WO2018161741A1

WO2018161741A1 - Ape1 inhibitor and application thereof in preparing drug for treating tumor and disease related to abnormal angiogenesis

Info

Publication number: WO2018161741A1
Application number: PCT/CN2018/074656
Authority: WO
Inventors: 袁岩
Original assignee: 中山大学
Priority date: 2017-03-09
Filing date: 2018-01-31
Publication date: 2018-09-13
Also published as: CN107184572B; CN107184572A

Abstract

An APE1 inhibitor and an application thereof in preparing a drug for treating a tumor and a disease related to abnormal angiogenesis. The APE1 inhibitor has the structure shown in the following formula 1 and selectively inhibits a redox end of APE1.

Description

APE1 inhibitor and application thereof in preparing medicine for treating tumor and vascular abnormal proliferative diseases

Technical field

The invention belongs to the fields of molecular biology, biochemistry and pathogens, and particularly relates to an APE1 inhibitor and its use in the preparation of a medicament for treating tumor and vascular aberrant proliferative diseases related to the function of APE1 redox end.

Background technique

Depurination of pyrimidine endonuclease/redox factor (APE-1) is an extremely important intracellular bifunctional protein. The C-terminus is involved in the base excision repair after DNA mismatching; the N-terminus contains redox activity, which can reduce the oxidized inactive transcription factor to a reduced active transcription factor, and the active transcription factor can bind downstream DNA. The promoter sequence initiates the expression of downstream genes and has an important influence on cell proliferation, apoptosis and differentiation. The transcription factors regulated downstream include HIF-1α, Egr-1, AP-1, NF-κB, CREB (cAMP response element binding protein), p53 and the like.

The study found that APE-1 is closely related to tumors. Protein expression levels of APE-1 have been found to increase in a variety of malignancies such as pancreatic cancer, cervical cancer, ovarian cancer, non-small cell lung cancer, head and neck cancer, and the like. The above tumors are often treated clinically by ordinary chemotherapy and radiotherapy. However, the recurrence rate is high and it is easy to develop drug resistance. This is closely related to the highly expressed APE1 at the tumor. APE1 can repair the DNA damage of tumor cells caused by radiotherapy, restore the superoxide ions produced by radiotherapy, and maintain the anti-oxidation state of the tumor. This opened up a new way for us to use APE1 as a target to treat the above tumors with APE1 inhibitors. At the cytological level, the redox-end inhibitor E3330 of APE1 can inhibit the proliferation of pancreatic cancer cells and glioma cells and promote cell apoptosis. The study is subject to further validation at the zoology level. In addition, the subcellular localization of APE-1 in some tumor tissues is different from that in normal tissues; for example, APE-1 is mainly distributed in the nucleus in cancer stem cells; APE-1 is in nucleus and patina in lung cancer and bladder cancer. There are distributions in the bladder cancer patients can detect the protein level of APE1 in the blood, suggesting that APE1 can be excreted in some form. Clinically, it has been reported that the expression level of APE-1 protein in serum can be used as a biomarker of bladder cancer for the diagnosis of malignant degree of bladder cancer, and the therapeutic effect can be evaluated according to the expression level thereof.

Studies have shown that the redox function of APE-1 is associated with abnormal vascular proliferation in vivo. The use of APE-1 N-terminal inhibitor E3330 can reduce the secretion of vascular-related cytokine VEGF and reduce the expression of VEGF-R on the cell surface to inhibit Newborn blood vessels. In addition, E3330 also inhibits the differentiation of endothelial progenitor cells into CD31+ endothelial cells and inhibits the proliferation of endothelial cells and endothelial progenitor cells. Age-related macular degeneration is the leading cause of blindness in the elderly, and age-related macular degeneration is mainly caused by abnormal proliferation of blood vessels at the base of the retina. Antagonists that use VEGF directly can only produce transient effects that do not fundamentally cure the disease. In vivo and in vitro studies have shown that the redox-end inhibitor E3330 of APE1 can block the ability of retinal cells to form tubes and is validated at the zoological level using an ocular vascular proliferation model. This suggests that our inhibitors of APE1 are beneficial for the treatment of diseases with vascular hyperplasia.

In addition, APE1 is also associated with inflammation. APE-1 can regulate the secretion of inflammatory cytokines such as IL-6 and IL-8 through AP-1, and affect the occurrence of inflammatory diseases by regulating the microenvironment.

Kaposi's sarcoma is a special type of tumor, and Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of the disease. Kaposi's sarcoma can not be called sarcoma in the early stage of the disease, because the tumor tissue removed from the patient does not have the ability to clone clones, and a cell line of Kaposi's sarcoma cannot be isolated. It is characterized by abundant angiogenic factors, disordered inflammatory cytokines and malignant proliferating spindle cells. Tumor cells with abnormal vascular proliferation, inflammatory infiltration and malignant proliferation are a good model for studying the relationship between APE1 and tumors, blood vessels and inflammatory diseases.

