WO2019015383A1 - Protein kinase polypeptide inhibitor - Google Patents

Abstract

The present invention provides a protein kinase polypeptide inhibitor PI-38, having an amino acid sequence of Ser-Gly-Arg-Val-Met-Arg-Gly-Pro-Arg-Ser-Ala. The polypeptide PI-38 can block the interaction between p38MAPK protein and an effector protein thereof and specifically inhibit p38 kinase activity.

Description

Protein kinase polypeptide inhibitor Technical field

The invention belongs to the field of biomedicine, and in particular relates to a protease polypeptide inhibitor.

Background technique

MAPKs are a class of protein kinases that are widely present in yeast and higher plants and animals. They are responsible for transmitting specific extracellular stimuli to the nucleus, mediating specific responses of cells, and thus occupy a very important position in cell signaling. . p38MAPK was discovered in 1993 by Brewster et al. when studying the effects of hypertonic environments on fungi. It was later found to be also present in mammalian cells and is also a subclass of MAPKs, which is similar in nature to JNK and belongs to the stress-activated protein kinase. Studies have shown that the activator of the p38MAPK pathway is similar to the JNK pathway. Some pro-inflammatory factors (TNFα, IL-1), stress stimuli (UV, H2O2, heat shock, hypertonic and protein synthesis inhibitors) that activate JNK also activate p38. In addition, p38 can also be activated by lipopolysaccharide and G+ bacterial cell wall components. Upstream kinases that activate p38 include MKK3, MKK4, MKK6, and MKK7, while p38 downstream effector proteins include MK2 (MAP kinase-activated protein kinase 2, MAPKAPK2), MK3, MNK1 (MAP Kinase Interaction protein kinase), PRAK (p38 regulated/activated protein) Kinase), CK2 (Casein Kinase 2) and nuclear transcription factor ATF ₂ (activating transcription factor-2), ATF ₁ , Sap1 (SRF accessory protein 1), CHOP (growth arrest and DNA damage inducible gene 153, GADD153), p53 , STAT1, C/EBPβ, MEF2C (myocyte enhance factor 2C), and MEF2A. Therefore, p38MAPK is widely involved in inflammatory response, stress, development, cell growth and apoptosis, cell cycle regulation, ischemia/reperfusion injury, and cardiac hypertrophy in physiological and pathological processes, thus becoming an important drug development target. .

Most of the existing p38 inhibitor molecules are small molecule compounds that competitively bind to the ATP binding site or other important active sites in the p38 molecule, thereby inhibiting the activity of the kinase. However, due to the strong conservation of the kinase active site, a large number of kinase molecules in the body have similar active sites, so these inhibitors lack specificity; many studies have shown that these inhibitors have different degrees of sensitivity to other kinases. Cross-inhibition, as well as drug off-target effects; in addition, in animal experiments, also showed some gene and liver toxicity. Therefore, there is currently no p38 inhibitor drug suitable for clinical treatment.

In recent years, biopharmaceuticals have become a hot area of drug development due to the in-depth research and development of antibody drugs and their widespread application. As an important component of biopharmaceuticals, peptide drugs have become the focus of pharmaceutical research and development because of their moderate molecular weight (smaller than small molecule compounds), high biological activity and small side effects. With the advancement of the peptide production process, the improvement of the structural modification means and the improvement of the preparation level, the bottleneck problem that restricts the drug-forming properties of the polypeptide drug is gradually solved, so that the approved polypeptide drugs are increased year by year. Taking the FDA's peptide drug data in 2012 as an example, a total of 6 peptide drugs were approved for marketing. There were 14 peptide drugs in the third phase, 74 in the second phase, and 40 in the first phase.

Summary of the invention

In view of the above-mentioned defects of the p38MAPK small molecule inhibitor, the present invention provides a polypeptide inhibitor PI-38 capable of blocking the interaction of the p38MAPK protein with its upstream and downstream effectors and effectively inhibiting the signaling of the p38MAPK signaling pathway.

