RESUMEN
Mechanisms of unassisted delivery of RNA therapeutics, including inhibitors of microRNAs, remain poorly understood. We observed that the hepatocellular carcinoma cell line SKHEP1 retains productive free uptake of a miR-21 inhibitor (anti-miR-21). Uptake of anti-miR-21, but not a mismatch (MM) control, induces expression of known miR-21 targets (DDAH1, ANKRD46) and leads to dose-dependent inhibition of cell growth. To elucidate mechanisms of SKHEP1 sensitivity to anti-miR-21, we conducted an unbiased shRNA screen that revealed tumor susceptibility gene 101 (TSG101), a component of the endosomal sorting complex required for transport (ESCRT-I), as an important determinant of anti-proliferative effects of anti-miR-21. RNA interference-mediated knockdown of TSG101 and another ESCRT-I protein, VPS28, improved uptake of anti-miR-21 in parental SKHEP1 cells and restored productive uptake to SKHEP1 clones with acquired resistance to anti-miR-21. Depletion of ESCRT-I in several additional cancer cell lines with inherently poor uptake resulted in improved activity of anti-miR-21. Finally, knockdown of TSG101 increased uptake of anti-miR-21 by cancer cells in vivo following systemic delivery. Collectively, these data support an important role for the ESCRT-I complex in the regulation of productive free uptake of anti-miRs and reveal potential avenues for improving oligonucleotide free uptake by cancer cells.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , MicroARNs/antagonistas & inhibidores , Neoplasias/metabolismo , Oligonucleótidos/metabolismo , Animales , Transporte Biológico , Línea Celular Tumoral , Proteínas de Unión al ADN/fisiología , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/fisiología , Femenino , Humanos , Ratones SCID , MicroARNs/metabolismo , Neoplasias/genética , Factores de Transcripción/fisiologíaRESUMEN
PSI-352938 is a novel cyclic phosphate prodrug of ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methylguanosine-5'-monophosphate with potent anti-HCV activity. In order to inhibit the NS5B RNA-dependent RNA polymerase, PSI-352938 must be metabolized to the active triphosphate form, PSI-352666. During in vitro incubations with PSI-352938, significantly larger amounts of PSI-352666 were formed in primary hepatocytes than in clone A hepatitis C virus (HCV) replicon cells. Metabolism and biochemical assays were performed to define the molecular mechanism of PSI-352938 activation. The first step, removal of the isopropyl group on the 3',5'-cyclic phosphate moiety, was found to be cytochrome P450 (CYP) 3A4 dependent, with other CYP isoforms unable to catalyze the reaction. The second step, opening of the cyclic phosphate ring, was catalyzed by phosphodiesterases (PDEs) 2A1, 5A, 9A, and 11A4, all known to be expressed in the liver. The role of these enzymes in the activation of PSI-352938 was confirmed in primary human hepatocytes, where prodrug activation was reduced by inhibitors of CYP3A4 and PDEs. The third step, removal of the O(6)-ethyl group on the nucleobase, was shown to be catalyzed by adenosine deaminase-like protein 1. The resulting monophosphate was consecutively phosphorylated to the diphosphate and to the triphosphate PSI-352666 by guanylate kinase 1 and nucleoside diphosphate kinase, respectively. In addition, formation of nucleoside metabolites was observed in primary hepatocytes, and ecto-5'-nucleotidase was able to dephosphorylate the monophosphate metabolites. Since CYP3A4 is highly expressed in the liver, the CYP3A4-dependent metabolism of PSI-352938 makes it an effective liver-targeted prodrug, in part accounting for the potent antiviral activity observed clinically.
