RESUMEN
Cyclic dinucleotides (CDNs) trigger the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, which plays a key role in cytosolic DNA sensing and thus in immunomodulation against infections, cell damage and cancer. However, cancer immunotherapy trials with CDNs have shown immune activation, but not complete tumor regression. Nevertheless, we designed a novel class of CDNs containing vinylphosphonate based on a STING-affinity screening assay. In vitro, acyloxymethyl phosphate/phosphonate prodrugs of these vinylphosphonate CDNs were up to 1000-fold more potent than the clinical candidate ADU-S100. In vivo, the lead prodrug induced tumor-specific T cell priming and facilitated tumor regression in the 4T1 syngeneic mouse model of breast cancer. Moreover, we solved the crystal structure of this ligand bound to the STING protein. Therefore, our findings not only validate the therapeutic potential of vinylphosphonate CDNs but also open up opportunities for drug development in cancer immunotherapy bridging innate and adaptive immunity.
Asunto(s)
Neoplasias , Nucleótidos Cíclicos , Animales , Ratones , Nucleótidos Cíclicos/farmacología , Nucleótidos Cíclicos/metabolismo , ADN , Neoplasias/tratamiento farmacológico , Inmunoterapia , Inmunidad InnataRESUMEN
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.
Asunto(s)
Proteínas de la Membrana , Nucleótidos Cíclicos , Ratones , Animales , Humanos , Nucleótidos Cíclicos/química , Ligandos , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas , Citocinas , InterferonesRESUMEN
Stimulator of interferon genes (STING) is an adaptor protein of the cGAS-STING signaling pathway involved in the sensing of cytosolic DNA. It functions as a receptor for cyclic dinucleotides (CDNs) and, upon their binding, mediates cytokine expression and host immunity. Besides naturally occurring CDNs, various synthetic CDNs, such as ADU-S100, have been reported to effectively activate STING and are being evaluated in clinical trials for the treatment of cancer. Here, we describe the preparation of a unique new class of STING agonists: isonucleotidic cyclic dinucleotides and the synthesis of their prodrugs. The presented CDNs stimulate STING with comparable efficiency to ADU-S100, whereas their prodrugs demonstrate activity up to four orders of magnitude better due to the improved cellular uptake. The compounds are very potent inducers of inflammatory cytokines by peripheral blood mononuclear cells (PBMCs). We also report the X-ray crystal structure of the lead inhibitor bound to the wild-type (WT) STING.
Asunto(s)
Nucleótidos Cíclicos , Profármacos , Citosol/metabolismo , Leucocitos Mononucleares/metabolismo , Proteínas de la Membrana/química , Nucleótidos Cíclicos/metabolismo , Nucleótidos Cíclicos/farmacologíaRESUMEN
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.
Asunto(s)
Inmunidad Innata/genética , Proteínas de la Membrana/ultraestructura , Nucleótidos/biosíntesis , Relación Estructura-Actividad , Bacillus thuringiensis/enzimología , Bacillus thuringiensis/ultraestructura , Cristalografía por Rayos X , Citocinas/química , Citocinas/genética , Escherichia coli/enzimología , Escherichia coli/ultraestructura , Humanos , Leucocitos Mononucleares/química , Leucocitos Mononucleares/enzimología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Nucleótidos/química , Nucleótidos/genética , Teoría Cuántica , Especificidad por Sustrato , Thermotoga maritima/enzimología , Thermotoga maritima/ultraestructura , Vibrio cholerae/enzimología , Vibrio cholerae/ultraestructuraRESUMEN
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.
Asunto(s)
Ésteres/química , Proteínas de la Membrana/agonistas , Fosfatos/química , Profármacos/síntesis química , Cristalografía por Rayos X , Teoría Funcional de la Densidad , Ésteres/farmacología , Ésteres/uso terapéutico , Células HEK293 , Humanos , Interferón gamma/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Espectroscopía de Resonancia Magnética , Proteínas de la Membrana/metabolismo , Fosfatos/metabolismo , Fosfatos/farmacología , Fosfatos/uso terapéutico , Profármacos/química , Profármacos/metabolismo , Profármacos/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de la Membrana/metabolismo , Nucleótidos Cíclicos/metabolismo , Mutación Puntual , Sitios de Unión , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Estructura Molecular , Nucleótidos Cíclicos/química , Conformación Proteica , Dominios ProteicosRESUMEN
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 25â 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.
