CN112076189B

CN112076189B - Application of amide compound in preparation of medicine for treating sepsis

Info

Publication number: CN112076189B
Application number: CN202011007200.1A
Authority: CN
Inventors: 唐怡庭; 吕奔
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-09-23
Filing date: 2020-09-23
Publication date: 2021-11-12
Anticipated expiration: 2040-09-23
Also published as: CN112076189A

Abstract

The invention discloses an application of an amide compound in preparing a medicament for treating sepsis, wherein the structure of the amide compound is as follows:

the amide compound can inhibit macrophage apoptosis induced by Caspase11, inhibit the release of proinflammatory cytokines IL-1 alpha and IL-1 beta, and inhibit macrophage apoptosis by inhibiting GSDMD protein oligomerization, so that the amide compound has a certain treatment effect on sepsis, and provides a new treatment method and a new treatment medicine for clinical treatment of sepsis.

Description

Application of amide compound in preparation of medicine for treating sepsis

Technical Field

The invention relates to the field of biological medicines, in particular to application of an amide compound in preparation of a medicine for treating sepsis.

Background

Sepsis (sepsis, also known as sepsis) is a critical condition characterized by multiple organ failure induced by infection, characterized by an acute onset and a severe condition with a mortality rate of up to 40%. Common clinical symptoms include fever, accelerated respiratory rate and heartbeat, and unconsciousness, and severe sepsis can cause insufficient blood flow to supply tissues and even cause organ failure and septic shock, which seriously threatens the life safety of patients. At present, early fluid resuscitation, early use of antibiotics and organ function support, etc. treatment, reduce the mortality rate of sepsis to some extent, but sepsis remains the leading cause of death in Intensive Care Unit (ICU) patients.

The pathogenesis of sepsis is quite complex and in recent years, new sepsis pathogenesis has been revealed in several studies: caspase-11 mediated activation of inflammatory bodies to induce apoptosis (Pyrtopsis) plays an important role in sepsis^[1-7]. These studies found that Caspase-11 is an intracellular receptor for bacterial endotoxins (LPS)^[1-4]After LPS enters into cytoplasm, the LPS can be directly combined with Caspase-11 and can activate the Caspase-11; the activated Caspase-11 shears downstream Gasderm D (GSDMD) protein into peptide segments with membrane breaking function, and the peptide segments are gathered on cell membranes to form cell membrane pore channels, so that cell swelling and cell lysis are caused; this process is accompanied by the release of a number of proinflammatory cytokines such as interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta), and the like^[5-7]. The IL-1 alpha or IL-1 beta gene is knocked out, so that the survival rate of an endotoxemia model mouse is not influenced; however, the knockout of Caspase-11 or GSDMD gene can obviously improve the sepsis and endotoxemia of miceSurvival rate in^[1-7]. The above studies show that: caspase-11 mediated cell apoptosis plays an extremely important role in the lethal link of sepsis and endotoxemia. The research of relevant mechanisms finds that: caspase-11 is mainly expressed in mononuclear macrophages and vascular endothelial cells. Activation of mononuclear macrophage Caspase-11 in sepsis induces cell apoptosis to generate a large amount of arachidonic acid, resulting in increase of systemic vascular permeability and massive extravasation of intravascular fluid^[1-7](ii) a Microcirculation disturbance caused by cell apoptosis induced by Caspase-11 activation of vascular endothelial cells^[8](ii) a Finally, the body is subjected to low volume shock, and multiple organ function failure and even death are caused.

Amide compounds are widely present in modern drugs and biologically active substances, such as peptides and proteins containing amide bonds, lactam antibiotics with antibacterial activity, and amide derivatives of sulfonamides and chloramides, which are widely used in the medical field.

At present, medicines for treating sepsis mainly take nonspecific treatment such as glucocorticoid to prevent and treat organ failure, shock and other symptomatic treatment. While treatment methods to control infection can alleviate the symptoms of sepsis and prolong the life of the patient, they are both temporary and permanent. Therefore, the development of new sepsis therapeutic drugs for application in clinical therapy is of great significance. There is no report of the use of amide compounds for the treatment of sepsis.

