RAC1 inhibitor and application thereof

Abstract

The invention discloses an RAC1 inhibitor and application thereof, and belongs to the technical field of medical preparations, and the RAC1 inhibitor comprises chlorogenic acid or a derivative thereof. The chlorogenic acid and the derivative thereof can be combined with RAC1, inhibit abnormal activation / activity of RAC1 and inhibit mesangial cell proliferation caused by abnormal activation of RAC1, have no toxic effect on cells, and are a safe and effective RAC1 inhibitor; the glomerular injury caused by RAC1 abnormal activation can be relieved.

Description

Technical Field

[0001] The present invention relates to the technical field of medical preparations, and in particular to a RAC1 inhibitor and application thereof. Background Art

[0002] The RAC1 protein is a plasma membrane-associated small GTPase that dynamically regulates a variety of cellular responses by cycling between an active GTP-bound state and an inactive GDP-bound state. In its active form, RAC1 regulates processes such as secretory function, phagocytosis, cell polarization, neuronal activity, and growth factor-induced membrane ruffle formation. Abnormal activation of RAC1 is closely related to the occurrence and development of a variety of diseases, including cancer, cardiovascular disease, and neurodegenerative diseases. In mesangial cells, the activation of RAC1 also plays an important role. Mesangial cells are an important component of the glomerulus, and their main function is to maintain the structure and filtration function of the glomerulus. Studies have shown that abnormal activation of RAC1 can lead to proliferation and inflammatory responses of mesangial cells, thereby inducing glomerular damage. Therefore, inhibiting abnormal activation of RAC1 is one of the main methods to alleviate glomerular damage.

[0003] The development and application of RAC1 inhibitors still face some limitations. Although a variety of RAC1 inhibitors have been developed, such as NSC23766 and EHT1864, they have problems such as high IC50 and do not meet the clinical use standards. Natural products, as an important source for drug development, have better biocompatibility and lower toxicity. Therefore, the research on screening safe and effective RAC1 inhibitors from natural compounds has attracted much attention. Summary of the invention

[0004] In view of the above technical problems existing in the prior art, the present invention provides a safe and effective RAC1 inhibitor and application thereof.

[0005] The first aspect of the present invention provides a RAC1 inhibitor, comprising chlorogenic acid or a derivative thereof.

[0006] Preferably, the inhibitor is used to bind to RAC1 and inhibit the activity of RAC1.

[0007] Preferably, the inhibitor is used to inhibit abnormal activation of RAC1.

[0008] Preferably, the inhibitor is used to inhibit the mRNA expression of Ki67 and PCNA caused by abnormal activation of RAC1.

[0009] Preferably, the inhibitor is used to improve and alleviate glomerular damage caused by RAC1 activation.

[0010] Preferably, the inhibitor is used in the preparation of a medicament.

[0011] The second aspect of the present invention provides a medicine comprising the above RAC1 inhibitor.

[0012] Preferably, the drug further comprises any one of the following ingredients or a combination thereof: excipients, salts and solvents.

[0013] Preferably, the drug is an injection or an oral drug.

[0014] Preferably, the concentration of chlorogenic acid or its derivatives is 35-140 μM.

[0015] Compared with the prior art, the beneficial effects of the present invention are: chlorogenic acid and its derivatives can combine with RAC1, and inhibit the abnormal activation / activity of RAC1, inhibit the proliferation of mesangial cells caused by the abnormal activation of RAC1, have no toxic effect on cells, and are a safe and effective RAC1 inhibitor; they can alleviate glomerular damage caused by abnormal activation of RAC1. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a comparison chart of cell viability test results; Figure 2 It is the secondary mass spectrum of Pulldown; Figure 3A This is the molecular docking diagram of CGA and RAC1; Figure 3B This is a simulation diagram of the amino acid sites of the docking of CGA and RAC1 molecules; Figure 4A This is a water box simulation diagram of the CGA and RAC1 complex; Figure 4B is the RMSD plot of the CGA and RAC1 complex; Figure 4C is the RMSF plot of the CGA and RAC1 complex; Figure 5 This is a comparison chart of RAC1 enzyme activity detection; Fig. 6A is a comparison chart of Ki67 mRNA expression levels; Figure 6B This is a comparison chart of PCNA mRNA expression levels. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] The present invention is further described in detail below in conjunction with the accompanying drawings: The present invention provides a RAC1 inhibitor, comprising chlorogenic acid CGA or a derivative thereof.

