CN109223793B - Compound C1 as histone methyltransferase NSD3 activity inhibitor and application thereof - Google Patents

Abstract

The invention discloses a compound C1 as an inhibitor of histone methyltransferase NSD3 activity and a pharmaceutical application thereof. The compound C1 has the chemical structure shown in formula I:

the chemical name is as follows: 6-amino-9- (2- (o-tolyloxy) ethyl) -9H-purine-8-thiol; the pharmaceutical application refers to that at least one of the compound C1 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 C1 can effectively inhibit the activity of NSD3 enzyme, and the enzymology level IC of the compound C1₅₀The value is 14.63 +/-3.85 mu mol/L, and the compound has good NSD3 enzyme inhibition effect, is used as an effective inhibitor of an important tumor target NSD3, is expected to be used as an active ingredient for preparing antitumor drugs, and has medicinal prospects.

Description

Compound C1 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 hydrate, a pharmaceutically acceptable salt, a tautomer, a stereoisomer and a precursor compound thereof

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

Preferred tumors of the invention are lung cancer, breast cancer, pancreatic cancer, osteosarcoma, head and neck cancer.

Preferably, the medicament of the invention is a medicament prepared from a compound of formula I or a hydrate, a pharmaceutically acceptable salt, a tautomer, a stereoisomer, a precursor compound and one or more pharmaceutically acceptable carriers thereof. 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 C1) in the formula I can inhibit the activity of NSD3 enzyme from a ChemDiv compound library by adopting a method of integrating virtual screening and experimental determination, and verifies the anti-tumor activity of the compound by combining a cell experiment.

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 C1 (chemical name: 6-amino-9- (2- (o-tolyloxy) ethyl) -9H-purine-8-thiol), the enzymology level IC50 value of which is 14.63 +/-3.85 mu mol/L, and the invention has good NSD3 enzyme inhibition effect.

Drawings

FIG. 1: IC50 value plot of C1 at NSD3 protein level.

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.

Test example one, C1 test for inhibiting NSD3 enzyme Activity

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 C1) 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 C1)

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 Compound C1 and 2.3. mu.g 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.

2. Results of the experiment

The inhibitory effect of C1 on the activity of histone methyltransferase NSD3 at the protein and cellular level was determined.

As shown in FIG. 1, C1 has good NSD3 enzyme inhibition effect on in vitro protease level, and IC thereof₅₀The value was 14.63. + -. 3.85. mu.M.

In conclusion, the compound C1 is a potent histone methyltransferase NSD3 inhibitor, and can effectively inhibit the enzymatic activity in vitro. Due to the important effect of NSD3 in various tumors, the compound C1 has potential anti-tumor effect as an NSD3 inhibitor, is expected to become an active compound for preparing anti-tumor drugs, especially for preparing anti-lung cancer drugs, 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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