CN109223794B - Compound C6 as histone methyltransferase NSD3 activity inhibitor and application thereof - Google Patents

Abstract

The invention discloses a compound C6 as an inhibitor of histone methyltransferase NSD3 activity and a pharmaceutical application thereof. The compound C6 has the chemical structure shown in formula I:

the chemical name is as follows: 6-amino-9- (2- (naphtalen-1-yloxy) ethyl) -9H-purine-8-thiol; the pharmaceutical application refers to that at least one of the compound C6 or hydrate, pharmaceutically acceptable salt, tautomer, stereoisomer and precursor compound thereof is used as an active ingredient for preparing the antitumor drug. Experiments show that the compound C6 can effectively inhibit the activity of NSD3 enzyme, and the enzymology level IC of the compound C6₅₀The value is 17.97 +/-2.75 mu mol/L, and the compound can obviously inhibit the growth and proliferation of non-small cell lung cancer cell lines H460, H1299 and H1650.

Description

Compound C6 as histone methyltransferase NSD3 activity inhibitor and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a histone methyltransferase NSD3 activity inhibitor and application thereof.

Background

Histone methylation is one of the most important modification mechanisms for epigenetic modification. The abnormal expression of histone methyltransferases of histone methylation and the regulation thereof is closely related to the occurrence of various diseases such as genetic diseases, autoimmune diseases, aging, cancers and the like, and has important significance particularly in the occurrence and development of tumors. Therefore, histone methyltransferases are considered as potential new targets for anti-tumor therapy.

In recent years, the development of inhibitors targeting histone methyltransferases has been carried out, the histone methyltransferases EZH2 inhibitor EPZ-6438(Epizyme company) enters a phase I/II clinical test in 2014 at present, is used for treating non-Hodgkin lymphoma, advanced solid tumors and malignant mesothelioma patients, the histone methyltransferases DOT 1L inhibitor EPZ-5676(Epizyme company) also enters a phase I clinical test in 2014 at present, is used for treating acute leukemia patients, and in addition, a plurality of small molecule inhibitors of histone lysine methyltransferases, such as SETD7 inhibitor PFI-2, G9a (EHMT2) and G L P (EHMT1) inhibitors UNC0638, A-366, SMYD 1 inhibitor 1Y-507, are in preclinical research stages, and have a wide application for the development of anti-targeting therapeutic drugs, the development of histone methyltransferases (NSCLC 1) and the development of malignant tumor cells of pancreatic cancer cells, such as breast cancer, the tumor cells, the tumor growth of NST-72, the tumor growth of NSCLC 1, the gene of the gene, the gene of the NSCLC 1, the gene, the CHCD 1, the gene of the gene, the gene of the.

NSD3 consists of 1 SET (Su (var), Enhancer of zeste, and Trithorax) domain, 2 PWWP (Pro-Trp-Trp-Pro motif) domains and 4 PHD domains (Plant Homeodomail). Wherein the SET domain is a catalytic center, the PWWP and PHD domains are generally involved in chromatin-related transcriptional regulation and DNA repair and other biological processes.

However, in the research field of histone lysine methyltransferase inhibitors, most of the research is mainly focused on histone lysine methyltransferases such as EZH1, EZH2, DOT 1L, SETD7, EHMT1 and EHMT2, and relatively few researches on other histone methyltransferases which are proved to be closely related to the development of human tumorigenesis.

Disclosure of Invention

The invention provides a histone methyltransferase NSD3 activity inhibitor, which is a compound of formula I or a pharmaceutically acceptable salt thereof

The invention also provides a new application of the compound in the formula I in preparing a medicine for treating tumors.

Preferably, the tumor of the invention is lung cancer, breast cancer, pancreatic cancer, osteosarcoma, head and neck cancer, and the delivered lung cancer is preferably non-small cell lung cancer.

Preferably, the medicament of the invention is a medicament prepared from a compound of formula I, a hydrate, a pharmaceutically acceptable salt, a tautomer, a stereoisomer or a precursor compound thereof and one or more pharmaceutically acceptable carriers. The carrier includes diluent, excipient, filler, binder, wetting agent, disintegrating agent, absorption enhancer, surfactant, adsorption carrier, lubricant, etc. which are conventional in the pharmaceutical field.

The medicine of the invention can be prepared into various forms such as injection, tablets, powder, granules, pills, capsules, oral liquid, ointment, cream and the like. The medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field.

The medicine is used for treating tumor by inhibiting the activity of histone methyltransferase NSD3, and can be introduced into organism such as muscle, intradermal, subcutaneous, vein, mucosal tissue by injection, spray, nasal drop, eye drop, penetration, absorption, physical or chemical mediated method; or mixed or coated with other materials and introduced into body.

The invention searches and discovers that the compound (hereinafter referred to as C6) in the formula I can inhibit the activity of NSD3 enzyme by adopting a method of integrating virtual screening and experimental determination from a ChemDiv compound library, and verifies the anti-tumor activity of the compound by combining cell experiments, although the research on the inhibitor C6 is not mature to enter the clinical stage, the research is crucial to finally developing the anti-NSD 3 medicament and has important application prospect.

