CN112263582B

CN112263582B - S100A8/A9 protein inhibitor Tepontinib and application thereof

Info

Publication number: CN112263582B
Application number: CN202011219787.2A
Authority: CN
Inventors: 池在龙; 李可馨
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2020-11-04
Filing date: 2020-11-04
Publication date: 2022-03-15
Anticipated expiration: 2040-11-04
Also published as: CN112263582A

Abstract

The invention relates to a new application of a MET kinase inhibitor Tepontinib as an S100A8/A9 protein inhibitor and application thereof, and discloses a new application of an old drug, wherein an FDA approved drug is subjected to computer virtual screening of a molecular docking model through a crystal structure based on S100A8/A9, and a molecular docking simulation process, so that the MET kinase inhibitor Tepontinib can be stably combined on a CHAPS combination site of an S100A9 dimer, and the formation and the stability of the S100A8/A9 oligomer are influenced by inhibiting S100A9 dimerization and simultaneously inhibiting S100A9 dimerization in an S100A8/A9 oligomer, and a new example is provided for the MET kinase inhibitor Tepontinib, and the rapid development of a novel S100A8/A9 protein inhibitor.

Description

S100A8/A9 protein inhibitor Tepontinib and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a new application of a MET kinase inhibitor Tepontinib as an S100A8/A9 protein inhibitor and an application thereof.

Background

The S100 protein comprises 25 known members and is an important family of multifunctional calcium binding proteins. The S100A8 and S100a9 proteins are major members of the S100 protein family, and can form heterodimers, heterotetramers, etc. in vitro and in vivo, and exist as oligomers (S100A8/a9, also known as Calprotectin), thereby playing an important role in the regulation of inflammation and immune response. At the site of infection or sterile injury, granulocytes and monocytes highly express the S100A8/A9 protein and secrete outside the cell. The S100A8/A9 protein acts as a risk associated molecular patterns (DAMPs) protein and an alarm (alarmin), interacts with a pattern recognition receptor Toll-like receptor 4(TLR4) and a advanced glycation end product (RAGE), activates intracellular NF-kB and MAPK pathways, aggregates inflammatory cells through chemotaxis on neutrophils, and secretes inflammatory cytokines and chemokines, thereby exerting a pro-inflammatory effect.

The serum concentration of S100A8/A9 is proved to reflect the severity of various inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus and psoriasis, the stability and the sensitivity of the serum concentration are superior to those of C-reactive protein (CRP), and the S100A8/A9 protein is considered to be a better inflammation-related biomarker.

In addition, other studies show that the S100A8/A9 inhibitor can achieve the purpose of anti-inflammation through interaction with the inhibitor, and has good effect in the treatment of systemic lupus erythematosus; S100A8/A9 is also involved in the pathophysiological processes of other diseases, including: S100A8/A9 induces BV-2 microglia to be activated by activating an NF-kB signal channel, promotes the generation of proinflammatory factors and further aggravates OPC injury; blocking the combination of the S100A8/A9 protein and a receptor thereof can inhibit the migration of neutrophils after myocardial infarction and improve the cardiac function; inhibition of S100A8/A9 can prevent the induction of proinflammatory cytokines and the activation of NF-kB after hypoxia, and delay the progress of heart failure; the S100A8/A9 mediates the accumulation of neutrophils in the development process of chronic tuberculosis, and the S100A8/A9 protein inhibitor controls the inflammation caused by mycobacterium tuberculosis (Mtb) of patients with non-acute tuberculosis; song G et al developed a new monoclonal antibody against S100A8/A9, which could effectively prevent the metastasis of lung cancer; in addition, research shows that Melanoma Cell Adhesion Molecules (MCAM) are important receptors of S100A8/A9, the combination of S100A8/A9 and MCAM leads to the metastasis of melanoma cells to lung tissues, and the combination of S100A8/A9 and cell surface receptor Melanoma Cell Adhesion Molecules (MCAM) accelerates the growth and metastasis of breast cancer and prostate cancer; S100A8/A9 mediates kidney injury and fibrosis, possibly through interaction with tubular epithelial cells resulting in irreversible injury, and inhibition of S100A8/A9 is a therapeutic strategy for preventing renal fibrosis in patients with chronic kidney disease. The above suggests that S100A8/A9 is a promising drug target. In view of the above, the project is designed to screen and develop small molecule compounds with S100A8/A9 as target proteins.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a novel small molecule compound which is combined with the S100A8/A9 protein in a targeted way.

