CN110950834A

CN110950834A - Identification and evaluation of novel EED-EZH2 interaction small molecule inhibitors

Info

Publication number: CN110950834A
Application number: CN201911177340.0A
Authority: CN
Inventors: 张华�; 刘凯璐; 闫薛; 朱孔凯
Original assignee: University of Jinan
Current assignee: University of Jinan
Priority date: 2019-11-26
Filing date: 2019-11-26
Publication date: 2020-04-03

Abstract

The invention relates to application of a compound 35 shown in figure 1 as an EED-EZH2 interaction small molecule inhibitor targeting the bottom of an EED and application thereof in preparing a medicament for preventing and/or treating malignant lymphoma. The malignant lymphoma is a disease related to diffuse large B cell lymphoma.

Description

Identification and evaluation of novel EED-EZH2 interaction small molecule inhibitors

Technical Field

The invention belongs to the field of medicines, and relates to application of a compound 35 shown in figure 1 as a small molecule inhibitor of EED-EZH2 interaction and application of the compound in preparation of a medicine for preventing and/or treating malignant lymphoma.

Background

Histone methylation is one of the important epigenetic modifications that mediate a variety of basic cellular processes. Polycomb inhibitory Complex 2 (PRC 2) has histone methyltransferase activity, catalyzes the trimethylation of 27 th lysine in histone H3, and plays an important role in various biological processes such as embryonic development, stem cell plasticity, cell differentiation and the like. PRC2 is composed of three key subunits, E2H2, EED and SUZ12, E2H2 is a catalytic subunit, and functions after forming a complex with EED and SUZ12, and PRC2 is inactivated by deletion of EED or SUZ 12.

Numerous studies have shown that abnormal activity of the core component of PRC2 is closely associated with a variety of diseases, particularly the invasion, development and progression of malignant tumors. In various human cancers, such as breast cancer, prostate cancer, liver cancer, colorectal cancer, glioma and lymphoma, the expression levels of EZH2, EED and SUZ12 are significantly increased, while the down-regulation of EZH2 can significantly inhibit the proliferation of these tumor cells. Thus, PRC2 is a very promising anti-tumor target.

Targeting EZH2 or EED-EZH2 protein-protein interactions (PPIs) are two strategies currently in development of PRC2 inhibitors. Among the reported EZH2 inhibitors, GSK126, EPZ6438 and CPI-1205 have entered clinical trials. However, on the one hand, because EZH2 inhibitors act directly on the binding site of its natural substrate, S-adenosylmethionine (SAM), whereas there are similar SAM binding pockets in more than 50 methyltransferases in humans, it takes a lot of experimental resources to test the selectivity of compounds for other homologous proteins; on the other hand, EZH2 needs to form a complex with EED and SUZ12 to exert catalytic activity, and thus, by inhibiting the interaction of EZH2 with its accessory proteins (e.g., EED), the catalytic activity of EZH2 can also be inhibited, and at the same time, since the specificity of the EZH2 protein-accessory protein interaction site is much higher than that of its binding site with SAM, a site inhibitor targeting protein-protein interaction can solve the problem of selectivity of the compound. In 2013, Woojinkim et al, Harvard medical college, designed and synthesized an EZH2 cyclic peptide compound (stabilized peptide) according to the compound crystal structure of EZH2 and EED, selectively inhibited the combination of EZH2 and EED, promoted the degradation of multiple components of PRC2, and further inhibited the activity of methyltransferase. Compared with an EZH2 inhibitor, the cyclic peptide compound has a killing effect on tumor cells depending on the catalytic activity of EZH2, and more importantly, has an obvious proliferation inhibition effect on some tumor cells (such as basal malignant breast cancer, androgen-resistant malignant prostate cancer and the like) depending on EZH2 but not depending on the activity of methyltransferase, so that the application range of the PRC2 compound inhibitor is greatly expanded. However, the use of polypeptides is severely restricted due to the disadvantages of low cell penetration, poor metabolic stability and expensive preparation. On the contrary, the small molecular compound has the characteristics of good permeability, high metabolic stability, low preparation difficulty and the like, and effectively makes up for the defects of the polypeptide compound. Therefore, the high-activity and high-drug-activity small molecule inhibitor specifically targeting the interaction of the EED-EZH2 has wider clinical application prospect, and can provide powerful theoretical support for the treatment of various malignant tumors depending on EZH 2.