According to an aspect of the present invention, the present invention provides a polypeptide PI-38 having an amino acid sequence as shown in SEQ ID NO: 1, specifically: SGRVMRGPRSA.

According to another aspect of the invention, the invention provides the use of the polypeptide PI-38 in the manufacture of a medicament for the treatment, prevention, amelioration and/or amelioration of p38 MAPK kinase-associated diseases.

According to another aspect of the present invention, the present invention provides the use of the polypeptide PI-38 for the preparation of a medicament for inhibiting an inflammatory response.

According to another aspect of the present invention, the present invention provides the use of the polypeptide PI-38 for the preparation of a medicament for inhibiting tumors.

The tumor includes bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, colorectal cancer, esophageal cancer, hepatocellular carcinoma, lymphoma, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, Lung cancer, prostate cancer, spleen cancer.

According to another aspect of the invention, the invention provides the use of the polypeptide PI-38 in the manufacture of a medicament for the treatment, prevention, amelioration and/or amelioration of rheumatoid arthritis.

According to another aspect of the present invention, there is provided a pharmaceutical composition comprising the active ingredient polypeptide PI-38 and a pharmaceutically acceptable carrier.

The pharmaceutical compositions include, but are not limited to, injections, tablets, granules, capsules, oral solutions, pills, and the like.

The pharmaceutically acceptable carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrating agents, absorption enhancers, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field, and the like. Flavoring agents, sweeteners and the like can also be added.

The polypeptide PI-38 of the present invention should be "effective amount" as an active ingredient, and the "effective amount" means a non-toxic, but sufficient amount of a drug or agent that provides a desired effect. In the pharmaceutical compositions or kits of the invention, an "effective amount" of an ingredient or formulation unit refers to an amount of the ingredient that is effective to provide the desired effect when used in combination with other ingredients. The "effective amount" will vary from subject to subject, depending on the age and general condition of the individual, the particular active drug, and the like. Thus, it is not always possible to refer to an accurate "effective amount", however, a suitable "effective amount" in any individual case can be determined by one of ordinary skill in the art using routine experimental methods.

The polypeptide PI-38 of the present invention can be prepared by methods known to those skilled in the art (e.g., solid phase synthesis), and can be isolated and purified by separation and purification methods known in the art (e.g., high performance liquid chromatography).

The present inventors have shown that the polypeptide PI-38 can block the interaction of the p38MAPK protein with its effector protein in vitro and specifically inhibit the p38 kinase activity in vitro. PI-38 can inhibit the secretion of tumor necrosis factor TNFα and interleukin-1β in human peripheral blood mononuclear cells induced by bacterial endotoxin LPS stimulation. PI-38 also specifically inhibits phosphorylation of effector molecules by p38MAPK in bacterial endotoxin LPS-stimulated human tumor cells. Animal experiments have also shown that PI-38 can inhibit the inflammatory response.

DRAWINGS

Figure 1 is a mass spectrometry diagram of polypeptide PI-38;

Figure 2 is a blockade effect of the peptide PI-38 on the interaction of the p38MAPK protein with its effector protein;

Figure 3 is a graph showing the inhibitory effect of the polypeptide PI-38 on p38MAPK protein kinase activity;

Figure 4 is a graph showing the inhibitory effect of polypeptide PI-38 on phosphorylation of p38 in tumor cells;

Figure 5 is a graph showing the inhibitory effect of the polypeptide PI-38 on the secretion of TNFα and IL-1β in human peripheral blood mononuclear cells;

Figure 6 is a graph showing the inhibitory effect of the polypeptide PI-38 on the secretion of inflammatory factors.

Detailed ways

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. In addition, it is to be understood that various changes and modifications may be made by those skilled in the art in the form of the invention.

Example 1 Synthesis and Purification of Polypeptide PI-38

1. The peptide PI-38 was synthesized according to the Fmoc solid phase peptide synthesis method. The synthesis steps are as follows:

1.1 In order to ensure that the C-terminus of the polypeptide is -CONH ₂ structure, Rink Amide resin is selected as the polymer solid phase carrier.

