock4.2 to score and evaluate the binding effect between the biomarker and various probe candidates using the fast gradient optimization conformation search algorithm. The scoring results show that 8-hydroxyquinoline-5-sulfonic acid nitrogen oxide is the optimal structure for docking results;

[0010] Step 4: Use quantum chemistry software Gaussian to optimize the nitrogen and oxygen of 8-hydroxyquinoline-5-sulfonic acid, using CAM-B3LYP functional and 6-311G* basis set no less than TDDFT;

[0011] Step 5: Take the structure obtained in the previous step, optimize the S1 state with a functional not lower than PBE1PBE and a 6-311G* basis set, calculate the information of the 5 lowest energy excited states, and obtain the wfn file containing the wave function information and the output result file;

[0012] Step 6: Use the wave function analysis software Multiwfn to plot the UV absorption spectrum and fluorescence spectrum of 8-hydroxyquinoline-5-sulfonic acid nitrogen oxide, and calculate the value of the Stokes shift. If the value is greater than 100, it is determined that the structure has good potential for application as a fluorescent probe;

[0013] Step 7: Use the wave function analysis software Multiwfn to analyze the average local ionization energy and electrostatic potential of the nitrogen oxide of 8-hydroxyquinoline-5-sulfonic acid to determine the specific binding site of the structure with the zinc finger protein;

[0014] Step 8: The probe was synthesized and used to detect the serum of breast cancer patients. The fluorescence area intensity was analyzed and calculated using a fluorescence analyzer to observe whether there were differences in the serum results of breast cancer patients with different malignancy levels. The results showed that when the fluorescence area was greater than 600, it indicated that the patient's tumor was most likely not to have metastasized, and when the fluorescence area was less than 500, the patient's tumor was most likely to have metastasized.

[0015] Furthermore, after the molecular model is successfully constructed in step 3, the molecular mechanics method should be used to preliminarily optimize the constructed geometric configuration under the MM2 force field to obtain the stable configuration with the lowest energy.

[0016] Furthermore, in step 4, the optimized molecular structure is taken to construct an input file of the quantum chemistry software Gaussian, and the structure of the cyclic compound is optimized using the CAM-B3LYP method and 6-311G* basis set which are not inferior to DFT, and the quantum chemistry parameters and checkpoint files are obtained.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1) A fluorescent probe method for quickly and conveniently discovering and detecting tumor preliminary screening indicators was invented. This method can also be applied to the selection of tumor markers, which is more conducive to promotion and use;

[0019] 2) The newly discovered tumor marker -- zinc finger protein total amount can help doctors predict the patient's condition as early as possible, help doctors determine a more accurate treatment plan, and reduce the patient's psychological pressure. It can also be used in routine health examinations;

[0020] 3) Apply quantum chemical calculations and wave function analysis, examine the binding energy between the candidate fluorescent structure and the target protein, and use parameters such as Stokes shift as prediction descriptors to establish the best prediction index, increase the accuracy of the prediction, and improve the prediction ability;

[0021] 4) By examining the electrostatic potential and the average local ionization energy and predicting the corresponding reaction sites, the binding mode of the candidate fluorescent structure to the target protein can be predicted more intuitively and clearly. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The present invention uses quinoline as the parent structure and attaches specific auxiliary groups thereto to obtain nine fluorescent structure candidates;

[0023] Figure 2 is the binding energy result of docking 9 fluorescent structure candidates with disease biomarkers in the present invention;

[0024] Figure 3is the spectral property of 8-hydroxyquinoline-5-sulfonic acid nitrogen oxide in the present invention;

[0025] Figure 4 is the average local ionization energy of the nitrogen oxide of 8-hydroxyquinoline-5-sulfonic acid in the present invention;

[0026] Figure 5 is the electrostatic potential of the nitrogen oxide of 8-hydroxyquinoline-5-sulfonic acid in the present invention;

[0027] Figure 6 The fluorescence intensity area of ​​the serum of breast cancer patients detected by 8-hydroxyquinoline-5-sulfonic acid nitrogen oxide in the present invention; 1 is the result of a fluorescence intensity area less than 400, 2 is the result of a fluorescence area between 400 and 600, and 3 is the result of a fluorescence area greater than 600; DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Specific implementation method:

[0030] Step 1: Through existing databases and literature, zinc finger protein was selected as a biomarker related to tumor invasion and migration. Based on the structural characteristics of this marker, it was found that quinoline has good selectivity for zinc ions in the zinc finger protein structure, so quinoline was selected as the fluorescent parent structure;

[0031] Step 2: Use the concept of combinatorial chemistry to modify it, that is, connect specific auxiliary groups to the structure of quinoline to obtain a group of probe candidates. The candidate structures are as follows Figure 1 As shown;

[0032] Step 3: Use ChemDraw chemical software to construct the molecular structure of the probe candidate, and use the Autodockvina program in Autodock4.2 to score and evaluate the binding effect of the biomarker and the probe candidate using the fast gradient optimization conformation search algorithm, and select the structure with the best score. The results are as follows: Figure 2 As shown: 8-Hydroxyquinoline-5-sulfonic acid nitrogen oxide has the lowest structural energy with the model protein and the best score;