Asunto(s)
Antivirales/metabolismo , Óxidos P-Cíclicos/metabolismo , Hepacivirus/efectos de los fármacos , Nucleósidos/metabolismo , Células Cultivadas , Citocromo P-450 CYP3A/metabolismo , Guanilato-Quinasas/metabolismo , Hepatocitos/metabolismo , Humanos , Nucleósido-Difosfato Quinasa/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismoRESUMEN
A phosphoramidate prodrug of 2'-deoxy-2'-α-fluoro-ß-C-methyluridine-5'-monophosphate, PSI-7851, demonstrates potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. PSI-7851 is a mixture of two diastereoisomers, PSI-7976 and PSI-7977, with PSI-7977 being the more active inhibitor of HCV RNA replication in the HCV replicon assay. To inhibit the HCV NS5B RNA-dependent RNA polymerase, PSI-7851 must be metabolized to the active triphosphate form. The first step, hydrolysis of the carboxyl ester by human cathepsin A (CatA) and/or carboxylesterase 1 (CES1), is a stereospecific reaction. Western blot analysis showed that CatA and CES1 are both expressed in primary human hepatocytes. However, expression of CES1 is undetectable in clone A replicon cells. Studies with inhibitors of CatA and/or CES1 indicated that CatA is primarily responsible for hydrolysis of the carboxyl ester in clone A cells, although in primary human hepatocytes, both CatA and CES1 contribute to the hydrolysis. Hydrolysis of the ester is followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the spontaneous elimination of phenol and the production of an alaninyl phosphate metabolite, PSI-352707, which is common to both isomers. The removal of the amino acid moiety of PSI-352707 is catalyzed by histidine triad nucleotide-binding protein 1 (Hint1) to give the 5'-monophosphate form, PSI-7411. siRNA-mediated Hint1 knockdown studies further indicate that Hint1 is, at least in part, responsible for converting PSI-352707 to PSI-7411. PSI-7411 is then consecutively phosphorylated to the diphosphate, PSI-7410, and to the active triphosphate metabolite, PSI-7409, by UMP-CMP kinase and nucleoside diphosphate kinase, respectively.
Asunto(s)
Antivirales/farmacocinética , Hepacivirus/fisiología , Profármacos/farmacocinética , Uridina Monofosfato/análogos & derivados , Proteínas no Estructurales Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Antivirales/farmacología , Hidrolasas de Éster Carboxílico/metabolismo , Catepsina A/metabolismo , Línea Celular , Hepatocitos/metabolismo , Hepatocitos/virología , Humanos , Hidrólisis , Nucleósido-Fosfato Quinasa/metabolismo , Profármacos/farmacología , ARN Viral/metabolismo , Sofosbuvir , Estereoisomerismo , Uridina Monofosfato/farmacocinética , Uridina Monofosfato/farmacología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/fisiologíaRESUMEN
Immune checkpoint blockade elicits durable anti-cancer responses in the clinic, however a large proportion of patients do not benefit from treatment. Several mechanisms of innate and acquired resistance to checkpoint blockade have been defined and include mutations of MHC I and IFNγ signaling pathways. However, such mutations occur in a low frequency of patients and additional mechanisms have yet to be elucidated. In an effort to better understand acquired resistance to checkpoint blockade, we generated a mouse tumor model exhibiting in vivo resistance to anti-PD-1 antibody treatment. MC38 tumors acquired resistance to PD-1 blockade following serial in vivo passaging. Lack of sensitivity to PD-1 blockade was not attributed to dysregulation of PD-L1 or ß2M expression, as both were expressed at similar levels in parental and resistant cells. Similarly, IFNγ signaling and antigen processing and presentation pathways were functional in both parental and resistant cell lines. Unbiased gene expression analysis was used to further characterize potential resistance mechanisms. RNA-sequencing revealed substantial differences in global gene expression, with tumors resistant to anti-PD-1 displaying a marked reduction in expression of immune-related genes relative to parental MC38 tumors. Indeed, resistant tumors exhibited reduced immune infiltration across multiple cell types, including T and NK cells. Pathway analysis revealed activation of TGFß and Notch signaling in anti-PD-1 resistant tumors, and activation of these pathways was associated with poorer survival in human cancer patients. While pharmacological inhibition of TGFß and Notch in combination with PD-1 blockade decelerated tumor growth, a local mRNA-based immunotherapy potently induced regression of resistant tumors, resulting in complete tumor remission, and resensitized tumors to treatment with anti-PD-1. Overall, this study describes a novel anti-PD-1 resistant mouse tumor model and underscores the role of two well-defined signaling pathways in response to immune checkpoint blockade. Furthermore, our data highlights the potential of intratumoral mRNA therapy in overcoming acquired resistance to PD-1 blockade.