1.2 to Fmoc protection group:

1.2.1 The desired polymer solid phase carrier is placed in a synthetic column, and then immersed in an organic solvent DMF (dimethylformamide) for 10 minutes, and then discharged; 20% piperidine/DMF solution is passed over the surface of the resin. The N ₂ reaction was discharged after 10 minutes; the reaction was repeated after passing through 20% piperidine/DMF and passing N ₂ for 10 minutes.

1.2.2 Under N ₂ protection, pass the DMF solvent 5 times to thoroughly wash the above resin.

1.3 condensation reaction:

Amino acid activation: Weigh 3 times the required molar amount of Fmoc-protected amino acid, dissolve in DMF solvent, prepare a saturated solution, add equimolar amount of 1-hydroxybenzotriazole (HOBT) and diisopropyl carbon Imine (DIC), if necessary, additional DMF is added until complete dissolution, forming an amino acid activation solution.

In the N ₂ environment, the amino acid activation solution is introduced into the synthesis column, and is sufficiently contacted with the polymer resin to cause (dehydration) condensation reaction between the active group on the polymer resin and the activated amino acid to form a peptide bond; condensation reaction time at room temperature 30 minutes to 1 hour; discharge waste liquid after reaction;

1.4 Repeat steps 1.2 through 1.3 according to the polypeptide sequence until the synthesis of the entire peptide chain is completed.

1.5 cleavage and removal of side chain protecting groups:

1.5.1 Preparation of trifluoroacetic acid (TFA) mixed lysate "K", the formula is as follows:

Trifluoroacetic acid (TFA) (82.5% v/v)

Phenol (5%)

Water (5%v/v)

Phenylmethyl sulfide (thioanisole) (5% v/v)

1,2-ethanedithiol (2.5% v/v)

1.5.2 Implementation steps:

1.5.2.1, according to step 1.2, first remove the N-terminal Fmoc protecting group of the polypeptide linked to the solid phase carrier, wash away the residual DMF with dichloromethane, dry, and transfer to a dry glass container;

1.5.2.2 The above TFA lysate "K" (30 ml / gram of resin) was introduced, and the reaction was gently stirred at room temperature for 4 hours.

1.5.2.3 After the reaction is completed, the resin is filtered off, and the lysate is directly poured into methylol isobutyl ether (30 ml: 5 ml v/v) cooled in advance (-20 ° C);

1.5.2.4 High-speed centrifugation (4000 rpm, 4 ° C) for 5 minutes, discard the supernatant, dissolve the bottom of the bottle with 95% TFA, and pour into cooled methyl isobutyl ether (30ml: 5ml v / v) precipitate;

1.5.2.5 Discard the clear solution, pass N2 to methyl isobutyl ether to evaporate substantially, dissolve the precipitate with a small amount of deionized water, freeze at -80 ° C, and dry on a freeze dryer to obtain a powdery yellowish thick Peptide.

2. Analysis and purification of peptides:

In Shimadzu LC-10ATVP infusion pump, Shimadzu DGU-14A degasser, Shimadzu SPD-10A UV detector, Rheodyne 7725I injection valve, Phenomenex Nucleosil C18 column (250 × 4.6mm ID), WDL The -95 chromatography station constitutes an HPLC separation system. After sample injection, linear gradient elution of 0.1% trifluoroacetic acid in 5% acetonitrile aqueous solution to 0.1% trifluoroacetic acid in 60% acetonitrile aqueous solution at a flow rate of 1.0 mL/min for 0-30 minutes, UV220nm detection chromatographic conditions , for separation monitoring. The chromatographic peak effluent was collected in a centrifuge tube for subsequent mass spectrometry analysis.

The instrument used for mass spectrometry is Bruker Daltonics BIFLEX type MALDI-TOF mass spectrometer, nitrogen laser, laser wavelength 337nm, using delay (extracted extraction) and reflection (Reflection) working mode, accelerating voltage 19.5kV, reflection voltage 20kV, delay The extraction voltage is 14.5kV, the delay time is 50~200ns, and the positive ion is detected. Mix the chromatographic peak collection solution with an appropriate amount of the matrix solution, take about 1 microliter of the solution, and add it to the sample target. After the solvent is evaporated, the sample is crystallized, sent to a mass spectrometer for mass spectrometry, and 30 times of a single scan signal is obtained to obtain mass spectrum. Figure (see Figure 1).

