Human stimulator of interferon genes STING Dotaz Zobrazit nápovědu
Chronic hepatitis B (CHB) remains a major public health problem worldwide, with limited treatment options, but inducing an antiviral response by innate immunity activation may provide a therapeutic alternative. We assessed the cytokine-mediated anti-hepatitis B virus (HBV) potential for stimulating the cyclic GMP-AMP synthase-stimulator of interferon genes (STING) pathway using STING agonists in primary human hepatocytes (PHH) and nonparenchymal liver cells (NPCs). The natural STING agonist, 2',3'-cyclic GMP-AMP, the synthetic analogue 3',3'-c-di(2'F,2'dAMP), and its bis(pivaloyloxymethyl) prodrug had strong indirect cytokine-mediated anti-HBV effects in PHH regardless of HBV genotype. Furthermore, STING agonists induced anti-HBV cytokine secretion in vitro, in both human and mouse NPCs, and triggered hepatic T cell activation. Cytokine secretion and lymphocyte activation were equally stimulated in NPCs isolated from control and HBV-persistent mice. Therefore, STING agonists modulate immune activation regardless of HBV persistence, paving the way toward a CHB therapy.
- MeSH
- cytokiny metabolismus MeSH
- hepatitida B * farmakoterapie MeSH
- hepatocyty MeSH
- herpesvirus B * MeSH
- interferony metabolismus MeSH
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Tento článek se zaměřuje na roli strukturní biologie ve vývoji malých molekul, sloužících jako ligandy proteinů, které by mohly být v budoucnosti využity jako léky. Pro získávání strukturních informací o proteinech jsou využívány experimentální metody, které následně umožňují virtuální screening rozsáhlých knihoven látek. Nalezené ligandy jsou dále experimentálně ověřovány, přičemž strukturní biologie hraje klíčovou roli v jejich dalším zdokonalení. Cílem je vyvinout ligand s vysokou afinitou a specifitou, který by se mohl stát účinným léčivem. Následuje optimalizace těchto látek z hlediska chemie a farmakologie. Celý tento proces je ilustrován na příkladech z nedávné minulosti, jako jsou kinasy lipidů, stimulátor interferonových genů (STING) a virové methyltransferasy.
This article focuses on the role of structural biology in the development of small molecules that serve as protein ligands, which could potentially be used as drugs in the future. Experimental methods are utilized to obtain structural information about proteins, which then allow for virtual screening of extensive libraries of substances. The discovered ligands are further experimentally verified, with structural biology playing a crucial role in their refinement. The goal is to develop a ligand with high affinity and specificity, which could become an effective medicine. This is followed by the optimization of these substances from a chemical and pharmacological standpoint. The entire process is illustrated with examples from the recent past, such as lipid kinases, the stimulator of interferon genes (STING), and viral methyltransferases.
Cyclic dinucleotides are second messengers in the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, which plays an important role in recognizing tumor cells and viral or bacterial infections. They bind to the STING adaptor protein and trigger expression of cytokines via TANK binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3) and inhibitor of nuclear factor-κB (IκB) kinase (IKK)/nuclear factor-κB (NFκB) signaling cascades. In this work, we describe an enzymatic preparation of 2'-5',3'-5'-cyclic dinucleotides (2'3'CDNs) with use of cyclic GMP-AMP synthases (cGAS) from human, mouse, and chicken. We profile substrate specificity of these enzymes by employing a small library of nucleotide-5'-triphosphate (NTP) analogues and use them to prepare 33 2'3'CDNs. We also determine affinity of these CDNs to five different STING haplotypes in cell-based and biochemical assays and describe properties needed for their optimal activity toward all STING haplotypes. Next, we study their effect on cytokine and chemokine induction by human peripheral blood mononuclear cells (PBMCs) and evaluate their cytotoxic effect on monocytes. Additionally, we report X-ray crystal structures of two new CDNs bound to STING protein and discuss structure-activity relationship by using quantum and molecular mechanical (QM/MM) computational modeling.
- MeSH
- biotest MeSH
- cytokiny metabolismus MeSH
- HEK293 buňky MeSH
- konformace proteinů MeSH
- leukocyty mononukleární účinky léků MeSH
- lidé MeSH
- membránové proteiny chemie metabolismus MeSH
- nukleotidy cyklické chemická syntéza farmakologie MeSH
- počítačová simulace MeSH
- regulace genové exprese účinky léků MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The 3'-5', 3'-5' cyclic dinucleotides (3'3'CDNs) are bacterial second messengers that can also bind to the stimulator of interferon genes (STING) adaptor protein in vertebrates and activate the host innate immunity. Here, we profiled the substrate specificity of four bacterial dinucleotide synthases from Vibrio cholerae (DncV), Bacillus thuringiensis (btDisA), Escherichia coli (dgcZ), and Thermotoga maritima (tDGC) using a library of 33 nucleoside-5'-triphosphate analogues and then employed these enzymes to synthesize 24 3'3'CDNs. The STING affinity of CDNs was evaluated in cell-based and biochemical assays, and their ability to induce cytokines was determined by employing human peripheral blood mononuclear cells. Interestingly, the prepared heterodimeric 3'3'CDNs bound to the STING much better than their homodimeric counterparts and showed similar or better potency than bacterial 3'3'CDNs. We also rationalized the experimental findings by in-depth STING-CDN structure-activity correlations by dissecting computed interaction free energies into a set of well-defined and intuitive terms. To this aim, we employed state-of-the-art methods of computational chemistry, such as quantum mechanics/molecular mechanics (QM/MM) calculations, and complemented the computed results with the {STING:3'3'c-di-ara-AMP} X-ray crystallographic structure. QM/MM identified three outliers (mostly homodimers) for which we have no clear explanation of their impaired binding with respect to their heterodimeric counterparts, whereas the R2 = 0.7 correlation between the computed ΔG'int_rel and experimental ΔTm's for the remaining ligands has been very encouraging.
