RESUMEN
Linearly homomorphic signature (LHS) allows the acquisition of a new legal signature using the homomorphic operation of the original signatures. However, the public composability of LHS also prevents it from being used in some scenarios where the combiner needs to be designated. The LZZ22 scheme designates a combiner and preserves the signature structure by having the signer and the designated combiner share a secret. However, LZZ22 is not secure enough because the secret is constant. Here, we first prove that there is a polynomial time adversary that can crack the secret in LZZ22 through multiple signature queries. Then, we propose a new scheme, which realizes all the functions of LZZ22 and fixes the security problem by changing the secret with the message. The proposed scheme is shown to be secure against existential forgery on adaptively chosen subspace attacks under the random oracle model. Finally, we detail how to apply our scheme to the proxy signature and perform it on a personal computer, and the results show that our scheme is efficient.
RESUMEN
OBJECTIVE: Glucagon-like peptide (GLP)-1 is an incretin hormone that acts after food intake to stimulate insulin production, enhance satiety, and promote weight loss. Here we describe the discovery and characterization of ecnoglutide (XW003), a novel GLP-1 analog. METHODS: We engineered a series of GLP-1 peptide analogs with an alanine to valine substitution (Ala8Val) and a γGlu-2xAEEA linked C18 diacid fatty acid at various positions. Ecnoglutide was selected and characterized in GLP-1 receptor signaling assays in vitro, as well as in db/db mice and a diet induced obese (DIO) rat model. A Phase 1, double-blind, randomized, placebo-controlled, single (SAD) and multiple ascending dose (MAD) study was conducted to evaluate the safety, tolerability, and pharmacokinetics of subcutaneous ecnoglutide injection in healthy participants. SAD doses ranged from 0.03 to 1.0 mg; MAD doses ranged from 0.2 to 0.6 mg once weekly for 6 weeks (ClinicalTrials.gov Identifier: NCT04389775). RESULTS: In vitro, ecnoglutide potently induced cAMP (EC50 = 0.018 nM) but not GLP-1 receptor internalization (EC50 > 10 µM), suggesting a desirable signaling bias. In rodent models, ecnoglutide significantly reduced blood glucose, promoted insulin induction, and led to more pronounced body weight reduction compared to semaglutide. In a Phase 1 trial, ecnoglutide was generally safe and well tolerated as a once-weekly injection for up to 6 weeks. Adverse events included decreased appetite, nausea, and headache. The half-life at steady state ranged from 124 to 138 h, supporting once-weekly dosing. CONCLUSIONS: Ecnoglutide showed a favorable potency, pharmacokinetic, and tolerability profile, as well as a simplified manufacturing process. These results support the continued development of ecnoglutide for the treatment of type 2 diabetes and obesity.
Asunto(s)
Diabetes Mellitus Tipo 2 , Péptido 1 Similar al Glucagón , Ratones , Ratas , Animales , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Peso Corporal , Obesidad/tratamiento farmacológico , Obesidad/inducido químicamente , Pérdida de Peso , Insulina/uso terapéuticoRESUMEN
BACKGROUND: With the increasing incidence of upper digestive tract tumors, more upper digestive tract surgeries are performed each year, and surgeons have difficulty in the postoperative management of gastrointestinal anastomotic fistula. The use of a new minimally invasive technique, endoluminal vacuum-assisted closure (E-VAC), has increased the success rate of the treatment of gastrointestinal fistula. METHODS: We present 6 cases of gastrointestinal fistula treated in our hospital in 2021: 3 cases of anastomotic fistula after esophageal cancer surgery, 2 cases of anastomotic fistula after gastric cancer surgery, and one case of esophageal rupture after trauma. With E-VAC and other adjuvant treatment measures, the gastrointestinal fistulas were eventually closed or significantly reduced. RESULTS: Both local and systemic infections in all 6 patients were controlled with the use of E-VAC device, resulting in significant reduction or closure of fistulas. CONCLUSION: E-VAC devices can effectively help in the removal of the exudate and necrotic tissue around the fistula, promote the proliferation of granulation tissue, and support closure of the fistula. However, further improvements to the device are needed to improve patient comfort and operational safety.
