Application of targeted P5CS and c-Myc binding site in indirect screening of targeted c-Myc protein inhibitor and method of targeted P5CS and c-Myc binding site

Abstract

The invention discloses a screening method of a small-molecule inhibitor targeting protein interacting with c-Myc protein and application of the small-molecule inhibitor. The screening method comprises the following steps: taking hotspot residues Ser79, Ser117, Arg159, Thr299, Ser301, Val303, Met305 and Lys311 of a P5CSGK structural domain as pockets; the invention relates to a polypeptide containing 15 amino acids of c-Myc, a binding interface of the c-Myc protein and P5CS is determined, the binding interface is used as a site for screening small-molecule inhibitors to carry out drug screening of indirect targeting c-Myc protein, nine small-molecule inhibitors are screened, and the screened inhibitors can be used as antitumor drugs.

Description

Technical Field

[0001] The present invention relates to the field of drug screening, and in particular to the application of targeting P5CS and c-Myc binding sites for screening targeted c-Myc protein inhibitors; and also relates to a method for screening small molecule inhibitors. Background Art

[0002] The transcription factor c-Myc protein, encoded by the proto-oncogene MYC, is abnormally expressed in more than 70% of human cancers, including colorectal carcinoma (CRC), lymphoma, and T-cell acute lymphoblastic leukemia. The c-Myc protein is involved in regulating the expression of many genes in cell growth, proliferation, and metabolic pathways, indicating the core pathogenic role of c-Myc in a variety of cancers. Inhibiting the expression or activity of c-Myc may have key clinical results. However, the crystal structure of the full-length c-Myc protein has not yet been resolved, and according to some research reports, c-Myc is a highly disordered protein with no obvious binding pocket for drug screening. It is also called an "undruggable" target for therapeutic intervention by many researchers. At present, there are few specific inhibitors that directly target c-Myc. Therefore, it is urgent to develop some strategies that directly or indirectly target c-Myc protein inhibitors from other angles.

[0003] In the case where c-Myc protein cannot be directly targeted, hindering the binding of c-Myc protein to its interacting proteins can indirectly inhibit the function of c-Myc protein. In view of this, we propose a strategy to use the interface between c-Myc protein and its interacting proteins as a target for screening small molecule inhibitors. P5CS (Delta-1-pyrroline-5-carboxylatesynthase) is a glutamate metabolic enzyme in the body. It can convert glutamate into proline through its enzymatic function and is an important enzyme in proline biosynthesis. Studies have shown that P5CS is highly expressed in tumors such as colorectal cancer and lymphoma. At the same time, it can interact with c-Myc protein and affect the process and progression of tumors.

[0004] Therefore, we first determined the interface where c-Myc protein binds to P5CS; secondly, we used this binding interface as a site for screening small molecule inhibitors to screen drugs that indirectly target c-Myc protein. By inhibiting the binding of c-Myc protein to its interacting proteins, the activity or function of c-Myc protein is affected, thereby affecting the occurrence and progression of tumors. Summary of the invention

[0005] In view of this, one of the objects of the present invention is to provide an application of targeting P5CS and c-Myc binding sites for screening targeted c-Myc protein inhibitors; a second object of the present invention is to provide a screening method for small molecule inhibitors targeting proteins that interact with c-Myc protein; a third object of the present invention is to provide small molecule inhibitors screened using the method; and a fourth object of the present invention is to provide an application of the small molecule inhibitors in the preparation of anti-tumor disease drugs.

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

[0007] 1. Targeting P5CS and c-Myc binding sites for screening of c-Myc protein inhibitors.

[0008] Preferably, in the P5CS and c-Myc binding site, the P5CS_GK domain uses hotspot residues Ser79, Ser117, Arg159, Thr299, Ser301, Val303, Met305, and Lys311 as pockets; the c-Myc is a 15-amino acid polypeptide of the c-Myc protein, and its amino acid sequence is shown in SEQ ID NO.1.