The interaction interface (top and bottom) of the EED with EZH2 can be targeted by PPI inhibitors. Researchers at noval pharmaceutical company have discovered a number of PPI inhibitors targeting the top of the EED and determined eutectic compound structures, while few are currently targeted to the bottom of the EED.

In view of the above, there is an urgent need in the art to develop PPI small molecule inhibitors that target the bottom of the EED.

Disclosure of Invention

The inventor of the invention aims to solve the problems, and discovers a novel EED-EZH2 interaction small-molecule inhibitor targeting the bottom of EED by combining virtual screening with biological evaluation, and measures the antiproliferative activity of the inhibitor on a Toledo cell of a diffuse large B cell lymphoma cell line. It is therefore an object of the present invention to provide a novel EED-EZH2 interaction small molecule inhibitor, Compound 35, as shown in FIG. 1.

Another object of the present invention is to provide a use of compound 35 shown in fig. 1 for the preparation of a medicament for the prevention and/or treatment of malignant lymphoma.

Preferably, the malignant lymphoma is a disease associated with diffuse large B-cell lymphoma.

The compound 35 shown in figure 1, as an inhibitor of the EED-EZH2 interaction and an inhibitor of PRC2, can effectively inhibit the EED-EZH2 interaction, thereby inhibiting the catalytic activity of PRC2, and further effectively prevent and treat malignant lymphoma diseases related to diffuse large B cell lymphoma.

Drawings

FIG. 1 novel EED-EZH2 interaction small molecule inhibitor, Compound 35.

Fig. 2 is a flow diagram of virtual screening.

Figure 3 predicted binding pattern of compound 35.

Figure 4 MACCS fingerprint similarity.

Figure 5 chemical structure and inhibition curve of compound 35.

Figure 6 anti-cell proliferation activity of compound 35.

Detailed Description

In the invention, a fluorescence polarization technology is adopted to carry out high-throughput screening on candidate compounds, the fluorescence polarization method is widely used for the high-throughput screening work of small molecule inhibitors of protein-protein or nucleic acid-protein interaction, the method is that EED protein, FITC-EZH2 polypeptide tracer substrate and a compound to be detected are added into buffer solution, after incubation for hours at room temperature, a microplate reader is used for detecting the fluorescence polarization value, and the result shows that the compound 35 can compete with the EZH2 polypeptide to be combined with the EED protein; the molecular docking technology is adopted to discuss the combination mode of the compound 35 and the EED; in vitro cell activity assays were performed to determine the antiproliferative activity of compound 35 on Toledo cells of the diffuse large B-cell lymphoma cell line, and the results indicated that compound 35 exhibited dose-dependent antiproliferative activity on Toledo cells.

The invention will now be further illustrated with reference to specific examples, which are not intended to be limiting but merely illustrative.

Experimental example 1: performing virtual screening based on molecular docking on the SPECS database, selecting a candidate compound, and predicting the binding mode of the head-of-seedling compound and the EED by adopting a molecular docking technology.

In previous studies, we found the first potent EED-EZH2 interaction inhibitor targeting the bottom of EED through virtual screening (astemizole IC)₅₀= 93.80 μ M), in order to obtain more PPI inhibitors targeting the bottom of the EED, a SPECS database containing 211838 compounds was subjected to molecular docking-based virtual screening.

Molecular docking uses the Glide 5.5 program of the software package Maestro 7.5. minimize protein coordinates using default settings in the protein preparation wizard workflow, centered on resolving binding free energy-determining key residues (R46 residues), construct a cubic box of length 15 Å as binding sites while generating lattice points within the box, introduce the compounds prepared in the SPECS database into pre-defined binding sites in Standard Precision (SP) mode, perform further docking validation and additional precision (XP) analysis on the top 500 compounds, then perform cluster analysis on the top 200 compounds using Pipeline pilot7.5, select 50 candidate compounds based on the cluster analysis results and purchase from the SPECS database supplier, then test their EED-EHZ2 interaction inhibition activity using fluorescence polarization experiments, select the top compounds, and virtually screen the flowsheet as shown in fig. 2.