The invention provides application of a MET kinase inhibitor Tepontinib in preparation of a S100A8/A9 protein targeted inhibition binding agent.

Further, the binding target of the MET kinase inhibitor Tepontinib is positioned in the hydrophobic amino acid or the hydrophobic side chain part of the hydrophilic amino acid of the S100A9 protein in the S100A8/A9 protein.

Further, the binding target of the MET kinase inhibitor Tepontinib comprises His61, Leu86, Leu49, Flq48, Asn47, Lys50 and Lys51 of one S100A9 protein in the S100A8/A9 hetero oligomer and His61, Lys57, Lys51, His61, Glu52 and Leu62 amino acid sites on the other S100A9 protein.

Further, a MET kinase inhibitor Tepontinib is used for preparing the S100A8/A9 protein targeted inhibition binding agent as an anti-inflammatory drug; or an anti-cancer drug; or a myocardial infarction prognostic drug; or a drug for relieving heart failure; or drugs for ameliorating chronic pulmonary tuberculosis; or a medicament for treating chronic kidney disease and renal fibrosis.

Further, the MET kinase inhibitor Tepotinib is used for preparing the S100A8/A9 protein targeted inhibition binding agent as an anti-inflammatory drug, wherein the inflammation comprises uveitis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus and psoriasis.

Further, the S100A9 dimer or S100A8/A9 protein targeted inhibition binding agent is used as an anticancer drug, and the cancer comprises lung cancer, breast cancer, prostate cancer and melanoma.

The invention has the following advantages: the invention carries out computer virtual screening of a molecular docking model on FDA approved drugs through a crystal structure based on S100A8/A9 based on the thought of old drugs, and a molecular docking simulation process finds that MET kinase inhibitor Teponinib can be stably combined on a CHAPS binding site of an S100A9 dimer, inhibits S100A9 dimerization in an S100A8/A9 heterotetramer while inhibiting S100A9 dimerization, or inhibits S100A9 dimerization in other S100A8/A9 oligomers, thereby influencing the formation and the stability of S100A8/A9 protein, and in vitro experiments also prove that the MET kinase inhibitor Tepontinib is a S100A9 dimer and a S100A8/A9 protein inhibitors, thereby proving that the MET kinase inhibitor Tepontinib is a S100A9 dimer and a S100A8/A9 protein inhibitor. The invention not only provides a new application of the MET kinase inhibitor Tepotinib, but also provides a paradigm for the rapid development of a novel S100A8/A9 protein inhibitor.

Drawings

FIG. 1 Interactive chemical model of MET kinase inhibitor Tepontinib;

FIG. 2(a) the tetrameric structure S100A8/A9, with the A/B chain being S100A8 and the C/D chain being S100A 9; (b) the binding site of S100a9 (at a); (c) crystal structure of S100a9 dimer, S100a9 dimer inhibitor CHAPS, steric hindrance (at B);

FIG. 3 is a molecular docking pattern diagram of MET kinase inhibitor Tepontinib;

FIG. 4(a) a graph of the interaction between the MET kinase inhibitor Tepontinib and the S100A9 protein; (b) specific experimental data for the interaction curve;

figure 5(a) the concentration of inflammatory cytokine IL-6 in cell supernatants was measured by ELISA kit and the data were mean ± SD, P < 0.05 compared to LPS group; (b) measuring the concentration of inflammatory cytokine TNF- α in the cell supernatants by ELISA kit, data as mean ± SD, P < 0.0005 compared to LPS group; FIG. 6(a) anterior ocular segment inflammation was not observed in the normal control group; (b) pupillary constriction, vasodilation, and even flexion into a spiral (arrow), anterior chamber exudate (thick arrow) were observed in LPS group under slit lamp 24 hours after LPS injection; (c) only slight vasodilation (arrow) was observed in 2752-65-0(10 μ M) + LPS group; (d) clinical scores of different groups after LPS injection, P < 0.0001 compared to LPS group.