Reference documents:

1.Kayagaki N,Warming S,Lamkanfi M,Vande Walle L,Louie S,Dong J,Newton K,Qu Y,Liu J,Heldens S,Zhang J,Lee WP,Roose-Girma M,Dixit VM.Non-canonical inflammasome activation targets caspase-11.Nature.2011,479(7371):117-121.

2.Hagar JA,Powell DA,Aachoui Y,Ernst RK,Miao EA.Cytoplasmic LPS activates caspase-11:implications in TLR4-independent endotoxic shock.Science.2013,341(6151):1250-1253.

3.Kayagaki N,Wong MT,Stowe IB,Ramani SR,Gonzalez LC,Akashi-Takamura S,Miyake K,Zhang J,Lee WP,Muszynski A,Forsberg LS,Carlson RW,Dixit VM.Noncanonical inflammasome activation by intracellular LPS independent of TLR4.Science.2013,341(6151):1246-1249.

4.Shi J,Zhao Y,Wang Y,Gao W,Ding J,Li P,Hu L,Shao F.Inflammatory caspases are innate immune receptors for intracellular LPS.Nature.2014,514(7521):187-192.

5.Kayagaki N,Stowe IB,Lee BL,O'Rourke K,Anderson K,Warming S,Cuellar T,Haley B,Roose-Girma M,Phung QT,Liu PS,Lill JR,Li H,Wu J,Kummerfeld S,Zhang J,Lee WP,Snipas SJ,Salvesen GS,Morris LX,Fitzgerald L,Zhang Y,Bertram EM,Goodnow CC,Dixit VM.Caspase-11cleaves gasdermin D for non-canonical inflammasome signalling.Nature.2015,526(7575):666-671.

6.Shi J,Zhao Y,Wang K,Shi X,Wang Y,Huang H,Zhuang Y,Cai T,Wang F,Shao F.Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death.Nature.2015,526(7575):660-665.

7.Liu X,Zhang Z,Ruan J,Pan Y,Magupalli VG,Wu H,Lieberman J.Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores.Nature.2016Jul 7；535(7610):153-8.

8.Cheng KT,Xiong S,Ye Z,Hong Z,Di A,Tsang KM,Gao X,An S,Mittal M,Vogel SM,Miao EA,Rehman J,Malik AB.Caspase-11-mediated endothelial pyroptosis underlies endotoxemia-induced lung injury.J Clin Invest.2017Nov 1；127(11):4124-4135.

disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides the application of the amide compound in preparing the medicament for treating the sepsis, and the medicament has good treatment effect on the sepsis.

The invention also provides application of the amide compound in preparation of an inhibitor in a sepsis-related pharmacological pathway.

Use of an amide compound according to an embodiment of the first aspect of the invention, having the formula:

the application of the amide compound in the preparation of the medicament for treating sepsis according to the embodiment of the invention has at least the following beneficial effects: the amide compound is used for preparing a medicine for treating sepsis, and proves that the amide compound can inhibit cell apoptosis and the production of proinflammatory cytokines IL-1 alpha, IL-1 beta and the like, and has a potential treatment effect on sepsis.

Sepsis also includes sepsis-associated diseases in the present invention, such as endotoxemia (endoxemia), severe sepsis (severe septis), and septic shock (septic shock). All belong to symptoms such as systemic infection caused by gram-negative bacteria and further systemic multi-organ dysfunction caused by gram-negative bacteria, and the pathogenic causes and the treatment methods of the symptoms are similar.

According to some embodiments of the invention, the sepsis therapeutic agent is an agent capable of inhibiting apoptosis of cells in an infected sepsis animal.

