RESUMEN
The efficacy of T cell-based immunotherapies is limited by immunosuppressive pressures in the tumor microenvironment. Here we show a predominant role for the interaction between BTLA on effector T cells and HVEM (TNFRSF14) on immunosuppressive tumor microenvironment cells, namely regulatory T cells. High BTLA expression in chimeric antigen receptor (CAR) T cells correlated with poor clinical response to treatment. Therefore, we deleted BTLA in CAR T cells and show improved tumor control and persistence in models of lymphoma and solid malignancies. Mechanistically, BTLA inhibits CAR T cells via recruitment of tyrosine phosphatases SHP-1 and SHP-2, upon trans engagement with HVEM. BTLA knockout thus promotes CAR signaling and subsequently enhances effector function. Overall, these data indicate that the BTLA-HVEM axis is a crucial immune checkpoint in CAR T cell immunotherapy and warrants the use of strategies to overcome this barrier.
Asunto(s)
Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos , Receptores Inmunológicos , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Microambiente Tumoral , Animales , Humanos , Inmunoterapia Adoptiva/métodos , Miembro 14 de Receptores del Factor de Necrosis Tumoral/metabolismo , Miembro 14 de Receptores del Factor de Necrosis Tumoral/inmunología , Miembro 14 de Receptores del Factor de Necrosis Tumoral/genética , Ratones , Microambiente Tumoral/inmunología , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Receptores Quiméricos de Antígenos/genética , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/genética , Linfocitos T Reguladores/inmunología , Transducción de Señal , Línea Celular Tumoral , Neoplasias/inmunología , Neoplasias/terapia , Ratones NoqueadosRESUMEN
Inhibition of LSD1 was proposed as promising and attractive therapies for treating osteoporosis. Here, we synthesized a series of novel TCP-(MP)-Caffeic acid analogs as potential LSD1 inhibitors to assess their inhibitory effects on osteoclastogenesis by using TRAP-staining assay and try to explore the preliminary SAR. Among them, TCP-MP-CA (11a) demonstrated osteoclastic bone loss both in vitro and in vivo, showing a significant improvement in the in vivo effects compared to the LSD1 inhibitor GSK-LSD1. Additionally, we elucidated a mechanism that 11a and its precursor that 11e directly bind to LSD1/CoREST complex through FAD to inhibit LSD1 demethylation activity and influence its downstream IκB/NF-κB signaling pathway, and thus regulate osteoclastic bone loss. These findings suggested 11a or 11e as potential novel candidates for treating osteoclastic bone loss, and a concept for further development of TCP-(MP)-Caffeic acid analogs for therapeutic use in osteoporosis clinics.
Asunto(s)
Ácidos Cafeicos , Osteoclastos , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Ácidos Cafeicos/farmacología , Ácidos Cafeicos/química , Ácidos Cafeicos/síntesis química , Animales , Relación Estructura-Actividad , Ratones , Estructura Molecular , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Humanos , Osteoporosis/tratamiento farmacológico , Resorción Ósea/tratamiento farmacológico , Células RAW 264.7 , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis químicaRESUMEN
Mertk, a type I receptor tyrosine kinase and member of the TAM family of receptors, has important functions in promoting efferocytosis and resolving inflammation under physiological conditions. In recent years, Mertk has also been linked to pathophysiological roles in cancer, whereby, in several cancer types, including solid cancers and leukemia/lymphomas. Mertk contributes to oncogenic features of proliferation and cell survival as an oncogenic tyrosine kinase. In addition, Mertk expressed on macrophages, including tumor-associated macrophages, promotes immune evasion in cancer and is suggested to act akin to a myeloid checkpoint inhibitor that skews macrophages towards inhibitory phenotypes that suppress host T-cell anti-tumor immunity. In the present study, to better understand the post-translational regulation mechanisms controlling Mertk expression in monocytes/macrophages, we used a PMA-differentiated THP-1 cell model to interrogate the regulation of Mertk expression and developed a novel Mertk reporter cell line to study the intracellular trafficking of Mertk. We show that PMA treatment potently up-regulates Mertk as well as components of the ectodomain proteolytic processing platform ADAM17, whereas PMA differentially regulates the canonical Mertk ligands Gas6 and Pros1 (Gas6 is down-regulated and Pros1 is up-regulated). Under non-stimulated homeostatic conditions, Mertk in PMA-differentiated THP1 cells shows active constitutive proteolytic cleavage by the sequential activities of ADAM17 and the Presenilin/γ-secretase complex, indicating that Mertk is cleaved homeostatically by the combined sequential action of ADAM17 and γ-secretase, after which the cleaved intracellular fragment of Mertk is degraded in a proteasome-dependent mechanism. Using chimeric Flag-Mertk-EGFP-Myc reporter receptors, we confirm that inhibitors of γ-secretase and MG132, which inhibits the 26S proteasome, stabilize the intracellular fragment of Mertk without evidence of nuclear translocation. Finally, the treatment of cells with active γ-carboxylated Gas6, but not inactive Warfarin-treated non-γ-carboxylated Gas6, regulates a distinct proteolytic itinerary-involved receptor clearance and lysosomal proteolysis. Together, these results indicate that pleotropic and complex proteolytic activities regulate Mertk ectodomain cleavage as a homeostatic negative regulatory event to safeguard against the overactivation of Mertk.