Asunto(s)
Inmunoterapia , Neoplasias , Animales , Presentación de Antígeno , Modelos Animales de Enfermedad , Humanos , Ratones , ARN Mensajero/genéticaRESUMEN
Local immunotherapy ideally stimulates immune responses against tumors while avoiding toxicities associated with systemic administration. Current strategies for tumor-targeted, gene-based delivery, however, are limited by adverse effects such as off-targeting or antivector immunity. We investigated the intratumoral administration of saline-formulated messenger (m)RNA encoding four cytokines that were identified as mediators of tumor regression across different tumor models: interleukin-12 (IL-12) single chain, interferon-α (IFN-α), granulocyte-macrophage colony-stimulating factor, and IL-15 sushi. Effective antitumor activity of these cytokines relied on multiple immune cell populations and was accompanied by intratumoral IFN-γ induction, systemic antigen-specific T cell expansion, increased granzyme B+ T cell infiltration, and formation of immune memory. Antitumor activity extended beyond the treated lesions and inhibited growth of distant tumors and disseminated tumors. Combining the mRNAs with immunomodulatory antibodies enhanced antitumor responses in both injected and uninjected tumors, thus improving survival and tumor regression. Consequently, clinical testing of this cytokine-encoding mRNA mixture is now underway.
Asunto(s)
Citocinas , Neoplasias , Citocinas/genética , Humanos , Neoplasias/genética , Neoplasias/terapia , ARN MensajeroRESUMEN
Hepatocellular carcinoma (HCC) represents a serious public health challenge with few therapeutic options available to cancer patients.Wnt/ß-catenin pathway is thought to play a significant role in HCC pathogenesis. In this study, we confirmed high frequency of CTNNB1 (ß-catenin) mutations in two independent cohorts of HCC patients and demonstrated significant upregulation of ß-catenin protein in the overwhelming majority of HCC patient samples, patient-derived xenografts (PDX) and established cell lines. Using genetic tools validated for target specificity through phenotypic rescue experiments, we went on to investigate oncogenic dependency on ß-catenin in an extensive collection of human HCC cells lines. Our results demonstrate that dependency on ß-catenin generally tracks with its activation status. HCC cell lines that harbored activating mutations in CTNNB1 or displayed elevated levels of non-phosphorylated (active) ß-catenin were significantly more sensitive to ß-catenin siRNA treatment than cell lines with wild-type CTNNB1 and lower active ß-catenin. Finally, significant therapeutic benefit of ß-catenin knock-down was demonstrated in established HCC tumor xenografts using doxycycline-inducible shRNA system. ß-catenin downregulation and tumor growth inhibition was associated with reduction in AXIN2, direct transcriptional target of ß-catenin, and decreased cancer cell proliferation as measured by Ki67 staining. Taken together, our data highlight fundamental importance of aberrant ß-catenin signaling in the maintenance of oncogenic phenotype in HCC.
