BMRF1-targeting small-molecule inhibitor and application thereof

Abstract

The invention relates to the field of biotechnology and medicine, in particular to a BMRF1-targeted small-molecule inhibitor and application thereof. According to the invention, 50 potential small-molecule inhibitors with targeted inhibition of BMRF1 are screened through computer-aided drug design, the small-molecule inhibitors are selected based on docking scores, and finally three small-molecule inhibitors are obtained through screening and can be combined with DNA binding sites of BMRF1 to prevent binding of DNA and BMRF1, so that EBV replication depending on BMRF1 is inhibited, and the BMRF1-dependent EBV replication is inhibited. A new strategy is provided for treatment of EBV infection and related diseases, and treatment and prognosis of EBV positive tumors are expected to be improved.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and medicine, and in particular to a small molecule inhibitor targeting BMRF1 and application thereof. Background Art

[0002] Epstein-Barr virus (EBV) belongs to the herpes virus family and is a ubiquitous double-stranded DNA virus with a linear double-stranded DNA genome size of approximately 172kb. EBV has the biological characteristics of specifically infecting human and certain primate B cells in vivo and in vitro. EBV infection is very common worldwide, and it is estimated that approximately 90% of the adult population is infected. Although most EBV infections are asymptomatic, it is also associated with the occurrence and development of a variety of diseases, especially certain types of lymphoma and epithelial cell cancer. Although there have been great advances in the diagnosis and treatment of lymphoma, patients with EBV-positive tumors generally respond worse to traditional chemotherapy and have a less than ideal prognosis than EBV-negative tumors. In addition, when the body's immune function is impaired, infected cells proliferate and activate on a large scale, and continuous antigen stimulation can cause an inflammatory cytokine storm. The high level of expression of these cytokines and the ability of EBV to escape immune recognition and killing through various mechanisms can cause the body to develop hemophagocytic lymphohistiocytosis (HLH), accelerate disease progression, and lead to multiple organ failure or even death. Therefore, the development of small molecule inhibitors against EBV replication is of great significance.

[0003] BMRF1 is a key protein encoded by EBV that is expressed in large quantities during the EBV lytic infection cycle. It can be called the DNA polymerase processivity factor of EBV, also known as the DNA polymerase auxiliary subunit of EBV. It plays an important role in the replication and life cycle of the virus. Its functions are mainly concentrated in two aspects: first, as a processivity factor of DNA polymerase, it enhances the activity of viral DNA polymerase; second, as a transcriptional activator, it activates multiple promoters of EBV. First, during the DNA replication of EBV, BMRF1, as a polymerase auxiliary factor, can bind to the DNA polymerase catalytic subunit BALF 5 to enhance the polymerase processivity and nuclease exonuclease activity of the whole enzyme, effectively promoting the extension of the DNA chain. In addition, BMRF1 may also act as a sliding clamp to prevent DNA polymerase from falling off the DNA template during replication, which is crucial to maintaining the continuity and efficiency of replication. Secondly, BMRF1 also regulates the expression of viral genes by transcriptionally activating specific EBV promoters. For example, BMRF1 can activate the oriLyt promoter, thereby regulating the lytic replication cycle of EBV. Therefore, BMRF1, as a key auxiliary subunit of EBV DNA polymerase, plays a very important role in the replication of EBV. Designing small molecule targeted drugs against BMRF1 has the value of inhibiting EBV replication.

