CN112656798B

CN112656798B - Application of CDK7 targeted inhibitor in preparation of medicine for treating cytokine release syndrome

Info

Publication number: CN112656798B
Application number: CN202011587308.2A
Authority: CN
Inventors: 苏锋涛; 陆雪官; 郭小毛; 魏也; 李冲; 卞慧芳
Original assignee: Fudan University Shanghai Cancer Center
Current assignee: Fudan University Shanghai Cancer Center
Priority date: 2020-12-28
Filing date: 2020-12-28
Publication date: 2023-03-07
Anticipated expiration: 2040-12-28
Also published as: CN112656798A

Abstract

The invention provides application of a CDK7 targeted inhibitor in preparation of a medicament for treating cytokine release syndrome, which is verified through biochemical and animal model experiments: (1) Under a proper concentration, the CDK7 targeted inhibitor can specifically regulate and control inflammatory signal channels of immune cells such as macrophages, T cells, endothelial cells and the like by inhibiting the expression of super enhancers and transcription factors related to inflammatory factors such as stat1, IL-6 and the like; (2) Can obviously reduce the death of mice caused by acute inflammatory storm caused by bacterial or viral infection, and reduce the functional failure caused by the inflammation of related organs, and has no obvious toxic or side effect in the period. Has great significance in the treatment of cytokine release syndrome caused by bacteria, virus or immunotherapy.

Description

Application of CDK7 targeted inhibitor in preparation of drug for treating cytokine release syndrome

Technical Field

The invention relates to the technical field of medicines, in particular to application of a CDK7 targeted inhibitor in preparation of a medicine for treating cytokine release syndrome.

Background

Cytokine Release Syndrome (CRS), also called "Cytokine storm", refers to a phenomenon in which a large amount of cytokines are rapidly produced in the body after the immune system of the body is strongly stimulated (such as by virus, bacteria, immune intervention and treatment), and is a severe inflammatory response syndrome. Infection diseases such as novel coronavirus (COVID-19), SARS, influenza virus, bacteria and the like, and immunotherapy such as CAR-T and the like are accompanied with a certain proportion of patients to have cytokine release syndrome, particularly critical patients. When the pathogen initially attacks the body, macrophage, neutrophil, etc. immune cells release cytokines that recruit phase immune cells (macrophages, granulocytes, DC cells, T cells, B cells) to the site of infection, and the cytokines further activate these cells, stimulating them to produce more cytokines. Usually, this positive feedback loop is controllable by the body itself. However, in cytokine storms, a large number of immune cells are activated and secrete more cytokines, which recruit to a larger number of immune cells, creating an uncontrolled cascade of amplification effects. Therefore, CRS is a disease that the immune system is over-activated, so that various cytokines are over-expressed in tissues and organs, and finally, single-organ or multiple-organ damage, function exhaustion and death are caused. Referring to the drawings fig. 1A shows the pattern of inflammatory storm formation, CRS may be responsible for a variety of conditions (including infection, acute respiratory distress syndrome ARDS, sepsis, acute pancreatitis, rheumatic diseases, etc.). Severe CRS, which causes systemic inflammatory reaction of the host, may cause damage to various tissues and organs, causing multiple organ failure and even death.

Studies on patients with the novel coronavirus COVID-19 have found that higher levels of inflammatory cytokines, such as IL-2, IL-7, IL-10, IL-6, G-SCF, and TNF- α, are present in the plasma of Intensive Care Unit (ICU) patients. A research published in a magazine of Lancet medical records on line discovers that the prognosis of most patients is good after receiving antiviral, antibiotic and oxygen inhalation treatment in 99 cases of confirmed coronary pneumonia in a jin Yin Tan Hospital; however, 17 critically ill patients developed cytokine storm induced Acute Respiratory Distress Syndrome (ARDS), of which 11 died of multiple organ failure. In fact, as early as No. 24/1 of 2020, the first clinical data report (Lancet) of new cases of coronavirus infection referred to the term "cytokine storm". Of the above cytokines, IL-6, IL-1 and TNF- α have the greatest effects on the induction of inflammatory storms.