Asunto(s)
Proteína ADAM17 , Secretasas de la Proteína Precursora del Amiloide , Proteolisis , Tirosina Quinasa c-Mer , Humanos , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa c-Mer/genética , Proteína ADAM17/metabolismo , Proteína ADAM17/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células THP-1 , Macrófagos/metabolismo , Proteína S/metabolismo , Monocitos/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
BACKGROUND: Commercial anti-CD19 chimeric antigen receptor T-cell therapies (CART19) are efficacious against advanced B-cell non-Hodgkin lymphoma (NHL); however, most patients ultimately relapse. Several mechanisms contribute to this failure, including CD19-negative escape and CAR T dysfunction. All four commercial CART19 products utilize the FMC63 single-chain variable fragment (scFv) specific to a CD19 membrane-distal epitope and characterized by slow association (on) and dissociation (off) rates. We hypothesized that a novel anti-CD19 scFv that engages an alternative CD19 membrane-proximal epitope independent of FMC63 and that is characterized by faster on- and off-rates could mitigate CART19 failure and improve clinical efficacy. METHODS: We developed an autologous CART19 product with 4-1BB co-stimulation using a novel humanized chicken antibody (h1218). This antibody is specific to a membrane-proximal CD19 epitope and harbors faster on/off rates compared to FMC63. We tested h1218-CART19 in vitro and in vivo using FMC63-CART19-resistant models. We conducted a first-in-human multi-center phase I clinical trial to test AT101 (clinical-grade h1218-CART19) in patients with relapsed or refractory (r/r) NHL. RESULTS: Preclinically, h1218- but not FMC63-CART19 were able to effectively eradicate lymphomas expressing CD19 point mutations (L174V and R163L) or co-expressing FMC63-CAR19 as found in patients relapsing after FMC63-CART19. Furthermore, h1218-CART19 exhibited enhanced killing of B-cell malignancies in vitro and in vivo compared with FMC63-CART19. Mechanistically, we found that h1218-CART19 had reduced activation-induced cell death (AICD) and enhanced expansion compared to FMC63-CART19 owing to faster on- and off-rates. Based on these preclinical results, we performed a phase I dose-escalation trial, testing three dose levels (DL) of AT101 (the GMP version of h1218) using a 3 + 3 design. In 12 treated patients (7 DLBCL, 3 FL, 1 MCL, and 1 MZL), AT101 showed a promising safety profile with 8.3% grade 3 CRS (n = 1) and 8.3% grade 4 ICANS (n = 1). In the whole cohort, the overall response rate was 91.7%, with a complete response rate of 75.0%, which improved to 100% in DL-2 and -3. AT101 expansion correlates with CR and B-cell aplasia. CONCLUSIONS: We developed a novel, safe, and potent CART19 product that recognizes a membrane-proximal domain of CD19 with fast on- and off-rates and showed significant efficacy and promising safety in patients with relapsed B-cell NHL. TRIAL REGISTRATION: NCT05338931; Date: 2022-04-01.
