CN117069657B - Compound targeting c-Src kinase SH3 structural domain and application thereof - Google Patents

Abstract

The invention discloses a quinoline compound containing an ethoxyphenyl structure shown in a formula (I) and application thereof as a novel Src kinase small molecule inhibitor targeting a c-Src protein SH3 structural domain. The invention also discloses application of the compound (I) in the aspect of targeting c-Src protein for preventing and/or treating cervical cancer, human mantle cell lymphoma, acute B cell leukemia, diffuse large B cell lymphoma and other tumor diseases.

Description

Compound targeting c-Src kinase SH3 structural domain and application thereof

Technical Field

The invention belongs to the field of medicines, and relates to a compound targeting a c-Src protein SH3 structural domain and application thereof. Specifically, the compound of the present invention has a structure represented by formula (I). The invention discloses application of the compound serving as a targeting c-Src protein in preventing and/or treating cervical cancer and other tumor diseases. The compound of the invention plays an anti-tumor activity by targeting the action mechanism of the SH3 domain for regulating and controlling the activity of c-Src kinase, so that the defect that the existing medicine which plays an anti-tumor activity clinically is easy to cause tumor cell drug resistance can be overcome.

Background

Src protein tyrosine kinase family members include Src, lyn, fyn, yes, lck, blk and Hck, where c-Src is the hub of a range of signal transduction pathways that affect cell proliferation, differentiation, movement, and survival. Functional regulation of Src is by tyrosine phosphorylation at two sites, but with opposite effects. Wherein phosphorylation at Tyr416 would up-regulate the catalytic activity of Src, whereas phosphorylation at Tyr527 would decrease the catalytic activity of Src. The activated c-Src is involved in normal physiological and cancer-generating processes including proliferation, differentiation, survival, angiogenesis, etc.

SRC acts as a classical proto-oncogene, encoding the c-SRC protein with tyrosine kinase activity. There have been many studies reporting that c-Src is in a highly expressed state or has high kinase activity in various human tumors and is involved in various processes of carcinogenesis and development, including cell proliferation, differentiation, angiogenesis and prognosis survival, etc., more than 50% of c-Src activation has been demonstrated in tumors from colon, liver, lung, breast and pancreas, and in addition, c-Src can promote cell proliferation, migration, invasion and angiogenesis; numerous studies have demonstrated that protein tyrosine kinases are important targets for the treatment of tumors.

Currently, ATP-competitive inhibitors targeting the Src protein catalytic domain have been used clinically for tumor therapy. However, tumor cells are very easy to generate drug resistance to the drugs, which is also a main problem faced by the existing antitumor drugs targeting the c-Src protein catalytic domain. The c-Src protein consists of four Src homology domains (SH 4, SH3, SH2, SH 1). The SH4 domain is located at the N-terminus, comprising a myristoylation sequence that anchors the upper membrane, thus targeting Src family kinases to the plasma membrane. SH3 domains bind to amino acid sequences rich in proline residues, thereby playing an important role in c-Src activity, intracellular localization and recruitment of c-Src substrates. SH2 domains bind short motifs (motif) that contain phosphorylated tyrosine. SH2 and SH3 domains co-operate to regulate the catalytic activity of Src family kinases. SH1 (catalytic domain) kinase Activity in an inactive conformation, tyrosine at position c-Src 527 of human origin is phosphorylated and interacts with its own SH2 domain. This action causes the SH3 domain to interact with the proline-rich linker domain, maintaining the c-Src in a tightly bound inactive state. Once tyrosine 527 is dephosphorylated, intramolecular interactions leave c-Src in an unstable state resulting in autophosphorylation of tyrosine 416. A series of events causes the molecule to open and release SH2 and SH3 domains, which in turn interact with other receptor tyrosine kinases, G protein-coupled receptors, and local adhesion kinases (FAKs), and the like. The protein contains two self-binding peptide (SBP) sites between SH3 domain and polyproline-II (PPII) helix and between SH2 domain and C-terminal phosphorylation tail (CTPT), which are potential targets for anticancer drugs to regulate kinase activity.

Traditional non-receptor tyrosine kinase drugs act on kinase catalytic domains such as sunitinib, dasatinib, nilotinib, bosutinib, vandetanib and the like, drug resistance is easy to generate, and SH3 domain is taken as an important part for regulating the catalytic activity of Src family kinase, so no related antitumor drugs targeting the sites are marketed at present.

Disclosure of Invention

High-throughput screening is carried out through a protein thermomigration experiment, and a novel Src kinase small molecule inhibitor targeting the Src protein SH3 domain is discovered; and its anti-cell proliferation activity against cervical cancer cell Hela was measured. It is therefore an object of the present invention to provide a novel small molecule inhibitor of Src kinase targeting the SH3 domain of c-Src protein, of formula (i):

(Ⅰ)。

The compound shown in the formula (I) provides a novel skeleton for the discovery of an anti-tumor small molecule lead compound targeting the SH3 domain of the Src protein, and provides a chemical structure teaching for the design of novel anti-tumor drugs targeting the Src protein.