The function of APE1 is critical for tumor survival and growth, and now there are only a handful of inhibitors targeting the redox end of APE1, most of which are structural analogs of E3330.

Summary of the invention

In order to solve the disadvantages and deficiencies in the prior art, it is a primary object of the present invention to provide an APE1 inhibitor.

Another object of the present invention is to provide an application of the above APE1 inhibitor.

The object of the invention is achieved by the following technical solutions:

An APE1 inhibitor having a structure represented by the following formula 1 (hereinafter referred to as a C10 compound):

The APE1 inhibitor has the structure shown in the following formula 2:

Wherein R ₁ and R ₂ are respectively a five-membered ring, a six-membered ring or a benzene-parallel compound, the five-membered ring is furan, thiophene, pyrrole, thiazole or imidazole, and the six-membered ring is pyridine, pyrazine, pyrimidine or pyridazine, and benzene is connected in parallel. The compound is an anthracene or isoindole compound. The R ₁ and R ₂ are each preferably a cyclohexyl group or a substituted cyclohexyl group.

The APE1 inhibitor has the structure shown in the following formula 3:

Wherein R is a C, N, S or O element; R ₁ , R ₂ , R ₃ , R ₄ , R ₅ or R ₆ are each hydrogen, or various saturated or unsaturated, ring-containing or ring-free, ring-shaped A substituted or unsubstituted alkyl compound.

The APE1 inhibitor has the structure shown in the following formula 4:

The use of the above APE1 inhibitor for the preparation of a medicament for the treatment of a related disease dependent on the function of the redox end of APE1. The APE1 inhibitor targets APE1 and can inhibit the redox end function of APE.

The use of the above APE1 inhibitor for the preparation of a medicament for the treatment of tumor and vascular aberrant proliferative diseases.

The tumor includes a tumor caused by a virus such as Kaposi's sarcoma, nasopharyngeal cancer or cervical cancer.

The tumor also includes pancreatic cancer, bladder cancer, ovarian cancer, glioma, osteosarcoma, ovarian cancer or breast cancer.

The vascular aberrant proliferative diseases include age-related macular degeneration.

The above APE1 inhibitors can be prepared into various types of drugs for the treatment of related tumors and vascular proliferative diseases by means of pharmaceutical preparations.

The above-mentioned APE1 inhibitor is used as an active ingredient, and the compound is made into a tablet, a gel, a capsule, a dispersing agent, an injection, a spray by a pharmaceutical preparation in a manner that inhibits the specificity of the redox end of APE1. Pharmaceutically acceptable carriers and/or excipients such as oral solutions are within the scope of protection of the present invention.

a pharmaceutically acceptable salt or ester of the above compound, such as the above-mentioned APE1 inhibitor is made into a hydrochloride, a phosphate, a sulfate, a carbonate, etc.; the anti-APE1 redox end is used as a mechanism for treating the above Tumor and vascular proliferative diseases are also within the scope of protection of the present invention.

Meanwhile, the present invention provides a pharmaceutical composition of the above APE1 inhibitor, or a combination preparation, and the above-mentioned APE1 inhibitor as a main active ingredient of the drug, for inhibiting the redox end of APE1 for treating the above diseases belongs to the protection scope of the present invention.

The above-mentioned APE1 inhibitors are combined with clinically available anti-tumor drugs, such as cisplatin, artemisinin and other anti-tumor drugs through other mechanisms of action, these compounds mainly act by inhibiting APE1, such administration The method should fall within the scope of protection of the present invention.

The principle of the invention is:

In an early large-scale drug screening, the inventors found that structural analogs of the C10 compound and the C10 compound have very good ability to inhibit the redox end of APE1 without affecting the DNA repair function of APE1. It is a characteristic inhibitor that selectively inhibits the redox end of APE1.

Further experiments at the cellular level revealed that C10 compounds can effectively inhibit KSHV and EBV cleavage replication with little cytotoxicity. Oral administration of mice with C10 compounds at the zoological level is effective in preventing replication of murine IFN herpesvirus in mice. C10 compounds can effectively inhibit KSHV-induced vascular hyperplasia and inflammatory cytokine secretion at the cellular and animal levels, and can simultaneously block the pathological process promoted by KSHV in many aspects. The efficacy of the C10 compound on other tumor cells was evaluated, and it was found that the C10 compound can simultaneously inhibit the proliferation of tumor cells such as nasopharyngeal carcinoma and osteosarcoma.