- MeSH
- Bacillus thuringiensis enzymologie ultrastruktura MeSH
- cytokiny chemie genetika MeSH
- Escherichia coli enzymologie ultrastruktura MeSH
- krystalografie rentgenová MeSH
- kvantová teorie MeSH
- leukocyty mononukleární chemie enzymologie MeSH
- lidé MeSH
- membránové proteiny chemie genetika ultrastruktura MeSH
- nukleotidy biosyntéza chemie genetika MeSH
- přirozená imunita genetika MeSH
- substrátová specifita MeSH
- Thermotoga maritima enzymologie ultrastruktura MeSH
- Vibrio cholerae enzymologie ultrastruktura MeSH
- vztahy mezi strukturou a aktivitou * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cyclic dinucleotides (CDNs) are second messengers that bind to the stimulator of interferon genes (STING) and trigger the expression of type I interferons and proinflammatory cytokines. Here we evaluate the activity of 3',3'-c-di(2'F,2'dAMP) and its phosphorothioate analogues against five STING allelic forms in reporter-cell-based assays and rationalize our findings with X-ray crystallography and quantum mechanics/molecular mechanics calculations. We show that the presence of fluorine in the 2' position of 3',3'-c-di(2'F,2'dAMP) improves its activity not only against the wild type (WT) but also against REF and Q STING. Additionally, we describe the synthesis of the acyloxymethyl and isopropyloxycarbonyl phosphoester prodrugs of CDNs. Masking the negative charges of the CDNs results in an up to a 1000-fold improvement of the activities of the prodrugs relative to those of their parent CDNs. Finally, the uptake and intracellular cleavage of pivaloyloxymethyl prodrugs to the parent CDN is rapid, reaching a peak intracellular concentration within 2 h.
- MeSH
- estery chemie farmakologie terapeutické užití MeSH
- fosfáty chemie metabolismus farmakologie terapeutické užití MeSH
- HEK293 buňky MeSH
- interferon gama metabolismus MeSH
- krystalografie rentgenová MeSH
- leukocyty mononukleární cytologie účinky léků metabolismus MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie MeSH
- membránové proteiny agonisté metabolismus MeSH
- prekurzory léčiv chemická syntéza chemie metabolismus farmakologie MeSH
- teorie funkcionálu hustoty MeSH
- TNF-alfa metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cyclic dinucleotides (CDNs) are second messengers that activate stimulator of interferon genes (STING). The cGAS-STING pathway plays a promising role in cancer immunotherapy. Here, we describe the synthesis of CDNs containing 7-substituted 7-deazapurine moiety. We used mouse cyclic GMP-AMP synthase and bacterial dinucleotide synthases for the enzymatic synthesis of CDNs. Alternatively, 7-(het)aryl 7-deazapurine CDNs were prepared by Suzuki-Miyaura cross-couplings. New CDNs were tested in biochemical and cell-based assays for their affinity to human STING. Eight CDNs showed better activity than 2'3'-cGAMP, the natural ligand of STING. The effect on cytokine and chemokine induction was also evaluated. The best activities were observed for CDNs bearing large aromatic substituents that point above the CDN molecule. We solved four X-ray structures of complexes of new CDNs with human STING. We observed π-π stacking interactions between the aromatic substituents and Tyr240 that are involved in the stabilization of CDN-STING complexes.
- MeSH
- cytokiny MeSH
- interferony MeSH
- lidé MeSH
- ligandy MeSH
- membránové proteiny * metabolismus MeSH
- myši MeSH
- nukleotidy cyklické * chemie MeSH
- nukleotidyltransferasy MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Human stimulator of interferon genes (hSTING) is a signaling adaptor protein that triggers innate immune system by response to cytosolic DNA and second messenger cyclic dinucleotides (CDNs). Natural CDNs contain purine nucleobase with different phosphodiester linkage types (3'-3', 2'-2' or mixed 2'-3'-linkages) and exhibit different binding affinity towards hSTING, ranging from micromolar to nanomolar. High-affinity CDNs are considered as suitable candidates for treatment of chronic hepatitis B and cancer. We have used molecular dynamics simulations to investigate dynamical aspects of binding of natural CDNs (specifically, 2'-2'-cGAMP, 2'-3'-cGAMP, 3'-3'-cGAMP, 3'-3'-c-di-AMP, and 3'-3'-c-di-GMP) with hSTINGwt protein. Our results revealed that CDN/hSTINGwt interactions are controlled by the balance between fluctuations (conformational changes) in the CDN ligand and the protein dynamics. Binding of different CDNs induces different degrees of conformational/dynamics changes in hSTINGwt ligand binding cavity, especially in α1-helices, the so-called lid region and α2-tails. The ligand residence time in hSTINGwt protein pocket depends on different contribution of R232 and R238 residues interacting with oxygen atoms of phosphodiester groups in ligand, water distribution around interacting charged centers (in protein residues and ligand) and structural stability of closed conformation state of hSTINGwt protein. These findings may perhaps guide design of new compounds modulating hSTING activity.Communicated by Ramaswamy H. Sarma.