[0004] The BMRF1 molecule is approximately 43KD in size and has a total length of 404 amino acids. Its functions in viral replication and transcriptional activation involve two different domains of BMRF1: the N-terminal domain (NTD; 1 to 300 residues) that plays an important role in dimerization, DNA binding, and DNA polymerase processivity, and the C-terminal domain (CTD; 300 to 404 residues) that mediates transcriptional activation. BMRF1 can form homodimers, and two dimers can form a tetrameric ring structure similar to human proliferating cell nuclear antigen (PCNA) and other herpes virus processivity factors. Previous studies have found that knocking out the BMRF1 gene affects the DNA replication of lytic viruses and the late synthesis of lytic proteins. Therefore, BMRF1 plays an important role in the replication of EBV, but small molecule inhibitors targeting BMRF1 have rarely been reported worldwide. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a small molecule inhibitor that targets and inhibits BMRF1, which has the function of inhibiting EBV replication with high efficiency and low toxicity.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides an application of a small molecule inhibitor in targeted inhibition of BMRF1 protein, wherein the small molecule inhibitor comprises at least one of compounds HY-Q48960, HY-Q09187 and HY-Q17227; the CAS number of the compound HY-Q48960 is 917217-17-5; the CAS number of the HY-Q09187 is 1340783-30-3; and the CAS number of the HY-Q17227 is 959535-31-0.

[0008] The present invention screened out 50 small molecule inhibitors with potential targeted inhibition of BMRF1 through computer-aided drug design, and selected the small molecule inhibitors based on docking scores, and finally screened out three small molecule inhibitors: HY-Q48960 (CAS: 917217-17-5), HY-Q09187 (CAS: 1340783-30-3) and HY-Q17227 (CAS: 959535-31-0), all of which have strong ability to inhibit EB virus DNA copy. These three small molecule inhibitors can prevent DNA from binding to BMRF1 by binding to the DNA binding site of BMRF1, thereby inhibiting BMRF1-dependent EBV replication.

[0009] Preferably, the small molecule inhibitor inhibits BMRF1 protein by targeting the binding site of BMRF1 with DNA.

[0010] Preferably, the binding sites include R256, K19, K99, and K29 ​​sites.

[0011] The key amino acid residues R256, K19, K99 and K29 ​​of the BMRF1 protein play a core role in its binding to DNA. These sites are located in the N-terminal domain of BMRF1, which is directly involved in the interaction with DNA, thereby ensuring the continuous synthetic activity of DNA polymerase. The positive charge characteristics of these amino acid residues enable BMRF1 to effectively bind to the negatively charged phosphate backbone of DNA, thereby stabilizing the complex structure during viral replication. The present invention precisely targets the above residues and designs small molecule inhibitors to block the binding of BMRF1 to DNA, cutting off the key steps of EBV replication, thereby effectively inhibiting the viral lytic cycle. The inhibition of these key sites can prevent viral replication and transmission, showing potential antiviral therapeutic value.

[0012] In a second aspect, the present invention provides an application of a small molecule inhibitor in the preparation of a drug for treating EBV-positive tumors or improving the prognosis of EBV-positive tumors, wherein the small molecule inhibitor comprises at least one of compounds HY-Q48960, HY-Q09187 and HY-Q17227; the CAS number of the compound HY-Q48960 is 917217-17-5; the CAS number of the HY-Q09187 is 1340783-30-3; and the CAS number of the HY-Q17227 is 959535-31-0.

[0013] The small molecule inhibitor of the present invention can prevent the interaction between BMRF1 and DNA by binding to the DNA binding site of BMRF1, thereby depriving it of its function as a DNA polymerase. EBV-DNA cannot replicate, thereby effectively blocking the replication and spread of EBV, and can be used as a targeted drug for inhibiting EBV replication with high efficiency and low toxicity. The small molecule inhibitor of the present invention provides a new strategy for the treatment of EBV infection and related diseases, and is expected to improve the treatment and prognosis of EBV-positive tumors.

[0014] Preferably, the small molecule inhibitor treats EBV-positive tumors or improves the prognosis of EBV-positive tumors by inhibiting EBV replication.

[0015] Preferably, the small molecule inhibitor inhibits EBV replication by targeting and inhibiting BMRF1 protein.

[0016] Preferably, the small molecule inhibitor inhibits BMRF1 protein by targeting the binding site of BMRF1 with DNA.

[0017] Preferably, the binding sites include R256, K19, K99, and K29 ​​sites.