This novel APE1 inhibitor C10 compound or a structural analog of C10 can be used to treat KSHV, EBV-infected related tumors, including: Kaposi's sarcoma (classic Kaposi's sarcoma, AIDS-related Kaposi's Sarcoma, organ transplant-related Kaposi's sarcoma, iatrogenic Kaposi's sarcoma, primary exudative lymphoma, multicenter Castleman's disease, Burkitt's lymphoma, Hodgkin's lymphoma, nasopharyngeal carcinoma Etc.; treatment of vascular abnormal proliferative diseases such as age-related macular degeneration; treatment of other tumors such as: bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, breast cancer, glioma, osteosarcoma and so on.

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

(1) The C10 compound or the C10 analog enriches the structural form of the existing APE1 redox terminal inhibitor.

(2) C10 compounds or C10 analogs are highly potent and less toxic; C10 has a significant improvement in various activities compared to existing APE1 inhibitors.

(3) The compound of C10 is simple in structure, easy to synthesize, and has good drug-forming properties;

Based on the findings of the present invention, the compounds of the present invention will have good application prospects in the following aspects:

1. Treatment of related malignant tumors caused by KSHV.

KSHV can cause a variety of malignant tumors, in which Kaposi's sarcoma is characterized by abundant vascular invasion, inflammatory factor secretion and malignant proliferation of spindle-shaped cells. First, the sustained latency of KSHV in spindle cells is dependent on viral cleavage replication. The lytic replication of the virus continuously releases infectious viral particles to maintain a latent infection of the virus. Latently infected cells can maintain the survival of tumor cells and promote the proliferation of tumor cells. The clinical use of drugs that inhibit viral lytic replication directly reduces the occurrence of KS. However, when clinical studies using antiviral lytic replication drugs alone, many of the molecules have low activity and are not effective. This is related to the efficacy of the compound against viral cleavage replication. KS also has vascular proliferation, strong invasive characteristics, etc., if it can simultaneously combine multiple characteristics of KS, this may provide a new way for KS targeted therapy. The redox activity of APE1 regulates the activity of multiple downstream transcription factors. These transcription factors are distributed in various processes in which KSHV promotes the development of disease. Therefore, the inhibitor of APE1 can target KS against the characteristics of KS from multiple aspects.

First, the redox-end inhibitor C10 of APE1 blocks the cleavage replication of KSHV. KSHV first expresses RTA in the process of cleavage and replication. RTA is a virus that is an early gene, and the smooth expression of RTA can ensure the smooth replication of virus cleavage. That is, the promoter of the early gene RTA has a binding sequence of the AP1 transcription factor. The lytic cycle of the virus proceeds smoothly depending on the expression of RTA. The activity of the transcription factor of AP1 downstream of the MAPK signaling pathway, and the activity of viral AP1 is directly related to the expression of RTA. APE1 regulates the smooth progression of the viral lysis life cycle by controlling the activity of the AP1 transcription factor.

Second, C10 compounds and C10 analogs can also efficiently inhibit KSHV-induced vascular proliferation. Block the nutrient supply at the tumor site and improve the microenvironment at the tumor. In the cell model, KSHV can induce differentiation of vascular endothelial cells into vascular endothelial cells, increase the angiogenic ability of cells, and promote angiogenesis by promoting secretion of angiogenic growth factors by oral mesenchymal stem cells. . Treatment of KSHV-infected oral mesenchymal stem cells with APE1 redox-end inhibitor C10 compound can block KSHV-induced angiogenesis, block KSHV through paracrine-induced angiogenesis, and block pro-angiogenic factors VEGF, IL -6, secretion of IL-8. This will facilitate the improvement of the vascular characteristics of local dysplasia of KS and improve the microenvironment of local angiogenesis when the compound is used clinically. Thereby inhibiting the progression of KS tumors.

The C10 compound can also affect the invasive ability of KSHV-promoted cells by inhibiting the redox end function of APE1. The invasive ability of tumor cells is closely related to the metastasis of tumors. After treatment of APE1 with C10 compounds, the invasion ability of cells promoted by KSHV infection can be blocked. This may be related to the promotion of VEGF secretion by APE1, which promotes the invasive ability of cells.

Therefore, the use of C10 compounds and structural analogs of C10 can efficiently inhibit the cleavage and replication of KSHV while also specifically blocking the cell-forming and invasive ability of the induced microenvironment during KS, compared with the simple use of antiviral replication. The compounds used to treat KS, C10 and C10 analogs have many advantages.

2. Treatment of related diseases caused by EBV:

EBV is a positive infection in more than 95% of adults, but often shows that latent infection does not produce symptoms. When the host's immunity declines, EBV can cause a variety of diseases, such as: infectious mononucleosis, AIDS-related lymphoma, transplanted lymphoproliferative disease, Hodgkin's lymphoma, Burkini Lymphoma, as well as endemic nasopharyngeal cancer and so on.

