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
• Názvy látek
• membránové proteiny MeSH
• nukleotidy cyklické MeSH
• STING1 protein, human MeSH Prohlížeč

STING (stimulator of interferon genes) is a key regulator of innate immunity that has recently been recognized as a promising drug target. STING is activated by cyclic dinucleotides (CDNs) which eventually leads to expression of type I interferons and other cytokines. Factors underlying the affinity of various CDN analogues are poorly understood. Herein, we correlate structural biology, isothermal calorimetry (ITC) and computational modeling to elucidate factors contributing to binding of six CDNs-three pairs of natural (ribo) and fluorinated (2'-fluororibo) 3',3'-CDNs. X-ray structural analyses of six {STING:CDN} complexes did not offer any explanation for the different affinities of the studied ligands. ITC showed entropy/enthalpy compensation up to 25 kcal mol-1 for this set of similar ligands. The higher affinities of fluorinated analogues are explained with help of computational methods by smaller loss of entropy upon binding and by smaller strain (free) energy.

• Klíčová slova
• conformational analysis, cyclic dinucleotides, entropy, quantum chemistry, strain energy,
• MeSH
• lidé MeSH
• ligandy MeSH
• membránové proteiny chemie   MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• nukleotidy cyklické chemie   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ligandy MeSH
• membránové proteiny MeSH
• nukleotidy cyklické MeSH
• STING1 protein, human MeSH Prohlížeč

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
• Názvy látek
• cytokiny MeSH
• membránové proteiny MeSH
• nukleotidy cyklické MeSH
• STING1 protein, human MeSH Prohlížeč

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
• Názvy látek
• cytokiny MeSH
• membránové proteiny MeSH
• nukleotidy MeSH
• STING1 protein, human MeSH Prohlížeč

The development of small molecule drugs that target protein binders is the central goal in medicinal chemistry. During the lead compound development process, hundreds or even thousands of compounds are synthesized, with the primary focus on their binding affinity to protein targets. Typically, IC50 or EC50 values are used to rank these compounds. While thermodynamic values, such as the dissociation constant (KD), would be more informative, they are experimentally less accessible. In this study, we compare isothermal calorimetry (ITC) with surface plasmon resonance (SPR) using human STING, a key protein of innate immunity, and several cyclic dinucleotides (CDNs) that serve as its ligands. We demonstrate that SPR, with recent technological advancements, provides KDs that are sufficiently accurate for drug development purposes. To illustrate the versatility of our approach, we also used SPR to estimate the KD of poxin binding to cyclic GMP-AMP (cGAMP) that serves as a second messenger in the innate immune system. In conclusion, SPR offers a high benefit-to-cost ratio, making it an effective tool in the drug design process.

• Klíčová slova
• Cyclic dinucleotide, ITC, Poxin, SPR, STING,
• MeSH
• kalorimetrie MeSH
• lidé MeSH
• ligandy MeSH
• membránové proteiny * metabolismus   chemie   MeSH
• nukleotidy cyklické * metabolismus   chemie   MeSH
• povrchová plasmonová rezonance * metody   MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cyclic guanosine monophosphate-adenosine monophosphate MeSH Prohlížeč
• ligandy MeSH
• membránové proteiny * MeSH
• nukleotidy cyklické * MeSH
• STING1 protein, human MeSH Prohlížeč

The stimulator of interferon genes (STING) protein plays a crucial role in the activation of the innate immune response. Activation of STING is initiated by cyclic dinucleotides (CDNs) which prompted the community to synthesize structural analogues to enhance their biological properties. We present here the synthesis and biological evaluation of four novel CDN analogues composed of an N-acylsulfonamide linkage. These CDNs were obtained in high overall yields via the sulfo-click reaction as a key step.