The peptide PI-38 was identified by HPLC purity, and its purity was greater than 98%, containing 11 amino acids (SGRVMRGPRSA), and the molecular weight was 1173 Da.

The above polypeptide was synthesized by Ningbo Kangbei Biochemical Co., Ltd.

Example 2 Blocking effect of PI-38 on the interaction of p38 MAPK protein with its effector protein.

Using the SPOT technique, the MKK3b and MEF2A proteins interacting with the p38MAPK protein were prepared into a polypeptide array chip, and the polypeptide was used to block the interaction between the protein on the array and the p38 recombinant protein.

MATERIALS: The peptide array was self-made in the laboratory. The amino acid was purchased from Jill Biochemical, the p38 recombinant protein was purchased from GeneTex, the human p38 protein antibody was purchased from CST, and the HRP-labeled anti-human p38 protein antibody was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. Chemiluminescence reaction solution was purchased from Thermo

The polypeptide chip taken out from the -20 degree refrigerator was placed in a dish and washed three times with absolute ethanol for 5 minutes each time. The polypeptide chip membrane was then placed in a dish and washed 3 times with TBS-T solution for 10 minutes each time. Subsequently, the polypeptide chip membrane was placed in a dish, and a blocking solution (5% skim milk powder solution) was added, and after blocking at room temperature for 4 hours, the membrane was washed once with a TBS-T solution for 5 minutes. The p38 protein solution was diluted with a blocking solution (5% skim milk powder solution) to a final concentration of 0.3 nM, and the polypeptide was incubated with the p38 protein reaction solution for 30 minutes at a final concentration of 0.9 nM, respectively. Or the p38 protein reaction solution containing no polypeptide was incubated at room temperature for 2 hours, and then washed with a TBS-T solution for 3 times for 10 minutes each time. The primary antibody (human p38 protein antibody) solution was diluted with the above blocking solution at a dilution ratio of 1:500. The polypeptide chip membrane was placed in the reaction solution and shaken for 2 hours, and then washed with a TBS-T solution for 3 times, each time 15 times. minute. Afterwards, the HRP-labeled secondary antibody solution is diluted with a blocking solution, usually at a dilution ratio of 1:5000 (the dilution can be adjusted according to the antibody specification), and the polypeptide chip membrane is placed in the reaction solution for 2 hours after shaking, and TBS-T is used. The solution was shaken and washed three times for 15 minutes each time. Then, the excess buffer solution on the polypeptide chip membrane was discarded, and the chemiluminescence reaction solution was added dropwise to the membrane. After the reaction solution completely covered the surface of the membrane, the reaction was fully reacted for 2 minutes to keep the membrane moist. The film is placed in a chemiluminescence detector to scan the spots on the film, and the scanned image is saved, as shown in FIG.

The polypeptides selected in the box are the sites where the polypeptide blocks the interaction between the p38 protein and the array polypeptide. It can be seen from the figure that the polypeptide blocks the interaction between p38 and MKK3b and MEF2A, respectively, indicating that the polypeptide can block at the molecular level. Interacting with recombinant p38 protein and effector protein.

Example 3 Inhibition of p38MAPK protein kinase activity by PI-38

The p38 kinase assay/inhibitor screening kit is a non-quantitative enzymatic assay for detecting p38 protein activity. The kit for detection in the kit is pre-coated with a substrate equivalent to ATF2, and ATF2 contains a Thr amino acid residue which can be efficiently phosphorylated by p38. The detection antibody can specifically detect the ATR2 phosphorylated by p38 kinase. . When an active polypeptide inhibitor is added and the inhibitory active polypeptide is capable of inhibiting the kinase activity of p38, the specific antibody does not recognize the phosphorylated Thr on the ATF2 protein.