The nasopharyngeal carcinoma caused by EBV is highly prevalent in Guangdong. The current treatment method is mainly based on traditional radiotherapy and chemotherapy. The side effects of such methods are large, and the treatment of the symptoms is not easy to cause the disease to recur. In view of the inflammatory factors and high antioxidant properties of nasopharyngeal carcinoma, the use of redox end inhibitors of APE1 can directly inhibit the cleavage and replication of EBV, which is the cause of nasopharyngeal carcinoma, and can also improve the tumor of nasopharyngeal carcinoma and the affected area. The microenvironment has a good application prospect together with other anti-tumor drugs.

3. Treatment of bladder cancer, pancreatic cancer, glioma and other malignant tumors.

APE1 is highly expressed in these malignancies. The reasons may be as follows: 1) The cells in the tumor are in a high-frequency split state, and more DNA mutations can be generated per unit time, so the up-regulated DNA repair protein contributes to the survival of tumor cells. 2) The hypoxic microenvironment of the tumor site can reduce the production of superoxide ions induced by cellular aerobic respiration, reduce the damage of superoxide ions on the DNA of tumor cells, reduce the apoptosis of tumor cells, and maintain the survival of tumor cells. The redox end of APE1 regulates the activity of the hypoxia-induced transcription factor HIF-1a, and promoting the normal expression of the gene under hypoxic conditions is essential for the normal growth of tumor cells. 3) The abundant vascular proliferation at the tumor provides nutrition for the growth of the tumor. APE1 can promote the secretion of vascular growth factors such as VEGF, regulate the proliferation of blood vessels, and maintain the growth of tumors. Therefore, the use of the redox-end inhibitor of APE1 can simultaneously block the growth of tumors from various aspects, thereby achieving the purpose of treating tumors.

4. Treatment of vascular proliferative diseases.

The proliferation of blood vessels on the surface can bring nutrients to the tissue parts, but abnormal vascular proliferation usually leads to a number of diseases. For example, vascular proliferation at the bottom of the retina can cause macular degeneration, which is the leading cause of blindness in the elderly. Due to the small amount of blood vessels in the eye, the blood concentration of conventional oral administration or injection administration is often unable to maintain an effective onset concentration in the eye, resulting in poor drug efficacy. If a conventional VEGF antagonist is administered by injection, the therapeutic effect is poor or the therapeutic time is short, and the administration mode is not as varied as the small molecule inhibitor. Cell level and animal level experiments have shown that APE1 inhibitors are very effective in animal models of macular degeneration. Thus, structural analogs of C10 and C10 can inhibit diseases of abnormal vascular proliferation such as macular degeneration by inhibiting the redox end function of APE1.

DRAWINGS

Figure 1 shows the interaction between C10 and APE1 proteins:

A. DSF test compound and APE1 interaction diagram; B. CD detection compound and APE1 interaction diagram; C. SPR detection compound and APE1 affinity constant map.

Figure 2 is a graph showing the inhibition of the redox end of APE1 by C10 and E3330:

A. C10 inhibits the activity map of the redox end of APE1; B. E3330 inhibits the activity map of the redox end of APE1.

The left panel in Figure 3 is a schematic diagram of DNA repair at the APE1C end; the right panel shows the DNA repair end activity of C10 without inhibiting APE1.

Figure 4 is a graph comparing C10 and E3330 inhibition of KSHV cleavage replication activity;

A.C10 inhibits the cleavage replication activity of KSHV; B.E3330 inhibits the cleavage replication activity of KSHV; C.C10 inhibits the activity map of intracellular transcription factor AP-1; D.E3330 inhibits the activity of intracellular transcription factor AP-1 Figure.

Figure 5 is a graph comparing the ability of C10 and E3330 to inhibit KSHV-induced cell angiogenesis;

A. C10 and E3330 inhibit KSHV-induced cell angiogenic ability comparison; B. C10 and E3330 inhibit KSHV paracrine-induced cell angiogenic ability comparison; C.C10 inhibits KSHV-PDLSC tube formation ability in mice.

Figure 6 is a graph comparing the ability of C10 and E3330 to inhibit the secretion of cytokines induced by KSHV-PDLSC;

A.C10 inhibits the secretion of VEGF-A, IL-6 and IL-8 by KSHV-PDLSC; B.E3330 inhibits the secretion of VEGF-A, IL-6 and IL-8 by KSHV-PDLSC.

Figure 7 is a graph showing the ability of C10 to inhibit KSHV-induced cell invasion;

A. KSHV promotes the invasion ability map of PDLSC; B. KSH promotes the invasion ability of PDLSC by paracrine; C. Inhibits the invasion ability of KSHV-PDLSC after knocking out APE1; D. Inhibits the secretion of KSHV-PDLSC after Knockout APE1 Invasion ability map; E.C10 inhibits the invasion ability map of KSHV-PDLSC.

Figure 8 is a graph showing the proliferation of C10 inhibiting osteosarcoma cells.

detailed description

The invention will now be further described with reference to the accompanying drawings and embodiments.