Apoptosis (Pyroptosis), also known as inflammatory necrosis, is a programmed cell death that is characterized by a constant swelling of cells until the cell membrane is ruptured, resulting in the release of cellular contents that in turn activate a strong inflammatory response. Cell apoptosis is an important natural immune response in the body and plays an important role in combating infection. Cell apoptosis is characterized by dependence on inflammatory caspases (primarily Caspase-1, 4, 5, 11) with release of a number of proinflammatory cytokines.

Further, inhibition of cellular apoptosis is inhibition of Caspase11 dependent cellular apoptosis.

Further, the inhibition of cell apoptosis is the inhibition of cell apoptosis induced by activating Caspase11 by CTB transfected LPS. Further, the cell is a macrophage.

According to some embodiments of the invention, the sepsis therapeutic agent is an agent capable of inhibiting the secretion of proinflammatory cytokines in an infected sepsis animal.

Further, the proinflammatory cytokines include IL-1 alpha, IL-1 beta.

According to some embodiments of the invention, the sepsis therapeutic agent is an agent capable of inhibiting oligomerization of the GSDMD protein in an animal infected with sepsis.

GSDMDM belongs to a family of proteins with unknown function called gasdermin, which also includes GSDMA, GSDMB, GSDMC, DFNA5, DFNB59, etc., the proteins of this family have about 45% homology, have two domains, gasdermin-N and-C domains, and can be cleaved by inflammatory Caspase.

Further, inhibition of GSDMD protein oligomerization inhibits Caspase11 dependent cell apoptosis.

Further, the cells that are burned out are macrophages.

According to some embodiments of the invention, the medicament further comprises a pharmaceutically acceptable carrier or excipient.

According to some embodiments of the invention, the medicament is formulated into any one of the pharmaceutically acceptable formulations as required, for example, into an oral administration formulation: tablets, capsules, pills, granules, dripping pills, oral preparations and the like; preparing a rectal administration preparation: suppositories, enemas; preparing an injection preparation: intramuscular injection preparations, intravenous injection preparations, and the like.

Use of an amide compound according to the second aspect of the present invention for the preparation of an inhibitor of a sepsis-related pharmacological pathway, said amide compound having the formula:

also provides the application of the amide compound in the preparation of the cell apoptosis inhibitor; or

The use of the amide compound in the preparation of a proinflammatory cytokine inhibitor; or

The amide compound is applied to the preparation of GSDMD protein oligomerization inhibitors.

According to some embodiments of the invention, the proinflammatory cytokines comprise IL-1 α and IL-1 β.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 shows the results of an LDH assay for the compound of example 1 of the present invention;

FIG. 2 shows the results of ELISA assay for detecting cytokine of the compound of example 1 of the present invention, wherein (a) is an IL-1. alpha. content test pattern, and (b) is an IL-1. beta. content test pattern;

FIG. 3 is an ECL development pattern of the compound of example 2 of the present invention to inhibit GSDMDM oligomer formation;

FIG. 4 is a 3D diagram of molecular docking of compounds and analogs with GSDMD protein in example 3 of the present invention;

FIG. 5 shows the results of a control experiment for LDH detection of compounds and analogues of example 3 of the present invention;

FIG. 6 shows the results of ELISA assay control experiments for cytokines of the compounds and analogs of example 3 of the present invention, wherein the left figure shows the cytokine IL-1. alpha. and the right figure shows the cytokine IL-1. beta.

Detailed Description

In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available. The compounds used in the following examples are amide compounds

Structural analogs thereof

Are purchased from selelck corporation.

Example 1: compound for inhibiting primary macrophage apoptosis induced by CTB transfected LPS activated Caspase11

1. The experimental steps are as follows: extracting primary abdominal cavity macrophage of WT mouse, re-suspending the cell in RPMI-1640 complete culture medium to 1 × 10⁶500. mu.l/we per mlThe cells were washed 2 times with DPBS after overnight adherence in the ll 24 well plates and replaced with serum-free 1640. The compound is added into cells at final concentrations of 5 mu M and 10 mu M respectively for pre-incubation for 1h, then a transfection reagent Cholera Toxin (CTB)5 mu g/ml + LPS 1 mu g/ml (pre-incubation is carried out for 20min at room temperature in advance) is added for stimulating the cells overnight, and supernatant is collected for 16-18 h to detect the content of Lactate Dehydrogenase (LDH) (judging the cell death rate) and ELISA (detecting proinflammatory cytokines IL-1 alpha and IL-1 beta).