Materials: The p38 kinase assay/inhibitor screening kit was purchased from Abnova, the small molecule inhibitor SB202190 was purchased from MCE, and the other reagents were all included in the kit.

Remove the appropriate number of plated wells from the plate provided on the kit and place on ice for later use. 10ul of purified p38 recombinant protein (final concentration 10munits/well), 10ul positive control SB202190 (final concentration 20uM), and 10ul gradient diluted peptide samples were added to different wells (final concentrations were 20nM, 200nM, 2μM, respectively). 20 μM, 200 μM), then 90 ul of Kinase Reaction Buffer was added to each well, and the lid was incubated at 30 ° C for 60 min. Clean each plate hole 5 times with Wash Buffer, pour off the remaining Wash Buffer, gently tap the plate to remove the remaining Wash Buffer, or draw the remaining liquid. 100 μl of Anti-phospho-ATF2 Thr71 polyclonal antibody (PPT-09) was added to each well, and the plate was placed on the plate for 30 min at room temperature. After washing the plate again (the method is the same as above), 100 ul of HRP-labeled anti-rabbit IgG was added to each well, and the cover plate was placed on the plate for 30 min at room temperature, and the unbound liquid portion was discarded. Wash the plate again (the method is the same as above), then add 100 ul Substrate Reagent to each well, cover the plate with the plate and leave it at room temperature for 5-15 min. Finally, 100 ul of stop solution was added to each well, and the plate was placed in a microplate reader and read at a wavelength of 450 nm.

The experimental results are shown in Fig. 3 and Table 1. The results showed that the absorbance of the plate was gradually decreased with the increase of the polypeptide concentration after adding different concentrations of the polypeptide, indicating that the p38 kinase activity was inhibited by the polypeptide.

Table 1 Inhibition of p38MAPK protein kinase activity by different concentrations of peptides

Example 4 PI-38 specifically inhibits phosphorylation activation of p38 in tumor cells

MATERIALS: Human breast cancer cells (MCF-7) were purchased from ATCC; cell culture fluid RPMI1640 was purchased from Hyclone; calf serum was purchased from Hangzhou Sijiqing Bioengineering Materials Co., Ltd.; anisomycin was purchased from MCE; PVDF membranes were purchased from PALL Phosphorylated ATF2, phosphorylated HSP27, phosphorylated p38 antibodies were purchased from Santa Cruz; Tublin antibodies were purchased from CST.

MCF-7 cells were cultured in RPMI1640 cell culture medium containing 10% FBS at 37 ° C, 5% CO 2 to 80% pellets. The cultured cells were transferred to a 24-well cell plate containing 0.2 ml of fresh RPMI 1640 cell culture medium containing 10% FBS at 5 × 10 5 cells/well, and cultured for one day to 80%. Then, fresh RPMI1640 cell culture medium containing 10% FBS was replaced and treated with different concentrations of peptide inhibitor (5 μM, 10 μM or 25 μM) for half an hour, and then stimulated with 10 μg/mL of anisomycin for 30 minutes. The culture supernatant was discarded, and total cell protein was collected, separated by 12% SDS-PAGE electrophoresis and electroporated to a PVDF membrane. Phosphorylated ATF2, phosphorylated HSP27, phosphorylated p38 antibody and Tublin antibody (control) were used for western blot to detect the corresponding phosphorylation level of p38 effector protein.

The results are shown in Figure 4, indicating that the polypeptide can inhibit phosphorylation of the p38 effector protein in tumor cell MCF-7 in a dose-dependent manner.

Example 5 PI-38 inhibits TNFα and IL-1β secretion in human peripheral blood mononuclear cells

MATERIALS: Healthy human blood was collected from laboratory staff. Lipopolysaccharide (LPS derived from E. coli 055:B5) was purchased from sigma, cell culture DMEM was purchased from Hyclone, buffer DPBS and PBS were purchased from Beijing. Laibao Biotechnology Co., Ltd., Elisa kit for human IL-1β and TNF-α was purchased from Beijing Baizhi Biotechnology Co., Ltd.