[0018] In a third aspect, the present invention provides a drug for treating EBV-positive tumors or improving the prognosis of EBV-positive tumors, the drug comprising at least one of compounds HY-Q48960, HY-Q09187 and HY-Q17227; the CAS number of the compound HY-Q48960 is 917217-17-5; the CAS number of the HY-Q09187 is 1340783-30-3; the CAS number of the HY-Q17227 is 959535-31-0.

[0019] The beneficial effects of the present invention are:

[0020] The present invention provides a new small molecule inhibitor targeting BMRF1 protein. 50 small molecule inhibitors with potential targeted inhibition of BMRF1 are screened out through computer-aided drug design, and the small molecule inhibitors are selected based on docking scores. Finally, three small molecule inhibitors are screened out, which can act on the key amino acid residues R256, K19, K99 and K29 ​​of BMRF1 protein, which are the key sites for BMRF1 to bind to DNA. The small molecule inhibitor targeting BMRF1 and DNA binding sites provided by the present invention inhibits BMRF1-dependent EBV replication by preventing the binding of DNA to BMRF1. It provides a new strategy for the treatment of EBV infection and related diseases, and is expected to improve the treatment and prognosis of EBV-positive tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The three-dimensional structure of BMRF1, where red indicates the DNA binding site.

[0022] Figure 2-4 These are the top 50 small molecule compounds with strong binding ability to BMRF1 protein screened from the MCE 50K Diversity Library in the present invention.

[0023] Figure 5 The docking score results of 50 small molecule compounds docked to the DNA binding site of BMRF1. DETAILED DESCRIPTION

[0024] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0025] The structural formulas of the 50 small molecule inhibitors used in the present invention are as follows Figure 2-4 As shown in the figure, all 50 small molecule inhibitors can be obtained from the MCE 50K Diversity Library according to Catalog_NO or CAS number. The CAS numbers of some compounds are shown in Table 1.

[0026] Table 1 CAS numbers of small molecule compounds

[0027]

[0028] Example 1: Screening for small molecule compounds with strong binding ability to BMRF1 protein

[0029] In this example, small molecule compounds with strong binding ability to BMRF1 protein were screened from MCE 50K Diversity Library. Through computer simulation, compounds that can effectively act on the key DNA binding sites (R256, K19, K99 and K29) of BMRF1 protein were identified.

[0030] The specific steps include:

[0031] 1. Protein preparation: Download the three-dimensional structure of Human herpesvirus 4BMRF1 (PDBID: 2Z0L) from the RCSB website. Figure 1 The Protein Preparation Wizard module was used to hydrogenate the protein, delete the BH chain, retain only the A chain, delete the water molecules, delete the chloride ions, and then perform energy optimization (OPLS2005 force field, RMSD is ). The Receptor Grid Generation module was used to create a grid file with amino acid residues R256 / K19 / K99 / K29 as the center, and the box sizes were set to

[0032] 2. Compound preparation: The 2D format of the MCE 50K Diversity Library (containing 50k compounds) is The software LigPrep Module performs hydrogenation, energy optimization (OPLS2005 force field), protonation and other processes, and outputs 3D structures for virtual screening.

[0033] 3. The receptor and ligand molecules are docked with each other through geometric matching and energy matching. The small molecule compound library of MCE50K Diversity Library is screened. The standard (SP) mode is used for the second round of screening and the high precision (XP) mode is used for the third round of screening. The molecular docking score (docking score) is calculated based on factors such as the intermolecular classical force field, electrostatic interaction, and hydrogen bond interaction. It is an internal algorithm of the Schrödinger software, which is related to the affinity of the molecular interaction, and the ranking of the small molecule compounds is obtained according to the docking score.

