CN114028368A - RhoC covalent binding inhibitor - Google Patents
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Abstract
The present invention relates to compounds that are useful as covalent binding inhibitors that directly target RhoC. The invention synthesizes two chemical micromolecular probes with the original activity and inactivation of the glaucocalyxin A through a 'click chemistry' specific chemical reaction, and finds a differential protein (namely a direct action target spot of the glaucocalyxin A for exerting biological functions) combined by the two probes. A key target, namely RhoC, is screened by methods such as bioinformatics analysis and the like, the compound is covalently bound with RhoC through Lys18 amino acid residue sites, and the influence of the glaucocalyxin A on the GTP activity of the compound is verified in mouse fibroblast NIH-3T 3. The molecular mechanism results show that: the glaucocalyxin A influences the activity of RhoC-GTPase and inhibits the generation of downstream ROCK kinase and MLC phosphorylation.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to a RhoC covalent binding inhibitor.
Background
Rho family protein is a monomeric G protein, has been identified as an important regulator in cells, and has been reported to participate in various biological processes, which play a crucial role in various diseases and related signaling pathways. In the course of research, Rho-GTPases, a subfamily of the Rho family, are highly expressed in many cells, and are the first GTPases to be discovered, including RhoA, RhoB and RhoC, which affect the dynamic changes of the cytoskeleton and play an important role in the biological processes of human beings. In vivo, Rho proteins cycle repeatedly between a state of binding to inactive Guanosine Diphosphate (GDP) (Rho-GDP) and a state of binding to active Guanosine Triphosphate (GTP) (Rho-GTP). When Rho binds to GTP, Rho-GTP interacts with its downstream kinases, phospholipases, actin regulators, adaptor proteins, etc. to perform specific biological functions.
In the cardiovascular context, RhoC is considered to be a downstream regulator of VEGF in endothelial cells, promoting angiogenesis (Wang W, Wu F, Fang F, et al, RhoC is the essential for angiogenesis induced by hepatic cells, cells vision regulation of endothelial cells, 2008,99(10):2012 2018), whose GTP activity may influence hypoxic pulmonary vasoconstriction (Robertson T P, Dipp M, Ward J P T, et al, inhibition of pathological vasoconstriction by Y-27632in isolated pulmonary constriction and permeability of the lung vessel, J J.tissue, British tissue of cardiovascular tissue, Ca < 1 > + 3580, Ca < 3 > + J.S. > 2, Ca < 3 > -2 >,83, Ca < 3 > -2 >,83, 1995,268(1) H301-H307). In The nervous system, when Rho-GTPases activity is inhibited, central nervous system apoptosis is prevented (Dubreuil C I, Winton M J, McKerracher L. Rho activation patterns after protein kinase in The nerve system [ J ]. The Journal of cellular biology,2003,162(2): 233-. In addition, there are many papers reporting that Rho activity is associated with diseases such as renal interstitial fibrosis, diabetic nephropathy, renal cancer (willow fly, Paiping. RhoA/Rho associated coiled coil formed protein kinase signaling pathway and chronic kidney disease [ J ]. Wahsi medicine 2011,26(5): 781-783; Highui, Yunming. Rho GTPase/Rho kinase system and renal interstitial fibrosis [ J ]. clinical collection, 2005,20(15): 896-898). Meanwhile, the Rho kinase signal pathway participates in the occurrence and development processes of many respiratory diseases, such as chronic obstructive pulmonary disease, bronchial asthma, pulmonary hypertension, idiopathic pulmonary fibrosis, lung cancer, etc. (Shulong, Jiwei Ping. Rho kinase and respiratory disease [ J ]. International journal of respiration, 2013,33(3): 232-. Rho is also reported to be a signaling protein that regulates actin cytoskeleton (Ridley A J. Rho family proteins: coordinating cell responses [ J ]. Trends in cell biology,2001,11(12): 471-477). Therefore, the development of Rho protein targeted inhibitors has great scientific significance and application value.
However, Rho-GTPases have extremely strong binding affinity to GDP and GTP as substrates, and because Rho-GTPases lack an ideal binding site, it is difficult to find a small molecule inhibitor targeting the binding pocket, and Rho-GTPases family proteins are generally regarded as "non-medicinal" targets, which is also an important reason for slow development of drugs targeting Rho-GTPases family proteins.