Asunto(s)
Linfoma no Hodgkin , Receptores de Antígenos de Linfocitos T , Receptores Quiméricos de Antígenos , Humanos , Anticuerpos , Antígenos CD19 , Epítopos/metabolismo , Inmunoterapia Adoptiva/efectos adversos , Linfoma no Hodgkin/terapia , Linfoma no Hodgkin/metabolismo , Recurrencia Local de Neoplasia/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidoresRESUMEN
In this study we observed that human GD1c/GT1a/GQ1b synthase (hST8Sia V) is particularly expressed in human glioblastoma cells. To address the mechanism regulating human glioblastoma-specific gene expression of the hST8Sia V, after the transcription start site (TSS) was identified by the 5'-rapid amplification of cDNA end with total RNA from human glioblastoma U87MG cells, the 5'-flanking region (2.5 kb) of the hST8Sia V gene was isolated and its promoter activity was examined. By luciferase reporter assay, this 5'-flanking region revealed strong promoter activity in only U-87MG cells, but not in other tissue-derived cancer cells. 5'-deletion mutant analysis showed that the region from -1140 to -494 is crucial for transcription of the hST8Sia V gene in U87MG cells. This region contains the activator protein-1 (AP-1) binding site, the main target of the c-Jun N-terminal kinase (JNK) downstream. The AP-1 binding site at -1043/-1037 was proved to be indispensable for the hST8Sia V gene-specific expression in U87MG cells by site-directed mutagenesis. Moreover, the transcriptional activation of hST8Sia V gene in U87MG cells was strongly inhibited by a specific JNK inhibitor, SP600125. These results suggest that the hST8Sia V gene-specific expression in U87MG cells is controlled by JNK/AP-1 signaling pathway.
Asunto(s)
Glioblastoma , Humanos , Glioblastoma/genética , Factor de Transcripción AP-1/genética , Regiones Promotoras Genéticas/genética , Activación TranscripcionalRESUMEN
Drug repurposing approach was applied to find a potent antiviral agent against RNA viruses such as SARS-CoV-2, influenza viruses and dengue virus with a concise strategy of small change in parent molecular structure. For this purpose, ß-D-N4-hydroxycytidine (NHC, 1) with a broad spectrum of antiviral activity was chosen as the parent molecule. Among the prepared NHC analogs (8a-g, and 9) from uridine, ß-D-N4-O-isobutyrylcytidine (8a) showed potent activity against SARS-CoV-2 (EC50 3.50 µM), Flu A (H1N1) (EC50 5.80 µM), Flu A (H3N2) (EC50 7.30 µM), Flu B (EC50 3.40 µM) and DENV-2 (EC50 3.95 µM) in vitro. Furthermore, its potency against SARS-CoV-2 was >5-fold, 3.4-fold, and 3-fold compared to that of NHC (1), MK-4482 (2), and remdesivir (RDV) in vitro, respectively. Ultimately, compound 8a was expected to be a potent inhibitor toward RNA viruses as a viral mutagenic agent like MK-4482.
Asunto(s)
COVID-19 , Subtipo H1N1 del Virus de la Influenza A , Humanos , SARS-CoV-2 , Subtipo H3N2 del Virus de la Influenza A , Replicación Viral , Antivirales/químicaRESUMEN
Cisplatin-induced ototoxicity leads to hearing impairment, possibly through reactive oxygen species (ROS) production and DNA damage in cochlear hair cells (HC), although the exact mechanism is unknown. Avenanthramide-C (AVN-C), a natural, potent antioxidant, was evaluated in three study groups of normal adult C57Bl/6 mice (control, cisplatin, and AVN-C+cisplatin) for the prevention of cisplatin-induced hearing loss. Auditory brainstem responses and immunohistochemistry of outer hair cells (OHCs) were ascertained. Cell survival, ROS production, Phospho-H2AX-enabled tracking of DNA damage-repair kinetics, and expression levels of inflammatory cytokines (TNF-α, IL-1ß, IL6, iNOS, and COX2) were assessed using House Ear Institute-Organ of Corti 1 (HEI-OC1 Cells). In the in vivo mouse model, following cisplatin-induced damage, AVN-C decreased the hearing thresholds and sheltered all cochlear turns' OHCs. In HEI-OC1 cells, AVN-C preserved cell viability and decreased ROS production, whereas cisplatin enhanced both ROS levels and cell viability. In HEI-OC1 cells, AVN-C downregulated IL6, IL-1ß, TNF-α, iNOS, and COX2 production that was upregulated by cisplatin treatment. AVN-C attenuated the cisplatin-enhanced nuclear H2AX activation. AVN-C had a strong protective effect against cisplatin-induced ototoxicity through inhibition of ROS and inflammatory cytokine production and DNA damage and is thus a promising candidate for preventing cisplatin-induced sensorineural hearing loss.