Asunto(s)
Fístula , Terapia de Presión Negativa para Heridas , Tracto Gastrointestinal Superior , Humanos , Terapia de Presión Negativa para Heridas/efectos adversos , Terapia de Presión Negativa para Heridas/métodos , Estómago , Anastomosis Quirúrgica/efectos adversos , Fístula/etiología , Fístula/cirugía , Fuga Anastomótica/etiología , Fuga Anastomótica/terapiaRESUMEN
Monophosphate prodrug analogs of 2'-deoxy-2'-fluoro-2'-C-methylguanosine have been reported as potent inhibitors of hepatitis C virus (HCV) RNA-dependent RNA polymerase. These prodrugs also display potent anti-dengue virus activities in cellular assays although their prodrug moieties were designed to produce high levels of triphosphate in the liver. Since peripheral blood mononuclear cells (PBMCs) are among the major targets of dengue virus, different prodrug moieties were designed to effectively deliver 2'-deoxy-2'-fluoro-2'-C-methylguanosine monophosphate prodrugs and their corresponding triphosphates into PBMCs after oral administration. We identified a cyclic phosphoramidate, prodrug 17, demonstrating well-balanced anti-dengue virus cellular activity and in vitro stability profiles. We further determined the PBMC concentration of active triphosphate needed to inhibit virus replication by 50% (TP50). Compound 17 was assessed in an AG129 mouse model and demonstrated 1.6- and 2.2-log viremia reductions at 100 and 300 mg/kg twice a day (BID), respectively. At 100 mg/kg BID, the terminal triphosphate concentration in PBMCs exceeded the TP50 value, demonstrating TP50 as the target exposure for efficacy. In dogs, oral administration of compound 17 resulted in high PBMC triphosphate levels, exceeding the TP50 at 10 mg/kg. Unfortunately, 2-week dog toxicity studies at 30, 100, and 300 mg/kg/day showed that "no observed adverse effect level" (NOAEL) could not be achieved due to pulmonary inflammation and hemorrhage. The preclinical safety results suspended further development of compound 17. Nevertheless, present work has proven the concept that an efficacious monophosphate nucleoside prodrug could be developed for the potential treatment of dengue virus infection.
Asunto(s)
Dengue , Guanosina/análogos & derivados , Profármacos , Amidas , Animales , Antivirales/farmacología , Dengue/tratamiento farmacológico , Perros , Femenino , Hepacivirus , Leucocitos Mononucleares , Masculino , Ácidos Fosfóricos , Profármacos/farmacología , Profármacos/uso terapéuticoRESUMEN
PURPOSE: Infection with hepatitis C virus is the leading cause of infectious disease mortality in the United States. BZF961 is a novel small molecule inhibitor of the hepatitis C virus NS3-4A protease. Here we present the results of a randomized, double-blinded, placebo-controlled, multicentered study in treatment-naïve patients with chronic hepatitis C virus genotype-1 infection. METHODS: Patients were enrolled sequentially in 2 parts and treated for 3days. BZF961 was administered as monotherapy (500mg BID for 3 days) or in combination with the cytochrome P450 3A4 inhibitor ritonavir to boost its exposure (BZF961 10, 20, or 50mg QD or BID). FINDINGS: BZF961 was safe and well tolerated in the patients studied with no serious adverse events. There were no appreciable differences in adverse events among patients who received BZF961, BZF961 with ritonavir, or placebo. There was a significant, clinically meaningful reduction in viral load from baseline in patients treated either with BZF961 500mg every 12hours alone or BZF961 50mg every 12hours in combination with ritonavir. Activity against the hepatitis C virus of the lower-dose regimens was apparent but more modest. There were no relevant changes from baseline viral loads in placebo-treated patients. IMPLICATIONS: Coadministration of ritonavir with BZF961 boosted BZF961 exposure (including Cmin, which is the clinically relevant parameter associated with antiviral activity) in a therapeutic range with less variability compared with BZF961 alone. For strategic reasons, BZF961 is no longer under development.