PBMCs (peripheral blood lymphocytes) were isolated from heparin anticoagulated whole blood of healthy people. After washing the cells three times with pH 7.2 concentration of 0.01 M Duns' phosphate buffer (DPBS), the cells were resuspended in DMEM medium containing 5% endotoxin-free fetal bovine serum and antibiotic (penicillin-streptomycin), followed by 2.5×10. ^{At 5} cells/well, the obtained PBMCs were placed in a 96-well cell culture plate. The polypeptide inhibitors prepared in PBS at a pH of 7.2 concentration of 0.01 M were added to the cells at different final concentrations (0.0001-25 μM), and after co-incubation with the cells for 1 hour, the cells were stimulated by adding LPS at a final concentration of 22 ng/ml for 18 hours. The cell supernatant after 24 hours of culture was collected, and the concentration of the cytokine IL-1β and TNF-a in the supernatant was measured by Elisa kit.

The results are shown in Figure 5 and Table 2. As can be seen from the figure, with the increase of the concentration of the peptide inhibitor, the secretion of IL-1β and TNF-a decreased compared with the control, indicating that the polypeptide inhibitor effectively inhibits monocytes. The activity of p38.

Table 2 Inhibition of TNFα and IL-1β secretion by human peripheral blood mononuclear cells

Example 6 Inhibition of PI-38 on secretion of inflammatory factors in a mouse model of acute inflammation

Fifty male Kunming mice (17-22 g) (China Concord Animal Center) were randomly divided into 5 groups, 10 in each group. Pretreatment was given separately: the positive drug control group was given indomethacin (China Hebei Yongfeng Pharmaceutical Co., Ltd.) by intragastric administration (25 mg/kg), the blank control group was given intraperitoneal injection of the same volume of normal saline, and the experimental group was given intraperitoneal injection. Polypeptide (PI38) low dose (5 mg/kg), intraperitoneal injection of active polypeptide (10 mg/kg) and intraperitoneal injection of active polypeptide (PI38) high dose (20 mg/kg). After half an hour, the mice were coated with xylene-induced inflammatory agent 20 μL/ear on both sides of the right ear, and the left ear was a blank control. Two hours later, the mice were sacrificed by cervical dislocation, the ears were cut along the baseline of the auricle, and the ears were placed on the same site with an 8 mm corneal trephine, and weighed with an electronic balance. The weight of the left ear was subtracted from the weight of the left ear, and the swelling degree (mg) of each group was calculated. The swelling rate (%) = (swelling degree of the blank control group - swelling degree of the administration group) / swelling degree of the blank control group.

The results are shown in Figure 6 and Table 3. As can be seen from the figure, the polypeptide is capable of inhibiting the release of inflammatory factors in an animal model of inflammation, indicating that the polypeptide has anti-inflammatory drug activity.

Table 3 Inhibition of inflammatory factor secretion by mouse polypeptide in acute inflammation model

Claims (8)

1. A polypeptide PI-38, characterized in that the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1.
2. The use of the polypeptide PI-38 according to claim 1 for the manufacture of a medicament for the treatment, prevention, amelioration and/or amelioration of a p38 MAPK kinase-associated disease.
3. The use of the polypeptide PI-38 according to claim 1 for the preparation of a medicament for inhibiting an inflammatory response.
4. The use of the polypeptide PI-38 according to claim 1 for the preparation of a medicament for inhibiting tumors.
5. The tumor according to claim 4, including bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, colorectal cancer, esophageal cancer, hepatocellular carcinoma, lymphoma, melanoma, myeloid leukemia, myeloma, oral cavity Cancer, ovarian cancer, lung cancer, prostate cancer, spleen cancer.
6. Use of the polypeptide PI-38 according to claim 1 for the manufacture of a medicament for the treatment, prevention, amelioration and/or amelioration of rheumatoid arthritis.
7. A pharmaceutical composition comprising the active ingredient polypeptide PI-38 and a pharmaceutically acceptable carrier.
8. The pharmaceutical composition according to claim 7 is any one of an injection, a tablet, a granule, a capsule, an oral solution or a pill.

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