Asunto(s)
Antineoplásicos , Pérdida Auditiva , Ototoxicidad , Ratones , Animales , Cisplatino/toxicidad , Cisplatino/metabolismo , Citocinas/metabolismo , Antineoplásicos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Ototoxicidad/etiología , Ototoxicidad/metabolismo , Interleucina-6/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ciclooxigenasa 2/metabolismo , Línea Celular , Apoptosis , Células Ciliadas Auditivas/metabolismo , Estrés Oxidativo , Pérdida Auditiva/inducido químicamente , Pérdida Auditiva/prevención & control , Pérdida Auditiva/metabolismo , Daño del ADNRESUMEN
The aim of this study was to examine whether rubrofusarin, an active ingredient of the Cassia species, has an antidepressive effect in chronic restraint stress (CRS) mouse model. Although acute treatment using rubrofusarin failed, chronic treatment using rubrofusarin ameliorated CRS-induced depressive symptoms. Rubrofusarin treatment significantly reduced the number of Fluoro-Jade B-positive cells and caspase-3 activation within the hippocampus of CRS-treated mice. Moreover, rubrofusarin treatment significantly increased the number of newborn neurons in the hippocampus of CRS-treated mice. CRS induced activation of glycogen synthase kinase-3ß and regulated development and DNA damage responses, and reductions in the extracellular-signal-regulated kinase pathway activity were also reversed by rubrofusarin treatment. Microglial activation and inflammasome markers, including nod-like receptor family pyrin domain containing 3 and adaptor protein apoptosis-associated speck-like protein containing CARD, which were induced by CRS, were ameliorated by rubrofusarin. Synaptic plasticity dysfunction within the hippocampus was also rescued by rubrofusarin treatment. Within in vitro experiments, rubrofusarin blocked corticosterone-induced long-term potentiation impairments. These were blocked by LY294002, which is an Akt inhibitor. Finally, we found that the antidepressant effects of rubrofusarin were blocked by an intracerebroventricular injection of LY294002. These results suggest that rubrofusarin ameliorated CRS-induced depressive symptoms through PI3K/Akt signaling.
Asunto(s)
Depresión/tratamiento farmacológico , Neuronas/efectos de los fármacos , Pironas/farmacología , Estrés Psicológico/tratamiento farmacológico , Animales , Antidepresivos/farmacología , Depresión/patología , Modelos Animales de Enfermedad , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Ratones , Neuronas/patología , Restricción Física/psicología , Transducción de Señal/efectos de los fármacos , Estrés Psicológico/patologíaRESUMEN
Alzheimer's disease (AD) is a neurodegenerative disease characterized by neurological dysfunction, including memory impairment, attributed to the accumulation of amyloid ß (Aß) in the brain. Although several studies reported possible mechanisms involved in Aß pathology, much remains unknown. Previous findings suggested that a protein regulated in development and DNA damage response 1 (REDD1), a stress-coping regulator, is an Aß-responsive gene involved in Aß cytotoxicity. However, we still do not know how Aß increases the level of REDD1 and whether REDD1 mediates Aß-induced synaptic dysfunction. To elucidate this, we examined the effect of Aß on REDD1-expression using acute hippocampal slices from mice, and the effect of REDD1 short hairpin RNA (shRNA) on Aß-induced synaptic dysfunction. Lastly, we observed the effect of REDD1 shRNA on memory deficit in an AD-like mouse model. Through the experiments, we found that Aß-incubated acute hippocampal slices showed increased REDD1 levels. Moreover, Aß injection into the lateral ventricle increased REDD1 levels in the hippocampus. Anisomycin, but not actinomycin D, blocked Aß-induced increase in REDD1 levels in the acute hippocampal slices, suggesting that Aß may increase REDD1 translation rather than transcription. Aß activated Fyn/ERK/S6 cascade, and inhibitors for Fyn/ERK/S6 or mGluR5 blocked Aß-induced REDD1 upregulation. REDD1 inducer, a transcriptional activator, and Aß blocked synaptic plasticity in the acute hippocampal slices. REDD1 inducer inhibited mTOR/Akt signaling. REDD1 shRNA blocked Aß-induced synaptic deficits. REDD1 shRNA also blocked Aß-induced memory deficits in passive-avoidance and object-recognition tests. Collectively, these results demonstrate that REDD1 participates in Aß pathology and could be a target for AD therapy.