The predicted binding pattern of compound 35 is shown in fig. 3, from which it is seen that compound 35 is able to occupy the binding site of R46 well, forming a hydrophobic interaction with residues L315, G316, D317, L318, W373, Q374, L391, V393, and P396.

Example 2: and calculating the similarity of the MACCS fingerprint by using the RDkit.

Smiles formats of compound 35 and reference compounds (astemizole and apomorphine hydrochloride) were converted to two-dimensional structures using molfrommemmiles, MACCS key fingerprint similarity was calculated and compared. The results are shown in fig. 4, from which it can be seen that compound 35 has little structural similarity to astemizole and apomorphine hydrochloride.

Example 3: and (3) expressing and purifying the protein.

Subcloning an EED gene (EED protein with an encoding amino acid sequence of 81-441) into a modified pET28a vector (with a 6 XHis SUMO tag at the N-terminal), overexpressing a recombinant protein at 16 ℃ by using an escherichia coli strain BL21 (DE3), collecting the strain, performing ultrasonic treatment, centrifuging (18000 rpm, 30 min, 4 ℃), and taking a supernatant; proteins were purified by nickel affinity chromatography (GE Healthcare) and size exclusion chromatography (Superdex 75; GE Healthcare). The proteins were stored in a buffer containing 25mM HEPES (pH 8.0), 150 mM NaCl and 1 mM DTT.

Example 4: the influence of candidate compounds on the EED-EZH2 interaction was examined by Fluorescence Polarization method (FP).

Fluorescence polarization experiments, using Fluorescein Isothiocyanate (FITC) labeled EHZ2 polypeptide as tracer substrate, preparing a series of test compounds 35 at diluted concentrations from 100 mM DMSO stock solutions, adding 625 nM EED protein, 20nM FITC-EZH2 polypeptide tracer substrate (residues 39-63) and different concentrations of test compounds 35 in 40. mu.L FP buffer (25mM HEPES, pH 8.0, 150 mM NaCl, 0.1 mg/mL BSA and 0.01% NP40), incubating at room temperature for 2 hours, detecting fluorescence polarization values with a fully automatic multifunctional microplate reader (POLAR horse Omega microplate reader, EnVision, Perkin Elmer), using excitation light and emission light with wavelengths of 485nM and 520nM, respectively; three sets of repeated experiments are set for each group, and experimental data are analyzed and charted by adopting GraphPad Prism 6.0 software. The results are shown in FIG. 5, Compound 35 (IC)₅₀= 63.88 ± 6.48 μ M) exhibits better than astemizole (IC)₅₀= 93.80 μ M), indicating that compound 35 may compete with EZH2 polypeptide for binding to EED protein, exhibiting potent inhibitory activity.

Example 5: in vitro cell viability assay experiments examined the effect of compound 35 on DLBCLs cancer cell proliferation caused by deregulated PRC2 activity.

2 × 10 per well on RPMI 1640 medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin⁴Density of individual cells exponentially growing cells were cultured, then incubated with 100. mu.M and 50. mu.M of Compound 35, and after three days of incubation, Cell Titer-Glo luminescence Cell Viability kit (Promega) was used to test Cell activity, and the results of the experiment are shown in FIG. 6, and Compound 35 exhibited dose-dependent antiproliferative activity on Toledo cells.

Combining the results of the above specific examples, compound 35 was able to occupy well the binding site of R49, had little similarity to the fingerprints of two known active compounds (astemizole and apomorphine hydrochloride) and was shown to be comparable to astemizole (IC)₅₀= 93.80 μ M) more potent inhibitory activity, exhibiting dose-dependent antiproliferative activity on Toledo cells. Our work provides a new backbone for the discovery of more PRC2 inhibitors and structural implications for the design of novel EED-EZH2 interaction inhibitors.

Claims

1. The compound 35 shown in figure 1 is used as an EED-EZH2 interaction small molecule inhibitor targeting the bottom of EED and an application thereof in preparing a medicament for preventing and/or treating malignant lymphoma.