1.C10 interaction with APE1

The inventors used a computer to virtually screen a library of small molecule compounds in Guangdong to find a small molecule C10 that is completely different in structure from the existing APE1 inhibitor. E3330 is now a recognized APE1 inhibitor. In the specific implementation process, we compare C10 and E3330 with each other. First, we evaluated the interaction of the small molecule compound C10 with APE1 at an in vitro level. C10 can reduce the melting temperature of APE1 by 1.3 degrees, and the melting temperature of APE1 is lowered by 1 degree compared with E3330. C10 can make the protein structure of APE1 unstable (Fig. 1A). C10 produced a deflected light for the APE1 protein at a drug concentration of 10 μM, while E3330 was at 500 mM (Fig. 1B). The affinity constant between C10 and APE1 was 170 nM, and the E3330 position was 1.63 μM (Fig. 1C). The above evidence indicates that by differential thermal scanning (DSF), circular dichroism (CD) and surface plasmon resonance (SPR), the inventors have not only verified that C10 can interact with APE1, and C10 can communicate with APE1 compared to E3330. Better combination.

Differential Thermal Scanning (DSF)

The purified APE1 protein was diluted to 2 μM and incubated with different concentrations of C10 for 30 min at 37 °C. The fluorescent dye SYPRO orange was added to the protein solution. Scan with Lightcycler 480 and run at 25-95 ° C min-1. use

The 480 software plots the melting curve and obtains the Tm value for each concentration. Thus real-time analysis of the interaction between C10 and APE1

Circular dichroism (CD)

A far-ultraviolet circular dichroism spectrum at intervals of 225-260 nm was obtained by a circular dichroism. In a 0.1 cm thick cuvette containing 10 mM phosphate buffer, 5 μM of APE1 was reacted with 10 μM of C10, respectively. The thermal denaturation curve at 230 nm was measured in the range of 20-90 °C. Thus, the change in the deflection spectrum of the APE1 protein before and after the addition of the drug interference was detected.

Surface plasmon resonance (SPR)

Analysis by C10 and interaction APE1 ProteOn XPR36 ^TM SPR apparatus. APE1 dissolved in sodium acetate buffer was immobilized on EDAC/Sulfo-NHS activated GLH biosensor chip channel L1 and its surface blocked with 1 M ethanolamine. The final fixed level of APE1 is 12,000 RU. The GLH biosensor chip channel L2 was activated with EDAC/Sulfo-NHS and blocked with 1 M ethanolamine. The compound dissolved in PBST was maintained at a flow rate of 20 ml/min for 240 s, and the surface of the chip was repaired with 0.85% H ₃ PO ₄ and running buffer. In each kinetic study, the interaction of C10 with APE1 was studied at six concentrations. Concentration data for compounds were collected using ProtedOn ManagerTM 2.0. Each sensor was analyzed using a 1:1 Langmuir binding model to obtain kinetic rate constants (Kon and Koff). Through each reaction, kinetic rate constants (ka and kd) were obtained, and the equilibrium dissociation constant KD=kd/ka was calculated. Thereby an affinity constant for the interaction of the compound with APE1 is obtained.

2.C10 is a specific inhibitor of the redox end of APE1 and does not affect the activity of the DNA repair end.

After confirming that C10 can bind to APE1, the inventors evaluated the activity of C10 on the C-terminus and the N-terminus of APE at the in vitro molecular level, respectively. The principle of DNA binding activity of the AP-1 transcription factor can be increased by APE1. Artificially synthesizing a fluorescently labeled AP-1 DNA sequence, using EMSA experiments, it was found that AP-1 binds to DNA and forms a larger molecular complex with DNA. Gel electrophoresis can distinguish unbound DNA from AP-1 binding. The DNA, thereby determining the activity of AP-1, indirectly reflects the redox activity of APE1. C10 inhibited APE1 from reducing AP-1 at a lower concentration than E3330 (Figure 2). APE1 DNA repair mode diagram (Figure 3 left). After APE1 cleaves the DNA substrate, the bands of 18Mer and 40Mer can be distinguished by gel electrophoresis to reflect the cutting efficiency of APE1. C10 does not affect DNA cleavage of APE1 (as shown in Figure 3, right), so compound C10 is a specific inhibitor of the redox end of APE1.

Expression of purified APE1 and AP-1 (c-jun and c-fos) in E.coli

The cDNAs of APE1 and c-fos were cloned into the N-terminal His-tagged pET-28a vector, and c-jun was cloned into the GST-tagged pGEX-4T-1 vector. Each plasmid was transferred to Roseta E. coli and cultured in LB medium. When the bacterial density OD reached 0.6, it was induced with 1 mM IPTG. E. coli transferred to APE-1 was incubated at 37 ° C for 4 h, and E. coli transfected with c-jun and c-fos was incubated at 25 ° C for 4 h. The bacterial solution was lysed with a lysate with PMSF. His-tagged APE-1 and c-fos were purified using Ni2+-NTA-resin and eluted with an imidazole-containing buffer. The GST-tagged c-jun was purified using glutathione magnetic beads and eluted with a low concentration of glutathione buffer. The concentration of the purified protein was determined using a BCA protein quantification kit.