The specific experimental operations were as follows:

1) extracting primary abdominal cavity macrophages of the mice: 3% thioglycollate medium (3 ml/mouse) is injected into the abdominal cavity, after 3-4 days, 10ml of RPMI1640 medium is injected into the abdominal cavity of the mouse, the abdominal cavity lavage fluid is pumped back by a syringe after the abdomen of the mouse is slightly kneaded, the operation is repeated for 1 time, the mouse is collected in a centrifuge tube, and the mouse is centrifuged at 800rpm for 5 minutes, and the supernatant is discarded. Resuspending cells in 5-10 ml of RPMI1640 complete medium, microscopically counting cells, and adjusting the cell concentration to 1 × 10⁶Pieces/ml, 100. mu.l/well seeded 96-well plate, attached overnight.

2) Adding medicine for stimulation: washing the cells 2 times by using DPBS to remove non-adherent suspension cells, replacing the cells with 1640 serum-free medium, performing pretreatment for 1h at the final concentration of the compound of 10 mu M/hole, and adding CTB of 5 mu g/ml + LPS of 1 mu g/ml (pre-incubation for 20min at room temperature) to stimulate the cells overnight (the experiment also comprises a control group, wherein the control group is not added with LPS and CTB, but only added with LPS of 1 mu g/ml, and only added with the compound);

3) collecting cell supernatant to detect the content of Lactate Dehydrogenase (LDH), cell factors IL-1 alpha and IL-1 beta.

a. Cell death rate assay (lactate dehydrogenase LDH assay):

the experimental steps are as follows: 100 μ l of LDH release reagent was added to the control group, and RPMI-1640 medium was added to make up the volume of the control group to the original volume and mixed well, followed by incubation at room temperature for 1 hour. The supernatant was collected into 1.5ml EP tubes, placed in a 4-degree centrifuge, centrifuged at 500rpm for 5min, and then transferred to new EP tubes. And (3) preparing an LDH detection working solution: lactic acid solution, iodonitrotetrazolium chloride (INT) solution (1X), and enzyme solution are mixed according to the ratio of 1:1: 1. Taking a 96-well plate, firstly adding 80 mu l of RPMI-1640 culture medium into each well, then adding 60 mu l of each sample to be detected, and then respectively adding 60 mu l of LDH detection working solution into each well. Mix well and incubate in the dark at room temperature (about 25 ℃) for 30 min. The absorbance was then measured at 490 nm. The two-wavelength measurement is performed using either 600nm or a wavelength greater than 600nm as a reference wavelength.

Data processing: calculated according to the following formula (the absorbance of each group should be subtracted by the absorbance of the background blank control well):

cell death (%) × (treated sample absorbance-sample control well absorbance)/(absorbance for maximum enzyme activity of cells-sample control well absorbance) × 100.

Elisa assay (cytokine assay): and respectively detecting the contents of IL-1 alpha and IL-1 beta in the supernatant sample by an ELISA detection kit. The ELISA plates were coated with IL-1 α and IL-1 β, respectively, overnight at 4 ℃. 0.05% PBST was washed 3 times for 1 min/time. 1 × Assay buffer was blocked for 1h at RT. Washing 0.05% PBST for 3 times, 1 min/time, adding corresponding sample to be tested and cytokine standard, and incubating for 2h at room temperature on a shaking table. 0.05% PBST was washed 3 times for 1 min/time. Adding corresponding detection antibody and incubating for 1h in a shaking table at room temperature. 0.05% PBST was washed 3 times for 1 min/time. Finally adding an HRP shaker to incubate for 30min at room temperature. TMB developed after 5 washes with 0.05% PBST, and the development was stopped with 2M sulfuric acid. Absorbance at 450nm was measured.