Asunto(s)
Proteínas de la Membrana/química , Nucleótidos Cíclicos/química , Sitios de Unión , Humanos , Ligandos , Modelos Moleculares , Conformación MolecularRESUMEN
Picornaviruses infect a wide range of mammals including livestock such as cattle and swine. As with other picornavirus genera such as Aphthovirus, there is emerging evidence of a significant economic impact of livestock infections caused by members of the genera Enterovirus and Kobuvirus. While the human-infecting enteroviruses and kobuviruses have been intensively studied during the past decades in great detail, research on livestock-infecting viruses has been mostly limited to the genomic characterization of the viral strains identified worldwide. Here, we extend our previous studies of the structure and function of the complexes composed of the non-structural 3A proteins of human-infecting enteroviruses and kobuviruses and the host ACBD3 protein and present a structural and functional characterization of the complexes of the following livestock-infecting picornaviruses: bovine enteroviruses EV-E and EV-F, porcine enterovirus EV-G, and porcine kobuvirus AiV-C. We present a series of crystal structures of these complexes and demonstrate the role of these complexes in facilitation of viral replication.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Infecciones por Enterovirus/metabolismo , Enterovirus Bovino/patogenicidad , Enterovirus Porcinos/patogenicidad , Kobuvirus/patogenicidad , Proteínas de la Membrana/metabolismo , Infecciones por Picornaviridae/metabolismo , Animales , Bovinos , Línea Celular , Infecciones por Enterovirus/veterinaria , Infecciones por Enterovirus/virología , Enterovirus Porcinos/genética , Células HEK293 , Humanos , Kobuvirus/genética , Infecciones por Picornaviridae/veterinaria , Infecciones por Picornaviridae/virología , Porcinos , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/genéticaRESUMEN
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.
Asunto(s)
Proteínas de la Membrana/metabolismo , Nucleótidos Cíclicos/síntesis química , Nucleótidos Cíclicos/farmacología , Bioensayo , Simulación por Computador , Citocinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Proteínas de la Membrana/química , Conformación Proteica , Relación Estructura-ActividadRESUMEN
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 (â¼10â 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.
Asunto(s)
GMP Cíclico/química , Magnesio/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Cristalografía por Rayos X , GMP Cíclico/metabolismo , Proteínas de la Membrana/genéticaRESUMEN
Enteroviruses, members of the family of picornaviruses, are the most common viral infectious agents in humans causing a broad spectrum of diseases ranging from mild respiratory illnesses to life-threatening infections. To efficiently replicate within the host cell, enteroviruses hijack several host factors, such as ACBD3. ACBD3 facilitates replication of various enterovirus species, however, structural determinants of ACBD3 recruitment to the viral replication sites are poorly understood. Here, we present a structural characterization of the interaction between ACBD3 and the non-structural 3A proteins of four representative enteroviruses (poliovirus, enterovirus A71, enterovirus D68, and rhinovirus B14). In addition, we describe the details of the 3A-3A interaction causing the assembly of the ACBD3-3A heterotetramers and the interaction between the ACBD3-3A complex and the lipid bilayer. Using structure-guided identification of the point mutations disrupting these interactions, we demonstrate their roles in the intracellular localization of these proteins, recruitment of downstream effectors of ACBD3, and facilitation of enterovirus replication. These structures uncovered a striking convergence in the mechanisms of how enteroviruses and kobuviruses, members of a distinct group of picornaviruses that also rely on ACBD3, recruit ACBD3 and its downstream effectors to the sites of viral replication.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Interacciones Huésped-Patógeno , Proteínas de la Membrana/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Picornaviridae/fisiología , Proteínas Virales/metabolismo , Replicación Viral , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Cristalización , Cristalografía por Rayos X , Células HEK293 , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Modelos Moleculares , Mutación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Unión Proteica , Conformación Proteica , Homología de Secuencia , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
We describe the expression and purification of an active recombinant Zika virus RNA-dependent RNA polymerase (RdRp). Next, we present the development and optimization of an in vitro assay to measure its activity. We then applied the assay to selected triphosphate analogs and discovered that 2'-C-methylated nucleosides exhibit strong inhibitory activity. Surprisingly, also carbocyclic derivatives with the carbohydrate locked in a North-like conformation as well as a ribonucleotide with a South conformation exhibited strong activity. Our results suggest that the traditional 2'-C-methylated nucleosides pursued in the race for anti-HCV treatment can be superseded by brand new scaffolds in the case of the Zika virus.