CN113527310B - Small molecule compound for relieving adverse inflammatory reaction of autoimmune disease patient and application thereof - Google Patents

Abstract

The invention provides a small molecular compound for relieving adverse inflammatory reaction of autoimmune disease patients and application thereof, wherein the small molecular compound is Milciclib or pharmaceutically acceptable salt thereof. The small molecular compound, namely the miciclib or the pharmaceutically acceptable salt thereof, provided by the invention can specifically recognize STING and inhibit a STING downstream signal pathway, so that the autoinflammatory reaction of an organism is reduced, the survival benefit of a patient is improved, the disease burden of the patient is reduced, and a new strategy is provided for preparing a medicament for relieving the adverse inflammatory reaction of the autoimmune disease patient.

Description

Small molecule compound for relieving adverse inflammatory reaction of autoimmune disease patient and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a small molecular compound for relieving adverse inflammatory reaction of autoimmune disease patients and application thereof.

Background

After the DNA receptor senses the foreign or self DNA accumulated in the cytoplasm, it triggers a strong immune response by activating downstream signals to cause the production of interferons and other proinflammatory cytokines. The discovery of the cytoplasmic DNA recognition receptor guanosine monophosphate adenosine synthase (cGAS) is an important milestone in the field of DNA recognition. cGAS, upon recognition of cytoplasmic DNA, catalyzes synthesis of cGAMP from Adenosine Triphosphate (ATP) and Guanosine Triphosphate (GTP), and subsequently, cGAMP binds to and induces STING dimerization of interferon-stimulating genes (stimulator of interferon gene, STING), activating TANK-binding kinase 1(TANK-binding kinase1, TBK1) and nuclear factor KB inhibitors (inhibitor of nuclear factor kappa-B kinase, IKK), which in turn activate interferon regulatory factor 3(interferon regulatory factor 3, IRF3) and nuclear factor KB (nuclear factor kappa-B kinase, NF- κ B), respectively, ultimately leading to rapid and intense interferon production. Aberrant DNA accumulation activates cGAS-STING pathway-induced production of downstream interferons and other cytokines, possibly leading to autoinflammation and autoimmune diseases.

At present, the aim of clinically treating RA is mainly to relieve joint inflammatory response, inhibit lesion development and irreversible bone destruction, protect functions of joints and muscles as far as possible and relieve pain of patients, wherein the medicament for treating RA is mainly composed of non-steroidal anti-inflammatory drugs (NASIDs), antirheumatic drugs (DMARDs), Glucocorticoids (GCs), biological agents and the like Broad-spectrum inflammatory reaction inhibiting drugs, improve the survival benefit of patients and reduce the disease burden of patients.

The cGAS plays an important role in the disease process of RA, in RA-FLS, TNF alpha stimulates cGAS-STING overexpression to promote RA-FLS proliferation, and STING remarkably increases the expression of main proinflammatory regulators IL-1 beta, IL-6, MMP-1 and MMP-3 through IRF3-TBK1 and MAPK-NF-kappa B pathways. Therefore, the screened small molecular drug capable of specifically inhibiting cGAS-cGAMP-STING can inhibit inflammatory reaction, improve the survival benefit of patients and has important clinical application value.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a small molecular compound, namely, micciclib (an effective CDK and Tropomosin Receptor Kinase (TRK)) for relieving adverse inflammatory reactions of patients with autoimmune diseases and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect the present invention provides a small molecule compound for use in alleviating an adverse inflammatory response in a patient with an autoimmune disease, which small molecule compound is micciclib or a pharmaceutically acceptable salt thereof.

Further, the chemical molecular structure of the above-mentioned miciclib is represented by formula (i):

further, the small molecule compound can specifically recognize STING and inhibit STING downstream signaling pathway.

In a second aspect, the invention provides the use of a small molecule compound according to the first aspect of the invention in the manufacture of a medicament for use in alleviating an adverse inflammatory response in a patient suffering from an autoimmune disease.