2. The experimental results are as follows: the experimental results of the cytotoxicity experiments of this example are shown in FIG. 1 (in the figure, L1 is LPS only, CL is CTB and LPS, CL + 5. mu.M is the group of compounds with 5. mu.M, LH + 10. mu.M is the group of compounds with 10. mu.M, and 10. mu.M is the group of compounds with 10. mu.M), it can be seen from FIG. 1 that the content of lactate dehydrogenase (LDH assay) in the cell supernatant is greatly reduced with the addition of the compounds, and the compound concentration of 5. mu.M has a good effect, indicating that the addition of the compound can reduce the cell death rate, i.e., inhibit macrophage apoptosis induced by Caspase 11. The ELISA assay of this example is shown in FIG. 2, (a) is an IL-1 alpha content test chart, and (b) is an IL-1 beta content test chart, and it can be seen from the chart that when the compound is added, the content of proinflammatory cytokines IL-1 alpha and IL-1 beta is significantly reduced, which indicates that the compound inhibits the release of proinflammatory cytokines induced by Caspase 11.

After LPS enters into cytoplasm, Caspase-11 can be directly combined and activated, and the activated Caspase-11 can trigger subsequent cascade reaction to cause cell apoptosis so as to generate a large amount of proinflammatory cytokines, and the mediated cell apoptosis plays an extremely important role in lethal links of sepsis and endotoxemia. The experiment proves that the contents of proinflammatory cytokines IL-1 alpha and IL-1 beta in a test group added with the compound are both reduced, which shows that the compound inhibits the primary macrophage apoptosis induced by activated Caspase 11. Meanwhile, the main pathogenesis of the sepsis also comprises the generation and release of a large amount of proinflammatory cytokines to cause immune disorder in an organism, so that the fact that the compound can reduce cytotoxicity, reduce cell death rate and further inhibit the release of the proinflammatory cytokines proves that the compound has a certain treatment effect on the sepsis.

Example 2: compounds for inhibiting GSDMD oligomerization and further inhibiting primary macrophage apoptosis

1. The experimental steps are as follows: extracting primary abdominal cavity macrophages of WT mice, and adjusting the concentration of the resuspended cells to 1x 10^ 6/ml and 2ml/well type 6-well plate by RPMI-1640 complete culture medium. Cells were washed 2 times with DPBS after overnight adherence and switched to serum-free 1640. The compound is added into cells at final concentrations of 5 mu M and 10uM respectively for pre-incubation for 1h, then CTB 5 mu g/ml + LPS 1 mu g/ml (pre-incubation is carried out for 20min at room temperature in advance) is added for stimulating the cells overnight, cell proteins are collected and added into protein lysate RIPA (a cocktail protease inhibitor and a phosphatase inhibitor are proportionally added into the RIPA), and the cell proteins are lysed on ice for 45 min. The ice box was placed on a shaker to ensure that the lysate was in sufficient contact with the cells. The cells were then collected into an EP tube with a precooled cell scraper, centrifuged at 13000g for 15min at 4 degrees, and the supernatant was collected as the cell protein from lysis. BCA was used to quantify proteins, and loading buffer (5 × loading buffer: Tris-HCl pH 6.8(60mM), SDS (2%), bromophenol blue (0.1%), and glycerol (25%)) containing no reducing agent was used to adjust the protein concentration, loading was performed at 20 μ g/well, electrophoresis was performed at 4 ℃ and the temperature was lowered, and membrane transfer was performed. The primary antibody of GSDMDM was incubated with PVDF membrane overnight at 4 degrees, and the membrane was washed 3 times with 0.5% TBST the next day, 15 min/time. And then incubating corresponding secondary antibodies, washing, and carrying out ECL development to detect the formation of the GSDMD oligomer.