[0009] Preferably, in the present invention, the amino acid sequence of the P5CS_GK domain is shown as SEQ ID NO.2; and the amino acid sequence of the c-Myc binding site is shown as SEQ ID NO.3.

[0010] 2. A method for screening small molecule inhibitors targeting proteins interacting with c-Myc protein, comprising the following steps:

[0011] (1) Molecular docking was performed based on the targeting of the P5CS_GK domain and the 15 amino acids of the c-Myc protein, and the conformation with the highest score was selected as the final docking result of the P5CS_GK domain and the 15 amino acids of the c-Myc protein to determine the binding site of the small molecule inhibitor;

[0012] (2) The predicted P5CS_GK domain model was defined as a receptor, and the Drugbank, Targetmol, and Specs small molecule libraries were used as virtual screening compound libraries for compound screening. The screening process first sorted the docking scores of all compounds through high-throughput rigid docking, then performed flexible docking on the top 10,000 compounds in the rigid docking, and selected and divided them into structural clusters through fingerprint-based clustering. Finally, 51 different subsets were determined.

[0013] (3) SPR was used to further screen the 51 small molecules obtained by virtual screening to detect the binding affinity between small molecules and proteins, and a total of 9 small molecule inhibitors were identified.

[0014] Preferably, in step (1), the docking process sets parameters so that AutoDock CrankPep performs 100 independent Monte Carlo searches, each time allocating 2.5 million evaluations of the scoring function.

[0015] 3. A small molecule inhibitor screened by the method, wherein the small molecule inhibitor is C 25 H 21 N7O3S3、C 27 H 30 N6O2S、C 25 H 37 NO4, C 20 H 20 N6O6、C 38 H 43 N2O + , C 32 H 22 O8S2、C 32 H 55 N4O + , C 14 H 23 N3O 10 , C 22 H 30 CL2N 10 .

[0016] 4. Use of the small molecule inhibitor in the preparation of anti-tumor disease drugs.

[0017] The beneficial effects of the present invention are as follows: the present invention provides a method for utilizing the fact that P5CS is highly expressed in tumors and can interact with c-Myc protein, so that small molecule inhibitors targeting P5CS can be screened by targeting the P5CS_GK domain and the c-Myc-15mer binding site, and c-Myc can be indirectly targeted to provide anti-tumor activity, thereby providing basic research assistance for the P5CS_GK domain and c-Myc, and more useful small molecule compounds can be screened for anti-tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0019] Figure 1 The expression of P5CS in pan-cancer and the results of Co-IP experiment with c-Myc protein.

[0020] Figure 2 This is the docking result of P5CS_GK domain and c-Myc-15mer.

[0021] Figure 3It is the hot spot amino acid residues and screening pocket of the binding interface.

[0022] Figure 4 Flow chart of virtual screening.

[0023] Figure 5 Surface plasmon resonance of a single concentration of a small molecule inhibitor.

[0024] Figure 6 It is the half-maximal inhibitory concentration of small molecule inhibitors on cells.

[0025] Figure 7 The effect of small molecule inhibitors on the stability of intracellular receptor proteins. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0027] Example 1: Co-IP experiment to determine the domain of interaction between P5CS and c-MYC protein

[0028] P5CS is an amino acid kinase that plays an important role in proline metabolism. As a metabolic enzyme, it is highly expressed in many tumor cells. In order to verify whether P5CS interacts with the protein c-MYC expressed by the proto-oncogene, thereby promoting the occurrence and development of tumors, a Co-IP experiment was performed to verify the results. Figure 1 As shown in A.