RESUMEN
UNLABELLED: Hepatocellular carcinoma (HCC) remains a significant clinical challenge with few therapeutic options available to cancer patients. MicroRNA 21-5p (miR-21) has been shown to be upregulated in HCC, but the contribution of this oncomiR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific single-stranded oligonucleotide inhibitors of miR-21 (anti-miRNAs) and used them to interrogate dependency on miR-21 in a panel of liver cancer cell lines. Treatment with anti-miR-21, but not with a mismatch control anti-miRNA, resulted in significant derepression of direct targets of miR-21 and led to loss of viability in the majority of HCC cell lines tested. Robust induction of caspase activity, apoptosis, and necrosis was noted in anti-miR-21-treated HCC cells. Furthermore, ablation of miR-21 activity resulted in inhibition of HCC cell migration and suppression of clonogenic growth. To better understand the consequences of miR-21 suppression, global gene expression profiling was performed on anti-miR-21-treated liver cancer cells, which revealed striking enrichment in miR-21 target genes and deregulation of multiple growth-promoting pathways. Finally, in vivo dependency on miR-21 was observed in two separate HCC tumor xenograft models. In summary, these data establish a clear role for miR-21 in the maintenance of tumorigenic phenotype in HCC in vitro and in vivo. IMPLICATIONS: miR-21 is important for the maintenance of the tumorigenic phenotype of HCC and represents a target for pharmacologic intervention.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Proliferación Celular/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Neoplasias Hepáticas/metabolismo , MicroARNs/metabolismo , Oligorribonucleótidos Antisentido/farmacología , Animales , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Xenoinjertos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Ratones Desnudos , MicroARNs/antagonistas & inhibidores , MicroARNs/química , Invasividad Neoplásica , Oligorribonucleótidos Antisentido/uso terapéuticoRESUMEN
Monoacylglycerol lipase (MAGL) represents a primary degradation enzyme of the endogenous cannabinoid (eCB), 2-arachidonoyglycerol (2-AG). This study reports a potent covalent MAGL inhibitor, SAR127303. The compound behaves as a selective and competitive inhibitor of mouse and human MAGL, which potently elevates hippocampal levels of 2-AG in mice. In vivo, SAR127303 produces antinociceptive effects in assays of inflammatory and visceral pain. In addition, the drug alters learning performance in several assays related to episodic, working and spatial memory. Moreover, long term potentiation (LTP) of CA1 synaptic transmission and acetylcholine release in the hippocampus, two hallmarks of memory function, are both decreased by SAR127303. Although inactive in acute seizure tests, repeated administration of SAR127303 delays the acquisition and decreases kindled seizures in mice, indicating that the drug slows down epileptogenesis, a finding deserving further investigation to evaluate the potential of MAGL inhibitors as antiepileptics. However, the observation that 2-AG hydrolysis blockade alters learning and memory performance, suggests that such drugs may have limited value as therapeutic agents.
Asunto(s)
Analgésicos/farmacología , Ácidos Araquidónicos/metabolismo , Carbamatos/farmacología , Endocannabinoides/metabolismo , Glicéridos/metabolismo , Aprendizaje/efectos de los fármacos , Memoria a Corto Plazo/efectos de los fármacos , Monoacilglicerol Lipasas/metabolismo , Sulfonamidas/farmacología , Acetilcolina/metabolismo , Administración Oral , Analgésicos/química , Analgésicos/uso terapéutico , Animales , Ácidos Araquidónicos/química , Sitios de Unión , Encéfalo/metabolismo , Antagonistas de Receptores de Cannabinoides/farmacología , Carbamatos/química , Carbamatos/uso terapéutico , Cromatografía Líquida de Alta Presión , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Estimulación Eléctrica , Endocannabinoides/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Glicéridos/química , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Hidrólisis , Técnicas In Vitro , Potenciación a Largo Plazo/efectos de los fármacos , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Monoacilglicerol Lipasas/antagonistas & inhibidores , Dolor/tratamiento farmacológico , Dolor/patología , Piperidinas/farmacología , Estructura Terciaria de Proteína , Pirazoles/farmacología , Rimonabant , Convulsiones/tratamiento farmacológico , Convulsiones/patología , Sulfonamidas/química , Sulfonamidas/uso terapéuticoRESUMEN
Response regulator proteins of two-component systems are usually activated by phosphorylation. The phosphorylated response regulator protein CheY-P mediates the chemotaxis response in Escherichia coli. We performed random mutagenesis and selected CheY mutants that are constitutively active in the absence of phosphorylation. Although a single amino acid substitution can lead to constitutive activation, no single DNA base change can effect such a transition. Numerous different sets of mutations that activate in synergy were selected in several different combinations. These mutations were all located on the side of CheY defined by alpha4, beta5, alpha5, and alpha1. Our findings argue against the two-state hypothesis for response regulator activation. We propose an alternative intermolecular mechanism that involves a dynamic interplay between response regulators and their effector targets.
