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This paper is from an article in "pharmaceutical frontier" column, Vol. 51, 2019, issue 1, Journal of China University of Pharmaceutical Sciences, the research progress of interferon gene stimulator agonists for tumor immunotherapy.
Abstract: tumor immunotherapy is a new research hotspot in the field of tumor therapy in recent years. In addition to the great success of immunocheckpoint inhibitors and cellular immunotherapy, small molecule immunotherapy is also receiving more and more attention. As a key signal transducer involved in innate immune response, interferon gene stimulator (sting) plays an important role in the defense of virus and intracellular bacterial infection, the production of interferon I and the regulation of spontaneous antitumor immune response in vivo. In this paper, the biological mechanism of sting signaling pathway is briefly introduced, and the research progress of sting agonists is summarized according to the structure classification, in order to provide reference for the design and discovery of sting agonists.
Key words: interferon gene stimulator; tumor immunotherapy; agonist; antitumor
Tumor immunotherapy is one of the new methods of tumor treatment after surgery, chemotherapy and targeted therapy. In recent years, the success of immunocheckpoint inhibitor and chimeric antigen receptor T cell immunotherapy (CAR-T), represented by CTLA-4 and PD-1 pathway, confirms the unique advantages and broad application prospects of tumor immunotherapy. Interferon gene stimulating factor (sting) is a key protein in DNA receptor pathway, which plays an important role in innate and adaptive immune activation. Sting, as a signal transduction adaptor protein, can raise tank binding kinase 1 (tbk1) and activate interferon regulatory factor 3 (IRF-3), then induce the production of type I interferon (IFN - α, IFN - β) and regulate the innate immune response to anti-tumor. As a therapeutic method, sting agonist has shown significant therapeutic effect in a variety of mouse tumor models. The biological mechanism of sting pathway
1.1 structure and function of sting protein
Sting protein is a signal transduction molecule closely related to innate immune response. It is expressed in a variety of endothelial cells, epithelial cells and hematopoietic stem cells, mainly located in the endoplasmic reticulum, mitochondria and the outer membrane of microsomes. The human sting protein consists of 379 amino acids, which can be divided into three functional domains, including four transmembrane domains (tm1-4), dimerization domains at N-terminus, and cytoplasmic domains at C-terminus. The structure of sting dimer in free state is relatively loose and in "open" state. When the U-shaped groove between dimers is occupied by 2 ′, 3 ′ - cgamp, dimers will form a tight "closed" state (Fig. 1-A), and four standard β - folded sheets will cover the binding groove, making dimers completely wrap the binding ligand. However, it has also been found that when amino benzimidazoles (DI abzi) activate sting, it always keeps sting "open", as shown in figure 1-b. This finding increases the possibility that sting can be activated without blocking the conformation. In conclusion, more studies are needed to understand how the sting conformation regulates the activation of signaling pathways.
1.2 sting signal path
The cgas-sting intracellular DNA sensing pathway mediated by sting protein participates in the innate immune response of the body and plays an important role in anti-virus infection, autoimmune diseases and tumor immunotherapy. Its signal transduction mechanism is as follows: after recognizing the DNA in the cytoplasm, CGAs dimerization catalyzes the reaction of GTP and ATP to generate cgamp. As a second messenger, cgamp binds and activates sting protein to form dimer. After dimerization, sting protein was transferred from endoplasmic reticulum to Golgi body and then to peripheral nuclear corpuscle, and then tank binding kinase 1 (tbk1) was recruited to induce phosphorylation of interferon regulatory factor 3 (IRF-3). Then, IRF-3 enters the nucleus and causes the transcription of interferon β (IFN - β) and other genes to play a biological effect (Figure 2).
1.3 the role of sting pathway in tumor immune monitoring
Sting pathway plays an important role in tumor immune monitoring. There is a continuous DNA damage reaction in tumor cells, which leads to the accumulation of DNA in their solutes. The tumor derived DNA was detected by APC and absorbed into the cytoplasm of APC, then the sting pathway was activated to mediate the production of sting dependent IFN type I. The activation of tumor specific CD8 + T cells and the infiltration of lymphocytes are closely related to the gene expression induced by type I IFN. Type I IFN signaling pathway defects, such as IFN - α / β receptor alpha chain or signal transduction and activator of transcription 1 (STAT1) defects, will inhibit the stimulation effect of IFN, resulting in the reduction of T cells targeting tumor associated antigens. Therefore, sting pathway can play an anti-tumor adaptive immune effect by mediating the production of type I interferon.
