CN105820058A - Novel inhibitor of arginine deiminase 4 - Google Patents

Abstract

The present invention provides a novel inhibitor of arginine deiminase 4 (PAD4), the K _D value of the dissociation constant between the compound and PAD4 is 112 μM.

Description

Novel inhibitors of arginine deiminase 4

technical field

The invention belongs to the field of pharmaceutical compounds, and more specifically relates to an inhibitor of arginine deiminase 4 (PAD4) and an application in combination with PAD4.

Background technique

Rheumatoid arthritis (rheumatoid arthritis, RA) is a serious chronic autoimmune disease that threatens human health. The current method of treating RA is to relieve symptoms, such as anti-inflammation, detumescence and pain relief. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the first-line anti-RA drugs that reduce tissue pain and inflammation, including aspirin, celecoxib, meloxicam, and cortisone (glucocorticoids). NSAIDs drugs can alleviate clinical symptoms but cannot cure inflammation, and cannot prevent joint destruction and tissue damage. They need to be used in combination with other antirheumatic drugs to effectively control the disease. Disease-modifying antirheumatic drugs (DMARDs), as second-line anti-RA drugs, are used to alleviate or prevent RA cartilage and joint damage, including methotrexate, hydroxychloroquine, etc. DMARDs usually have a slow onset of action and affect the immune process of RA patients. The current therapeutic drugs have no decisive effect on the immunopathological mechanism of RA, and cannot treat RA fundamentally. Therefore, it is necessary to develop drugs targeting the pathogenesis of RA.

In recent years, it has been discovered that PAD4 is a new target for the treatment of rheumatoid arthritis. PAD4 is one of the modifying enzymes that catalyzes arginine in protein polypeptide chains to citrulline. Studies have shown (ZhangY.2013) that PAD4 protein is significantly expressed in patients with rheumatoid arthritis, which causes citrullination of various proteins in RA patients, such as citrullination of fibrin and antithrombin in synovium. And then form citrullinated protein/peptide antigen (citrullinated protein/peptideantigen, CPA), stimulate RA patients to autoimmune to form anti-citrullinated protein antibody. Anti-citrullinated protein antibodies target a variety of proteins and peptides that play a pathogenic role in autoimmune arthritis. PAD4 is a calcium ion-dependent enzyme that contains five Ca ²⁺ binding sites, consists of 663 amino acid residues, and has a molecular weight of 74kDa. PAD4 has 2 domains, an N-domain and a C-domain. The catalytically active site of PAD4 is located in the C-domain, which contains four key amino acid residues Asp350, His471, Asp473 and Cys645. Studies have shown (Arita K., 2004) that the binding of Ca ²⁺ causes a conformational change of PAD4, thereby forming an active site cleft that accommodates histone N-terminal peptide and two Ca ²⁺ binding sites. PAD4 can catalyze the deimination of monomethylarginine residues and peptidylarginine to citrulline, but not dimethylarginine.

At present, the development of targeted PAD4 inhibitors is aimed at the active catalytic site, which can be divided into irreversible inhibitors and reversible inhibitors. Irreversible inhibitors are mainly haloamidines, including F-amidine and Cl-amidine, with IC ₅₀ ranging from 1.9 to 22 μM. The hydrogen amidine inhibitors are reversible inhibitors of PAD4, indicating that the active site of PAD4 requires an electron-withdrawing leaving group (halogen) to react with a thiol, thereby increasing the electrophilicity of the amidine carbon atom. Existing reversible inhibitors (paclitaxel, streptomycin, minocycline, etc.), except GSK199 and GSK484 (IC ₅₀ is 200nM and 50nM) (Lewis, HD, 2015), all have weak inhibitory activity, IC ₅₀ Values are at the millimolar level.

Contents of the invention

The object of the present invention is to provide compound NSC299189 and its application in combination with PAD4 protein.

The invention provides a compound with the following structural formula:

The above compounds are naphthoquinone compounds and contain a 2-[2-(2-hydroxyethylamino)ethylamino] substituent.

The present invention provides the use of the above compound in combination with PAD4.

The hydroxyl group of the above compound forms two hydrogen bonds with the Asp473 and His471 residues in the active pocket of PAD4. The amino groups of the above compounds form hydrogen bonds with Asp350 in the active pocket of PAD4. The naphthoquinone group of the above compound extends to the solvation region and forms two hydrogen bonds with the Arg374 and Arg345 residues in the active pocket of PAD4.