[0019] Chlorogenic acid CGA can bind to RAC1 and inhibit the abnormal activation of RAC1, inhibit the proliferation of mesangial cells caused by abnormal activation of RAC1, and has no toxic effect on cells. It is a safe and effective RAC1 inhibitor; it can alleviate glomerular damage caused by abnormal activation of RAC1.

[0020] Cytotoxicity assay of chlorogenic acid.

[0021] Detection method: Step 101: Cell culture. Primary human renal mesangial cells (HRMCs) (ScienCell, Catalog No. 4200) were seeded in 96-well plates and cultured in a cell culture medium (ScienCell, Catalog No. 4201) containing 2% fetal bovine serum (ScienCell, Catalog No. 0010), 1% penicillin / streptomycin solution (ScienCell, Catalog No. 0503) and 1% cell growth factor (ScienCell, Catalog No. 4252) in a cell culture incubator containing 5% carbon dioxide at 37°C.

[0022] Step 102: After HRMCs adhered to the wall, CGA 0, 4.375, 8.75, 17.5, 35, 70, 140, 280 μM was administered to the corresponding wells, and the stimulation lasted for 48 hours.

[0023] Step 103: CCK8 cytotoxicity assay. Figure 1 The results showed that there was no statistically significant difference in cell viability among the groups, and CGA had no toxicity to HRMC.

[0024] Target protein detection.

[0025] Detection method: Step 201: Protein extraction and concentration determination. Cell lysis: Add 500 μl of lysis buffer to the cell EP tube, lyse at 4℃ for 2h, vortex every 30 min. Protein extraction: Centrifuge at 13000 rpm for 20 min at 4℃, take the supernatant; centrifuge again at 13000 rpm for 5 min, take the supernatant. Dilute 2 μl 50 times to measure the concentration, and store the rest at 4℃. Concentration determination: Measure the protein concentration using the BCA kit.

[0026] Step 202: Pull-down. Equilibration gel: Add 50 μl of streptavidin gel suspension to the spin column tube, equilibrate with 250 μl TBS, and centrifuge at 1300 g for 60 s. Biotinylated small molecule fixation: Add 300 μl of biotinylated CGA to the spin column tube, incubate at 4°C, and centrifuge at 1300 g for 60 s. Biotin blocking: Add 250 μl of biotin blocking solution, incubate at room temperature for 5 min, and centrifuge at 1300 g for 60 s. Capture protein: Add 300 μl of protein sample, incubate at 4°C, and centrifuge at 1300 g for 60 s.

[0027] Step 203: Enzymatic protein digestion. Reduction and alkylation: DTT reduction, IAA alkylation, NH4HCO3 washing, centrifugation at 1000 g for 2 min each time. Enzymatic digestion: Trypsin digestion, incubation at 37°C, centrifugation at 12000 rpm for 5 min, and supernatant. Peptide extraction: NH 4 HCO 3 Wash, centrifuge at 12000 rpm for 5 min, take the supernatant, mix and desalt.

[0028] Step 204: Sample loading. The sample was vacuum dried, re-dissolved in 0.1% FA, and loaded onto the machine with an equal volume. HPLC separation, flow rate 300 nL / min. DDA mode mass spectrometry detection, full scan resolution 60000 FWHM, mass-to-charge ratio 350-1800 m / z, HCD fragmentation mode, collision energy 30%.

[0029] See the secondary mass spectrum for Figure 2 , which matches the amino acid sequence of CPNTPIIILVGTK-HH, a unique peptide of RAC1. Pulldown analysis of mesangial cells combined with mass spectrometry showed that CGA can directly bind to the RAC1 protein on mesangial cells.

[0030] Molecular dynamics simulation. The root mean square deviation (RMSD) value of the protein α-carbon atom relative to the initial structure was calculated to detect dynamic changes to further study the binding mode and stability of CGA and RAC1 proteins. The Desmond module of the Schrödinger drug design software package was used to perform molecular dynamics simulation studies on the small molecule protein complex obtained by molecular docking.

[0031] The results are as follows Figure 3A and Figure 3BAs shown in the figure, CGA can interact with the amino acid sites ASP118A, SER158A, LYS16A, ALA159A, ILE33A, ALA13A, LEU160A, LYS116A, GLY15A, VAL14A, THR17A in the GTP / GDP binding pocket of RAC1 through hydrogen bonds, which include all the amino acid sites where the RAC1 inhibitor EHT1864 interacts with RAC1 through hydrogen bonds. The key amino acid sites are determined based on the hydrogen bond interaction formed between them and the ligand molecules and meet the spatial distance threshold (<5 Å).