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/farmacología , Hipocampo/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Trastornos de la Memoria/metabolismo , Sinapsis/metabolismo , Factores de Transcripción/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Animales , Anisomicina/farmacología , Dactinomicina/farmacología , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Masculino , Trastornos de la Memoria/genética , Trastornos de la Memoria/patología , Pruebas de Memoria y Aprendizaje , Ratones , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-fyn/metabolismo , ARN Interferente Pequeño , Receptor del Glutamato Metabotropico 5/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/genética , Sinapsis/patología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
Human access to safe water has become a major problem in many parts of the world as increasing human activities continue to spill contaminants into our water systems. To guarantee the protection of the public as well as the environment, a rapid and sensitive way to detect contaminants is required. In this work, a paper-based microbial fuel cell was developed to act as a portable, single-use, on-site water quality sensor. The sensor was fabricated by combining two layers of paper for a simple, low-cost, and disposable design. To facilitate the use of the sensor for on-site applications, the bacterial cells were pre-inoculated onto the device by air-drying. To eliminate any variations, the voltage generated by the microorganism before and after the air-drying process was measured and calculated as an inhibition ratio. Upon the addition of different formaldehyde concentrations (0%, 0.001%, 0.005%, and 0.02%), the inhibition ratios obtained were 5.9 ± 0.7%, 6.9 ± 0.7%, 8.2 ± 0.6%, and 10.6 ± 0.2%, respectively. The inhibition ratio showed a good linearity with the formaldehyde concentrations at R2 = 0.931. Our new sensor holds great promise in monitoring water quality as a portable, low-cost, and on-site sensor.
Asunto(s)
Técnicas Biosensibles , Monitoreo del Ambiente , Contaminantes Químicos del Agua/aislamiento & purificación , Agua/química , Fuentes de Energía Bioeléctrica , Humanos , Aguas Residuales/análisis , Contaminantes Químicos del Agua/toxicidad , Calidad del Agua/normasRESUMEN
Nucleoside, nucleotide, and base analogs have been in the clinic for decades to treat both viral pathogens and neoplasms. More than 20% of patients on anticancer chemotherapy have been treated with one or more of these analogs. This review focuses on the chemical synthesis and biology of anticancer nucleoside, nucleotide, and base analogs that are FDA-approved and in clinical development since 2000. We highlight the cellular biology and clinical biology of analogs, drug resistance mechanisms, and compound specificity towards different cancer types. Furthermore, we explore analog syntheses as well as improved and scale-up syntheses. We conclude with a discussion on what might lie ahead for medicinal chemists, biologists, and physicians as they try to improve analog efficacy through prodrug strategies and drug combinations.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Nucleósidos/química , Nucleótidos/química , Antineoplásicos/química , Línea Celular , HumanosRESUMEN
Nucleoside analog inhibitors (NAIs) are an important class of antiviral agents. Although highly effective, some NAIs with activity against hepatitis C virus (HCV) can cause toxicity, presumably due to off-target inhibition of host mitochondrial RNA polymerase (POLRMT). The in vitro nucleotide substrate specificity of POLRMT was studied in order to explore structure-activity relationships that can facilitate the identification of nontoxic NAIs. These findings have important implications for the development of all anti-RNA virus NAIs.