During the formation of the cytokine storm, macrophages play a key role, being the engine of the cytokine storm and also being the amplifier of the cytokine storm, as shown in fig. 1B. The 3 rd month dynasty professor "nature" of 2020 reports the pathological anatomy research result of severe recurrent coronary pneumonia. The affected organ with the greatest pathological changes is the lung, which is diffuse lung injury, extensive exudation and bleeding, destruction of alveolar epithelium and reactive hyperplasia, inflammatory reaction and fibrosis mainly caused by macrophages. In patients with new coronary pneumonia, these activated macrophages may play an important role in a serious series of "inflammatory factor storms". The results of analyses conducted by the middle sciences research team on 33 patients with COVID-19 showed a significant increase in the proportion and number of inflammatory monocytes/macrophages (CD 14+ CD16 +) in the peripheral blood of patients with COVID-19. These macrophages are capable of secreting more pro-inflammatory factors IL-6 and GM-CSF.

Currently, the means for treating CRS mainly include the following aspects: (1) IL-6 receptor antagonists: IL-6 is mainly secreted by macrophages, and has pleiotropic activity, and becomes a biomarker for judging the severity of a cytokine storm and a prognostic index, so that the function of blocking IL-6 becomes a key strategy for inhibiting CRS. Clinically, the Touzumab is used as an inhibitor to block key cell factors of inflammatory storm induced by new coronavirus infection, so that the damage of inflammatory reaction to lung tissues and multiple organs of a patient is effectively reduced. It should be noted that IL-6 is only one of many inflammatory cytokines in cytokine storms, and IL-1, a chemokine, is expressed higher in some kinds of cytokine storms. Therefore, this method has certain limitations; (2) glucocorticoids: in clinic, nonspecific combination treatment measures such as anti-infective drugs, glucocorticoids, nutritional support and the like are mostly adopted for the cytokine storm. Among them, glucocorticoids are commonly used clinically because of their potent anti-inflammatory effects, but glucocorticoids have large side effects. The clinical CRS treatments described above have different limitations or side effects. Therefore, the key problem to be solved urgently is to deeply research the relevant molecular action mechanism, develop safe and efficient targeted drugs, more effectively treat CRS and simultaneously reduce side effects.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention is realized by the following technical scheme:

the invention provides an application of a CDK7 targeted inhibitor in preparation of a medicine (namely CRS) for treating cytokine release syndrome;

further, the targeted inhibitor of CDK7 is THZ1, BS-181 or SY-1365; further preferably, the targeted inhibitor of CDK7 is THZ1;

further, the cytokine release syndrome is caused by bacterial infection or viral infection.

Drawings

FIG. 1 is a left diagram of the pattern of formation of a cytokine storm; the right diagram is the key action pattern diagram of macrophage in cytokine storm

FIG. 2 is a schematic representation of the CDK7 regulation mode; wherein A represents a pattern of CDK 7-regulated DNA transcription into mRNA and CDK 7-regulated cell cycle progression; b represents a structural formula of a compound THZ1 of the formula I; c represents a schematic representation of the irreversible covalent binding of THZ1 to a cysteine residue (Cys 312) outside the kinase domain of CDK7 proteins

FIG. 3 shows the results of comparative experiments on the inhibition of THZ1, SY-1365 and BS-181 against related target molecules in an inflammation model

FIG. 4 is a schematic diagram of THZ1 targeting CDK7-RNA Polymerase II complex regulated mRNA transcription and a western diagram of molecular signaling pathway

FIG. 5 shows the results of qRT-PCR experiments for a model of bacterial infection with THZ1 targeting CDK7-RNA Polymerase II complex

FIG. 6 shows the qRT-PCR experimental results of THZ1 targeting CDK7-RNA Polymerase II complex H1N 1-influenza virus infection model