Asunto(s)
Proteínas Bacterianas , Quimiotaxis , Escherichia coli/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Quimiotaxis/fisiología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/química , Proteínas Quimiotácticas Aceptoras de Metilo , Modelos Moleculares , Mutagénesis , Mutación , Fosforilación , Estructura Terciaria de Proteína , Alineación de Secuencia , Transducción de Señal/fisiologíaRESUMEN
PSI-353661, a phosphoramidate prodrug of 2'-deoxy-2'-fluoro-2'-C-methylguanosine-5'-monophosphate, is a highly active inhibitor of genotype 1a, 1b, and 2a HCV RNA replication in the replicon assay and of genotype 1a and 2a infectious virus replication. PSI-353661 is active against replicons harboring the NS5B S282T or S96T/N142T amino acid alterations that confer decreased susceptibility to nucleoside/tide analogs as well as mutations that confer resistance to non-nucleoside inhibitors of NS5B. Replicon clearance studies show that PSI-353661 was able to clear cells of HCV replicon RNA and prevent a rebound in replicon RNA. PSI-353661 showed no toxicity toward bone marrow stem cells or mitochondrial toxicity. The metabolism to the active 5'-triphosphate involves hydrolysis of the carboxyl ester by cathepsin A (Cat A) and carboxylesterase 1 (CES1) followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the elimination of phenol and the alaninyl phosphate metabolite, PSI-353131. Histidine triad nucleotide-binding protein 1 (Hint 1) then removes the amino acid moiety, which is followed by hydrolysis of the methoxyl group at the O(6)-position of the guanine base by adenosine deaminase-like protein 1 (ADAL1) to give 2'-deoxy-2'-fluoro-2'-C-methylguanosine-5'-monophosphate. The monophosphate is phosphorylated to the diphosphate by guanylate kinase. Nucleoside diphosphate kinase is the primary enzyme involved in phosphorylation of the diphosphate to the active triphosphate, PSI-352666. PSI-352666 is equally active against wild-type NS5B and NS5B containing the S282T amino acid alteration.
Asunto(s)
Antivirales/farmacología , Guanosina Monofosfato/análogos & derivados , Hepacivirus/efectos de los fármacos , Profármacos/farmacología , Replicación Viral/efectos de los fármacos , Biotransformación , Catepsina A/metabolismo , Cromatografía Líquida de Alta Presión , Clonación Molecular , Evaluación Preclínica de Medicamentos , Guanosina Monofosfato/antagonistas & inhibidores , Guanosina Monofosfato/farmacología , Guanilato-Quinasas/metabolismo , Células Hep G2 , Hepacivirus/genética , Hepacivirus/fisiología , Hepatocitos/efectos de los fármacos , Humanos , Ácido Láctico/metabolismo , Luciferasas/metabolismo , Pruebas de Sensibilidad Microbiana , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mutación , Proteínas del Tejido Nervioso/metabolismo , Fenol/metabolismo , Fosforilación , Profármacos/química , Replicón , Proteínas no Estructurales Virales/antagonistas & inhibidoresRESUMEN
BACKGROUND: To reduce the incidence of drug resistance and to maintain viral suppression, patients chronically infected with HBV might require combination therapy using two or more drugs with different resistance profiles. We investigated the activity of clevudine (CLV) in combination with other nucleoside/nucleotide analogues to determine if these combinations were compatible in vitro. METHODS: Using the HepAD38 cell line, which expresses wild-type HBV, and a real-time PCR assay, we tested the anti-HBV activity of CLV in combination with entecavir, lamivudine, adefovir, tenofovir and telbivudine (TBV). We evaluated the uptake and phosphorylation of CLV in the presence of TBV, using HepAD38 cells and primary hepatocytes to determine the effect of TBV on the phosphorylation of CLV and vice versa. Phosphorylation of TBV and CLV to their corresponding monophosphate by deoxycytidine kinase, thymidine kinase-1 and thymidine kinase-2, and the phosphorylation of TBV monophosphate and CLV monophosphate by thymidylate kinase was evaluated and compared. RESULTS: When CLV was combined with entecavir, lamivudine, adefovir or tenofovir, a synergistic antiviral effect was observed; however, the combination of CLV and TBV gave an antagonistic antiviral response. The results of in vitro metabolism and enzyme studies suggest that the antagonism observed with the CLV/TBV combination involves competition for uptake and phosphorylation. CONCLUSIONS: The results of our studies demonstrate that combination treatments can provide enhanced antiviral activity and, when used in conjunction with appropriate metabolic investigations, provide a rational basis for the design and development of combination regimens for treating chronic HBV infection.