Asunto(s)
Antivirales/farmacología , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Amidas/efectos adversos , Amidas/farmacología , Antivirales/efectos adversos , Dominio Catalítico/efectos de los fármacos , Humanos , Mitocondrias/genética , Nucleósidos/farmacología , Ácidos Fosfóricos/efectos adversos , Ácidos Fosfóricos/farmacología , Sofosbuvir/efectos adversos , Sofosbuvir/farmacología , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Ribonucleoside analog inhibitors (rNAI) target the hepatitis C virus (HCV) RNA-dependent RNA polymerase nonstructural protein 5B (NS5B) and cause RNA chain termination. Here, we expand our studies on ß-d-2'-C-methyl-2,6-diaminopurine-ribonucleotide (DAPN) phosphoramidate prodrug 1 (PD1) as a novel investigational inhibitor of HCV. DAPN-PD1 is metabolized intracellularly into two distinct bioactive nucleoside triphosphate (TP) analogs. The first metabolite, 2'-C-methyl-GTP, is a well-characterized inhibitor of NS5B polymerase, whereas the second metabolite, 2'-C-methyl-DAPN-TP, behaves as an adenosine base analog. In vitro assays suggest that both metabolites are inhibitors of NS5B-mediated RNA polymerization. Additional factors, such as rNAI-TP incorporation efficiencies, intracellular rNAI-TP levels, and competition with natural ribonucleotides, were examined in order to further characterize the potential role of each nucleotide metabolite in vivo Finally, we found that although both 2'-C-methyl-GTP and 2'-C-methyl-DAPN-TP were weak substrates for human mitochondrial RNA (mtRNA) polymerase (POLRMT) in vitro, DAPN-PD1 did not cause off-target inhibition of mtRNA transcription in Huh-7 cells. In contrast, administration of BMS-986094, which also generates 2'-C-methyl-GTP and previously has been associated with toxicity in humans, caused detectable inhibition of mtRNA transcription. Metabolism of BMS-986094 in Huh-7 cells leads to 87-fold higher levels of intracellular 2'-C-methyl-GTP than DAPN-PD1. Collectively, our data characterize DAPN-PD1 as a novel and potent antiviral agent that combines the delivery of two active metabolites.
Asunto(s)
Adenosina/análogos & derivados , Antivirales/farmacología , Guanosina Monofosfato/análogos & derivados , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Profármacos/farmacología , Sofosbuvir/farmacología , Adenosina/farmacología , Línea Celular , ARN Polimerasas Dirigidas por ADN/metabolismo , Guanosina Monofosfato/farmacología , Humanos , ARN/metabolismo , ARN Mitocondrial , ARN Viral/metabolismo , Ribonucleósidos/metabolismo , Transcripción Genética/efectos de los fármacos , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
New carbocyclic nucleoside analogs with five-membered heterocyclic nucleobases were synthesized and evaluated as potential anti-HIV and anti-HCV agents. Among the synthesized carbocyclic nucleoside analogs, the pyrazole amide 15f exhibited modest selective anti-HIV-1 activity (EC50 = 24 µM).
RESUMEN
The synthesis of 9-(ß-d-1,3-dioxolan-4-yl)2,6-diaminopurine nucleoside phosphoramidate prodrugs as well as various 2-amino-6-carbamoylpurine dioxolane derivatives and their phosphoramidates prodrugs is reported. Their ability to block HIV and HBV replication along with their cytotoxicity toward HepG2, human lymphocyte, CEM and Vero cells was also assessed.
Asunto(s)
Adenosina/análogos & derivados , Amidas/farmacología , Antivirales/farmacología , Dioxolanos/farmacología , VIH-1/efectos de los fármacos , Virus de la Hepatitis B/efectos de los fármacos , Nucleósidos/farmacología , Ácidos Fosfóricos/farmacología , Profármacos/farmacología , Adenosina/síntesis química , Adenosina/química , Adenosina/farmacología , Amidas/síntesis química , Amidas/química , Antivirales/síntesis química , Antivirales/química , Dioxolanos/síntesis química , Dioxolanos/química , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Nucleósidos/síntesis química , Nucleósidos/química , Ácidos Fosfóricos/síntesis química , Ácidos Fosfóricos/química , Profármacos/síntesis química , Profármacos/química , Relación Estructura-ActividadRESUMEN
In this study, the effects of 3,5,7,3',4'-pentamethoxyflavone (KP1), a major bioactive ingredient isolated from the Kaempferia parviflora rhizomes, on a neurite outgrowth in Neuro2a cells and its mechanism have been investigated. KP1 increased concentration-dependently the percentage of neurite-bearing cells. KP1 showed a remarkable capability to elicit neurite outgrowth in Neuro2a cells, as evidenced by morphological alterations and immunostaining using anti-class III ß-tubulin and anti-NeuN antibodies. KP1 also displayed a higher neurogenic activity than retinoic acid (RA), a promoter of neurite outgrowth in Neuro2a cells. KP1 treatment caused significant elevation in phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK) and glycogen synthase kinase-3ß (GSK-3ß). However, KP1-triggered neurite outgrowth was markedly inhibited by treatment with the ERK inhibitor U0126, whereas p38 MAPK inhibitor SB203580 and GSK-3ß inhibitor SB216763 did not influence KP1-induced neurite outgrowth. These results demonstrate that KP1 elicits neurite outgrowth and triggers cell differentiation of Neuro2a cells through ERK signal pathway.
