CN108853110B - Compound A8 as histone methyltransferase NSD3 activity inhibitor and application thereof - Google Patents

Abstract

The invention discloses a compound A8 as an inhibitor of histone methyltransferase NSD3 activity and a pharmaceutical application thereof. The compound A8 has the chemical structure shown in formula I:

the chemical name of the compound is 6-amino-9- (2- (p-tolyloxy) ethyl) -9H-purine-8-thiol, and the medicinal application refers to that at least one of the compound A8 or hydrate, pharmaceutically acceptable salt, tautomer, stereoisomer and precursor compound thereof is used as an active ingredient for preparing the antitumor medicament.

Description

Compound A8 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 provides a new application of an inhibitor in preparing a medicine for treating tumors.

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 and head and neck cancer, and the lung cancer is preferably non-small cell lung cancer.

Preferably, the medicament of the invention is 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 adopts a method of integrating virtual screening and experimental determination to search and find a compound (hereinafter referred to as A8) in a formula I from a ChemDiv compound library, which can inhibit the activity of NSD3 enzyme, and verify the anti-tumor activity of the compound by combining cell experiments, although the research on an inhibitor A8 is not mature to enter the clinical stage, the research is crucial to finally develop an 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 an effective NSD3 enzyme inhibitor A8 (chemical name: 6-amino-9- (2- (p-tolyloxy) ethyl) -9H-purine-8-thiol) with an enzymology level IC50 value of 0.69 +/-0.06 mu mol/L, has the effect of inhibiting growth on H460 non-small cell lung cancer cell lines with IC50 values of 5-10 mu mol/L, and has good NSD3 enzyme inhibition effect.

Drawings

FIG. 1: IC50 value plot of A8 at NSD3 protein level.

FIG. 2: a8 inhibited NSD3 enzyme activity in cells.

FIG. 3: a8 has growth inhibiting effect on H460 non-small cell lung cancer cell.

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 possible ionization states and tautomerism of compounds at pH 7.4A structure body.

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 A8) 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 abbreviated as A8)

1.2NSD3 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 adding histone H3K36me1 polypeptide fragment respectively

(ATKAARKSAPATGGV-K (Me1) -KPHRYRPG-GK (Biotin)) (final concentration of 0.2. mu.M) and S-adenosyl methionine SAM (final concentration of 1.0. mu.M) were reacted at 30 ℃ for 1 hour in a reaction solution of 50mM Tris-HCl pH8.5, 50mM NaCl, 5mM MgCl2, 1mM DTT and 0.01% Tween;

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) 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; western blot experiments determined H3K36me3 and H3 protein expression.

1.4 cell level tumor suppression assay

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 ℃; adding Dojindo cellcounting kit-8 reagent after 72 hours, reacting at 37 ℃ for 1 hour, measuring the absorbance at 450nm, and finally measuring the tumor cell inhibition effect of the compound.

2. Results of the experiment

The inhibitory effect of A8 on the activity of histone methyltransferase NSD3 at protein and cell level and the inhibitory effect of A8 on the growth and proliferation of H460 non-small cell lung cancer cells at cell level were determined.

A8 has good NSD3 enzyme inhibition effect on in vitro protease level, and IC thereof₅₀The value was 0.69. + -. 0.06. mu.M, as shown in FIG. 1.

H460 and H1299 cells were treated with 0, 2.5, 5. mu.M Compound A8 for 48 hours, respectively, and the inhibitory effect of the compound on the modification of H3K36me3 in the cells was examined, with histone H3 as the reference protein. The results show that the inhibition rate of the compound on the modification of H3K36me3 in H460 and H1299 cell lines is 37.3% and 38.1% respectively at a concentration of 2.5. mu.M compared with the control group without the compound; the inhibition rate of the compound on the H3K36me3 modification in H460 and H1299 cell lines is 87.7% and 70.0% at a concentration of 5 μ M, respectively, which shows that A8 can effectively inhibit NSD3 enzyme activity and reduce H3K36me3 modification at a cellular level, and see figure 2.

Tumor growth inhibition experiments of A8 on H460, a non-small cell lung cancer cell, were performed at different concentrations, with A8 concentrations in gradients of 0, 2.5, 5.0, 10.0 and 20.0. mu.M, respectively. The results show that A8 has obvious inhibition effect on cell growth and proliferation in H460 non-small cell lung cancer cell lines, and the inhibition effect is enhanced with the increase of action concentration, compared with the control group, the inhibition rate of A8 on tumor cells with different concentrations is 21.7%, 41.3%, 48.3% and 54.5%, respectively, and P is less than 0.05), which is shown in figure 3.

In conclusion, the compound A8 is an effective histone methyltransferase NSD3 inhibitor, A8 can obviously inhibit the activity of NSD3 enzyme in cells, reduce the level of H3K36me3 and also has obvious inhibition effect on the growth and proliferation of non-small cell lung cancer cell lines. The A8 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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