FIG. 7 shows the results of ELISA experiments in which THZ 1-targeted CDK7-RNA Polymerase II complex inhibited release of relevant cytokines in cytokine storm

FIG. 8 is a diagram of genome-wide expression profile analysis of THZ 1-targeted CDK7-RNA Polymerase II complex in inflammatory model

FIG. 9 shows the results of analysis of THZ 1-targeted CDK7-RNA Polymerase II complex whole genome CHIP-Seq (RNA Poly II, H3K27 AC)

FIG. 10 is an animal level experiment of THZ1 targeting CDK7-RNA Polymerase II complex

FIG. 11 shows the results of the mouse survival experiment

Advantageous effects

The invention is proved by experiments that:

the targeted inhibitor of CDK7 can specifically inhibit acute inflammatory reaction caused in the processes of bacterial infection, viral infection and the like, and particularly has the best THZ1 effect; the biochemical and animal model experiments prove that: (1) Under a proper concentration, the CDK7 targeted inhibitor THZ1 can specifically regulate and control inflammatory signal pathways of immune-related cells such as macrophages, monocyte endothelial cells, fibroblasts and the like, and can inhibit the expression of inflammatory factors such as IL-1, IL-6, stat1 and the like and related super enhancers and transcription factors; (2) THZ1 can remarkably reduce the death of mice caused by acute inflammatory storm caused by bacterial or viral infection, and reduce the functional failure caused by inflammation of related organs, and no obvious toxic or side effect is found in the period.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

The THZ1 is a covalent inhibitor of the targeted CDK7 developed by Syros, and can prevent the THZ1 from

phosphorylating serine residues

2,5 and 7 of RNA polymerase II by targeted covalent modification of Cys312 residues outside a CDK7 kinase domain (see figure 1), inhibit the activity of the RNA polymerase II and further inhibit the transcription of mRNA.

The CASNO of THZ1 is 1604810-83-4, and the chemical name is: (E) -N- (3- ((5-chloro-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) -4- (4- (dimethyl a mine) but-2-enamido) benzamide with molecular formula C ₃₁ H ₂₈ ClN ₇ O ₂ The structural formula is shown as formula I:

BS-181 (CASNO.: 1397219-81-6) is a highly selective CDK7 inhibitor, IC ₅₀ 21nmol/L, more than 40 times more selective than CDK1,2,4,6 and 9. In human gastric cancer cell BGC-823, BS-181 significantly reduces CDK7 activity, and simultaneously down-regulates the level of anti-apoptotic proteins (cyclinD 1, XIAP and Bcl-2), thereby further inhibiting proliferation, invasion and migration of gastric cancer cells.

SY-1365 (CASNO.: 1816989-16-8) is a highly selective CDK7 inhibitor that inhibits cell growth in vitro for a number of different cancer types. SY-1365 treatment reduces MCL1 protein levels, BCL2L1 (BCL-XL) low-expressing cancer cells are more sensitive to SY-1365, and SY-1365 shows significant anti-tumor effect as a single drug in multiple AML xenograft models; SY-1365-induced growth inhibition was enhanced in combination with the BCL2 inhibitor venetoclax. Antitumor activity was also observed in the ovarian cancer xenograft model, indicating that SY-1365 has potential application prospects in hematology and clinical studies of solid tumors. Our findings support CDK7 as a novel approach to the treatment of transcription dependent cancers.

Example 1: CDK-7 targeted small molecule drugs of THZ1, SY-1365 and BS-181 in comparative inhibition experiment of related target molecules in inflammation model

Mononuclear cells (THP-1, a human monocyte cell line) in an exponential growth phase are treated with 100ng/ml PMA for 24 hours to form macrophages (mTHP-1, human monocyte induced macrophages), then 30nM THZ1, 30nM SY-1365 and 10 mu M BS-181 are respectively used for treating 500ng/ml LPS induced inflammation models, the cells are collected and the western detection of related proteins is carried out, and the results are shown in figure 3, THZ1, SY-1365 and BS-181 have the effect of inhibiting LPS induced inflammation (shown in figure 3A), and THZ1-CDK7, SY-1365-CDK7 and BS-181-CDK7 complexes have a certain effect of inhibiting RNA Poly II2,7 site serine phosphorylation (shown in figure 3B); in conclusion, THZ1 has the best anti-inflammatory effect.

Example 2: THZ 1-targeted CDK7-RNA Polymerase II complex regulation gene transcription and western experiment

Monocytes in exponential growth phase were treated with 100ng/ml PMA for 24 hours to form macrophages, then treated with different concentrations of THZ1 (0, 20, 40, 100, 200 nM) for 30 minutes, then sampled at different time points (8, 24 hours), the cells collected and subjected to western detection of the relevant proteins. FIG. 4 is a schematic representation of THZ1 targeting CDK7-RNA Polymerase II complex regulated gene transcription. Wherein, A represents a schematic diagram of mRNA transcription complex RNA Poly of THZ1 targeted regulation CDK 7; b represents the inhibition of the THZ1-CDK7 complex on the serine phosphorylation of RNA Poly II2,7 sites under different concentrations and different action times of THZ1 in the western experiment: over time, the inhibitory effect diminishes; the inhibitory effect increases with increasing concentration.

Example 3: bacterial infection model of THZ1 targeting CDK7-RNA Polymerase II complex

FIG. 5 is the qRT-PCR experimental result of the bacterial infection model of THZ1 targeting CDK7-RNA Polymerase II complex, wherein A shows that THZ1 can significantly inhibit the expression of related inflammatory genes such as interleukin IL1B, interleukin IL6, interleukin IL8, tumor necrosis factor TNFA and chemokine CXCL10 in mTHP-1-PMA; b shows that THZ1 can obviously inhibit the expression of related inflammatory genes such as interleukin IL1B, interleukin IL6, interleukin IL8, tumor necrosis factor TNFA, chemokine CXCL10 and the like in THP-1; c shows that THZ1 can remarkably inhibit the expression of related inflammatory genes such as interleukin IL1B, interleukin IL6, interleukin IL8, tumor necrosis factor TNFA, chemokine CXCL10 and the like in HFF-1 (skin fibroblasts); d shows that THZ1 can obviously inhibit the expression of interleukin IL1B, interleukin IL6, interleukin IL8, tumor necrosis factor TNFA, chemokine CXCL10 and other related inflammatory genes in HUVEC (endothelial cells).

Example 4: HZ1 targeting CDK7-RNA Polymerase II complex H1N 1-influenza virus infection model

FIG. 6 shows the qRT-PCR experimental results of THZ1 targeting CDK7-RNA Polymerase II complex H1N 1-influenza virus infection model, wherein A shows that THZ1 can significantly inhibit the expression of related inflammatory genes such as interleukin IL1B, interleukin IL6, interleukin IL8, tumor necrosis factor TNFA and chemokine CXCL10 in mTHP-1-PMA; b shows that THZ1 can obviously inhibit the expression of related inflammatory genes such as interleukin IL1B, interleukin IL6, interleukin IL8, tumor necrosis factor TNFA, chemokine CXCL10 and the like in THP-1-PMA; c shows that THZ1 can obviously inhibit the expression of related inflammatory genes such as interleukin IL1B, interleukin IL6, interleukin IL8, tumor necrosis factor TNFA, chemokine CXCL10 and the like in HFF-1; d shows that THZ1 can obviously inhibit the expression of related inflammatory genes such as interleukin IL1B, interleukin IL6, interleukin IL8, tumor necrosis factor TNFA, chemokine CXCL10 and the like in HUVEC.

Example 5: ELISA (enzyme-Linked immuno sorbent assay) experiment for inhibiting release of relevant cytokines in cytokine storm by THZ1 targeting CDK7-RNA Polymerase II complex

FIG. 7 shows the result of ELISA experiments in which THZ1 targeting CDK7-RNA Polymerase II complex inhibits the release of relevant cytokines in cytokine storm induced by LPS, wherein A shows that THZ1 can significantly inhibit the release of cytokine IL-1 β from macrophages; b shows that THZ1 can obviously inhibit the release of cytokine IL-6 in macrophages; c shows that THZ1 can obviously inhibit the release of a cytokine IL-8 in macrophages; d shows that THZ1 can obviously inhibit the release of tumor cell necrosis factor TNF-alpha in macrophages.

Example 6: whole genome expression profiling of THZ 1-targeted CDK7-RNA Polymerase II complex in inflammation model

Peripheral blood of healthy volunteers was isolated by Ficoll method and gradient time adherence, cultured monocytes were isolated and then treated with 100ng/ml PMA for 24 hours to form macrophages (MDMs), then treated with 30nM THZ1 for 30 minutes and then treated with 500ng/ml LPS for 8 hours, sampled, RNA extracted and RNA transcriptome sequenced. FIG. 8 is the results of genome-wide expression profiling of THZ 1-targeted CDK7-RNA Polymerase II complex, A: the mRNA sequencing analysis result shows that the gene expression difference of the transcriptome sequencing analysis of different treatment groups of the macrophage (in an inflammation model, the gene expression of related inflammatory factors is up-regulated, and THZ1 is added into the group, the gene expression of related inflammatory factors is inhibited), and the result shows that the relevant gene expression is remarkably activated by LPS (for example, the expression of inflammatory genes of different groups in LPS/Control is shown in figure 8A (the upper half part of the figure), but after the THZ1 is added, the genes with up-regulated expression are remarkably inhibited, and the expression of THZ1/LPS group (the lower half part of the figure) is reduced compared with that of the corresponding group); b: GO enrichment analysis shows that the differentially expressed genes are mainly enriched in an RNAPoint II signal pathway and an inflammatory stress regulation signal pathway; c: through comparison of genome-wide differential expression analysis, THZ1 is found to inhibit acute inflammatory response mainly by regulating activation of chemokines (CCL 8,15,5,2,7,4,1, CXCL9,10,11,23, 8), cytokines (IL 12B,27,7,23A,1B, 1A,6, TNF, CD70, etc.) and receptors (CCL 15,2, etc.), thereby realizing inhibition of cytokine storm.

Example 7: THZ 1-Targeted CDK7-RNA Polymerase II Complex Whole genome CHIP-Seq analysis

Monocytes were treated with 100ng/ml PMA for 24 hours to form macrophages, then treated with 0 or 30nM THZ1 for 30 minutes, then co-treated with 0 or 500ng/ml LPS for 8 hours, crosslinked with 1% formaldehyde at room temperature for 10 minutes, terminated with 0.125M glycine for 5 minutes, and then the cells were harvested by scraping. The collected samples were subjected to DNA fragmentation and enrichment of related antibody-bound DNA fragments as required by the Kit (SimpleChIP Enzymatic protein IP Kit, CST). Then, the Library was constructed with the Kit (ChIP-seq Library materials with construction of NEBNext Ultra II DNA Library Prep Kit for Illumina, NEB), and finally, sequencing and analysis were performed. FIG. 9 shows the analysis results of THZ1 targeting CDK7-RNA Polymerase II complex whole genome CHIP-Seq (RNA Poly II, H3K27 AC), FIG. 9A1 shows that THZ1 targeting inhibits CDK7 and RNA Poly II from binding, and the comparison of the abundance of the enriched DNA fragments is described as a whole, and the comparison shows that the abundance of DNA bound by LPS treatment group and RNA Poly II antibody is increased, which indicates that more genes are being transcribed; after THZ1 treatment, the abundance of the enriched DNA is reduced, which indicates that the transcribed gene is reduced; FIG. 9A2 shows statistics for DNA overall at transcription initiation and transcription extension; FIG. 9B1 shows that THZ1 inhibits the binding of RNA Poly II to inflammatory transcriptional regulators STAT1, IRF1, inhibiting the expression of the genes involved; fig. 9B2 shows, by CHIP-Seq data analysis of super enhancer marker molecule H3K27ac, that THZ1 has significant inhibitory activity on super enhancers upstream of transcriptional regulators STAT1, IRF1, and fig. 9B2 shows, by CHIP-Seq data analysis of super enhancer marker molecule H3K27ac, that THZ1 has significant inhibitory activity on super enhancers upstream of inflammatory factors IL- α, IL-1 β, which suggests that we target THZ1 to CDK7-RNA Poly II complex, inhibiting the binding ability of inflammatory-related genes, super enhancers upstream of inflammatory-related transcription factor genes, and directly inhibiting the binding of RNA Poly II to inflammatory-related factor genes, transcription factor genes, inhibiting the expression of genes, the above-mentioned recombination, thereby altering the expression of transcriptional regulators, ultimately affecting the expression of inflammatory cytokines, thereby specifically regulating cytokine production, inhibiting the occurrence of inflammatory storm.