Asunto(s)
Antivirales/uso terapéutico , Hepatitis C Crónica/tratamiento farmacológico , Compuestos Orgánicos/uso terapéutico , Ritonavir/uso terapéutico , Adulto , Antivirales/administración & dosificación , Antivirales/efectos adversos , Método Doble Ciego , Quimioterapia Combinada , Femenino , Genotipo , Hepacivirus/genética , Humanos , Masculino , Persona de Mediana Edad , Compuestos Orgánicos/administración & dosificación , Compuestos Orgánicos/efectos adversos , Estados Unidos , Carga Viral/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidoresRESUMEN
To identify a potent and selective nucleoside inhibitor of dengue virus RNA-dependent RNA polymerase, a series of 2'- and/or 4'-ribose sugar modified uridine nucleoside phosphoramidate prodrugs and their corresponding triphosphates were synthesized and evaluated. Replacement of 2'-OH with 2'-F led to be a poor substrate for both dengue virus and human mitochondrial RNA polymerases. Instead of 2'-fluorination, the introduction of fluorine at the ribose 4'-position was found not to affect the inhibition of the dengue virus polymerase with a reduction in uptake by mitochondrial RNA polymerase. 2'-C-ethynyl-4'-F-uridine phosphoramidate prodrug displayed potent anti-dengue virus activity in the primary human peripheral blood mononuclear cell-based assay with no significant cytotoxicity in human hepatocellular liver carcinoma cell lines and no mitochondrial toxicity in the cell-based assay using human prostate cancer cell lines.
Asunto(s)
Antivirales/farmacología , Inhibidores Enzimáticos/farmacología , Profármacos/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Uridina Monofosfato/análogos & derivados , Uridina Monofosfato/farmacología , Antivirales/química , Antivirales/toxicidad , Virus del Dengue/enzimología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/toxicidad , Células Hep G2 , Humanos , Leucocitos Mononucleares/virología , Estructura Molecular , Sistema Mononuclear Fagocítico/virología , Profármacos/química , Profármacos/toxicidad , Relación Estructura-ActividadRESUMEN
Influenza virus uses a unique mechanism to initiate viral transcription named cap-snatching. The PB2 subunit of the viral heterotrimeric RNA polymerase binds the cap structure of cellular pre-mRNA to promote its cleavage by the PA subunit. The resulting 11-13 capped oligomer is used by the PB1 polymerase subunit to initiate transcription of viral proteins. VX-787 is an inhibitor of the influenza A virus pre-mRNA cap-binding protein PB2. This clinical stage compound was shown to bind the minimal cap-binding domain of PB2 to inhibit the cap-snatching machinery. However, the binding of this molecule in the context of an extended form of the PB2 subunit has remained elusive. Here we generated a collection of PB2 truncations to identify a PB2 protein representative of its structure in the viral heterotrimeric protein. We present the crystal structure of VX-787 bound to a PB2 construct that recapitulates VX-787's biological antiviral activity in vitro. This co-structure reveals more extensive interactions than previously identified and provides insight into the observed resistance profile, affinity, binding kinetics, and conformational rearrangements induced by VX-787.
Asunto(s)
Antivirales/química , Virus de la Influenza A/enzimología , Subunidades de Proteína/química , ARN Polimerasa Dependiente del ARN/química , Antivirales/farmacología , Sitios de Unión , Humanos , Virus de la Influenza A/efectos de los fármacos , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Unión Proteica , Subunidades de Proteína/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
Nucleoside or nucleotide inhibitors are a highly successful class of antivirals due to selectivity, potency, broad coverage, and high barrier to resistance. Nucleosides are the backbone of combination treatments for HIV, hepatitis B virus, and, since the FDA approval of sofosbuvir in 2013, also for hepatitis C virus (HCV). However, many promising nucleotide inhibitors have advanced to clinical trials only to be terminated due to unexpected toxicity. Here we describe the in vitro pharmacology of compound 1, a monophosphate prodrug of a 2'-ethynyluridine developed for the treatment of HCV. Compound 1 inhibits multiple HCV genotypes in vitro (50% effective concentration [EC50], 0.05 to 0.1 µM) with a selectivity index of >300 (50% cytotoxic concentration [CC50], 30 µM in MT-4 cells). The active triphosphate metabolite of compound 1, compound 2, does not inhibit human α, ß, or γ DNA polymerases but was a substrate for incorporation by the human mitochondrial RNA polymerase (POLRMT). In dog, the oral administration of compound 1 resulted in elevated serum liver enzymes and microscopic changes in the liver. Transmission electron microscopy showed significant mitochondrial swelling and lipid accumulation in hepatocytes. Gene expression analysis revealed dose-proportional gene signature changes linked to loss of hepatic function and increased mitochondrial dysfunction. The potential of in vivo toxicity through mitochondrial polymerase incorporation by nucleoside analogs has been previously shown. This study shows that even moderate levels of nucleotide analog incorporation by POLRMT increase the risk of in vivo mitochondrial dysfunction. Based on these results, further development of compound 1 as an anti-HCV compound was terminated.