Figure 7 effect of MET kinase inhibitor Tepotinib on concentration of infiltrating cells and proteins in aqueous humor, P < 0.0001 compared to LPS group.

Detailed Description

The present invention will be further described in detail with reference to the following examples and effect examples, which are not intended to limit the scope of the present invention, but are not intended to limit the scope of the present invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The molecular formula of MET kinase inhibitor Tepontinib is C₂₉H₂₈N₆O₂The molecular weight is 492.6g/mol, and the molecular weight is represented by the following structural formula and an interactive chemical model as shown in figure 1.

Example 1: MET kinase inhibitor Tepontinib and S100A9 dimer molecule docking virtual model

The crystal structure (ID: 1XK4) of S100A8/A9 is shown in FIG. 2a, the A chain and B chain are S100A8, and the C chain and D chain are S100A 9. Because it forms a stable tetrameric configuration, it is difficult to optimize reasonable binding sites by molecular dynamics simulations. Further analysis shows that the crystal structure (ID: 1IRJ) of S100A9 has a binding site of a small molecule CHAPS, which can inhibit the activity of S100A9 and provide the possibility of virtual screening.

By analysis of the S100a9 dimerization crystal structure, the binding site of CHAPS (S100a9 dimerization inhibitor) corresponds to the binding sites of two S100a9 in the S100A8/a9 tetramer (fig. 2b indicates at arrow a). The S100A9 dimer crystal structure (PDB ID: 1IRJ) was congruent with the S100A8/A9 heterotetramer crystal structure (PDB ID: 1XK4), and from FIG. 2C it can be seen that the S100A9 dimerization inhibitor, CHAPS, can inhibit S100A9 dimerization in the S100A8/A9 tetramer (steric hindrance with the C-terminal Trp88, His91, Glu92 of S100A9, arrow B). Therefore, the inhibitor for S100A9 dimerization can inhibit S100A9 dimerization independently, and also can obviously inhibit S100A9 dimerization in the S100A8/A9 oligomer, thereby influencing the formation and stability of the S100A8/A9 oligomer. In view of this, we determined that the CHAPS binding site of the S100A9 dimer was used as a virtual screening binding site, and the crystal structure adopted was 1 IRJ.

Through screening compounds from different data, the binding effect of the MET kinase inhibitor Tepontinib and the CHAPS binding site of the S100A9 dimer is found to be good, and the molecular docking mode of the MET kinase inhibitor Tepontinib is shown in figure 3. In the figure, a mark represents a MET kinase inhibitor Tepontinib compound. Two identical S100a9 have some critical amino acid residues on the interacting surface, and it is these amino acids that are involved in binding to small molecule compounds. The MET kinase inhibitor Tepotinib is integrally bound to a hydrophobic oral cavity formed by a large number of hydrophobic amino acids (or hydrophobic side chain portions of hydrophilic amino acids) such as His61, Leu86, Leu49, Flq48, Asn47, Lys50, Lys51 on one S100a9 and His61, Lys57, Lys51, His61, Glu52, Leu62 on the other S100a 9.

Example 2: MET kinase inhibitor Tepontinib and S100A9 dimer molecule interaction

Biomolecular interactions were detected and analyzed using an Octet platform based on biofilm interference technology (BLI). The experimental Data was processed through Data Analysis software 9.0. Selecting a fitting model: 1: 1 model, namely the fitting mode of 1S 100A8/A9 protein combined with 1 small molecule MET kinase inhibitor Tepontinib: global, i.e. 6 concentrations were analyzed as a set of correlations. Preparing 125, 62.5, 31.3, 15.6, 7.81 and 3.91 mu M Tepotinib solution, detecting Tepotinib with different concentration with 100 mu L6 group and CaCl with 100 mu L concentration of 200 mu M after S100A8/A9 protein solidification of the chip₂Signals and data interacting with 100 μ L of 5% DMSO + PBST buffer; detecting blank chip, Tepotinib with different concentrations and 100 muL CaCl with concentration of 200 muM₂Signals and Data from the interaction with 100 μ L of 5% DMSO + PBST buffer were processed through Data Analysis software 9.0.