It is another object of the present invention to provide the use of a compound of formula (I) as targeting Src protein for anti-tumour.

The compounds of formula (I) are capable of targeting the SH3 domain of the c-Src protein and are effective in inhibiting activation of c-Src, thereby inhibiting the proliferation, migration, invasion and angiogenesis promoting activity of Src protein on cells. Thereby effectively preventing and treating cervical cancer, human mantle cell lymphoma, acute B cell leukemia and diffuse large B cell lymphoma.

FIG. 1 shows the structural formula of the compound 2- [2- (3, 4-diethoxyphenyl) vinyl ] -1-methylquinoline.

FIG. 2 shows the thermal migration assay of compounds with SH3 domain proteins.

FIG. 3 is a graph showing IC50 assay of compounds inhibiting cervical cancer Hela cell activity; FIG. 4 is a graph of IC50 assay of compounds inhibiting activity of human mantle cell lymphoma Z-138 cells; FIG. 5 IC50 assay curves for compounds that inhibit the activity of RCH-ACV in acute lymphoblastic B cell leukemia cells; FIG. 6 IC50 assay curves for compounds inhibiting diffuse large B cell lymphoma DB cell activity.

FIG. 7 anti-tumor cell proliferation activity of the compounds.

FIG. 8A test of the compound inhibiting the migration of cervical cancer Hela cells; FIG. 9 is a bar graph drawn by quantifying FIG. 8, and it is clearly observed that compound 81 (51) has a remarkable ability to inhibit migration of cervical cancer Hela cells.

FIG. 10 effect of compounds on Hela cell c-Src protein phosphorylation.

Description of the embodiments

In the invention, the candidate compounds are subjected to high-throughput screening by adopting a protein thermomigration method, and the protein thermomigration experiment is a convenient and reliable experimental method for determining whether the small molecular compound is combined with the target protein or not, and is widely applied to high-throughput screening of combining the protein with the small molecule. The basic principle of the protein thermomigration experiment is that a fluorescent dye is added into a buffer solution of a target protein, the temperature of the system is gradually increased from 25 ℃ to 80 ℃ by using a PCR instrument, hydrophobic groups of the target protein are exposed to the surface from the inside in the heating process, the combination degree of the fluorescent dye and the exposed hydrophobic groups is increased, a fluorescent signal is increased, and the fluorescent signal is gradually quenched after the maximum value is reached. The Tm (melting temperature) value can be determined by fitting a signal curve by boltzmann's equation, corresponding to the temperature at which the proteins half melt. After adding small molecules into the system, if the small molecules are combined with the protein, the stability of the protein is increased, namely the corresponding Tm value is increased, so that the melting curve of the dosing group is shifted rightward compared with that of the control group.

In the present invention, we have found through protein thermomigration experiments that the compound 2- [2- (3, 4-diethoxyphenyl) vinyl ] -1-methylquinoline binds to the SH3 domain of c-Src protein. The proliferation inhibition experiment at the cellular level shows that the compound 80 (14) has stronger anti-cell proliferation activity on cervical cancer Hela cells, human mantle cell lymphoma Z138 cells, acute lymphoblastic B cell leukemia cells RCH-ACV cells and diffuse large B cell lymphoma DB cells and has dose dependence.

The invention provides a small molecule inhibitor of c-Src kinase capable of targeting c-Src protein SH3 domain and the influence thereof on proliferation of cervical cancer Hela cells, human mantle cell lymphoma Z138 cells, acute lymphoblastic leukemia cells RCH-ACV cells and diffuse large B cell lymphoma DB cells.

The compound I of the invention exerts tumor cell proliferation inhibition activity by binding to the SH3 domain of c-Src protein.

The invention is further illustrated below in connection with specific embodiments, which are not intended to be limiting, but are merely illustrative of the invention.

Example 1: expression and purification of the protein.

Src-SH3 is constructed on a Rosetta (DE 3) expression vector, fusion protein expressed by the vector is provided with an N-terminal Trx tag and a His tag, recombinant protein is expressed at 16 ℃, strains are collected, and the supernatant is obtained by ultrasonic centrifugation (18000 rpm, 30min, 4 ℃). Purifying protein by nickel affinity chromatography (GE HEALTHCARE), collecting protein eluate, measuring concentration, adding thrombin according to its concentration, enzyme cutting at 25deg.C for 12-16 hr, concentrating in 3Kd concentrating tube in the next morning, and passing through nickel column with peristaltic pump to obtain target protein. Proteins were stored in a buffer containing 20mM Tris pH8.0, 100mM NaCl.

Example 2: protein thermomigration assay (Protein THERMAL SHIFT ASSAY) detects the binding of small molecules to proteins.