Asunto(s)
Antivirales/farmacocinética , Antivirales/toxicidad , ARN Polimerasas Dirigidas por ADN/metabolismo , Hepacivirus/efectos de los fármacos , Nucleósidos/farmacocinética , Animales , Antivirales/administración & dosificación , Línea Celular , ARN Polimerasas Dirigidas por ADN/genética , Perros , Hepacivirus/genética , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Polifosfatos/metabolismo , Profármacos/farmacocinética , Profármacos/toxicidad , Ratas Wistar , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Pruebas de Toxicidad/métodos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismoRESUMEN
Human cytomegalovirus (HCMV) infection is usually benign in healthy individuals but can cause life-threatening disease in those with compromised immune systems. Approved drugs available to treat HCMV disease, including ganciclovir, cidofovir, and foscarnet, have significant toxicities that limit their use in certain patient populations. LJP538 and LJP539 are human monoclonal antibodies that are being evaluated as immunoglobulin therapeutics. The antibodies target glycoproteins gB and the gH/gL/UL128/UL130/UL131a pentameric complex, respectively. Here we present an in vitro characterization of these antibodies. We show that LJP538 and LJP539 are more potent than a marketed immunoglobulin at inhibiting HCMV infection of various cell lines relevant to pathogenesis. We find that LJP538 and LJP539 are active against a panel of clinical isolates in vitro and demonstrate minor-to-moderate synergy in combination. Passage of HCMV in the presence of LJP538 or LJP539 alone resulted in resistance-associated mutations that mapped to the target genes. However, no loss of susceptibility to the combination of antibodies was observed for >400 days in culture. Finally, the binding regions of LJP538 and LJP539 are conserved among clinical isolates. Taken together, these data support the use of LJP538 and LJP539 in combination for clinical trials in HCMV patients.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Antivirales/farmacología , Antivirales/farmacología , Citomegalovirus/efectos de los fármacos , Citomegalovirus/patogenicidad , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/inmunología , Antivirales/inmunología , Línea Celular , Citomegalovirus/genética , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/prevención & control , Humanos , Glicoproteínas de Membrana/inmunología , Mutación , Proteínas del Envoltorio Viral/inmunología , Internalización del Virus/efectos de los fármacosRESUMEN
Chronic hepatitis B virus (HBV) infection affects 240 million people worldwide and is a major risk factor for liver failure and hepatocellular carcinoma. Current antiviral therapy inhibits cytoplasmic HBV genomic replication, but is not curative because it does not directly affect nuclear HBV closed circular DNA (cccDNA), the genomic form that templates viral transcription and sustains viral persistence. Novel approaches that directly target cccDNA regulation would therefore be highly desirable. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications (PTMs). Here, using a new cccDNA ChIP-Seq approach, we report, to our knowledge, the first genome-wide maps of PTMs in cccDNA-containing chromatin from de novo infected HepG2 cells, primary human hepatocytes, and from HBV-infected liver tissue. We find high levels of PTMs associated with active transcription enriched at specific sites within the HBV genome and, surprisingly, very low levels of PTMs linked to transcriptional repression even at silent HBV promoters. We show that transcription and active PTMs in HBV chromatin are reduced by the activation of an innate immunity pathway, and that this effect can be recapitulated with a small molecule epigenetic modifying agent, opening the possibility that chromatin-based regulation of cccDNA transcription could be a new therapeutic approach to chronic HBV infection.