[0032] In the molecular dynamics simulation, the water box constructed for the RAC1 and CGA small molecule complex is shown in Figure 4 A. After 100 ns of molecular dynamics, the molecular dynamics trajectory was analyzed to extract the RMSD of the trajectory RAC1 protein and CGA small molecule complex. The track showed that the overall RMSD of the protein complex fluctuated slightly, fluctuating between 1.2-3.2 Å, and the binding was relatively stable. It reached equilibrium after about 50 ns, and the RMSD in the equilibrium stage was about 2.8 Å, see Figure 4 B. The RMSF of the extracted trajectory protein, as shown in Figure 4 C, showed that the overall RMSF of the RAC1 protein and CGA small molecule complex changed little, both within 0.5-4.5 Å. The two regions with larger fluctuations were the N-terminus and C-terminus of the protein, which had no effect on the binding of small molecule compounds. The MM-GBSA calculation results and energy decomposition were performed on the last frame of the stable state of the molecular dynamics trajectory. The total binding energy was -9.79 kcal / mol, indicating that the small molecule and the protein had good binding. Among them, RMSD (Root Mean Square Deviation) is expressed as root mean square deviation; RMSF (Root Mean Square Fluctuation) is expressed as root mean square fluctuation, and the horizontal axis is the residue index (Residue Index).

[0033] Inhibition of abnormal activation of RAC1. Detection method: HRMCs were used as negative control; ML-097 (50 μM) was used as a stimulating factor to induce activation of RAC1 in mesangial cells, and activated cells and model groups were established; the activated cells were treated with 140 μM CGA (treatment group 1), 50 μM EHT1864 (treatment group 2), and a mixture of 140 μM CGA and 50 μM EHT1864 (treatment group 3). The activation status of RAC1 in each group was detected by the Rac1 G-LISA kit.

[0034] The steps for detecting the activation state of the small GTPase RAC1 using the Rac1 G-LISA kit (BK128) are as follows: Step 301: Remove the strips required for the test from the RAC1 kit, install them in the strip holder, and place them on ice. Step 302: Add 100 μl of ice water to each well to dissolve the powder at the bottom of the well, and tap the plate vigorously to remove the solution in the well. Step 303: Thaw the human renal mesangial cell lysate and tissue lysate in a water bath and immediately place them on ice. Step 304: Add the blank control (lysis buffer), positive control, and the sample to be tested to the well plate, 50 μl / well, and incubate at 400 rpm at 4°C for 30 min. Step 305: After the incubation is completed, remove the liquid in the well plate, add PBST 200 μl / well, wash twice at room temperature, and tap the plate vigorously to keep no liquid residue in the well plate. Step 306: Add the antigen presentation buffer to the well plate, 200 μl / well, and incubate at room temperature for 2 min. Step 307: After the incubation, remove the liquid from the plate, add 200 µl / well of PBST, wash three times at room temperature, and tap the plate vigorously to keep no liquid remaining in the plate. Step 308: Add anti-RAC1 primary antibody (diluted 1:300 with dilution buffer) to the plate, 50 µl / well, and incubate at 400 rpm for 30 min at 4°C on an oscillator. Step 309: After the incubation, remove the liquid from the plate, add 200 µl / well of PBST, wash three times at room temperature, and tap the plate vigorously to keep no liquid remaining in the plate. Step 310: Add anti-RAC1 secondary antibody (diluted 1:100 with dilution buffer) to the plate, 50 µl / well, and incubate at 400 rpm for 45 min at room temperature. Step 311: After the incubation, remove the liquid from the plate, add 200 µl / well of PBST, wash three times at room temperature, and tap the plate vigorously to keep no liquid remaining in the plate. Step 312: Thaw HRP detection reagents A and B in a room temperature water bath, mix equal volumes and protect from light, add 50 μl / well to the plate, and incubate at room temperature at 400 rpm for 20 minutes. Step 313: After the incubation, add 50 μl HRP stop buffer to each well to end the reaction. Step 314: Eliminate bubbles in the plate, read the absorbance at 490 nm using a microplate reader and count.

[0035] like Figure 5, the RAC1-GTP / RAC1 ratio in mesangial cells increased significantly after ML-097 stimulation, and the RAC1-GTP / RAC1 ratio in CGA treatment group 1, EHT1864 treatment group 2, and CGA+EHT1864 treatment group 3 all decreased significantly (P<0.05). However, the combined treatment group 3 of EHT1864 and CGA could not further reduce the RAC1-GTP / RAC1 ratio, which may be due to the competitive binding of CGA and EHT1864 to the RAC1 active site. The above results show that CGA can inhibit the abnormal activation of RAC1 in mesangial cells, and the effect is similar to that of EHT1864. Figure 5 In the figure, the ordinate is the relative activity of RAC1 (RAC1 activity of control).

[0036] Inhibition of mesangial cell proliferation caused by abnormal activation of RAC1. Detection method: HRMCs were used as negative control; ML-097 (50 μM) was used as a stimulating factor to induce RAC1 activation in mesangial cells, and activated cells and model groups were established; different doses of CGA (35 μM, 70 μM, 140 μM) were added to the activated cells, and the mRNA expression levels of proliferation markers Ki67 and PCNA were detected based on qRT-PCR.

[0037] The specific operation of detecting mRNA level is as follows: Step 401: Total RNA was extracted from cells / kidney tissues using the Trizol method. Step 402: Reverse transcription was performed using a cDNA synthesis kit (New England Biolabs, Cat. No. E6560S). Step 403: qRT-PCR detection was performed using PowerUp SYBR Green Master Mix (Applied Biosystems) and corresponding primers. Step 404: Analysis was performed using the QuantStudioTM 5 Real-Time System (ThermoFisher Scientific). Step 405: The expression levels of 18S and GAPDH genes were used as internal references, and relative gene expression was determined by the comparative threshold cycle (Delta Ct) method.

[0038] like Fig. 6A , CGA concentrations of 70μM and 140μM could significantly reduce the Ki67 mRNA level in mesangial cells after abnormal activation of RAC1 (P<0.001); Figure 6B CGA at a concentration of 140 μM could significantly reduce the PCNA mRNA level in mesangial cells after abnormal activation of RAC1 (P<0.01).

[0039] Through the above tests, it can be found and confirmed that CGA, as a natural RAC1 inhibitor, inhibits RAC1 activity by competitively binding to the GTP / GDP binding pocket of RAC1, thereby inhibiting mesangial cell proliferation. It is non-toxic to cells, can directly bind to RAC1 protein, inhibit RAC1 abnormal activation and RAC1 activity, inhibit the mRNA expression of Ki67 and PCNA caused by abnormal RAC1 activation, and inhibit mesangial cell proliferation caused by abnormal RAC1 activation.

[0040] The RAC1 inhibitor of the present invention can be used to prepare a drug for inhibiting abnormal activation of RAC1 and RAC1 activity, inhibiting the mRNA expression of Ki67 and PCNA generated by abnormal activation of RAC1, inhibiting mesangial cell proliferation caused by abnormal activation of RAC1, and improving and alleviating glomerular damage caused by RAC1 activation.

[0041] In the drug, the concentration of chlorogenic acid is 35-140 μM. The drug can be an injection or an oral drug; a chlorogenic acid solution can be used, and can also include excipients, salts, solvents, and inevitable impurities. Excipients can appropriately use the following components: diluents, adhesives, wetting agents, disintegrants, lubricants, cosolvents, etc. If necessary, colorants, preservatives, spices, flavoring agents or other additives can also be added to the drug preparation.

[0042] The dosage form of a drug can be a liquid dosage form, a solid dosage form or a semi-solid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o / w types, w / o types and multiple emulsions), suspensions, injections (including water injections, powder injections and infusions), etc.; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, micropills, dripping pills, suppositories, films, patches, aerosols (powders), sprays, etc.; semi-solid dosage forms can be ointments, gels, pastes, etc.

[0043] The above dosage forms can be made into ordinary preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle delivery systems, such as liposome preparations.

[0044] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A RAC1 inhibitor, characterized in that Including chlorogenic acid or its derivatives.

2. The RAC1 inhibitor according to claim 1, characterized in that Used to bind to RAC1 and inhibit RAC1 activity.

3. The RAC1 inhibitor according to claim 1, characterized in that Used to inhibit abnormal activation of RAC1.

4. The RAC1 inhibitor according to claim 3, characterized in that Used to inhibit the mRNA expression of Ki67 and PCNA caused by abnormal activation of RAC1.

5. The RAC1 inhibitor according to claim 3, characterized in that Used to improve and alleviate glomerular damage caused by RAC1 activation.

6. The RAC1 inhibitor according to any one of claims 1 to 5, characterized in that Used to prepare medicines.

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