[0029] Resuscitate 293T cells and passage them into three 10cm culture dishes. On the night before transfection, change the cell medium to serum-free DMEM medium. Observe the cell state the next day and prepare the transfection complex. The plasmids used were c-MYC plasmid with HA tag, full-length P5CS with His tag, and P5CS_GK domain plasmid. The transfection experiment was divided into three groups: the first group was a control group without HA-c-MYC plasmid, but only full-length His-P5CS was added; the second group was an experimental group with HA-c-MYC plasmid and full-length His-P5CS plasmid; the third group was an experimental group with HA-c-MYC plasmid and His-P5CS_GK domain plasmid. Let the prepared transfection complex stand for 15 minutes, then gently add it to the corresponding group, shake it slowly in a cross to mix, and then put it into a 37°C incubator for culture. After 6 hours of transfer, the cells were replaced with a new medium and cultured for another 48 hours before the cells were collected for Co-IP experiment verification. The experimental results are shown in Figure 1As shown in B. The results show that the full-length P5CS protein can interact with the c-MYC protein, and the P5CS_GK domain can also interact with the c-MYC protein. Since the P5CS_GK domain has a substrate binding site and an ATP binding site, this domain will be studied as the main domain that interacts with MYC in the future.

[0030] Example 2: Confirmation of the binding site between the P5CS_GK domain and the c-Myc protein

[0031] The protein structure of P5CS AF-P54886-F1 and the structure of c-Myc protein AF-P01106-F1 predicted by AlphaFold were downloaded from the uniprot website, and the GK domain of P5CS and a 15-amino acid polypeptide of c-Myc protein (LPTPPLSPSRRSGLC, SEQ ID NO.1) were intercepted using pymol software. The P5CS_GK domain and c-Myc-15mer were molecularly docked to confirm their binding sites. The details are as follows:

[0032] The amino acid sequence of the receptor protein P5CS_GK domain is shown in SEQ ID NO.2:

[0033] MLSQVYRCGFQPFNQHLLPWVKCTTVFRSHCIQPSVIRHVRSWSNIPFITVPLSRTHGKSFAHRSELKHAKRIVVKLGSAVVTRGDECGLALGRLASIVEQVSVLQNQGREMMLVTSGAVAFGKQRLRHEILLSQSVRQALHSGQNQLKEMAIPVLEARACAAAGQSGLMALYEAMFTQYS ICAAQILVTNLDFHDEQKRRNLNGTLHELLRMNIVPIVNTNDAVVPPAEPNSDLQGVNVISVKDNDSLAARLAVEMKTDLLIVLSDVEGLFDSPPGSDDAKLIDIFYPGDQQSVTFGTKSRVGMGGMEAKVKAALWALQGGTSVVIANGTHPKVSGHVITDIVEGKKVGTFFSEVKPAGPT

[0034] The amino acid sequence of the ligand protein c-Myc is shown in SEQ ID NO.3:

[0035] MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGD MVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPS SDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDS VRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRNSCA

[0036] First, the AutoGridFR (ADFR) suite was used to generate a zip file for docking, which contained affinity maps of all atom types in c-Myc_15-mer and metadata about the docking box. Later, the AutoDockCrankPep (ADCP) module was used to perform protein-peptide docking. The parameters were set so that ADCP performed 100 independent Monte Carlo searches, each with 2.5 million evaluations of the scoring function. The docking poses were then clustered to generate the final result. Later, the conformation with the highest score was selected as the final docking result of the P5CS_GK domain and c-Myc-15mer for subsequent analysis ( Figure 2 ). The docking results were analyzed by pymol software, and it was found that Ser79, Ser117, Arg159, Thr299, Ser301, Val303, Met305, Lys311 on the P5CS_GK domain formed hydrogen bond interactions with Thr3, Pro4, Leu6, Ser7, Ser9 and Ser12 on the c-Myc_15-mer peptide. The results are shown in Figure 3 As shown, the hotspot residues Ser79, Ser117, Arg159, Thr299, Ser301, Val303, Met305, and Lys311 on the P5CS_GK domain were used as pockets to screen inhibitors targeting the binding site of P5CS and c-Myc protein.