Further, the medicine also comprises a pharmaceutically acceptable carrier or excipient.

A third aspect of the present invention is to provide a medicament for treating autoimmune diseases, which comprises, as an effective ingredient, micciclib or a pharmaceutically acceptable salt thereof.

Further, the above-mentioned Milciclib or a pharmaceutically acceptable salt thereof is inhibited by

The cGAS-cGAMP-STING signaling pathway thereby alleviates adverse inflammatory responses in patients with autoimmune diseases.

In a fourth aspect of the present invention, there is provided a method for screening a small molecule compound according to the first aspect of the present invention, comprising the steps of:

step one, sealing the isocyanate groups on the positions of the small molecules which are not spotted on the substrate by adopting BSA solution on the small molecule microarray containing the active compound;

step two, after isocyanate groups at positions of the small molecules which are not spotted on the substrate are closed, target protein STING is adopted to react with the small molecule microarray;

and step three, scanning the microarray, and then collecting and analyzing data.

Further, the concentration of the BSA solution was 0.5 mg/mL.

Furthermore, the concentration of the target protein STING is 10 μ g/mL.

Further, the reaction time in the second step is 50-70 min.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

the small molecular compound Milciclib provided by the invention can specifically recognize STING and inhibit STING downstream signal channels, so that the autoinflammatory reaction of an organism is reduced, the survival benefit of a patient is improved, the disease burden of the patient is reduced, and a new strategy is provided for preparing a medicine for relieving adverse inflammatory reaction of an autoimmune disease patient.

Drawings

FIG. 1 is an OI-RD difference image of a small molecule microarray after reaction with STING in accordance with an embodiment of the present invention;

FIG. 2 is a difference image of the small molecule microarray OI-RD image in the HEPES buffer solution after reaction with STING and the small molecule microarray OI-RD image of the HEPES buffer solution before reaction in one embodiment of the present invention;

FIG. 3 shows the results of IFN- β expression levels of C57 mouse peritoneal macrophages (stimulated with exogenous ISD) in the presence of a primary screened small molecule compound in one embodiment of the invention;

FIG. 4 shows the results of IFN- β expression levels of C57 mouse peritoneal macrophages stimulated with exogenous cGAMP in the presence of a primary small molecule compound in an example of the invention.

Detailed Description

The STING pathway is very critical in the process of generating autoimmune diseases and becomes a very important intervention and treatment target, and the invention aims to screen a small molecule inhibitor aiming at the STING target, inhibit the autoimmune reaction of patients, improve the quality of life and improve the survival expectation. Based on the above purposes, the invention provides a small molecule compound for alleviating adverse inflammatory reactions of patients with autoimmune diseases and an application thereof, wherein the small molecule compound is micciclib or pharmaceutically acceptable salts thereof, and the chemical molecular structures of the micciclib are respectively shown as formula (I):

wherein the small molecule compound can specifically recognize STING and inhibit STING downstream signaling pathway.

The screening method of the small molecule compound comprises the following steps:

step one, on a small molecule microarray containing an active compound, adopting a BSA solution to seal isocyanate groups on a substrate at positions where small molecules are not spotted;

step two, after isocyanate groups at positions of the small molecules which are not spotted on the substrate are closed, target protein STING is adopted to react with the small molecule microarray;

and step three, scanning the microarray, and then collecting and analyzing data.

In a preferred embodiment of the invention, the concentration of BSA solution is 0.5 mg/mL.

In a preferred embodiment of the present invention, the concentration of the target protein STING is 10 μ g/mL.

In a preferred embodiment of the present invention, the reaction time in step two is 50-70 min.

The present invention will now be described in detail and with reference to specific examples and figures to provide a better understanding of the invention, but the following examples do not limit the scope of the invention.

In the examples, the conventional methods were used unless otherwise specified, and reagents used were those conventionally commercially available or formulated according to the conventional methods without specifically specified.