Asunto(s)
Cromatina/metabolismo , ADN Viral/genética , Epigénesis Genética , Virus de la Hepatitis B/genética , Histonas/metabolismo , Plásmidos/metabolismo , Células Hep G2 , Humanos , Transcripción GenéticaRESUMEN
Tegobuvir (GS-9190) is a non-nucleoside inhibitor of HCV RNA replication with proven antiviral activity in HCV-infected patients. The in vitro antiviral activity of Tegobuvir, when combined with one or two other direct acting antivirals (DAA) was assessed. When Tegobuvir was combined with either interferon α-2b, ribavirin, the protease inhibitor (PI) VX-950, the nucleoside polymerase inhibitor (NI) 2'-C-methylcytidine or various non-nucleoside polymerase inhibitors, an overall additive antiviral activity was observed. Adding Tegobuvir (at concentrations of 6, 30 or 150nM) to replicon-containing cells in the presence of suboptimal concentrations of the PI or of the various polymerase inhibitors either markedly delayed or completely prevented resistance development against these latter compounds. Tegobuvir (15nM), when combined with the PI, was able to cure replicon-containing cells from their replicon after a single passage, whereas either compound alone (at 2-fold higher concentration) was not. The triple combination of Tegobuvir (10nM), the PI and the NI resulted in clearance of replicon RNA after only two passages. In contrast, the inhibitors when used alone at 3-fold higher concentrations were not able to cure the cells from the replicon, after as long as 6 passages. Combinations containing low concentrations of Tegobuvir are thus highly effective in curing cells from HCV replicon and in delaying or preventing the development of resistance against other DAA.
Asunto(s)
Antivirales/farmacología , Farmacorresistencia Viral , Sinergismo Farmacológico , Hepacivirus/efectos de los fármacos , Purinas/farmacología , Piridazinas/farmacología , Línea Celular , Hepatocitos/virología , Humanos , Interferón-alfa/farmacología , Oligopéptidos/farmacología , ARN Viral/análisis , ARN Viral/genética , Ribavirina/farmacologíaRESUMEN
In this paper, we have proposed a new electrochemical aptasensor based on a novel ligase-assisted Exo III-catalyzed degradation reaction (LAECDR), which consists of DNA ligase-catalyzed ligation of thrombin-binding aptamer (TBA) with an extension strand (E-strand) and Exo III-catalyzed selective degradation of probe DNA, by using an improved target-induced strand displacement strategy. As a result of LAECDR, methylene blue (MB)-labeled mononucleotides can be released from the 3'-terminal of probe DNA and captured by cucurbit[7]uril-functionalized electrode to induce noticeable electrochemical response. Nevertheless, in the presence of the target protein, thrombin, the TBA that is partially complementary to probe DNA is preferentially binding with the target protein, thereby inhibiting LAECDR from taking place. The remaining intact probe DNA will prevent the terminal-attached MB from approaching to the electrode surface due to strong electrostatic repulsion, so the electrochemical response will be changed by thrombin. By tracing the electrochemical response of adsorbed MB, our aptasensor can exhibit high sensitivity for thrombin detection with a wide linear range from 100 fM to 1 nM and an extremely low detection limit of 33 fM, which can also easily distinguish thrombin in the complex serum samples with high specificity. Therefore, our aptasensor might have great potential in clinical applications in the future.
Asunto(s)
Aptámeros de Nucleótidos/química , ADN Ligasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Hidrocarburos Aromáticos con Puentes/química , Sondas de ADN/metabolismo , Técnicas Electroquímicas , Electrodos , Exodesoxirribonucleasas/química , Oro/química , Imidazoles/química , Nanopartículas del Metal/química , Azul de Metileno/química , Unión Proteica , Trombina/análisis , Trombina/metabolismoRESUMEN
Widely used chemical genetic screens have greatly facilitated the identification of many antiviral agents. However, the regions of interaction and inhibitory mechanisms of many therapeutic candidates have yet to be elucidated. Previous chemical screens identified Daclatasvir (BMS-790052) as a potent nonstructural protein 5A (NS5A) inhibitor for Hepatitis C virus (HCV) infection with an unclear inhibitory mechanism. Here we have developed a quantitative high-resolution genetic (qHRG) approach to systematically map the drug-protein interactions between Daclatasvir and NS5A and profile genetic barriers to Daclatasvir resistance. We implemented saturation mutagenesis in combination with next-generation sequencing technology to systematically quantify the effect of every possible amino acid substitution in the drug-targeted region (domain IA of NS5A) on replication fitness and sensitivity to Daclatasvir. This enabled determination of the residues governing drug-protein interactions. The relative fitness and drug sensitivity profiles also provide a comprehensive reference of the genetic barriers for all possible single amino acid changes during viral evolution, which we utilized to predict clinical outcomes using mathematical models. We envision that this high-resolution profiling methodology will be useful for next-generation drug development to select drugs with higher fitness costs to resistance, and also for informing the rational use of drugs based on viral variant spectra from patients.