[0037] Example 3: Virtual screening of the binding sites of P5CS_GK domain and c-Myc protein

[0038] Structure-based virtual screening studies were performed using the UCSF DOCK6 software package. The P5CS_GK domain model predicted by AlphaFold was defined as a receptor, and a mixed compound (Drugbank, Targetmol, Specs) library was used as a compound library for virtual screening. All compounds were desalted and hydrogenated before virtual screening. The docking sites included several hot residues (Ser79, Ser117, Arg159, Thr299, Ser301, Val303, Met305, Lys311) of the GK domain of the P5CS protein. The docked molecules were characterized by the ligand center, which was a volume-filled sphere calculated by spgen. The ligand center and receptor center were matched by comparing the distances of the ligand center / ligand center and the receptor center / receptor center. DOCK6 includes GB / SA solvation scores of Hawkins-Cramer-Truhlar and PB / SA solvation scores through the Zap library of OpenEye. Additional flexibility was added to the scoring options during energy minimization. First, all compounds were ranked by high-throughput rigid docking with DOCK6. Then, the top 10,000 compounds in rigid docking were subjected to flexible docking and selected and divided into structural clusters by fingerprint-based clustering. Finally, 51 different subsets were identified. Later, small molecules were identified by SPR experiments and in vitro and in vivo experiments. The overall screening process is as follows: Figure 4 shown.

[0039] Example 4: Surface plasmon resonance of a single concentration small molecule inhibitor

[0040] SPR is a label-free method for studying the binding affinity between ligands and target proteins and is widely used in drug screening. Therefore, SPR was used to further screen the 51 small molecules obtained through virtual screening. All compounds were purchased from Beijing Zhongda Weixin Biotechnology Co., Ltd. During the experiment, the target protein P5CS was fixed on the chip, and PBS was used as the running buffer. Under the conditions of a flow rate of 20 μL / min, a binding time of 90 s, and a dissociation time of 120 s, 51 small molecules with a final concentration of 50 μm / L were injected onto the chip to detect the binding affinity between the small molecules and the protein. The experimental results were analyzed using Biacore T200 analysis software (Cytiva, Marlborough, MA, USA). The experimental results are shown in the figure. Figure 5 As shown, a total of 9 small molecules were identified that could bind to P5CS protein, and the specific analysis information is shown in Table 1.

[0041] Table 1. Information of 9 small molecules identified

[0042]

[0043]

[0044] Example 5: Half-maximal inhibitory concentration of small molecule inhibitors on cells

[0045] In order to determine whether the small molecule inhibitors obtained by SPR experiments have a killing effect on tumor cells, CCK8 experiments were performed to determine the maximum half-effective inhibitory concentration (IC 50 ). First, the HCT116 cells in the logarithmic growth phase were digested with trypsin, and then 10 μL was taken for cell counting. The cell concentration was adjusted to 7000 / mL with complete culture medium and then inoculated into a 96-well transparent plate, 100 μL was inoculated in each well, and 5 replicates were added in each group. In order to reduce the impact of experimental errors on the results, PBS was added around the wells where cells were added to reduce evaporation. The inoculated cells were placed in an incubator and cultured until the cells adhered to the wall. The cells after adhesion were added with 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively. In order to prevent the absorbance value of the culture medium itself from being affected, a group was set up to add only complete culture medium for subsequent zeroing. After 72 hours, 10 μL of CCK8 working solution was added to each well. Be careful to avoid light when adding it, and then quickly put it back into the cell culture incubator for about 2 hours. The absorbance value at a wavelength of 450 nm was detected as soon as possible using an ELISA reader. The size of the detected OD value represents the viability of the cells. Then use GraphPad Prism to finally calculate the IC of different cells 50 , the results are as follows Figure 6 shown.