[0034] In this example, the top 50 small molecule compounds with strong binding ability to BMRF1 protein were screened from the MCE 50K Diversity Library, which are:

[0035] HY-Q37361, HY-Q33452, HY-Q05325, HY-Q34513, HY-Q20800,

[0036] HY-Q01466, HY-Q28019, HY-Q16075, HY-Q06468, HY-Q37011,

[0037] HY-Q11075, HY-Q30590, HY-Q30030, HY-Q31948, HY-Q43009,

[0038] HY-Q24816, HY-Q10674, HY-Q42519, HY-Q14895, HY-Q05498,

[0039] HY-Q12079, HY-Q43250, HY-Q09187, HY-Q44258, HY-Q27055,

[0040] HY-Q05578, HY-Q00640, HY-Q15308, HY-Q39012, HY-Q19111,

[0041] HY-Q01690, HY-Q43861, HY-Q90007, HY-Q11875, HY-Q50011,

[0042] HY-Q38848, HY-Q26923, HY-Q48960, HY-Q17227, HY-Q49888,

[0043] HY-W062674, HY-W581129, HY-Q25272, HY-Q24834, HY-Q30618,

[0044] HY-Q38848, HY-Q09541, HY-Q21622, HY-Q11226, HY-Q45236.

[0045] These small molecule compounds can be used as small molecule inhibitors of BMRF1, and their structures and corresponding docking scores are as follows Figure 2-4 The higher the absolute value of the molecular docking score, the stronger the binding force between the compound and the protein.

[0046] Example 2: Exploring the inhibitory effect of small molecule compounds

[0047] B95.5 cells were cultured in 10 cm dishes with 10% FCS RPMI-1640 at an initial cell density of 1 × 10 6Cells / mL, after the cells were cultured to the logarithmic growth phase (about 48 hours), 50 small molecule inhibitors (10 μM) screened in Example 1 were added and cultured for 72 hours. After the culture was completed, the cell precipitate was removed by centrifugation, and the supernatant was collected to extract DNA. The ability of different small molecule inhibitors to inhibit the copy number of EBV was detected using qPCR technology, the Ct value was recorded, and the copy number was calculated according to the standard curve, and the inhibitory effect of 50 small molecule inhibitors was analyzed.

[0048] The specific steps are as follows:

[0049] 1. Under sterile conditions, prepare a 10 cm culture dish and add 10 mL of 10% FCS RPMI-1640 medium. 6 Inoculate the culture dish with an initial cell density of 10 cells / mL, place the culture dish in a 37°C, 5% CO2 incubator, and culture for about 48 hours until the cells reach the logarithmic growth phase. Observe the cell growth status regularly to ensure that the cells are healthy and free of contamination.

[0050] 2. After the cells reach the logarithmic growth phase, prepare 50 small molecule inhibitors and dissolve each inhibitor in DMSO to a concentration of 10 μM. Add each inhibitor to the corresponding culture dish and gently shake to ensure uniform distribution. Continue to culture in a 37°C, 5% CO2 incubator for 72 hours.

[0051] 3. After the culture is completed, remove the cell pellet by centrifugation (1200rpm, 5 minutes) and collect the supernatant. Carefully transfer the supernatant to an RNase-free centrifuge tube to avoid disturbing the cell pellet. Use a DNA extraction kit (QIAGEN, 51304) to extract DNA from the supernatant according to the instructions. Use an ultra-micro UV spectrophotometer (Thermo / NanoD2000C) to detect the DNA concentration to ensure that a consistent starting amount is used in qPCR. Use real-time fluorescence quantitative PCR (LC480) to analyze the expression of EBV DNA. Record the Ct value and calculate the EBV copy number in the sample based on the standard curve. Compare the EBV copy number between the different small molecule inhibitor treatment groups and the control group, and analyze the inhibitory effect of each inhibitor. The test results are as follows Figure 5 And as shown in Table 2.