2. The experimental results are as follows: the experimental results of this example are shown in FIG. 3, wherein 2370 represents the compound of this example. FIG. 3 is an ECL development diagram of a compound for inhibiting the formation of a GSDMD oligomer, and (a) in FIG. 3 indicates that the compound has no inhibition on the GSDMD splicing body and inhibits the occurrence of cell apoptosis by inhibiting GSDMD oligomerization (multimers formed by oligomerization are positions indicated by arrows in (b) in FIG. 3). As can be seen in the figure, the compound inhibited the formation of GSDMD multimers, but the compound had no inhibitory effect on GSDMD cleavage, suggesting that multimer formation may be inhibited by inhibiting GSDMD oligomerization.

In the pathogenesis of sepsis, activated Caspase-11 shears downstream GSDMD protein into peptide fragments with membrane breaking function, and the peptide fragments are oligomerized and aggregated on cell membranes (polymers) to form cell membrane pore channels, thereby causing cell apoptosis and releasing a large amount of proinflammatory cytokines. Thus, inhibition of downstream GSDMD multimer formation may inhibit the onset of sepsis.

Example 3: molecular docking and analog control experiments

1. The experimental steps are as follows: the compound obtained by virtual screening is butted with GSDMD, and another analogue obtained by screening is obtained, wherein the analogue has a structural formula:

and reducing the binding energy of the jointed GSDMDM, and determining the jointing active site. Wherein, the molecular docking 3D diagram of the compound and the analogue with the GSDMD protein is shown in figure 4.

2. Structural analogue control experiment: cell apoptosis inhibition experiments were carried out using the same method as in example 1, using the above structural analogs in place of the compound in example 1, at each of the drug concentrations of 10. mu.M.

3. The experimental results are as follows: the control results of the LDH detection experiment are shown in FIG. 5, CON is the control group, and the compound and the analogue are added in an amount of 10 μ M in each group. The cell death rate is greatly reduced after the compound is added, and the influence on the cell death rate is little after the analogue is added, which shows that the analogue has no inhibiting effect on the primary macrophage apoptosis induced by CTB transfected LPS activated Caspase 11. The ELISA detection (cytokine detection) result is shown in FIG. 6 (the left figure is IL-1 alpha, the right figure is IL-1 beta), and it can be seen that when the compound is added, the contents of proinflammatory cytokines IL-1 alpha and IL-1 beta are both reduced, which indicates that the compound inhibits the secretion of proinflammatory cytokines, but the contents of proinflammatory cytokines IL-1 alpha and IL-1 beta are not reduced after the analogue is added, which indicates that the analogue cannot inhibit the secretion of proinflammatory cytokines.

Compared with biological activity, the compound and the analogue have a plurality of superposition modes and the most similar conformations, wherein the similarity is about 70 percent, and the electric regions of the compound and the analogue are greatly different, and the hydrophobic regions are partially similar. The difference in electrical area may be one of the causes of the change in biological activity.

In conclusion, the amide compound provided by the invention can inhibit macrophage apoptosis induced by Caspase11, can inhibit the release of proinflammatory cytokines IL-1 alpha and IL-1 beta, and can inhibit macrophage apoptosis by inhibiting GSDMD protein oligomerization, so that the amide compound has a certain treatment effect on sepsis, and provides a new treatment method and a new treatment medicine for clinical treatment of sepsis.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.

Claims

1. The application of an amide compound in preparing a medicament for treating sepsis is characterized in that the structure of the amide compound is as follows:

2. the use according to claim 1, wherein the sepsis therapeutic agent is an agent capable of inhibiting apoptosis of cells in an animal infected with sepsis.

3. The use according to claim 1, wherein the sepsis therapeutic agent is an agent capable of inhibiting the secretion of pro-inflammatory cytokines in an animal infected with sepsis.

4. The use according to claim 3, wherein the proinflammatory cytokines comprise IL-1 α and IL-1 β.

5. The use according to claim 1, wherein the sepsis therapeutic agent is an agent capable of inhibiting oligomerization of the GSDMD protein in an animal infected with sepsis.

6. The use according to any one of claims 1 to 5, wherein the medicament for the treatment of sepsis further comprises a pharmaceutically acceptable carrier or adjuvant.