Proteins are typically present in a native state and heating causes the protein to convert to Denatured state, exposing hydrophobic groups. The exposed hydrophobic group can be combined with the fluorescent dye SYPRO Orange, so that the emitted light of the fluorescent dye is improved, and the stability of the protein can be reflected by detecting the fluorescence intensity. Because the amount of hydrophobic groups exposed by the protein is different at different temperatures, the intensity of fluorescence is also different, and thus a fluorescence-temperature graph can be drawn. When a ligand is bound to a protein, the stability of the protein increases such that the exposed hydrophobic groups at the same temperature are reduced, and a higher temperature is required to fully expose the hydrophobic groups to the protein. Thus causing the fluorescence-temperature graph to shift to the right. A series of diluted concentrations of the test compounds were prepared from the 100mM mother liquor. The buffer (20 mM Tris PH8.0, 100mM NaCl), c-Src SH3 protein, the compound to be tested and 50 Xfluorescent dye are respectively added to form a20 mu L reaction system, the whole process of protein denaturation is prevented from being operated at low temperature, bio-rad fluorescent quantitative PCR is used, the temperature of the system is gradually increased from 25 ℃ to 80 ℃ at a 1% heating rate, and meanwhile, the change condition of the fluorescent intensity along with the temperature is recorded at 20 second intervals. Further, in the Bio-Rad CFX program, the melting temperature (Tm) of SH3 at various concentrations of the compound was calculated using the Boltzmann fitting method.

Example 3: in-vitro cell activity analysis experiments test the influence of the compound on proliferation activity of cervical cancer Hela cells, human mantle cell lymphoma Z138 cells, acute lymphoblastic B cell leukemia cells RCH-ACV cells and diffuse large B cell lymphoma DB cells caused by c-Src activity inhibition.

Logarithmic growth cells were cultured in DMEM medium, 1640 medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin, seeded at a density of 8 x 103 cells per well in 96 well plates, and then cells were incubated with 6.25 μm and 3.125 μm of the seeding compound. After three days of incubation, the cell activity was measured by MTT assay, and the experimental results are shown in FIG. 3, FIG. 4, FIG. 5, FIG. 6, and FIG. 7. The results indicate that compound 80 (14) exhibits dose-dependent antiproliferative activity on Hela cells, Z138 cells, RCH-ACV cells, DB cells. Our work provides a new framework for finding more c-Src inhibitors and a new idea for overcoming the defect that the existing drugs which clinically exert anti-tumor activity by targeting the c-Src kinase catalytic domain are easy to cause tumor cell drug resistance.

Example 4: cell scratch experiments analyze the effect of compounds on Hela cell migration capacity.

Cells in the log phase are inoculated into a six-hole plate at the density of 1.5 multiplied by 106 cells per hole, serum-free DMEM is added to dilute 80 (14) compounds in each hole of the original culture medium after 24 hours, the culture is carried out after IC50 concentration and DMSO control hole scribing is set, observation and photographing are carried out in 6 hours, 12 hours, 24 hours and 36 hours, and experimental data show that 80 (14) has obvious migration inhibition activity on Hela cells by image J and GRAPHPAD PRISM processing results (figures 8 and 9).

Example 5: the effect of the compound on the phosphorylation of Hela cell c-Src protein Tyr416 was analyzed by Western Blot.

Taking logarithmic growth phase Hela cells, inoculating the Hela cells to a 6-hole plate, 1X 106/hole, adding 80 (14) compound diluted by DMSO with different concentrations until the final concentration is 2 mu mol/L, 4 mu mol/L and 8 mu mol/L respectively, taking the panatinib as a positive control to act for 24 hours, collecting the cells, washing 3 times by PBS (phosphate buffered saline) precooled at 4 ℃, adding 75 mu L of cell lysate, performing on-ice lysis for 30 min, extracting total cell proteins, and measuring the protein concentration by a bradford method. Taking 25 mug protein samples, transferring the protein to a PVDF membrane after 10% SDS-PAGE electrophoresis separation, sealing the protein for 1h at room temperature by using 5% BSA/skimmed milk powder sealing liquid, adding anti-C-Src, p-C-Src (Tyr 416, tyr 527) and beta-actin antibodies after washing the membrane, incubating overnight at 4 ℃, washing the membrane for 3 times by TBST, adding secondary antibody for incubating at room temperature for 2 h, washing the membrane for 3 times by TBST, uniformly mixing the chemiluminescent enhancing liquids A and B in equal volumes, and smearing the mixture on the PVDF membrane, and obtaining images by using a gel imaging system. In the activation loop of the kinase domain, phosphorylation of the Tyr416 site upregulates enzyme activity. The results of the experiment (FIG. 10) show that the decrease in phosphorylation of Tyr416 site at concentrations of 2. Mu. Mol/L, 4. Mu. Mol/L, 8. Mu. Mol/L for the 80 (14) compound for 24 hours demonstrates that 80 (14) can inhibit c-Src activity and thus inhibit cancer cell proliferation, migration and invasion activity.

Claims (2)

1. The application of a pharmaceutical composition containing a compound shown in a formula I or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier in preparing a medicament for preventing and/or treating cervical cancer, human mantle cell lymphoma, acute lymphoblastic B cell leukemia and diffuse large B cell lymphoma is characterized in that the structural formula of the compound shown in the formula I is as follows:

。

2. use according to claim 1, characterized in that the medicament acts by targeting the mechanism of action of the SH3 domain in regulating c-Src kinase activity.