[0046] Example 6: Stability of small molecule inhibitors on intracellular receptor proteins in vivo

[0047] In order to detect whether the small molecule inhibitor directly acts on the target protein P5CS, a representative small molecule No. 41 was selected for a cellular thermal shift assay (Cellular Thermal Shift Assay, CETSA). If the small molecule directly binds to the P5CS protein in vivo, it will make the P5CS protein stable and not easily degraded under thermal conditions. The specific method is to passage HCT116 cells into two 10cm dishes, one dish as the experimental group and the other as the control group. When the cell confluence reaches 90%, the cells are treated with a final concentration of 4μM of small molecule No. 41 or the same volume of DMSO, and the cells are collected after continuing to be cultured in the incubator for 2h. First, wash the cells twice with pre-cooled PBS, then suspend the cells in 1mL PBS, and repeatedly blow and aspirate with a pipette until the cell pellet is completely dissolved. The cell suspension is repeatedly frozen and thawed in liquid nitrogen for 3 times to cause cell lysis, and then the cell lysate is centrifuged at 4°C and 14000x g for 30min. The cell supernatant after centrifugation was quantified by BCA method, and then the protein concentration of the control group and the experimental group was adjusted to be consistent. The adjusted supernatants of the two groups were added to 6 PCR tubes, 50 μL per tube, and heated for 5 minutes at different temperatures (30℃, 36℃, 42℃, 48℃, 54℃, 60℃) on the PCR instrument. 5x loading buffer was added to the heated samples, and then western blotting analysis was performed. The experimental results are shown in Figure 7 As shown, it can be seen that compared with the control group, the stability of P5CS in the drug-added group is significantly improved, indicating that the small molecule can directly bind to the target P5CS protein in vivo.

[0048] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. Targeting P5CS and c-Myc binding sites for screening of inhibitors targeting c-Myc protein.

2. The use according to claim 1, characterized in that: The P5CS_GK domain in the P5CS and c-Myc binding site uses hotspot residues Ser79, Ser117, Arg159, Thr299, Ser301, Val303, Met305, and Lys311 as pockets; the c-Myc is a 15-amino acid polypeptide in the c-Myc protein, and its amino acid sequence is shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that: The amino acid sequence of the P5CS_GK domain is shown in SEQ ID NO.2; the amino acid sequence of the c-Myc binding site is shown in SEQ ID NO.

3.

4. A method for screening small molecule inhibitors targeting proteins interacting with c-Myc protein, characterized in that: The steps include: (1) Molecular docking was performed based on the targeting of the P5CS_GK domain and the 15 amino acids of the c-Myc protein, and the conformation with the highest score was selected as the final docking result of the P5CS_GK domain and the 15 amino acids of the c-Myc protein to determine the binding site of the small molecule inhibitor; (2) The predicted P5CS_GK domain model was defined as a receptor, and three small molecule libraries, Drugbank, Targetmol, and Specs, were used as virtual screening compound libraries for compound screening. The screening process first ranked the docking scores of all compounds through high-throughput rigid docking, then performed flexible docking on the top 10,000 compounds in the rigid docking, and selected and divided them into structural clusters through fingerprint-based clustering. Finally, 51 different subsets were determined. (3) SPR was used to further screen the 51 small molecules obtained by virtual screening to detect the binding affinity between small molecules and proteins, and a total of 9 small molecule inhibitors were identified.

5. The method for screening small molecule inhibitors targeting proteins interacting with c-Myc protein according to claim 4, characterized in that: In step (1), the docking process was set with parameters such that AutoDock CrankPep performed 100 independent Monte Carlo searches, each assigning 2.5 million evaluations of the scoring function.

6. The small molecule inhibitor screened by the method of claim 5, characterized in that: The small molecule inhibitor is C 25 H 21 N7O3S3、C 27 H 30 N6O2S、C 25 H 37 NO4, C 20 H 20 N6O6、C 38 H 43 N2O + , C 32 H 22 O8S2、C 32 H 55 N4O + , C 14 H 23 N3O 10 , C 22 H 30 CL2N 10 .

7. Use of the small molecule inhibitor according to claim 6 in the preparation of anti-tumor disease drugs.

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