N-terminal activity detection of APE1

Using prokaryotic expressed proteins, APE1 was first treated with 0.25 mM DTT overnight to reduce it, followed by AP-1 and APE1 at 37 degrees for 20 min in EMSA binding buffer, during which different concentrations of drug were added. Then, the DNA substrate sequence of AP-1 was added at 37 degrees and incubation was continued for 30 minutes. TBE-PAGE gel electrophoresis was performed using an Odessy instrument to detect the fluorescent signal of DNA.

C-terminal activity detection of APE1

In vitro synthesis of a DNA sequence containing a mutation site, first using a UDG enzyme to treat the mismatched base, exposing the AP site (APE-1C end has the characteristics of an endonuclease, can recognize the AP site, and cleave) . The treatment with APE-1 enzyme was followed by the addition of an APE-1 inhibitor without a drug gradient. The efficiency of cutting was evaluated by PAGE gel electrophoresis, and the effect of APE-1 inhibitor on the activity of APE-1 C-terminus was estimated.

3. Comparison of various cell activities of C10 and E3330

1) Determination of KSHV cleavage replication inhibitory activity (intracellular lytic replication, extracellular viral particle release and cytotoxicity assay)

We then compared the activities of C10 and E3330 at the cytological level. First we examined the effect of C10 and E3330 on KSHV cleavage replication. BCBL-1 (KSHV positive cells) were induced with 20 ng/ml Tetradecanoyl phorbol acetate (TPA) for 3 hours, and the cells were treated with different concentrations of compounds (the above compounds), and 3 parallel wells were set for each concentration. TPA induction was compared to controls that were not treated with the compound. The virus was lysed and replicated intracellularly 2 days after drug induction. The release of intact viral particles into the body was detected 5 days after drug induction (Fig. 4A, B). In addition, we also tested the binding ability of intracellular C10 and E3330 to inhibit AP-1, and the results showed that C10 is more effective than E3330 in inhibiting the DNA binding activity of intracellular AP-1 (Fig. 4C, D), of which AP-1 Required for viral lytic replication of intracellular KSHV.

Intracellular lysis replication detection method

After the cells were induced for 2 days, the cells were collected and the total DNA of the cells was extracted. The real-time quantitative PCR technique was used to detect the copy number of LANA and GAPDH in the total DNA of the above cells, and the relative ratio of LANA/GAPDH was calculated to evaluate the relative replication of KSHV viral DNA in the cells. Calculate the relative inhibition number of the KSHV cleavage replication = (LANA/GAPDH _{TPA+&compound+} -LANA/GAPDH TPA- _&compound+ )/(LANA/GAPDHTPA _+&compound- -LANA/GAPDH TPA- _&compound- ) to calculate the concentration of each compound at different concentrations The relative inhibition number of KSHV cleavage replication. Taking the relative inhibition number of the virus as the ordinate, the drug concentration as the abscissa plotted the inhibition curve of each compound on viral cleavage replication, and calculating the half inhibitory amount (IC ₅₀ ) of each drug replication to evaluate the inhibition of viral cleavage replication by each compound. active.

Extracellular lysis replication detection method

After the cells were treated with TPA for 5 days, the collected cell culture medium was centrifuged at 1,000 rpm for 5 min, and the cell pellet was discarded. The supernatant was filtered through a 0.45 uM filter, centrifuged again at 100,000 g for 1 h, the supernatant was discarded, and the virus particles were resuspended in 200 ul PBS. The concentrated virus solution was treated with DNase I at 37 ° C for 1 hour to remove the impurity DNA molecules outside the virus particles. The virus concentrate was then treated with a lysate of proteinase K and split virus particles, and the DNA of the KSHV virus was extracted by phenol chloroform. The extracted viral DNA was precipitated by ice ethanol and finally dissolved in 50 ul of TE buffer. Using real-time quantitative PCR technology, LANA primers were used to detect the viral DNA copy number in the cell culture supernatant to evaluate the herpesvirus production level. According to the formula: KSHV toxicity relative inhibition number = (LANA _{TPA + & compound +} -LANA _{TPA + & compound -} ) / (LANA _{TPA + & compound - -} LANA _{TPA - & compound -} ) EBV toxi production relative inhibition number = (EBNA-1 _{TPA + & compound +} - EBNA-1 _{TPA+&compound-} )/(EBNA-1 _{TPA+&compound-} -EBNA-1 TPA- _&compound- ) The relative toxin inhibition number of KSHV at different concentrations of each compound was calculated. Taking the relative inhibition number of toxin as the ordinate, the drug concentration as the abscissa plotted the inhibition curve of each compound on toxicity, and calculating the half-inhibitory inhibitor (EC ₅₀ ) of each compound to evaluate the release of herpes virus particles by each compound. Inhibition activity.