• MeSH
• click chemie metody   MeSH
• membránové proteiny agonisté   chemie   MeSH
• nukleotidy cyklické * chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH
• nukleotidy cyklické * MeSH
• STING1 protein, human MeSH Prohlížeč

SAMHD1 was previously characterized as a dNTPase that protects cells from viral infections. Mutations in SAMHD1 are implicated in cancer development and in a severe congenital inflammatory disease known as Aicardi-Goutières syndrome. The mechanism by which SAMHD1 protects against cancer and chronic inflammation is unknown. Here we show that SAMHD1 promotes degradation of nascent DNA at stalled replication forks in human cell lines by stimulating the exonuclease activity of MRE11. This function activates the ATR-CHK1 checkpoint and allows the forks to restart replication. In SAMHD1-depleted cells, single-stranded DNA fragments are released from stalled forks and accumulate in the cytosol, where they activate the cGAS-STING pathway to induce expression of pro-inflammatory type I interferons. SAMHD1 is thus an important player in the replication stress response, which prevents chronic inflammation by limiting the release of single-stranded DNA from stalled replication forks.

• MeSH
• checkpoint kinasa 1 metabolismus   MeSH
• cytosol metabolismus   MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• helikasy RecQ metabolismus   MeSH
• homologní protein MRE11 metabolismus   MeSH
• interferon typ I imunologie   metabolismus   MeSH
• jednovláknová DNA metabolismus   MeSH
• lidé MeSH
• membránové proteiny metabolismus   MeSH
• nukleotidyltransferasy metabolismus   MeSH
• protein SAMHD1 nedostatek   metabolismus   MeSH
• replikace DNA * MeSH
• zánět imunologie   metabolismus   prevence a kontrola   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cGAS protein, human MeSH Prohlížeč
• checkpoint kinasa 1 MeSH
• CHEK1 protein, human MeSH Prohlížeč
• helikasy RecQ MeSH
• homologní protein MRE11 MeSH
• interferon typ I MeSH
• jednovláknová DNA MeSH
• membránové proteiny MeSH
• MRE11 protein, human MeSH Prohlížeč
• nukleotidyltransferasy MeSH
• protein SAMHD1 MeSH
• RECQL protein, human MeSH Prohlížeč
• SAMHD1 protein, human MeSH Prohlížeč
• STING1 protein, human MeSH Prohlížeč

Stimulator of interferon genes (STING) binds cyclic dinucleotides (CDNs), which induce a large conformational change of the protein. The structural basis of activation of STING by CDNs is rather well understood. Unliganded STING forms an open dimer that undergoes a large conformational change (∼10 Å) to a closed conformation upon the binding of a CDN molecule. This event activates downstream effectors of STING and subsequently leads to activation of the type 1 interferon response. However, a previously solved structure of STING with 3',3'-c-di-GMP shows Mg atoms mediating the interaction of STING with this CDN. Here, it is shown that no Mg atoms are needed for this interaction; in fact, magnesium can in some cases obstruct the binding of a CDN to STING.

• Klíčová slova
• 3′,3′-c-di-GMP, CDN, STING, cGAS, crystal structure,
• MeSH
• guanosinmonofosfát cyklický chemie   metabolismus   MeSH
• hořčík metabolismus   MeSH
• krystalografie rentgenová MeSH
• membránové proteiny chemie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• guanosinmonofosfát cyklický MeSH
• hořčík MeSH
• membránové proteiny MeSH
• STING1 protein, human MeSH Prohlížeč

BK polyomavirus (BKPyV) infection in humans is usually asymptomatic but ultimately results in viral persistence. In immunocompromised hosts, virus reactivation can lead to nephropathy or hemorrhagic cystitis. The urinary tract serves as a silent reservoir for the virus. Recently, it has been demonstrated that human bladder microvascular endothelial cells (HBMVECs) serve as viral reservoirs, given their unique response to infection, which involves interferon (IFN) production. The aim of the present study was to better understand the life cycle of BKPyV in HBMVECs, uncover the molecular pathway leading to IFN production, and to identify the connection between the viral life cycle and the activation of the IFN response. Here, in the early stage of infection, BKPyV virions were found in internalized monopinocytic vesicles, while later they were detected in late endosomes, lysosomes, tubuloreticular structures, and vacuole-like vesicles. The production of viral progeny in these cells started at 36 h postinfection. Increased cell membrane permeability and peaks of virion release coincided with the leakage of viral and cellular DNA into the cytosol at approximately 60 h postinfection. Leaked DNA colocalized with and activated cGAS, leading to the activation of STING and the consequent transcription of IFNB and IFN-related genes; in contrast, the IFN response was attenuated by exposure to the cGAS inhibitor, G140. These findings highlight the importance of the cGAS-STING pathway in the innate immune response of HBMVECs to BKPyV.