- MeSH
- dinukleosidfosfáty * chemie MeSH
- DNA MeSH
- lidé MeSH
- ligandy MeSH
- oligonukleotidy MeSH
- simulace molekulární dynamiky * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
This article focuses on the role of structural biology in the development of small molecules that serve as protein ligands, which could potentially be used as drugs in the future. Experimental methods are utilized to obtain structural information about proteins, which then allow for virtual screening of extensive libraries of substances. The discovered ligands are further experimentally verified, with structural biology playing a crucial role in their refinement. The goal is to develop a ligand with high affinity and specificity, which could become an effective medicine. This is followed by the optimization of these substances from a chemical and pharmacological standpoint. The entire process is illustrated with examples from the recent past, such as lipid kinases, the stimulator of interferon genes (STING), and viral methyltransferases.
STING protein (stimulator of interferon genes) plays an important role in the innate immune system. A number of potent compounds regulating its activity have been reported, mostly derivatives of cyclic dinucleotides (CDNs), natural STING agonists. Here, we aim to provide complementary information to large-scale "ligand-profiling" studies by probing the importance of STING-CDN protein-ligand interactions on the protein side. We examined in detail six typical CDNs each in complex with 13 rationally devised mutations in STING: S162A, S162T, Y167F, G230A, R232K, R232H, A233L, A233I, R238K, T263A, T263S, R293Q, and G230A/R293Q. The mutations switch on and off various types of protein-ligand interactions: π-π stacking, hydrogen bonding, ionic pairing, and nonpolar contacts. We correlated experimental data obtained by differential scanning fluorimetry, X-ray crystallography, and isothermal titration calorimetry with theoretical calculations. This enabled us to provide a mechanistic interpretation of the differences in the binding of representative CDNs to STING. We observed that the G230A mutation increased the thermal stability of the protein-ligand complex, indicating an increased level of ligand binding, whereas R238K and Y167F led to a complete loss of stabilization (ligand binding). The effects of the other mutations depended on the type of ligand (CDN) and varied, to some extent. A very good correlation (R2 = 0.6) between the experimental binding affinities and interaction energies computed by quantum chemical methods enabled us to explain the effect of the studied mutations in detail and evaluate specific interactions quantitatively. Our work may inspire development of high-affinity ligands against the common STING haplotypes by targeting the key (sometimes non-intuitive) protein-ligand interactions.
- MeSH
- bodová mutace * MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- membránové proteiny chemie genetika metabolismus MeSH
- molekulární struktura MeSH
- nukleotidy cyklické chemie metabolismus MeSH
- proteinové domény MeSH
- vazebná místa MeSH
- vodíková vazba MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Sensing of cytoplasmic DNA by cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) results in production of the dinucleotide cGAMP and consecutive activation of stimulator of interferon genes (STING) followed by production of type I interferon (IFN). Although cancer cells contain supra-normal concentrations of cytoplasmic DNA, they rarely produce type I IFN spontaneously. This suggests that defects in the DNA-sensing pathway may serve as an immune escape mechanism. We find that cancer cells produce cGAMP that is transferred via gap junctions to tumor-associated dendritic cells (DCs) and macrophages, which respond by producing type I IFN in situ. Cancer-cell-intrinsic expression of cGAS, but not STING, promotes infiltration by effector CD8+ T cells and consequently results in prolonged survival. Furthermore, cGAS-expressing cancers respond better to genotoxic treatments and immunotherapy. Thus, cancer-cell-derived cGAMP is crucial to protective anti-tumor CD8+ T cell immunity. Consequently, cancer-cell-intrinsic expression of cGAS determines tumor immunogenicity and makes tumors hot. These findings are relevant for genotoxic and immune therapies for cancer.
- MeSH
- CD8-pozitivní T-lymfocyty imunologie MeSH
- dendritické buňky metabolismus MeSH
- imunoterapie MeSH
- interferon typ I metabolismus MeSH
- lidé MeSH
- membránové proteiny MeSH
- mikrosatelitní repetice genetika MeSH
- myši inbrední C57BL MeSH
- nádorové buněčné linie MeSH
- nádory farmakoterapie imunologie patologie MeSH
- nukleotidy cyklické metabolismus MeSH
- nukleotidyltransferasy metabolismus MeSH
- poškození DNA MeSH
- progrese nemoci MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