Asunto(s)
Farmacorresistencia Viral , Perfilación de la Expresión Génica , Aptitud Genética , Hepacivirus/fisiología , Hepatitis C , Imidazoles/farmacología , Replicación Viral , Carbamatos , Línea Celular , Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/genética , Hepatitis C/tratamiento farmacológico , Hepatitis C/genética , Hepatitis C/metabolismo , Hepatitis C/patología , Humanos , Pirrolidinas , Valina/análogos & derivados , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
Alisporivir (ALV), a cyclophilin inhibitor, is a host-targeting antiviral (HTA) with multigenotypic anti-hepatitis C virus (HCV) activity and a high barrier to resistance. Recent advances have supported the concept of interferon (IFN)-free regimens to treat chronic hepatitis C. As the most advanced oral HTA, ALV with direct-acting antivirals (DAAs) represents an attractive drug combination for IFN-free therapy. In this study, we investigated whether particular DAAs exhibit additive, synergistic, or antagonistic effects when combined with ALV. Drug combinations of ALV with NS3 protease, NS5B polymerase, and NS5A inhibitors were investigated in HCV replicons from genotypes 1a, 1b, 2a, 3, and 4a (GT1a to -4a). Combinations of ALV with DAAs exerted an additive effect on GT1 and -4. A significant and specific synergistic effect was observed with ALV-NS5A inhibitor combination on GT2 and -3. Furthermore, ALV was fully active against DAA-resistant variants, and ALV-resistant variants were fully susceptible to DAAs. ALV blocks the contact between cyclophilin A and domain II of NS5A, and NS5A inhibitors target domain I of NS5A; our data suggest a molecular basis for the use of these two classes of inhibitors acting on two distinct domains of NS5A. These results provide in vitro evidence that ALV with NS5A inhibitor combination represents an attractive strategy and a potentially effective IFN-free regimen for treatment of patients with chronic hepatitis C. Due to its high barrier and lack of cross-resistance, ALV could be a cornerstone drug partner for DAAs.
Asunto(s)
Antivirales/farmacología , Ciclosporina/farmacología , Hepacivirus/efectos de los fármacos , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C/tratamiento farmacológico , Proteínas no Estructurales Virales/antagonistas & inhibidores , Ciclofilina A/metabolismo , Ciclofilinas/antagonistas & inhibidores , Farmacorresistencia Viral , Sinergismo Farmacológico , Quimioterapia Combinada , Genotipo , Humanos , Replicón/efectos de los fármacos , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
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
Hepatitis C virus (HCV) infection presents an unmet medical need requiring more effective treatment options. Nucleoside inhibitors (NI) of HCV polymerase (NS5B) have demonstrated pan-genotypic activity and durable antiviral response in the clinic, and they are likely to become a key component of future treatment regimens. NI candidates that have entered clinical development thus far have all been N-nucleoside derivatives. Herein, we report the discovery of a C-nucleoside class of NS5B inhibitors. Exploration of adenosine analogs in this class identified 1'-cyano-2'-C-methyl 4-aza-7,9-dideaza adenosine as a potent and selective inhibitor of NS5B. A monophosphate prodrug approach afforded a series of compounds showing submicromolar activity in HCV replicon assays. Further pharmacokinetic optimization for sufficient oral absorption and liver triphosphate loading led to identification of a clinical development candidate GS-6620. In a phase I clinical study, the potential for potent activity was demonstrated but with high intra- and interpatient pharmacokinetic and pharmacodynamic variability.