2 antineoplastic sting agonist
2.1 cyclic dinucleotide agonists
C-di-GMP (1), c-di-AMP (2), 3 ', 3' - cgamp (3) produced by microorganisms and 2 ', 3' - cgamp (4) synthesized in human body are natural agonists of sting, which can activate the innate immune response of host after combining sting. Through the analysis of the crystal structure of sting protein and 2 ', 3' - cgamp complex and the structure of compound 2 ', 3' - cgamp (Fig. 3), the results show that two purine bases are the core group binding to sting protein, which binds to sting through multiple hydrogen bonds and π - π stacking. Therefore, the cyclic structure of ribose phosphate can be used as the molecular skeleton of these compounds, and two purine bases can be used as the pharmacophore, which provides a theoretical basis for the design of new cyclic dinucleotide agonists. The compound ml-rr-s2cda (5) has high stability and antitumor effect, and can activate all the hsting variants. In B16 melanoma tumor model of mice, the tumor growth was inhibited by intratumoral injection of ml-rr-s2cda.
Compound 6 and compound 7 showed better ability of inducing IFN type I than 2 ', 3' - cgamp in THP-1 cells in vitro, and EC50 of compound 8 reached 1 nmol / L. The free hydroxyl groups on ribose in 2 ', 3' - cgamp were substituted and modified. Compound 9 was obtained by introducing F atom. Compound 9 and cgamp can regulate the immune response of HBsAg in vivo.
Compound 10 showed a high affinity for wild-type, HAQ (HBsAg mutation, r71h, g230a and r293q) and ref (sting mutation, r232h mutation) HBsAg. In luciferase reporter gene assay, compound 10 can activate HAQ sting in THP-1 cells, EC50 is 9.5 μ mol / L. A class of cyclic dinucleotide analogues, which are used as histing agonists, have shown high affinity for histing in fluorescence tests, representing compound 11. In IRF induced secretory embryo alkaline phosphatase reporter gene assay, compound 11 activated the hsting activity of thp-1blueisg cells with EC50 of 0.17 μ mol / L.
2.2 non CDN small molecule agonists
2.2.1 DMXAA
FAA (compound 12) has been proved to have a significant inhibitory effect on colon tumor in mice by destroying blood vessels. However, the failure of the subsequent phase I clinical trial and the lack of activity in the rat tumor model suggest that there may be species specificity. In order to obtain the similar drugs that can cause tumor hemorrhagic necrosis, the researchers further modified the molecular structure of FAA and synthesized a series of compounds, among which 5,6-dimethylxanthone-4-acetic acid (DMXAA, compound 13) had the best effect.
2.2.2 aminobenzimidazoles
The inhibition rate of compound 14 was (59 ± 8)% at 10 μ mol / L, and the apparent inhibition constant IC50 was (14 ± 2) μ mol / L. Crystal structure analysis showed that the C-terminal domain (CTD) dimer of sting protein formed a pocket containing two adjacent compounds 14, each small molecule binding one sting subunit. The 1-ethyl-3-methyl-1h-pyrazol-5-formamide part of compound 15 is bound to the bottom of the pocket, the key hydrogen bond is formed between the pyrazol nitrogen part and the hydroxy group of ser162, the hydrogen bond is formed between the carboxyamide part and thr263, and the two pairs of H-bond network are formed between the terminal amide and ser241 to maintain the structural stability of the complex. Compound 16 was further optimized on the basis of compound 15, and its affinity and intracellular efficiency were further improved. EC50 was about 130 nmol / L, more than 20 times of compound 15.
2.2.3 benzothiophene compounds
The EC50 of compound 17 in THP-1 cells reached 531 nmol / L in vitro. In the experiment of C57BL / 6J female mice with mc-38 cell transplantation tumor, compound 18 combined with anti-PD-1 monoclonal antibody migg1 (mouse immunoglobulin G subtype 1) significantly inhibited tumor growth compared with migg1 alone.
3 summary and Prospect
Sting protein is a relatively novel drug target at present, and the number of drugs in research and patent applications is relatively limited. Although the exact mechanism of Sting has not been fully understood, more and more evidences show that sting agonists are expected to be used in immunotherapy of tumor. However, there are still many problems in the study of sting agonists. In short, although there are many limitations and bottlenecks in the research and development of sting agonists, it is still possible to become another emerging hot spot of immunotherapy.
Cited in this paper: Ji Liyang, Hao Jing, Wang Guocheng, Xie Weijia. Research progress of interferon gene stimulator agonists for tumor immunotherapy [J]. Journal of China Pharmaceutical University, 2020,51 (1): 1-9
He Huiqin
Chen Ling, Gu Kai, Zou Xu
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