In the existing reports, there have been some small molecule inhibitors developed by indirectly targeting Rho-GTPases. Such as FTI-277 and GGTI-298, the mechanism of action of this class of molecules requires that Rho proteins be localized to the plasma membrane. Or simvastatin, a lipid-lowering drug, which exerts biological activity by inhibiting modification of Rho protein. However, none of the small molecules or drug inhibitors described above directly targets Rho protein. For small molecule inhibitors targeting Rho-GTPases, most of the inhibitor drugs under investigation or developed to the market are by targeting the Rho and Rho-GEF sites of action, their downstream kinase substrates and the carboxy-terminal lipid modification region. It has been reported that a small molecule inhibitor DC-Rhoins that directly targets RhoA, by binding to the cysteine residue Cys107 of RhoA, disrupts the interaction between Rho and Rho-GEF or Rho-GDI, and thereby affects RhoA-GTPase activity, has been found. However, there has been no report on finding a targeted inhibitor that acts directly on RhoC.
Disclosure of Invention
The invention aims to provide a RhoC inhibitor which can be used as a covalent binding inhibitor for directly targeting RhoC.
The name is as follows: glaucocalyxin A has a molecular formula as follows: c20H28O4Purified from Rabdosia rubescens (Rabdosia rubescens) of Labiatae.
The invention aims to find a small-molecule inhibitor drug directly targeting RhoC. Since RhoC plays an important role in cardiovascular, genitourinary, nervous and respiratory diseases, the invention can be used for treating various diseases with RhoC activity abnormality.
The invention finds that the structure of the formula (I) directly targets RhoC, and the structure is covalently combined with RhoC to specifically influence RhoC-GTPase activity.
In order to make the features and advantages of the present invention comprehensible, preferred embodiments accompanied with figures are described in detail below.
The invention synthesizes two chemical micromolecular probes with the original activity and inactivation of the glaucocalyxin A through a 'click chemistry' specific chemical reaction, and finds a differential protein (namely a direct action target spot of the glaucocalyxin A for exerting biological functions) combined by the two probes. A key target, namely RhoC, is screened by methods such as bioinformatics analysis and the like, the compound is covalently bound with RhoC through Lys18 amino acid residue sites, and the influence of the glaucocalyxin A on the GTP activity of the compound is verified in mouse fibroblast NIH-3T 3. The molecular mechanism results show that: the glaucocalyxin A influences the activity of RhoC-GTPase and inhibits the generation of downstream ROCK kinase and MLC phosphorylation.
Drawings
FIG. 1 Glaucocalyxin A inhibits RhoC activity. Lanes from left to right are: blank group, stimulation group, and 3 concentration administration groups (0.5. mu.M, 1. mu.M, 2. mu.M).
FIG. 2 amino acid sites of glaucocalyxin A binding to RhoC. Panel A identifies the K18 amino acid residue binding site, panel B identifies the C20 amino acid residue binding site, panel C identifies the C107 amino acid residue binding site, and panel D identifies the C159 amino acid disability binding site.
FIG. 3 the effect of each amino acid site on the binding of glaucocalyxin A to RhoC.
FIG. 4A shows the variation of heat in isothermal titration reaction of RhoC protein and glaucocalyxin A, and FIGS. 4B-E show the variation of heat in isothermal titration reaction of protein with glaucocalyxin A after four amino acid mutations of RhoC.
FIG. 5 is a simulation diagram of the binding of glaucocalyxin A to each site of the RhoC molecule, yellow glaucocalyxin A, green RhoC protein, red amino acid site of the binding of the two, and purple amino acid residue of the bound RhoC. In the A, the left picture is a simulation picture of covalent docking of the glaucocalyxin A and the RhoC at the 18 th amino acid, and the right picture is an enlarged view of the docking part at the K18 site; the left picture in the B picture is a simulation picture of covalent docking of the glaucocalyxin A and the RhoC at the 20 th amino acid, and the right picture is an enlarged view of the docking part at the C20 site; the left picture in the figure C is a simulation diagram of covalent docking of the glaucocalyxin A and the RhoC at the 107 th amino acid, and the right picture is an enlarged view of the docking part at the C107 site; in the diagram D, the left diagram is a simulation diagram of covalent docking of the glaucocalyxin A and the RhoC at the 159 th amino acid, and the right diagram is an enlarged diagram of the docking position of the C159 th amino acid.
FIG. 6 Glaucocalyxin A inhibits the conduction of the downstream pathway of RhoC, in lanes from left to right: blank group, stimulation group, and 4 concentration administration groups (0.5. mu.M, 1. mu.M, 2. mu.M, 5. mu.M). Panel A shows Western blot results of Rho downstream ROCK kinase, and panel B shows detection of the level of downstream MLC phosphorylation of ROCK.
Detailed Description
The preparation method of RhoC protein comprises the following steps: the Gene encoding RhoC protein having Gene Bank accession number 389 was inserted into pGEX-KG vector, introduced into E.coli, induced to express using IPTG, and purified using glutathione agarose purification resin (GST-beads, available from GE Healthcare) to obtain RhoC protein.
The preparation method of the mutant protein with four sites of RhoC-K18R, RhoC-C20A, RhoC-C107A and RhoC-C159A comprises the following steps: the 18 th amino acid of the RhoC protein is mutated from lysine to arginine by utilizing a point mutation cloning technology, the 20 th, 107 th and 159 th amino acids are mutated from cysteine to alanine, the amplified fragments are inserted into a pGEX-KG vector, and the RHOC protein purification step is repeated to obtain the four-site mutant proteins of RhoC-K18R, RhoC-C20A, RhoC-C107A and RhoC-C159A.
The chemical probe Q1-26 has a structure shown in formula (II).
Chemical probe Q1-27, structure see formula (III).
The preparation process of Q1-26 is shown in formula (IV):
the preparation process of Q1-27 is shown in formula (V):
example 1: glaucocalyxin A inhibits RhoC activity
The mouse fibroblast NIH-3T3 cells were cultured in adherence in DMEM high-sugar medium supplemented with 10% (volume percentage) fetal bovine serum, and then transferred to 5 culture dishes of 10cm diameter, each dish containing about 1.5X 10 cells6The culture volume was 10mL, and each of the plates was numbered as a blank group, a stimulation group, and 3 concentration administration groups. When the cell density is increased to 80%, discarding culture solution from each culture dish, adding 0.5 μ M, 1 μ M and 2 μ M glaucocalyxin A solution (DMSO as solvent) into 3 administration groups with concentration, respectively, adding DMSO solvents with the same volume as the glaucocalyxin A into the blank group and the stimulation group, intervening for 24 hours, and discarding supernatant; changing the culture solution in each culture dish into serum-free DMEM high-sugar culture medium, continuing starvation culture for 24 hours, and then directly adding 100 μ L fetal calf in the stimulation group and the administration group respectivelySerum (final concentration 10%) was stimulated for 30 minutes, and no serum was added to the blank group. Total protein of cells in each culture dish was extracted and adjusted to protein concentration using protein lysate, and the same volume was removed and transferred to a new centrifuge tube for use. Taking a one tenth volume of sample from the centrifuge tube as Total protein, and performing SDS-PAGE electrophoresis to detect the Total amount of RhoC (abbreviated as Total-RhoC) in the protein; meanwhile, Rho family specific agar gel beads (hereinafter abbreviated as RBD beads) are rapidly added into the remaining nine volume samples for immunoprecipitation, and are mixed and spun at 4 ℃ overnight; washing RBD beads to remove non-specific binding protein; to each sample was added a double volume of 2 × protein loading buffer of RBD beads, and after vortexing, the supernatant was aspirated for western blot assay to detect RhoC activity (i.e., RhoC-GTP).
As shown in FIG. 1, after 24 hours of starvation treatment, RhoC has no GTP activity, while RhoC-GTP is activated after 30 minutes of serum stimulation, while glaucocalyxin A decreases Rho-GTP activity and is concentration-dependent. Thus, glaucocalyxin A may target RhoC, inhibiting RhoC-GTPase activity.
Example 2: identification of glaucocalyxin A and RhoC binding site
Adding a solution containing 0.24nM of RhoC pure protein and 12nM glaucocalyxin A to a 1.5mL Ep tube, adding 10. mu.L of 0.5mM EDTA aqueous solution, then filling the system to 50. mu.L with 1 XPBS solution, placing on a shaker, and incubating at 600rpm and 37 ℃ for 8 hours; adding 5 times of sample loading buffer solution into each tube, and boiling for 5 minutes; preparing SDS-PAGE protein gel, and stopping gel running until one third of the length of the whole gel; after destaining by staining, an in-gel digestion was performed using trypsin. And (3) mass spectrum detection, wherein the binding sites of the glaucocalyxin A and the RhoC pure protein are identified by analyzing the change of the molecular weight.
FIG. 2 shows that in the presence of glaucocalyxin A, the mass of peptide fragment KLVIVGDGACGK containing Lys18 (FIG. 2A), peptide fragment TCLLIVFSK containing Cys20 (FIG. 2B), peptide fragment HFCPNVPIILVGNK containing Cys107 (FIG. 2C) and peptide fragment ISAFGYLECSAK containing Cys159 (FIG. 2D) all increased by 332.44Da, a mass shift that is exactly consistent with the addition of one molecular unit of glaucocalyxin A. Taken together, the result shows that the glaucocalyxin A can be combined with RhoC through four amino acid residue sites of K18, C20, C107 and C159.
Example 3: verification of glaucocalyxin A and RhoC binding site
(1) Immunoprecipitation experiments: the co-immunoprecipitation experiment was performed by using a chemical probe Q1-26 having glaucocalyxin activity and an inactive glaucocalyxin chemical probe Q1-27. Four point mutation cloning plasmids (the 18 th amino acid of RhoC protein is mutated from lysine to arginine, the 20 th, 107 th and 159 th amino acids are mutated from cysteine to alanine, the amplified fragments are inserted into a PCDH-puro vector and a Flag tag is inserted) are obtained by utilizing a point mutation cloning technology in advance. Then transferring the protein into cells for stable expression, performing intervention by using 2 mu M Q1-26 and Q1-27 respectively, and taking cell lysate with equal concentration as a total protein sample (namely a WCL sample) after 24 hours; simultaneously adding agar gel particles (beads, hereinafter referred to as beads, purchased from Sigma) with biotin into the remaining equivalent lysate as a precipitate sample (namely an IP sample), and carrying out mixed spinning at room temperature for more than 8 h; washing beads, adding 2 Xprotein loading buffer solution, mixing, boiling, and taking supernatant for immunoblotting experiment. After the mutation of the four sites, 18 th, 20 th, 107 th and 159 th, of the RhoC protein, the binding efficiency was examined using RhoC antibody (purchased from CST), and the effect of each site on the binding of glaucocalyxin A to RhoC was evaluated.
FIG. 3 shows: the RhoC-K18R, RhoC-C20A and RhoC-C107A mutants can partially block the combination of the glaucocalyxin A and the RhoC, while the RhoC-C159A mutant still retains the original combination strength of the glaucocalyxin A, and preliminarily determines that K18, C20 and C107 are amino acid residues combined by the glaucocalyxin A and the RhoC, so that the mass spectrum detection result is met.
(2) And (3) thermodynamic analysis: first, the appropriate reactant concentrations are determined by the following specific method: weighing glaucocalyxin A powder, dissolving the glaucocalyxin A powder in DMSO (dimethylsulfoxide) to prepare a stock solution with the concentration of 100mM, and diluting the stock solution to 250 mu M by PBS containing 5% DMSO to obtain a glaucocalyxin A solution; respectively diluting the RhoC protein, the four-point mutant RhoC-K18R, RhoC-C20A, RhoC-C107A and RhoC-C159A mutant to 5 mu M to obtain each protein solution; titrating the glaucocalyxin A solution into each protein solution respectively until the binding reaction reaches the balance, namely the curve reaches the plateau phase; correcting the original data; recording a binding constant (K), an enthalpy change (delta H), an entropy change (delta S) and the like in the reaction of the glaucocalyxin A and each protein; and analyzing the model.
As can be seen from FIG. 4A, Δ H<0，△S>0, the binding between glaucocalyxin A and RhoC is not driven by single action force, and the binding constant of the interaction of the glaucocalyxin A and the RhoC is 6.28 multiplied by 106±2.03×106M-1(ii) a FIGS. 4B-E show: an exothermic reaction (. DELTA.H) occurs when the 18 th amino acid is mutated>0) The binding constant is reduced by 30 times, which is far inferior to the binding capacity between wild-type RhoC protein (i.e., non-mutated RhoC protein) and glaucocalyxin A; when the 20 th amino acid and the 107 th amino acid are mutated, the binding capacity is reduced to different degrees, and when the 159 th amino acid is mutated, the difference of the binding capacity is not changed significantly. Thus, the amino acid at position 18 is the binding site for the interaction of glaucocalyxin A with RhoC.
(3) Simulating an eutectic structure to carry out molecular docking: the physiological conditions were simulated with MOE software at pH 7.4 and ionic strength 0.1. Under the condition, the three-dimensional structure of the RhoC protein is optimized, hydrogenated, dehydrated and residue corrected to obtain a protein 3D structure model with minimum energy. The results of simulating the docking of glaucocalyxin A with each site of RhoC molecule are shown in FIG. 5.
As can be seen from FIG. 5, when glaucocalyxin A is combined with the 18 th amino acid of RhoC, the free energy is lower, the monomolecular structure formed by the glaucocalyxin A and the rhoC is tighter, the Gibbs free energy before and after combination is changed from-772.57 kcal/mol to-738.28 kcal/mol, and the accessible surface area is changed from
Become into
And when combined with position 20, the Gibbs free energy is changed from-772.57 kcal/mol to-754.33 kcal/mol, and the accessible surface area is changed from
Become into
When combined with amino acid 107, the Gibbs free energy is changed from-772.57 kcal/mol to-718.87 kcal/mol, and the accessible surface area is changed from
Become into
When the glaucocalyxin A is combined with the 159 th site, the Gibbs free energy is changed from-772.57 kcal/mol to-740.94 kcal/mol, and the accessible surface area is changed from
Become into
In conclusion, the glaucocalyxin A can be combined with the RhoC at the Lys18 residue site to form a sphere; whereas at Cys20, Cys107 and Cys159 residue sites, the glaucocalyxin A binding conformation is outside the RhoC protein. Thus, the 18 th amino acid is a potential binding site for glaucocalyxin A and RhoC.
Example 4: glaucocalyxin A inhibits RhoC downstream signaling pathway conduction
Cells were plated at 5X 10 per well5The seeds were planted in 6-well plates and were set as a blank group, a stimulated group and 4 concentration administration groups, respectively. When the cell density is increased to 80%, adding 0.5 mu M, 1 mu M, 2 mu M and 5 mu M of glaucocalyxin A solution (prepared by DMSO) into the 4 administration groups with the final concentration respectively, and adding the DMSO solvents with the same volume as the glaucocalyxin A solution into the blank group and the stimulation group respectively for intervention for 24 hours; the supernatant was discarded from each group, the culture medium was replaced with serum-free medium to continue the starvation culture, and after 24 hours, 20. mu.L of fetal bovine serum (final concentration: 10%) was added directly to the stimulated group and each administered group and stimulated for 30 minutes, and the blank group was left untreated. Extracting total cell protein of each group, and detecting RhoC downstream ROC by Western blottingThe expression of K protein kinase and the activation of MLC protein are referenced to Tubulin.
As can be seen from FIG. 6, glaucocalyxin A effectively down-regulates the protein levels of the downstream kinases RhoC ROCK1 and ROCK2, thereby inhibiting the occurrence of serum-induced MLC protein phosphorylation (p-MLC) in a concentration-dependent manner.
Claims (3)
1. Use of glaucocalyxin A as a RhoC covalent binding inhibitor.
2. A medicine with RhoC inhibiting effect contains glaucocalyxin A as active ingredient.
3. The medicament according to claim 2, further comprising a pharmaceutically acceptable carrier.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103601641A (en) * | 2013-11-01 | 2014-02-26 | 中国人民解放军第二军医大学 | Glaucocalyxin derivatives and application thereof in preparation of antitumor drugs |
| CN107432873A (en) * | 2017-04-17 | 2017-12-05 | 南京工业大学 | Application of the glaucocalyxin A in the medicine for preparing anti-human osteosarcoma |
| CN111840263A (en) * | 2020-08-31 | 2020-10-30 | 上海交通大学医学院附属第九人民医院 | Application of glaucocalyxin A in preparation of products for preventing and treating periodontal diseases |
| CN112972479A (en) * | 2021-03-08 | 2021-06-18 | 天津医科大学 | New application of Rho inhibitor fasudil hydrochloride |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103601641A (en) * | 2013-11-01 | 2014-02-26 | 中国人民解放军第二军医大学 | Glaucocalyxin derivatives and application thereof in preparation of antitumor drugs |
| CN107432873A (en) * | 2017-04-17 | 2017-12-05 | 南京工业大学 | Application of the glaucocalyxin A in the medicine for preparing anti-human osteosarcoma |
| CN111840263A (en) * | 2020-08-31 | 2020-10-30 | 上海交通大学医学院附属第九人民医院 | Application of glaucocalyxin A in preparation of products for preventing and treating periodontal diseases |
| CN112972479A (en) * | 2021-03-08 | 2021-06-18 | 天津医科大学 | New application of Rho inhibitor fasudil hydrochloride |
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