Asunto(s)
Antivirales/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Hepacivirus/enzimología , Hepatitis C/tratamiento farmacológico , Nucleósidos/farmacología , Compuestos Organofosforados/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/farmacocinética , Antivirales/farmacología , Perros , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Hepatitis C/enzimología , Hepatitis C/virología , Humanos , Nucleósidos/química , Compuestos Organofosforados/química , Ratas , Carga ViralRESUMEN
One of the most challenging goals of hepatitis C virus (HCV) research is to develop well-tolerated regimens with high cure rates across a variety of patient populations. Such a regimen will likely require a combination of at least two distinct direct-acting antivirals (DAAs). Combining two or more DAAs with different resistance profiles increases the number of mutations required for viral breakthrough. Currently, most DAAs inhibit HCV replication. We recently reported that the combination of two distinct classes of HCV inhibitors, entry inhibitors and replication inhibitors, prolonged reductions in extracellular HCV in persistently infected cells. We therefore sought to identify new inhibitors targeting aspects of the HCV replication cycle other than RNA replication. We report here the discovery of the first small-molecule HCV infectivity inhibitor, GS-563253, also called HCV infectivity inhibitor 1 (HCV II-1). HCV II-1 is a substituted tetrahydroquinoline that selectively inhibits genotype 1 and 2 HCVs with low-nanomolar 50% effective concentrations. It was identified through a high-throughput screen and subsequent chemical optimization. HCV II-1 only permits the production and release of noninfectious HCV particles from cells. Moreover, infectious HCV is rapidly inactivated in its presence. HCV II-1 resistance mutations map to HCV E2. In addition, HCV-II prevents HCV endosomal fusion, suggesting that it either locks the viral envelope in its prefusion state or promotes a viral envelope conformation change incapable of fusion. Importantly, the discovery of HCV II-1 opens up a new class of HCV inhibitors that prolong viral suppression by HCV replication inhibitors in persistently infected cell cultures.
Asunto(s)
Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Antivirales/química , Línea Celular , Farmacorresistencia Viral , Hepacivirus/metabolismo , Hepatitis C/tratamiento farmacológico , Humanos , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Theiler's disease is an acute hepatitis in horses that is associated with the administration of equine blood products; its etiologic agent has remained unknown for nearly a century. Here, we used massively parallel sequencing to explore samples from a recent Theiler's disease outbreak. Metatranscriptomic analysis of the short sequence reads identified a 10.5-kb sequence from a previously undescribed virus of the Flaviviridae family, which we designate "Theiler's disease-associated virus" (TDAV). Phylogenetic analysis clusters TDAV with GB viruses of the recently proposed Pegivirus genus, although it shares only 35.3% amino acid identity with its closest relative, GB virus D. An epidemiological survey of additional horses from three separate locations supports an association between TDAV infection and acute serum hepatitis. Experimental inoculation of horses with TDAV-positive plasma provides evidence that several weeks of viremia preceded liver injury and that liver disease may not be directly related to the level of viremia. Like hepatitis C virus, the best characterized Flaviviridae species known to cause hepatitis, we find TDAV is capable of efficient parenteral transmission, engendering acute and chronic infections associated with a diversity of clinical presentations ranging from subclinical infection to clinical hepatitis.
Asunto(s)
Infecciones por Flaviviridae/veterinaria , Flaviviridae/genética , Hepatitis Viral Animal/virología , Caballos/virología , Animales , Toxinas Botulínicas/metabolismo , Análisis por Conglomerados , Brotes de Enfermedades , Infecciones por Flaviviridae/virología , Biblioteca de Genes , Genoma Viral , Metagenómica , Datos de Secuencia Molecular , Filogenia , ARN Viral/metabolismo , Análisis de Secuencia de ADNRESUMEN
Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by off target effects. Development of antiviral ribonucleosides for treatment of hepatitis C virus (HCV) infection has been hampered by appearance of toxicity during clinical trials that evaded detection during preclinical studies. It is well established that the human mitochondrial DNA polymerase is an off target for deoxyribonucleoside reverse transcriptase inhibitors. Here we test the hypothesis that triphosphorylated metabolites of therapeutic ribonucleoside analogues are substrates for cellular RNA polymerases. We have used ribonucleoside analogues with activity against HCV as model compounds for therapeutic ribonucleosides. We have included ribonucleoside analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents that are non-obligate chain terminators of the HCV RNA polymerase. We show that all of the anti-HCV ribonucleoside analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Unexpectedly, analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents were inhibitors of POLRMT and Pol II. Importantly, the proofreading activity of TFIIS was capable of excising these analogues from Pol II transcripts. Evaluation of transcription in cells confirmed sensitivity of POLRMT to antiviral ribonucleosides, while Pol II remained predominantly refractory. We introduce a parameter termed the mitovir (mitochondrial dysfunction caused by antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity.