RESUMEN
The oligoadenylate synthetase-ribonuclease L pathway is a major player in the interferon-induced antiviral defense mechanism of cells. Upon sensing viral dsRNA, 5'-phosphorylated 2',5'-oligoadenylates are synthesized, and subsequently activate latent RNase L. To determine the influence of 5'-phosphate end on the activation of human RNase L, four sets of 5'-phosphonate modified oligoadenylates were prepared on solid-phase. The ability of these 5'-modified oligoadenylates bearing shortened, isosteric and prolonged phosphonate linkages to activate RNase L was explored. We found that isosteric linkages and linkages prolonged by one atom were in general well tolerated by the enzyme with the EC50 values comparable to that of the natural activator. In contrast, linkages shortened by one atom or prolonged by two atoms exhibited decrease in the activity.
Asunto(s)
Nucleótidos de Adenina/farmacología , Endorribonucleasas/metabolismo , Oligorribonucleótidos/farmacología , Organofosfonatos/farmacología , Nucleótidos de Adenina/síntesis química , Nucleótidos de Adenina/química , Relación Dosis-Respuesta a Droga , Humanos , Conformación de Ácido Nucleico , Oligorribonucleótidos/síntesis química , Oligorribonucleótidos/química , Organofosfonatos/síntesis química , Organofosfonatos/química , Relación Estructura-ActividadRESUMEN
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
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
Analogs of nucleosides and nucleotides represent a promising pool of potential therapeutics. This work describes a new synthetic route leading to 2'-deoxy-2'-fluorotetradialdose D-nucleoside phosphonates. Moreover, a new universal synthetic route leading to tetradialdose d-nucleosides bearing purine nucleobases is also described. All new compounds were tested as triphosphate analogs for inhibitory potency against a variety of viral polymerases. The fluorinated nucleosides were transformed to phosphoramidate prodrugs and evaluated in cell cultures against various viruses including influenza and SARS-CoV-2.
RESUMEN
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
Delivery of pharmacologically active nucleoside triphosphate analogs to sites of viral infection is challenging. In prior work we identified a 2'-C-methyl-1'-cyano-7-deaza-adenosine C-nucleotide analog with desirable selectivity and potency for the treatment of hepatitis C virus (HCV) infection. However, the prodrug selected for clinical development, GS-6620, required a high dose for meaningful efficacy and had unacceptable variability due to poor oral absorption as a result of suboptimal solubility, intestinal metabolism, and efflux transport. While obtaining clinical proof of concept for the nucleotide analog, a more effective prodrug strategy would be necessary for clinical utility. Here, we report an alternative prodrug of the same nucleoside analog identified to address liabilities of GS-6620. A phosphoramidate prodrug containing the nonproteinogenic amino acid methylalanine, an isopropyl ester and phenol in the (S) conformation at phosphorous, GS2, was found to have improved solubility, intestinal stability, and hepatic activation. GS2 is a more selective substrate for hepatically expressed carboxyl esterase 1 (CES1) and is resistant to hydrolysis by more widely expressed hydrolases, including cathepsin A (CatA) and CES2. Unlike GS-6620, GS2 was not cleaved by intestinally expressed CES2 and, as a result, was stable in intestinal extracts. Levels of liver triphosphate following oral administration of GS2 in animals were higher than those of GS-6620, even when administered under optimal conditions for GS-6620 absorption. Combined, these properties suggest that GS2 will have better oral absorption in the clinic when administered in a solid dosage form and the potential to extend the clinical proof of concept obtained with GS-6620.
Asunto(s)
Antivirales/uso terapéutico , Hepacivirus/patogenicidad , Nucleótidos/uso terapéutico , Profármacos/uso terapéutico , Triazinas/uso terapéutico , Adenosina/análogos & derivados , Administración Oral , Alanina , Animales , Antivirales/administración & dosificación , Antivirales/farmacocinética , Células CACO-2 , Células Cultivadas , Perros , Hepacivirus/efectos de los fármacos , Hepatitis C/virología , Humanos , Masculino , Nucleótidos/administración & dosificación , Nucleótidos/farmacocinética , Profármacos/administración & dosificación , Profármacos/farmacocinética , Ratas , Triazinas/administración & dosificación , Triazinas/farmacocinética , Replicación Viral/efectos de los fármacosRESUMEN
A series of 2'-fluorinated C-nucleosides were prepared and tested for anti-HCV activity. Among them, the triphosphate of 2'-fluoro-2'-C-methyl adenosine C-nucleoside (15) was a potent and selective inhibitor of the NS5B polymerase and maintained activity against the S282T resistance mutant. A number of phosphoramidate prodrugs were then prepared and evaluated leading to the identification of the 1-aminocyclobutane-1-carboxylic acid isopropyl ester variant (53) with favorable pharmacokinetic properties including efficient liver delivery in animals.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Nucleósidos/química , Nucleósidos/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Amidas/química , Amidas/farmacocinética , Amidas/farmacología , Animales , Antivirales/farmacocinética , Células CACO-2 , Línea Celular , Cricetinae , Descubrimiento de Drogas , Farmacorresistencia Viral , Halogenación , Hepacivirus/genética , Hepacivirus/fisiología , Hepatitis C/tratamiento farmacológico , Humanos , Metilación , Simulación del Acoplamiento Molecular , Nucleósidos/farmacocinética , Ácidos Fosfóricos/química , Ácidos Fosfóricos/farmacocinética , Ácidos Fosfóricos/farmacología , Mutación Puntual , Profármacos/química , Profármacos/farmacocinética , Profármacos/farmacología , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Tenofovir alafenamide fumarate (TAF) is an oral phosphonoamidate prodrug of the HIV reverse transcriptase nucleotide inhibitor tenofovir (TFV). Previous studies suggested a principal role for the lysosomal serine protease cathepsin A (CatA) in the intracellular activation of TAF. Here we further investigated the role of CatA and other human hydrolases in the metabolism of TAF. Overexpression of CatA or liver carboxylesterase 1 (Ces1) in HEK293T cells increased intracellular TAF hydrolysis 2- and 5-fold, respectively. Knockdown of CatA expression with RNA interference (RNAi) in HeLa cells reduced intracellular TAF metabolism 5-fold. Additionally, the anti-HIV activity and the rate of CatA hydrolysis showed good correlation within a large set of TFV phosphonoamidate prodrugs. The covalent hepatitis C virus (HCV) protease inhibitors (PIs) telaprevir and boceprevir potently inhibited CatA-mediated TAF activation (50% inhibitory concentration [IC50] = 0.27 and 0.16 µM, respectively) in vitro and also reduced its anti-HIV activity in primary human CD4(+) T lymphocytes (21- and 3-fold, respectively) at pharmacologically relevant concentrations. In contrast, there was no inhibition of CatA or any significant effect on anti-HIV activity of TAF observed with cobicistat, noncovalent HIV and HCV PIs, or various prescribed inhibitors of host serine proteases. Collectively, these studies confirm that CatA plays a pivotal role in the intracellular metabolism of TAF, whereas the liver esterase Ces1 likely contributes to the hepatic activation of TAF. Moreover, this work demonstrates that a wide range of viral and host PIs, with the exception of telaprevir and boceprevir, do not interfere with the antiretroviral activity of TAF.
Asunto(s)
Adenina/análogos & derivados , Fármacos Anti-VIH/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Profármacos/metabolismo , Tenofovir/metabolismo , Adenina/metabolismo , Adenina/farmacología , Alanina , Fármacos Anti-VIH/farmacología , Biotransformación , Linfocitos T CD4-Positivos/enzimología , Linfocitos T CD4-Positivos/virología , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Catepsina A/antagonistas & inhibidores , Catepsina A/genética , Catepsina A/metabolismo , Cobicistat/farmacología , Interacciones Farmacológicas , Expresión Génica , Células HEK293 , VIH-1/efectos de los fármacos , VIH-1/crecimiento & desarrollo , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Oligopéptidos/farmacología , Cultivo Primario de Células , Profármacos/farmacología , Prolina/análogos & derivados , Prolina/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Tenofovir/farmacologíaRESUMEN
We report on an extensive structure-activity relationship study of novel PI4K IIIß inhibitors. The purine derivative of the potent screening hit T-00127-HEV1 has served as a suitable starting point for a thorough investigation of positions 8 and 2. While position 8 of the purine scaffold can only bear a small substituent to maintain the inhibitory activity, position 2 is opened for extensive modification and can accommodate even substituted phenyl rings without the loss of PI4K IIIß inhibitory activity. These empirical observations nicely correlate with the results of our docking study, which suggests that position 2 directs towards solution and can provide the necessary space for the interaction with remote residues of the enzyme, whereas the cavity around position 8 is strictly limited. The obtained compounds have also been subjected to antiviral screening against a panel of (+)ssRNA viruses.
Asunto(s)
Antivirales/farmacología , Enterovirus Humano B/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Rhinovirus/efectos de los fármacos , Antivirales/síntesis química , Antivirales/química , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Purinas/síntesis química , Purinas/química , Relación Estructura-ActividadRESUMEN
Novel 4'-substituted ß-d-2'-deoxy-2'-α-fluoro (2'd2'F) nucleoside inhibitors of respiratory syncytial virus (RSV) are reported. The introduction of 4'-substitution onto 2'd2'F nucleoside analogs resulted in compounds demonstrating potent cell based RSV inhibition, improved inhibition of the RSV polymerase by the nucleoside triphosphate metabolites, and enhanced selectivity over incorporation by mitochondrial RNA and DNA polymerases. Selectivity over the mitochondrial polymerases was found to be extremely sensitive to the specific 4'-substitution and not readily predictable. Combining the most potent and selective 4'-groups from N-nucleoside analogs onto a 2'd2'F C-nucleoside analog resulted in the identification of ß-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a promising nucleoside lead for RSV.
Asunto(s)
Adenosina/química , Antivirales/química , ADN Polimerasa Dirigida por ADN/química , Inhibidores de la Síntesis del Ácido Nucleico/química , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN/química , Virus Sincitiales Respiratorios/enzimología , Virus Sincitiales Respiratorios/fisiología , Adenosina/síntesis química , Adenosina/farmacología , Antivirales/síntesis química , Antivirales/farmacología , Compuestos Aza/química , ADN Polimerasa Dirigida por ADN/metabolismo , Evaluación Preclínica de Medicamentos , Inhibidores de la Síntesis del Ácido Nucleico/síntesis química , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , ARN/metabolismo , ARN Mitocondrial , ARN Polimerasa Dependiente del ARN/metabolismo , Virus Sincitiales Respiratorios/efectos de los fármacos , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
Many xenobiotics including the pharmacoenhancer cobicistat increase serum creatinine by inhibiting its renal active tubular secretion without affecting the glomerular filtration rate. This study aimed to define the transporters involved in creatinine secretion, applying that knowledge to establish the mechanism for xenobiotic-induced effects. The basolateral uptake transporters organic anion transporter OAT2 and organic cation transporters OCT2 and OCT3 were found to transport creatinine. At physiologic creatinine concentrations, the specific activity of OAT2 transport was over twofold higher than OCT2 or OCT3, establishing OAT2 as a likely relevant creatinine transporter and further challenging the traditional view that creatinine is solely transported by a cationic pathway. The apical multidrug and toxin extrusion transporters MATE1 and MATE2-K demonstrated low-affinity and high-capacity transport. All drugs known to affect creatinine inhibited OCT2 and MATE1. Similar to cimetidine and ritonavir, cobicistat had the greatest effect on MATE1 with a 50% inhibition constant of 0.99 µM for creatinine transport. Trimethoprim potently inhibited MATE2-K, whereas dolutegravir preferentially inhibited OCT2. Cimetidine was unique, inhibiting all transporters that interact with creatinine. Thus, the clinical observation of elevated serum creatinine in patients taking cobicistat is likely a result of OCT2 transport, facilitating intracellular accumulation, and MATE1 inhibition.
Asunto(s)
Carbamatos/farmacología , Creatinina/sangre , Creatinina/metabolismo , Túbulos Renales/efectos de los fármacos , Túbulos Renales/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/antagonistas & inhibidores , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Tiazoles/farmacología , Animales , Transporte Biológico Activo/efectos de los fármacos , Células CHO , Línea Celular , Células Cultivadas , Cimetidina/farmacología , Cobicistat , Cricetulus , Perros , Células HEK293 , Humanos , Cinética , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Proteínas de Transporte de Catión Orgánico/metabolismo , Transportador 2 de Cátion Orgánico , Proteínas Recombinantes/metabolismoRESUMEN
Human rhinovirus type C (HRV-C) is a newly discovered enterovirus species frequently associated with exacerbation of asthma and other acute respiratory conditions. Until recently, HRV-C could not be propagated in vitro, hampering in-depth characterization of the virus replication cycle and preventing efficient testing of antiviral agents. Herein we describe several subgenomic RNA replicon systems and a cell culture infectious model for HRV-C that can be used for antiviral screening. The replicon constructs consist of genome sequences from HRVc15, HRVc11, HRVc24, and HRVc25 strains, with the P1 capsid region replaced by a Renilla luciferase coding sequence. Following transfection of the replicon RNA into HeLa cells, the constructs produced time-dependent increases in luciferase signal that can be inhibited in a dose-dependent manner by known inhibitors of HRV replication, including the 3C protease inhibitor rupintrivir, the nucleoside analog inhibitor MK-0608, and the phosphatidylinositol 4-kinase IIIß (PI4K-IIIß) kinase inhibitor PIK93. Furthermore, with the exception of pleconaril and pirodavir, the other tested classes of HRV inhibitors blocked the replication of full-length HRVc15 and HRVc11 in human airway epithelial cells (HAEs) that were differentiated in the air-liquid interface, exhibiting antiviral activities similar to those observed with HRV-16. In summary, this study is the first comprehensive profiling of multiple classes of antivirals against HRV-C, and the set of newly developed quantitative HRV-C antiviral assays represent indispensable tools for the identification and evaluation of novel panserotype HRV inhibitors.
Asunto(s)
Antivirales/farmacología , Rhinovirus/efectos de los fármacos , Resfriado Común/tratamiento farmacológico , Resfriado Común/virología , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Técnicas In Vitro , Isoxazoles/farmacología , Oxadiazoles/farmacología , Oxazoles , Fenilalanina/análogos & derivados , Piperidinas/farmacología , Piridazinas/farmacología , Pirrolidinonas/farmacología , ARN Viral/genética , Replicón/efectos de los fármacos , Rhinovirus/genética , Tubercidina/análogos & derivados , Tubercidina/farmacología , Valina/análogos & derivados , Replicación Viral/efectos de los fármacosRESUMEN
The anti-hepatitis C virus nucleotide prodrug GS-6620 employs a double-prodrug approach, with l-alanine-isopropyl ester and phenol moieties attached to the 5'-phosphate that release the nucleoside monophosphate in hepatocytes and a 3'-isobutyryl ester added to improve permeability and oral bioavailability. Consistent with the stability found in intestinal homogenates, following oral administration, intact prodrug levels in blood plasma were the highest in dogs, followed by monkeys, and then were the lowest in hamsters. In contrast, liver levels of the triphosphate metabolite at the equivalent surface area-adjusted doses were highest in hamsters, followed by in dogs and monkeys. Studies in isolated primary hepatocytes suggest that relatively poor oral absorption in hamsters and monkeys was compensated for by relatively efficient hepatocyte activation. As intestinal absorption was found to be critical to the effectiveness of GS-6620 in nonclinical species, stomach pH, formulation, and food effect studies were completed in dogs. Consistent with in vitro absorption studies in Caco-2 cells, the absorption of GS-6620 was found to be complex and highly dependent on concentration. Higher rates of metabolism were observed at lower concentrations that were unable to saturate intestinal efflux transporters. In first-in-human clinical trials, the oral administration of GS-6620 resulted in poor plasma exposure relative to that observed in dogs and in large pharmacokinetic and pharmacodynamic variabilities. While a double-prodrug approach, including a 3'-isobutyryl ester, provided higher intrinsic intestinal permeability, this substitution appeared to be a metabolic liability, resulting in extensive intestinal metabolism and relatively poor oral absorption in humans.
Asunto(s)
Antivirales/farmacocinética , Profármacos/farmacocinética , Administración Oral , Animales , Antivirales/farmacología , Células CACO-2 , Línea Celular , Cricetinae , Perros , Hepacivirus/efectos de los fármacos , Humanos , Macaca fascicularis , Masculino , Mesocricetus , Profármacos/farmacologíaRESUMEN
As a class, nucleotide inhibitors (NIs) of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase offer advantages over other direct-acting antivirals, including properties, such as pangenotype activity, a high barrier to resistance, and reduced potential for drug-drug interactions. We studied the in vitro pharmacology of a novel C-nucleoside adenosine analog monophosphate prodrug, GS-6620. It was found to be a potent and selective HCV inhibitor against HCV replicons of genotypes 1 to 6 and against an infectious genotype 2a virus (50% effective concentration [EC50], 0.048 to 0.68 µM). GS-6620 showed limited activities against other viruses, maintaining only some of its activity against the closely related bovine viral diarrhea virus (EC50, 1.5 µM). The active 5'-triphosphate metabolite of GS-6620 is a chain terminator of viral RNA synthesis and a competitive inhibitor of NS5B-catalyzed ATP incorporation, with Ki/Km values of 0.23 and 0.18 for HCV NS5B genotypes 1b and 2a, respectively. With its unique dual substitutions of 1'-CN and 2'-C-Me on the ribose ring, the active triphosphate metabolite was found to have enhanced selectivity for the HCV NS5B polymerase over host RNA polymerases. GS-6620 demonstrated a high barrier to resistance in vitro. Prolonged passaging resulted in the selection of the S282T mutation in NS5B that was found to be resistant in both cellular and enzymatic assays (>30-fold). Consistent with its in vitro profile, GS-6620 exhibited the potential for potent anti-HCV activity in a proof-of-concept clinical trial, but its utility was limited by the requirement of high dose levels and pharmacokinetic and pharmacodynamic variability.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Nucleósidos/química , Nucleósidos/farmacología , Profármacos/farmacología , Replicación Viral/efectos de los fármacos , Antivirales/efectos adversos , Línea Celular Tumoral , Supervivencia Celular , Células Hep G2 , Humanos , Nucleósidos/efectos adversos , Profármacos/efectos adversos , Profármacos/química , Proteínas no Estructurales Virales/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Nucleótidos Cíclicos , Nucleótidos Cíclicos/química , Nucleótidos Cíclicos/metabolismo , Proteínas de la Membrana/agonistas , Proteínas de la Membrana/química , Química Clic/métodosRESUMEN