Example 1

The starting point and key step of novel drug research and development is the activity screening of compounds, and this example screens small molecule compounds specifically bound by STING protein by high throughput, and the specific experimental steps and results are as follows:

after sealing the isocyanate groups at the non-spotted small molecule sites on the substrate with 0.5mg/mL BSA solution on the FDA-approved 3375 active compound small molecule microarray, the small molecule microarray was reacted with 1.5mL target protein STING at a concentration of 10. mu.g/mL for 1 hour to obtain the OI-RD difference image of the small molecule microarray after reaction with the STING protein (the difference between the OI-RD image in the protein solution after reaction and the OI-RD image in the buffer solution before reaction), as shown in FIG. 1.

In order to correctly select the small molecules reacting with STING from all the bright spots in fig. 1, two OI-RD images were sequentially taken in the buffer solution after washing the reacted small molecule microarray with HEPES buffer solution. FIG. 2 is a difference image of the OI-RD image of the small molecule microarray in HEPES buffer solution after reaction and the OI-RD image of the small molecule microarray in HEPES buffer solution before reaction, where adjacent double highlights include small molecules with slower dissociation rates that can react with STING, and also include small molecules with signal changes due to various causes. The adjacent double bright spots appearing in fig. 1 but weakened or disappeared in fig. 2 correspond to small molecules that bind to STING but with faster off-rates.

Therefore, from FIGS. 1-2, a part of molecules bound to STING (faster off-rate) can be obtained initially. Then 3 independent STING screens were performed, and out of 3 experiments, at least 2 small molecules that became positive spots were the final positive spots of authentic positive compounds. According to the analysis, the final positive compound of STING was a6 small molecule-micciclib (corresponding to the compound indicated by the arrow, 8J 19).

Example 2

This example verifies the function of the small molecules screened in example 1 at the cytological level, and the specific experimental methods and conclusions are as follows:

the exogenous ISD (cGAS activator) with the concentration of 1 mu M and the cGAMP (STING activator) with the concentration of 1 mu M are respectively used for stimulating macrophages in abdominal cavities of C57 mice for 6h, small molecular compounds such as A2, A3, A7, B2 and B5 are respectively added to the macrophages in parallel control, and then the IFN-beta expression levels of the different stimulated tumor cells of the groups are detected by the RT-RCR technology, and the results are shown in FIGS. 3-4.

As can be seen from FIGS. 3-4, the A6 small molecule (Milciclib) can specifically inhibit the IFN-beta expression level of mouse peritoneal macrophages stimulated by ISD and cGAMP. The results show that the Milciclib can specifically inhibit the cGAS-STING pathway through STING, and up-regulate the expression of IFN-beta by tumor cells.

The embodiments of the present invention have been described in detail, but the embodiments are only examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent alterations and modifications are intended to be included within the scope of the present invention, without departing from the spirit and scope of the invention.

Claims (7)

1. Use of a small molecule compound in the manufacture of a medicament for use in alleviating an adverse inflammatory response in a patient with an autoimmune disease, wherein the small molecule compound is miciclib or a pharmaceutically acceptable salt thereof;

the chemical molecular structure of the Milciclib is shown as the formula (I):

2. the use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier or excipient.

3. The use of claim 1, wherein the small molecule compound is capable of specifically recognizing STING and inhibiting STING downstream signaling pathways.

4. The use according to claim 1, wherein the method for screening small molecule compounds comprises the steps of:

step one, sealing the isocyanate groups on the positions of the small molecules which are not spotted on the substrate by adopting BSA solution on the small molecule microarray containing the active compound;

step two, after isocyanate groups at positions of the small molecules which are not spotted on the substrate are closed, target protein STING is adopted to react with the small molecule microarray;

and step three, scanning the microarray, and then collecting and analyzing data.

5. The use of claim 4, wherein the concentration of the BSA solution is 0.5 mg/mL.

6. The use according to claim 4, wherein the concentration of the target protein STING is 10 μ g/mL.

7. The use according to claim 4, wherein the reaction time in step two is 50-70 min.

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