Through the analysis, the interaction between the small molecule drug and the S100A8/9 protein is verified, the result is shown in figure 4, the binding signal between the S100A8/A9 protein and the MET kinase inhibitor Tepontinib is increased along with the increase of the concentration of the MET kinase inhibitor Tepontinib, the binding signal and the MET kinase inhibitor Tepontinib are in positive correlation, and the interaction between the S100A8/A9 protein and the MET kinase inhibitor Tepontinib is verified.

Example 3: in vitro cell anti-inflammatory effect of MET kinase inhibitor Tepotinib

Preparing a Raw264.7 cell inflammation model stimulated by LPS, selecting MET kinase inhibitor Tepotinib with 0.2 mu M drug concentration, adding the MET kinase inhibitor Tepotinib into a culture medium, setting a normal Raw264.7 cell group and a Raw264.7 cell inflammation model control group not added with the MET kinase inhibitor Tepotinib and measuring the concentration of inflammatory cytokines in cell supernatant through an ELISA kit after setting an experimental group and the control group for 24 hours, wherein the result is shown in figure 5, the LPS stimulation promotes the obvious up-regulation of IL-6 and TNF-alpha in cell supernatant, and the MET kinase inhibitor Tepotinib can inhibit the secretion of IL-6 and TNF-alpha and shows better in-vitro anti-inflammatory effect.

Example 4: anti-inflammatory effect of MET kinase inhibitor Tepontinib on treatment of anterior uveitis

An LPS-induced anterior uveitis rat model (EIU) was prepared by injecting LPS into Waistar rat hindfoot pads, 100. mu.l of LPS at a concentration of 1mg/ml per footpad to set up the EIU model, a normal control group was prepared by injecting 100. mu.l of sterile saline into Waistar rat hindfoot pads, an experimental group was prepared by taking 10mM Tepotinib and diluting the Tepotinib to 100. mu.M with sterile saline, 2. mu.l of MET kinase inhibitor Tepotinib was injected into the vitreous humor of the LPS-induced anterior uveitis rat model, and after 24 hours, inflammation conditions of the normal control group, LPS group and experimental group were observed to evaluate clinical scores of inflammation reactions of the normal control group, LPS group and experimental group, respectively, and the results are shown in FIG. 6, and clinical scores of inflammation reactions of the experimental group were significantly reduced.

And the number of infiltrating cells in the aqueous humor was measured to confirm the anti-inflammatory effect of the MET kinase inhibitor Tepotinib in EIU. 24 hours after LPS injection, the number of aqueous humor cells in LPS group was significantly increased, but almost no cells were found in normal group. The number of infiltrating inflammatory cells in the aqueous humor of the tepotiib (100 μ M) administered group was significantly reduced compared to the LPS group, and the results are shown in fig. 7.

Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.

Claims

Use of the MET kinase inhibitor Tepotinib in the preparation of an anti-inflammatory medicament for the treatment of uveitis.
2. The use according to claim 1, characterized in that the MET kinase inhibitor Tepotinib, the binding target of which is located in the hydrophobic amino acid or the hydrophobic side chain portion of the hydrophilic amino acid of the S100a9 protein in the S100A8/a9 protein, acts as a S100A8/a9 protein targeted inhibitor.
3. The use according to claim 2, wherein the binding target of the MET kinase inhibitor Tepotinib comprises His61, Leu86, Leu49, Flq48, Asn47, Lys50, Lys51 of one of the S100a9 proteins and His61, Lys57, Lys51, His61, Glu52, Leu62 amino acid sites of the other S100a9 protein.