[0052] Table 250 Relative EBV DNA copy number ratios of small molecule inhibitors

[0053] Compound DNA copy number ratio Compound DNA copy number ratio HY-Q37361 0.92 HY-Q05578 0.88 HY-Q33452 0.57 HY-Q00640 0.19 HY-Q05325 0.9 HY-Q15308 0.14 HY-Q34513 0.74 HY-Q39012 0.33 HY-Q20800 0.93 HY-Q19111 0.15 HY-Q01466 0.08 HY-Q01690 0.6 HY-Q28019 0.95 HY-Q43861 0.14 HY-Q16075 0.74 HY-Q90007 0.59 HY-Q06468 0.74 HY-Q11875 0.19 HY-Q37011 0.64 HY-Q50011 0.2 HY-Q11075 0.52 HY-Q38848 0.14 HY-Q30590 0.47 HY-Q26923 0.22 HY-Q30030 0.22 HY-Q48960 0.05 HY-Q31948 0.53 HY-Q17227 0.04 HY-Q43009 0.77 HY-Q49888 0.32 HY-Q24816 0.41 HY-W062674 0.54 HY-Q10674 0.33 HY-W581129 0.5 HY-Q42519 0.37 HY-Q25272 0.63 HY-Q14895 0.77 HY-Q24834 0.27 HY-Q05498 0.54 HY-Q30618 0.07 HY-Q12079 0.24 HY-Q38848 0.41 HY-Q43250 0.33 HY-Q09541 0.46 HY-Q09187 0.03 HY-Q21622 0.95 HY-Q44258 0.82 HY-Q11226 0.55 HY-Q27055 0.92 HY-Q45236 0.12

[0054] The ability of the 50 small molecule inhibitors screened in Example 1 to inhibit the number of EBV copies varies greatly, among which HY-Q48960 (CAS: 917217-17-5), HY-Q09187 (CAS: 1340783-30-3) and HY-Q17227 (CAS: 959535-31-0) all have strong ability to inhibit EB virus DNA copies. After further screening, it was found that these three small molecule inhibitors can bind to the DNA binding site of BMRF1, prevent BMRF1 from interacting with DNA, and thus deprive it of its function as a DNA polymerase. Therefore, EBV-DNA cannot be replicated, thereby effectively blocking the replication and spread of EBV.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Use of a small molecule inhibitor in targeted inhibition of BMRF1 protein, characterized in that: The small molecule inhibitor includes at least one of compounds HY-Q48960, HY-Q09187 and HY-Q17227; the CAS number of the compound HY-Q48960 is 917217-17-5; the CAS number of the HY-Q09187 is 1340783-30-3; and the CAS number of the HY-Q17227 is 959535-31-0.

2. The use according to claim 1, characterized in that The small molecule inhibitor inhibits BMRF1 protein by targeting the binding site of BMRF1 to DNA.

3. The use according to claim 2, characterized in that The binding sites include R256, K19, K99, and K29 ​​sites.

4. Use of a small molecule inhibitor in the preparation of a drug for treating EBV-positive tumors or improving the prognosis of EBV-positive tumors, characterized in that: The small molecule inhibitor includes at least one of compounds HY-Q48960, HY-Q09187 and HY-Q17227; the CAS number of the compound HY-Q48960 is 917217-17-5; the CAS number of the HY-Q09187 is 1340783-30-3; and the CAS number of the HY-Q17227 is 959535-31-0.

5. The use according to claim 4, characterized in that The small molecule inhibitor treats EBV-positive tumors or improves the prognosis of EBV-positive tumors by inhibiting EBV replication.

6. The use according to claim 5, characterized in that The small molecule inhibitor inhibits EBV replication by targeting and inhibiting BMRF1 protein.

7. The use according to claim 6, characterized in that The small molecule inhibitor inhibits BMRF1 protein by targeting the binding site of BMRF1 to DNA.

8. The use according to claim 7, characterized in that The binding sites include R256, K19, K99, and K29 ​​sites.

9. A drug for treating EBV-positive tumors or improving the prognosis of EBV-positive tumors, characterized in that: The drug comprises at least one of compounds HY-Q48960, HY-Q09187 and HY-Q17227; the CAS number of the compound HY-Q48960 is 917217-17-5; the CAS number of the HY-Q09187 is 1340783-30-3; and the CAS number of the HY-Q17227 is 959535-31-0.

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