• Klíčová slova
• BK polyomavirus, BKPyV reservoir cells, STING, cGAS, interferon response,
• MeSH
• endoteliální buňky * virologie   MeSH
• interferony metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• membránové proteiny metabolismus   genetika   MeSH
• močový měchýř * virologie   MeSH
• nukleotidyltransferasy metabolismus   genetika   MeSH
• polyomavirové infekce virologie   imunologie   MeSH
• replikace viru MeSH
• signální transdukce * MeSH
• virion MeSH
• virus BK * fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cGAS protein, human MeSH Prohlížeč
• interferony MeSH
• membránové proteiny MeSH
• nukleotidyltransferasy MeSH
• STING1 protein, human MeSH Prohlížeč

OBJECTIVE: The TLR3/cGAS-STING-IFN signaling has recently been reported to be disturbed in colorectal cancer due to deregulated expression of the genes involved. Our study aimed to investigate the influence of potential regulatory variants in these genes on the risk of sporadic colorectal cancer (CRC) in a Czech cohort of 1424 CRC patients and 1114 healthy controls. METHODS: The variants in the TLR3, CGAS, TMEM173, IKBKE, and TBK1 genes were selected using various online bioinformatic tools, such as UCSC browser, HaploReg, Regulome DB, Gtex Portal, SIFT, PolyPhen2, and miRNA prediction tools. RESULTS: Logistic regression analysis adjusted for age and sex detected a nominal association between CRC risk and three variants, CGAS rs72960018 (OR: 1.68, 95% CI: 1.11-2.53, P-value = .01), CGAS rs9352000 (OR: 2.02, 95% CI: 1.07-3.84, P-value = .03) and TMEM173 rs13153461 (OR: 1.53, 95% CI: 1.03-2.27, P-value = .03). Their cumulative effect revealed a threefold increased CRC risk in carriers of 5-6 risk alleles compared to those with 0-2 risk alleles. Epistatic interactions between these genes and the previously genotyped IFNAR1, IFNAR2, IFNA, IFNB, IFNK, IFNW, IRF3, and IRF7 genes, were computed to test their effect on CRC risk. Overall, we obtained nine pair-wise interactions within and between the CGAS, TMEM173, IKBKE, and TBK1 genes. Two of them remained statistically significant after Bonferroni correction. Additional 52 interactions were observed when IFN variants were added to the analysis. CONCLUSIONS: Our data suggest that epistatic interactions and a high number of risk alleles may play an important role in CRC carcinogenesis, offering novel biological understanding for the CRC management.

• Klíčová slova
• CRC, interaction, polygenic-risk-score, risk,
• MeSH
• dospělí MeSH
• genetická epistáze * MeSH
• genotypizační techniky MeSH
• interferony genetika   MeSH
• jednonukleotidový polymorfismus MeSH
• karcinogeneze genetika   MeSH
• kinasa I-kappa B genetika   MeSH
• kohortové studie MeSH
• kolon diagnostické zobrazování   patologie   MeSH
• kolonoskopie MeSH
• kolorektální nádory diagnóza   genetika   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• membránové proteiny genetika   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• nukleotidyltransferasy genetika   MeSH
• protein-serin-threoninkinasy genetika   MeSH
• regulace genové exprese u nádorů * MeSH
• rektum diagnostické zobrazování   patologie   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• signální transdukce genetika   MeSH
• studie případů a kontrol MeSH
• toll-like receptor 3 genetika   MeSH
• výpočetní biologie MeSH
• zdraví dobrovolníci pro lékařské studie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• pozorovací studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cGAS protein, human MeSH Prohlížeč
• IKBKE protein, human MeSH Prohlížeč
• interferony MeSH
• kinasa I-kappa B MeSH
• membránové proteiny MeSH
• nukleotidyltransferasy MeSH
• protein-serin-threoninkinasy MeSH
• STING1 protein, human MeSH Prohlížeč
• TBK1 protein, human MeSH Prohlížeč
• TLR3 protein, human MeSH Prohlížeč
• toll-like receptor 3 MeSH