Cytotoxicity assay

The cell density of BCBL-1 cells was adjusted to 3×10 ⁵ cells/ml. Cells were treated with different concentrations of each compound. Three parallel wells were set for each concentration. After 2 days, trypan blue staining was used, and the number of viable cells was counted under light microscope. The result is processed. According to the formula: Relative toxicity = 1 - live cell _{compound +} / live cell _{compound -} The relative toxicity and median lethal dose (CC ₅₀ ) of each compound at different concentrations were calculated to evaluate the cytotoxicity of each compound. Further, the selection inhibition constant of each compound was calculated according to the formula: selection inhibition constant (SI) = CC ₅₀ /EC ₅₀ to evaluate the drug safety of each compound.

2) KSHV-PDLSC tube-forming experiment and pharmacodynamics determination of drug-forming ability

Abundant vascular proliferation in tumor tissues, strong cell invasion ability of tumor cells, and angiogenic factors and inflammatory factors in tumor microenvironment are essential for tumor development and malignant transformation and metastasis. KSHV is a tumor-causing virus. KSHV can significantly increase the angiogenic ability, cell invasion ability and cytokine secretion of PDLSC after infection with PDLSC. These cellular activities induced by the virus are critical for the KSHV to promote the development of KS. We used the KSHV-infected PDLSC model to evaluate the potential of small molecule compounds for the treatment of KS and other tumors.

The experimental results show that C10 can effectively inhibit the angiogenic activity induced by KSHV infection of PDLSC at 1 μM. The E3330 requires at least 50 μM to achieve a similar effect (Figure 5A). In addition, the conditioned medium of K10-treated KSHV-PDLSC reduced the angiogenic ability of PDLSC compared to the conditional culture of KSHV-PDLSC (Fig. 5B), indicating that C10 can affect KSHV-PDLSC to affect microenvironment by secreting cytokines. The angiogenesis of the cells. We also evaluated C10 inhibition of KSHV-induced tube formation directly in animals. Animal experiments showed that C10 can efficiently inhibit the vascularization of KSHV-PDLSC in vivo (Fig. 5C). Since there are a large number of cytokines and chemokines suitable for tumor growth in the tumor microenvironment, we directly evaluated the ability of C10 and E3330 to affect the release of cytokines by KSHV-PDLSC. The results indicate that C10 can block VEGF, IL-6, IL. The secretion of cytokines such as -8, C10 inhibited the ability of KSHV-PDLSC to release cytokines better than E3330 (Fig. 6A, B). AIDS-KS often exhibits strong diffusivity, and the invasive ability of KSHV-PDLSC is significantly increased after infection with PDNSC by KSHV (Fig. 7A, B). Knockdown of APE1 can inhibit KSHV-promoted cell invasion (Fig. 7C, D). The invasive ability of cells was significantly reduced after treatment of KSHV-PDLSC with C10 (Fig. 7E). We treated C10 on osteosarcoma cell lines and found that C10 inhibited the proliferation of osteosarcoma cell lines at 1 μM (Figure 8). In summary, we have discovered a new structure of APE1 inhibitor C10, C10 has the potential to become an effective small molecule compound for the treatment of KS.

Preparation of KSHV virus

Using the iSLK.219 cell line (KSHV.219 latently infected cell line), the latently infected virus in iSLK.219 was introduced into the lytic replication using DOX and sodium butyrate salt. On the fifth day, the induced cell supernatant was collected, and the supernatant was filtered using a 0.45 uM filter to remove cell debris. After 10,000 g of ultracentrifugation for 1 hour, the supernatant was discarded and the virus particles were resuspended in PBS and stored frozen at -80 °C.

KSHV virus infection

PDKSC was infected with the prepared KSHV virus. KSHV infected PSLDC with MOI=20. After the virus was added to the cell culture medium, polybrene was added at a final concentration of 5 ug/ml to increase the infection efficiency of the virus. After centrifugation at 2500 rpm for 60 minutes, the solution was changed.

Angiovascular test method

The matrigel was pre-coated in a 48-well plate and coagulated at 37 degrees for 1 hour. The KSHV-PDLSC and PDLSC were applied to the matrigel on the same cell number, and the actual tube formation was performed after 4-8 hours. Use a microscope to take pictures. To determine the effect of a compound on KSHV-induced tube-forming ability, a corresponding concentration of compound was added during the tube-forming process to observe the effect of the presence or absence of the compound on the KSHV-induced tube forming ability.