The invention adopts a computer simulation method to carry out advanced evaluation on the effectiveness of drug molecules, and adopts a biological experiment method to detect and verify the activity, so as to obtain the effective NSD3 enzyme inhibitor C6 (chemical name: 6-amino-9- (2- (naphthalene-1-yloxy) ethyl) -9H-purine

8-thiol), the value of the enzymology level IC50 is 17.97 +/-2.75 mu mol/L, the anti-proliferation effect on a plurality of non-small cell lung cancer cell lines is achieved, the IC50 values are respectively 50-100 mu mol/L, and the good NSD3 enzyme inhibition effect is achieved.

Drawings

FIG. 1: IC50 value plot of C6 at NSD3 protein level.

FIG. 2: expression of H3K36me3 in non-small cell lung cancer cell lines.

FIG. 3: c6 inhibited NSD3 enzyme activity in cells.

FIG. 4: growth inhibition by C6 on non-small cell lung cancer cell lines.

FIG. 5: c6 inhibits growth and proliferation of non-small cell lung cancer cells.

Detailed Description

In order to confirm the antitumor effect of the compounds of the present invention, the present invention will be further described with reference to the accompanying drawings and specific examples.

1. Experimental methods

1.1 virtual receptor-based screening

First adopt

The Protein preparation module in the software package, Protein prepatio Wizard, processed the crystal structure of NSD3 (PDB: 4YZ 8). The ChemDiv database was subjected to pretreatment of compounds, including de-weighting, removal of salt ions and inorganics, using Discov ery Studio 2.5, and structure normalization. By using

The L igPrep module in 9.0 produces the possible ionization states and tautomers of the compounds under pH 7.4 conditions.

Before virtual screening by adopting a molecular docking method, firstly, the effectiveness of the adopted Glide docking method needs to be verified, an NSD3 active site is defined, and the centroid of a ligand molecule S-adenosyl methionine (SAM) in a crystal structure is set as the center

In the cube region, using the SP (Standard precision) parameter setting of Glide software, ligand molecule SAM was treated with L igPrep and then docked into the NSD3 active pocket, and Glide was found to better replicate the binding conformation in the crystal structure.

Adopting a Glide HTVS (High through Virtual Screening) mode to carry out butt joint and scoring on ChemDiv library compounds, and selecting thirty-one-hundred-thousand compounds which are scored at the top; and then, carrying out re-docking and scoring by adopting a Glide SP mode, and keeping a small molecule binding mode of 3 ten thousand before scoring. In the crystal structure of the SAM binding to NSD3, three key hydrogen bonding interactions are formed with the active domain residues HIS1224, HIS 1274. For 3 ten thousand small molecule binding modes generated by docking, a hydrogen bond standard is adopted as a screening condition, and binding modes forming more than 2 hydrogen bonds with HIS1224 and HIS1274 are selected, so that 697 qualified compound molecules are obtained. In order to fully consider the structural diversity of the compounds, a Canvas module in Schrodinger is adopted for clustering analysis, and a batch of compounds are selected for experimental determination. The compound of the formula I (hereinafter referred to as C6) is found to have better histone methyltransferase inhibitory activity.

The chemical structural formula of the compound of formula I (the compound of formula I is hereinafter referred to as C6)

1.2 NSD3 in vitro enzyme Activity detection assay procedure

1) Expression and purification of NSD3 protein: a part of NSD3 gene (1021-1320aa) is obtained by PCR amplification from HEK293 cell line cDNA, and is subcloned in a prokaryotic expression vector pGEX-4T1 to construct a recombinant plasmid with a GST tag, and after sequencing and identification, the recombinant plasmid is transformed in Escherichia coli Rosseta to induce expression (after induction by 0.5mM IPTG, continuous culture at 16 ℃ for 20 hours), and purified by GST resin to obtain GST-tagged NSD3 protein.

2) NSD3 in vitro enzyme activity detection step:

① the compound and 2.3. mu.g of NSD3 protein were mixed well and left at 30 ℃ for 15 minutes;

② histone H3K36Me1 polypeptide fragment (ATKAARKSAPATGGV-K (Me1) -KPHRYRPG-GK (Biotin)) (final concentration: 0.2. mu.M) and S-adenosyl methionine SAM (final concentration: 1.0. mu.M) were added to the reaction mixture and reacted at 30 ℃ for 1 hour in 50mM Tris-HCl pH8.5, 50mM NaCl, 5mM MgCl2, 1mM DTT and 0.01% Tween reaction solution;

③ and combining with the cisbio HTRF histone methyltransferase detection kit, detecting corresponding fluorescence values at the wavelengths of 620nm and 665nm by using a multifunctional enzyme-labeling instrument, and calculating the activity of NSD 3.

1.3 intracellular NSD3 enzyme Activity detection assay

1) Protein immunoblotting (Western Blot) assay to determine the protein expression of H3K36me3 in various non-small cell lung cancer cell lines, and selecting a H3K36me3 high expression cell line and a low or no expression cell line to determine the intracellular NSD3 enzyme inhibition activity of the compound.