Experiments have shown that the above compounds can bind to PAD4 protein, and the dissociation constant is K _D =218 μM, and can be used to develop anti-RA inhibitors.

Description of drawings

Attached is the structure diagram of NSC299189.

detailed description

In order to understand the present invention, the present invention is further illustrated below with examples, but it is not intended to limit the protection scope of the present invention.

The NSC299189 compound (2-[2-(2-hydroxyethylamino)ethylamino]naphthalene-1,4-dione) was obtained from the NCI database. The present invention discovers a new structural type of PAD4 protein inhibitor NSC299189 through molecular dynamics simulation, pharmacophore model, molecular docking and in vitro compound screening based on the transition state structure of the reaction between fluoroamidine and PAD4. The structure of the NSC299189 compound is shown below:

Molecular formula of NSC299189: C ₁₄ H ₁₆ N ₂ O ₃ .

Molecular weight of NSC299189: 260.

NSC299189 is a naphthoquinone compound and contains a 2-[2-(2-hydroxyethylamino)ethylamino] substituent.

The NSC299189 compound prevents its ability to convert arginine to citrulline by binding to the active site of PAD4.

The dissociation constant K _D of the NSC299189 compound and PAD4 is 112 μM, and has the potential to be further optimized into an anti-RA candidate drug.

The ability of NSC299189 to bind PAD4 protein is determined by its own molecular structure characteristics. The hydroxyl group in the NSC299189 structure forms two hydrogen bonds with the Asp473 and His471 residues in the active pocket of PAD4. The amino group forms a hydrogen bond with Asp350 in the active pocket of PAD4. And the naphthoquinone group of the compound extends to the solvation region, forming two hydrogen bonds with the Arg374 and Arg345 residues in the active pocket of PAD4. Thus, the affinity of compound NSC299189 to PAD4 protein was enhanced.

In the evaluation of the biological activity of anti-PAD4 protein, since we did not have a direct method for detecting PAD4 activity, we used Surface Plasmon Resonance (SPR) technology to detect the binding activity between the compound and PAD4 protein.

1. Test material

BiacoreT200 optical biosensor (BiacoreLifeSciences, GE Healthcare) CM5 biosensor chip

10mM sodium acetate (pH5.0)

Buffer: 50 mm HEPES, pH 8.0, 300 mM NaCl, 2 mM DTT, 20 mM CaCl2, and 5% DMSO.

2. Test method

2.1 Drug preparation

Drug: --- (DMSO stock solution, 10mM)

The HEPES buffer solution containing 5% DMSO was used for 2-fold concentration gradient dilution. The final concentrations of the test drugs were 500 μM, 250 μM, 62.5 μM, 31.25 μM, 15.63 μM, and 7.81 μM. The final concentration of DMSO in the system was 5%.

2.2 Test procedure

2.2.1 The PAD4 protein was diluted to a concentration of 50 μg/ml with 10 mM sodium acetate (pH 5.0), and the diluted PAD4 was immobilized on the CM5 biosensor chip by amino coupling.

2.2.2 Compounds of each concentration (7.81-500 μM) were injected onto the surface of PAD4 protein at a constant flow rate of 30 μL/min for 60 s.

2.2.3 The buffer solution (50mmHEPES, pH8.0, 300mMNaCl, 2mMDTT, 20mMCaCl2, and 5% DMSO) was continuously washed on the surface of the chip for 300s to dissociate the compound.

2.2.4 Select a concentration of 31.25 μM to do repeated experiments.

2.2.5 Collect experimental data, and use BiacoreT200 evaluation software for data analysis, and calculate the dissociation constant K _D value of the compound and PAD4.

Claims (6)

1. A compound of the following structural formula: 2. The compound according to claim 1, characterized in that the compound is a naphthoquinone compound and contains a 2-[2-(2-hydroxyethylamino)ethylamino] substituent. 3. The use of the compound according to claim 1 in binding to PAD4 protein. 4. The use according to claim 3, characterized in that, the hydroxyl group of the compound forms two hydrogen bonds with the Asp473 and His471 residues in the active pocket of PAD4. 5. The use according to claim 3, characterized in that the amino group of the compound forms a hydrogen bond with Asp350 in the active pocket of PAD4. 6. The use according to claim 3, characterized in that the naphthoquinone group of the compound extends to the solvation region and forms two hydrogen bonds with the Arg374 and Arg345 residues in the active pocket of PAD4.