Evaluation of invasion ability

Using the Transwell system, Matrigel was pre-plated into a Transwell chamber, cells were seeded in the upper chamber with serum-free medium, and the lower chamber was subjected to a concentration gradient difference with a medium containing 10%-20% FBS. After 16h-24h, the cells invading the lower layer were stained with crystal violet.

Conditional medium of KSHV-PDLSC induces angiogenesis in PDLSC

After KSHV infection of PDLSC, conditioned medium of KSHV-PDLSC and PDLSC was collected separately. The matrigel was pre-plated into a 48-well plate and coagulated at 37 degrees for one hour. The collected conditioned mediums of KSHV-PDLSC and PDLSC were resuspended in the same amount of PDLSC, respectively, and the PDLSC was observed in the conditioned medium. Ability changes.

Detection of drugs on the bypass regulation of KSHV-PDLSC

KSHV-PDLSC and PDLSC were treated with C10 or E3330 for 24h respectively. The upper drug-containing medium was discarded, washed with PBS, replaced with normal medium of low blood, and 24 hours of conditioned medium was collected again. According to the experimental operation of the influence of conditioned medium on PDLSC tube forming ability, the influence of conditioned medium after drug treatment and conditioned medium of untreated group on PDLSC tube forming ability was evaluated.

ELISA detection of cytokine secretion assay

KSHV-PDLSC was treated with the drug for 24 hours, and the drug-containing medium was removed. The cells were cultured for 24 hours, and the supernatant was collected. The ELISA kit was used to detect the cytokine secretion levels of cells treated with different drug concentrations. The change.

Matrigel angiogenesis experiment

The same amount of 5-10X10^7 PDLSC or KSHV-PDLSC was mixed with 500 μl of Matrigel and injected into the groin of mice. The matrigel in the mice was collected for 7 days to observe the formation of blood vessels in the matrigel. The corresponding concentration of the drug can be mixed in the Matrigel to observe the effect of the drug on KSHV-induced tube formation in vivo.

C10 inhibits osteosarcoma and proliferation of nasopharyngeal carcinoma cells

The osteosarcoma cells or nasopharyngeal carcinoma cell lines were combined with 96-well plates, and the corresponding concentrations of the drug were added. After the cells were treated with C10 or the like for 48 hours, the supernatant was discarded and the serum-free medium was added to the MTT, 37 degrees. Incubation for 4 h, then discarding the supernatant again, adding 150 ul of DMSO to dissolve the colored precipitate, and measuring the absorbance of the colored substance at 590 nm to evaluate the number of viable cells to examine the effect of the drug on inhibiting tumor cell proliferation.

The above is a preferred embodiment of the present invention, but the embodiments of the present invention are not limited to the above, and any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and scope of the present invention. Equivalent replacement means are included in the scope of protection of the present invention.

Claims

An APE1 inhibitor characterized in that the APE1 inhibitor has the structure shown in the following formula 1:
The APE1 inhibitor according to claim 1, wherein the APE1 inhibitor has a structure represented by the following formula 2:

Wherein R 1 and R 2 are respectively a five-membered ring, a six-membered ring or a benzene-parallel compound, the five-membered ring is furan, thiophene, pyrrole, thiazole or imidazole, and the six-membered ring is pyridine, pyrazine, pyrimidine or pyridazine, and benzene is connected in parallel. The compound is an anthracene or isoindole compound.
The APE1 inhibitor according to claim 2, wherein the R 1 and R 2 are each a cyclohexyl group or a substituted cyclohexyl group.
The APE1 inhibitor according to claim 1, wherein the APE1 inhibitor has a structure represented by the following formula 3:

Wherein R is a C, N, S or O element; R 1 , R 2 , R 3 , R 4 , R 5 or R 6 are each hydrogen, or various saturated or unsaturated, ring-containing or ring-free, ring-shaped A substituted or unsubstituted alkyl compound.
The APE1 inhibitor according to claim 1, wherein the APE1 inhibitor has a structure represented by the following formula 4:

Wherein R is a C, N, S or O element; R 1 , R 2 , R 3 , R 4 , R 5 or R 6 are each hydrogen, or various saturated or unsaturated, ring-containing or ring-free, ring-shaped A substituted or unsubstituted alkyl compound.
The use of an APE1 inhibitor according to claim 1 for the preparation of a medicament for the treatment of a related disease dependent on the APE1 redox end function.
The use of an APE1 inhibitor according to claim 1 for the preparation of a medicament for the treatment of tumor and vascular aberrant proliferative diseases.
The use according to claim 7, wherein the tumor comprises Kaposi's sarcoma, nasopharyngeal carcinoma or cervical cancer.
The use according to claim 7, wherein the tumor comprises pancreatic cancer, bladder cancer, ovarian cancer, glioma, osteosarcoma, ovarian cancer or breast cancer.
The use according to claim 7, wherein said abnormal vascular proliferative disorder comprises age-related macular degeneration.