Example 8: animal level experiments with THZ1 targeting CDK7-RNA Polymerase II complex

As shown in fig. 10, the experimental results show that: the THZ1 targets a CDK7-RNA Polymerase II complex, specifically inhibits the release of cytokines, reduces the damage of organs, and obviously improves the survival rate of mice killed by cytokine storm (LPS bacterial inflammation storm model). FIG. 10A: the experimental process comprises the following steps: mice were given an intraperitoneal injection of THZ1 mg/kg, LPS40mg/kg 0.5h later, and then the mice were sacrificed 20 hours for the following analysis: (1) Serum was taken for cytokine analysis, (2) peritoneal lavage: cell analysis, (3) organ toxicity assessment; FIG. 10B: the multi-factor flow quantitative detection experiment shows that when LPS is used for treating a mouse, the immune stress of the mouse can be obviously activated, cytokine storm occurs, MCP-1, IL-1 beta, IL-6 and related cytokines are highly expressed, and after THZ1 is treated, the expression of the related cytokines is obviously reduced; FIG. 10C: peripheral blood flow type cell subtype analysis shows that the LPS treated mouse can obviously activate the immune stress of the mouse, induce the increase of the number of macrophages, treat THZ1 and obviously reduce the ratio of the macrophages, in addition, qRT-PCR experiments of mouse blood samples also show that the LPS treated mouse can obviously activate the immune stress of the mouse, generate cytokine storm, and highly express related cytokines (IL-1B and TNFA), wherein the high expression of the factors is realized by the regulation and control of STAT1 transcription factors, and the treatment of THZ1 can obviously inhibit the expression of STAT1 so as to inhibit the expression of the related factors; FIG. 10D: tissue anatomy HE staining shows that LPS (lipolysis of tissues) can remarkably cause inflammatory reaction (tissue congestion) of related organs (lung, liver, kidney and spleen) when a mouse is treated by the LPS; THZ1 treatment can significantly reduce the tissue congestion and reduce acute inflammatory response.

Example 9: mouse survival experiment

As shown in fig. 11, the survival experiments of mice (LPS bacterial inflammation storm model) showed that THZ1 targeted CDK7-RNA Polymerase II complex significantly increased the survival of mice lethal to cytokine storm. The survival curve shows that in the LPS inflammation group, all mice die within 72 hours due to the inflammatory storm induced by LPS compared with the control group, and the survival rate is 0%; by THZ1 administration, the death of mice caused by acute inflammatory storm can be remarkably reduced, and the survival rate is 68.75%.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the prior art according to the concepts of the present invention should be within the scope of protection determined by the claims.

Claims

1. Use of THZ1, a targeted inhibitor of CDK7, in the preparation of a medicament for the treatment of a cytokine release syndrome caused by bacterial or viral infection, wherein THZ1 is according to formula (I):

（I）。