The cGAS-STING pathway is a crucial part of innate immunity; it serves to detect DNA in the cytoplasm and to defend against certain cancers, viruses, and bacteria. We designed and synthesized fluorinated carbocyclic cGAMP analogs, MD1203 and MD1202D (MDs), to enhance their stability and their affinity for STING. These compounds demonstrated exceptional activity against STING. Despite their distinct chemical modifications relative to the canonical cyclic dinucleotides (CDNs), crystallographic analysis revealed a binding mode with STING that was consistent with the canonical CDNs. Importantly, MDs were resistant to cleavage by viral poxin nucleases and MDs-bound poxin adopted an unliganded-like conformation. Moreover, MDs complexed with poxin showed a conformation distinct from cGAMP bound to poxin, closely resembling their conformation when bound to STING. In conclusion, the development of MD1203 and MD1202D showcases their potential as potent STING activators with remarkable stability against poxin-mediated degradation-a crucial characteristic for future development of antivirals.
Asunto(s)
Neoplasias , Nucleótidos Cíclicos , Humanos , Nucleótidos Cíclicos/química , Nucleótidos Cíclicos/metabolismo , Nucleotidiltransferasas/química , Inmunidad InnataRESUMEN
A once-daily single-tablet antiretroviral regimen containing tenofovir (TFV) disoproxil fumarate, emtricitabine (FTC), elvitegravir (EVG), and cobicistat (COBI) is an approved combination for the treatment of patients infected with HIV. COBI and TFV have been reported to interact with distinct transporters in renal proximal tubules; while TFV is renally eliminated by a combination of glomerular filtration and tubular secretion via anion transporters OAT1, OAT3, and MRP4, COBI inhibits renal cation transporters, particularly MATE1, resulting in a measurable decrease in the tubular secretion of creatinine. To investigate the potential for a renal drug-drug interaction between TFV and COBI in vitro, the uptake of TFV in the presence and absence of COBI was determined in fresh human renal cortex tissue and in cells expressing the relevant renal transporters. At concentrations exceeding clinical protein-unbound plasma levels, COBI did not significantly inhibit the transport of TFV by the anion transporters OAT1, OAT3, and MRP4 (50% inhibitory concentrations [IC50s] of >15, 6.6, and 8.5 µM, respectively). Conversely, TFV had little or no effect on the cation transporters OCT2 and MATE1 (IC50 > 100 µM). Consistent with studies using individual transporters, no increase in the accumulation of TFV in freshly isolated human renal cortex tissue or renal proximal tubule cells (RPTECs) was observed in the presence of COBI. Finally, COBI alone or in combination with FTC and EVG did not affect the sensitivity to TFV of cultured primary RPTECs or cells coexpressing OAT1 and MRP4. These results illustrate that COBI and TFV interact primarily with distinct renal transporters and indicate a low potential for pharmacokinetic renal drug-drug interaction.
Asunto(s)
Adenina/análogos & derivados , Fármacos Anti-VIH/farmacología , Carbamatos/farmacología , Organofosfonatos/farmacología , Tiazoles/farmacología , Adenina/farmacología , Adulto , Línea Celular , Cobicistat , Interacciones Farmacológicas , Humanos , Técnicas In Vitro , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Transportador 2 de Cátion Orgánico , TenofovirRESUMEN
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.
Asunto(s)
Hepatitis B , Herpesvirus Cercopitecino 1 , Humanos , Animales , Ratones , Citocinas/metabolismo , Hepatocitos , Hepatitis B/tratamiento farmacológico , Interferones/metabolismoRESUMEN
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.