2) Planting cells on a 6-hole plate, adding a compound after 16 hours, continuously culturing for 48 hours, and then cracking the cells by using RIPA lysate to obtain a protein sample; the protein immunoblotting experiment measures the expression of H3K36me3 protein.

1.4 cell level antitumor Activity test

1) Tumor cell growth inhibition rate: planting 5000 cells in a 96-well plate, adding control DMSO or compounds with different concentrations after 16 hours, and continuously culturing in a 5% carbon dioxide incubator at 37 ℃; dojindo cellcounting is added after 72 hourskit-8 reagent, reacting at 37 ℃ for 1 hour, measuring absorbance at 450nm, and finally obtaining the tumor cell inhibition rate IC of the compound₅₀.

2) Cell proliferation assay: the 96-well plates were seeded with 500 cells (H460 and H1299) and 2000 cells (H1650), respectively, and after 16 hours the culture was continued by adding control DMSO or 25. mu. M C6, and the medium was changed every 2 days (containing the corresponding control DMSO or 25. mu. M C6). The corresponding values were determined with Dojindo cell counting kit-8 reagent at days 0, 1, 3, 5, 7 (H460 cell line), 0, 1, 3, 5, 7, 9 (H1299 cell line), and 0, 1, 4, 7, 10, 13, and 16 (H1650 cell line), respectively, after planting.

3) Plate clone formation test comprises planting 5000 cells in 6-well plate, adding control DMSO or compounds with different concentrations after 16 hours, culturing at 37 deg.C in 5% carbon dioxide incubator, changing liquid every 2 days, standing for 7-10 days, discarding supernatant, carefully washing with 1m L PBS for 2 times, adding 1m L pure methanol for fixation for 20 minutes, removing fixation liquid, adding 1m L Giemsa staining solution, standing for 10-30 minutes, slowly washing off staining solution with ultrapure water, and air drying.

2. Results of the experiment

The activity of C6 against histone methyltransferase NSD3 at protein and cell level, and the inhibition effect and inhibition rate of C6 against growth and proliferation of non-small cell lung cancer cells at cell level were determined.

IC at the level of in vitro proteomics₅₀The curve results show that C6 has good NSD3 enzyme inhibition effect on protein level and IC thereof₅₀The value was 17.97. + -. 2.75. mu.M, as shown in FIG. 1.

In the test of the expression level of the most main substrate H3K36me3 of NSD3 in a non-small cell lung cancer cell line, H460, H1299 and H1650 cell lines with higher expression level are selected to test the inhibitory effect of C6 on the activity of NSD3 enzyme in cells, and the result is shown in figure 2.

Inhibition assay the inhibition of substrate H3K36me3 by C6 treatment of H460, H1299 and H1650 cells at 30 μ M concentration for 48 hours was 55.89%, 48.96% and 34.64%, respectively; under the same conditions, the 60 μ M concentration produced inhibition rates of 32.25%, 23.58% and 18.84%, respectively, indicating that C6 was also effective at inhibiting NSD3 enzyme activity at the cellular level, as shown in fig. 3.

Tumor growth inhibition rate experiments of different concentrations of C6 on A549, H460, H1299 and H1650 non-small cell lung cancer cell lines show that the C6 concentration gradients are 0, 12.5, 25.0 and 50.0 mu M respectively. In an A549 cell line with low expression of H3K36me3, the concentration of 50.0 mu M has no obvious inhibition effect on the growth of the A549 lung cancer cell; in contrast, C6 showed significant inhibitory effects on cell growth proliferation in three other lung cancer cell lines (H460, H1299, H1650) that highly expressed H3K36me3, as shown in fig. 4.

The plate clone formation assay (fig. 5) also revealed that C6 has a significant inhibitory effect on the growth and proliferation of non-small cell lung cancer cells.

In conclusion, the compound C6 is an effective histone methyltransferase NSD3 inhibitor, C6 can obviously inhibit NSD3 enzyme activity in cells, reduce the level of H3K36me3 and has a remarkable inhibiting effect on the growth and proliferation of non-small cell lung cancer cell lines. The C6 compound can obviously inhibit the proliferation of tumor cells when being used as a histone methyltransferase inhibitor, has potential anti-tumor effect, is expected to be used as an active ingredient for preparing anti-tumor medicaments, is especially expected to be used for preparing anti-lung cancer medicaments, and has medicinal prospect.

Claims (4)

1. The application of an inhibitor of the activity of histone methyltransferase NSD3 in preparing an anti-tumor medicament is characterized in that the inhibitor is a compound shown as a formula I or a pharmaceutically acceptable salt

The tumor is non-small cell lung cancer.

2. The use of claim 1, wherein the medicament is a compound of formula I or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers.

3. The use according to claim 2, wherein the medicament is for the treatment of a tumor by inhibiting histone methyltransferase, NSD3, activity.

4. The use according to claim 2, wherein the medicament is in the form of tablets, capsules, granules or pills.

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