[0033] Step 4: Take the best scoring structure and optimize it using the quantum chemistry software Gaussian, using the CAM-B3LYP functional and 6-311G* basis set no less than TDDFT;

[0034] Step 5: Take the structure obtained in the previous step, optimize the S1 state with a functional not lower than PBE1PBE and a 6-311G* basis set, calculate the information of the 5 lowest energy excited states, and obtain the wfn file containing the wave function information and the output result file;

[0035] Step 6: Use the wave function analysis software Multiwfn to plot the UV absorption spectrum and fluorescence spectrum of the structure and calculate the value of the Stokes shift. The Stokes shift value of 8-hydroxyquinoline-5-sulfonic acid nitrogen oxide is greater than 100, indicating that the structure has good potential for application as a fluorescent probe;

[0036] Step 7: Use the wave function analysis software Multiwfn to analyze the average local ionization energy and electrostatic potential of the structure, such as Figure 4 , 5 As shown: Both figures have a minimum point at the nitrogen-oxygen bond on the structure, so it is judged that the structure is combined with the zinc ion in the biomarker in the form of an ionic bond at the nitrogen-oxygen bond on the structure;

[0037] Step 8: Synthesize probes and use them to detect 44 sera from breast cancer patients, collect 18 sera from healthy people as controls, and use a fluorescence analyzer to analyze and calculate the fluorescence area intensity, such as Figure 6 As shown: when the fluorescence area is less than 400, it indicates that the patient is most likely healthy and has no tumor in the body; when the fluorescence area is between 400 and 600, the possibility of tumor metastasis is slightly higher; and when the fluorescence area is greater than 600, it indicates that the patient has a tumor and it is highly likely that metastasis has not occurred.

[0038] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for rapidly designing fluorescent probes for discovering and detecting preliminary screening indicators of tumors, characterized in that: The steps include: Step 1: Select zinc finger proteins that are closely related to tumor invasion and migration through existing databases and literature, and consider them as preliminary screening indicators for tumors. Based on the structural characteristics of zinc finger proteins, select quinoline as the parent structure of the fluorescent probe; Step 2: Use the concept of combinatorial chemistry to modify it and connect specific auxiliary groups to the parent structure to obtain a probe candidate library; Step 3: Use ChemDraw chemical software to construct the molecular structure of the probe candidate, and use the Autodock vina program in Autodock 4.2 to score and evaluate the binding effect between a matrix metalloproteinase in zinc finger protein and various probe candidates using a gradient optimized conformation search algorithm to screen out the 8-hydroxyquinoline-5-sulfonic acid nitrogen oxygen structure; Step 4: Use quantum chemistry software Gaussian to optimize the selected nitrogen-oxygen structure of 8-hydroxyquinoline-5-sulfonic acid with CAM-B3LYP functional and 6-311G* basis set with calculation accuracy not less than TDDFT; Step 5: Take the nitrogen-oxygen structure of 8-hydroxyquinoline-5-sulfonic acid in the lowest energy state obtained in the previous step, and optimize the S1 state using the functional with a calculation accuracy not lower than PBE1PBE and the 6-311G* basis set, calculate the information of the 5 lowest energy excited states, and obtain the wfn file containing the wave function information and the output result file; Step 6: Use the wave function analysis software Multiwfn to plot the optimized UV absorption spectrum and fluorescence spectrum of 8-hydroxyquinoline-5-sulfonic acid nitrogen oxide, and calculate the value of the Stokes shift. If the value is greater than 100, it is determined that the optimized structure has good potential for application as a fluorescent probe; Step 7: Use the wave function analysis software Multiwfn to analyze the average local ionization energy and electrostatic potential of the optimized structure to determine the specific binding site between the optimized structure and the biomarker; Step 8: Synthesize the probe and use it for serum testing of breast cancer patients. Use a fluorescence analyzer to obtain fluorescence intensity and fluorescence integrated area data to compare whether there are differences in serum results of breast cancer patients with different malignancy levels.

2. A method for rapid design of fluorescent probes for discovering and detecting preliminary screening indicators of tumors as claimed in claim 1, characterized in that: After the molecular model is successfully constructed in step 3, the molecular mechanics method should be used to perform preliminary optimization of the constructed geometric configuration under the MM2 force field to obtain the stable configuration with the lowest energy.

3. A method for rapid design of fluorescent probes for discovering and detecting preliminary screening indicators of tumors as claimed in claim 1, characterized in that: Step 4: Take the screened molecular structures and construct the input file of the quantum chemistry software Gaussian. Use the CAM-B3LYP functional and 6-311G* basis set with a calculation accuracy not less than TDDFT to optimize the structure of the probe candidate and obtain the quantum chemistry parameters and checkpoint files.

4. A method for rapid design of fluorescent probes for discovering and detecting preliminary screening indicators of tumors as claimed in claim 1, characterized in that: The total amount of detectable zinc finger proteins in clinical samples is used as a preliminary screening indicator for tumor detection.

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