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Sistema de Señalización de MAP Quinasas , Proyección Neuronal , Animales , Proyección Neuronal/efectos de los fármacos , Ratones , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Neuritas/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Fosforilación/efectos de los fármacos , Flavonoides/farmacología , Flavonas/farmacología , Flavonas/química , Línea Celular Tumoral , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Línea CelularRESUMEN
A direct synthetic strategy of aryloxy phosphonamidate nucleotide prodrugs (A, G, C, and U) was developed with the CM reaction assisted by ultrasonic irradiation and partitioned addition of 12 mol % of Hoveyda-Grubbs (H-G) II catalyst in 61-82% yields as a mixture of E-/Z-isomers (â¼2:1) from aryloxy vinylphosponamidate and 5'-vinyl nucleoside moieties.
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Profármacos , Profármacos/química , Profármacos/síntesis química , Profármacos/farmacología , Estructura Molecular , Catálisis , Nucleótidos/química , Nucleótidos/síntesis química , Ultrasonido , Ondas UltrasónicasRESUMEN
Bispecific T-cell engagers (BiTEs) bring together tumour cells and cytotoxic T cells by binding to specific cell-surface tumour antigens and T-cell receptors, and have been clinically successful for the treatment of B-cell malignancies. Here we show that a BiTE-sialidase fusion protein enhances the susceptibility of solid tumours to BiTE-mediated cytolysis of tumour cells via targeted desialylation-that is, the removal of terminal sialic acid residues on glycans-at the BiTE-induced T-cell-tumour-cell interface. In xenograft and syngeneic mouse models of leukaemia and of melanoma and breast cancer, and compared with the parental BiTE molecules, targeted desialylation via the BiTE-sialidase fusion proteins enhanced the formation of immunological synapses, T-cell activation and T-cell-mediated tumour-cell cytolysis in the presence of the target antigen. The targeted desialylation of tumour cells may enhance the potency of therapies relying on T-cell engagers.
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Neuraminidasa , Animales , Neuraminidasa/metabolismo , Humanos , Ratones , Línea Celular Tumoral , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , Linfocitos T/inmunología , Femenino , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Activación de Linfocitos , Ácido N-Acetilneuramínico/metabolismo , Ácido N-Acetilneuramínico/química , Ensayos Antitumor por Modelo de Xenoinjerto , Linfocitos T Citotóxicos/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Rationale: Promotion of mitophagy is considered a promising strategy for the treatment of neurodegenerative diseases including Alzheimer's disease (AD). The development of mitophagy-specific inducers with low toxicity and defined molecular mechanisms is essential for the clinical application of mitophagy-based therapy. The aim of this study was to investigate the potential of a novel small-molecule mitophagy inducer, ALT001, as a treatment for AD. Methods: ALT001 was developed through chemical optimization of an isoquinolium scaffold, which was identified from a chemical library screening using a mitophagy reporter system. In vitro and in vivo experiments were conducted to evaluate the potential of ALT001 as a mitophagy-targeting therapeutic agent and to investigate the molecular mechanisms underlying ALT001-induced mitophagy. The therapeutic effect of ALT001 was assessed in SH-SY5Y cells expressing mutant APP and mouse models of AD (5×FAD and PS2APP) by analyzing mitochondrial dysfunction and cognitive defects. Results: ALT001 specifically induces mitophagy both in vitro and in vivo but is nontoxic to mitochondria. Interestingly, we found that ALT001 induces mitophagy through the ULK1-Rab9-dependent alternative mitophagy pathway independent of canonical mitophagy pathway regulators such as ATG7 and PINK1. Importantly, ALT001 reverses mitochondrial dysfunction in SH-SY5Y cells expressing mutant APP in a mitophagy-dependent manner. ALT001 induces alternative mitophagy in mice and restores the decreased mitophagy level in a 5×FAD AD model mouse. In addition, ALT001 reverses mitochondrial dysfunction and cognitive defects in the PS2APP and 5×FAD AD mouse models. AAV-mediated silencing of Rab9 in the hippocampus further confirmed that ALT001 exerts its therapeutic effect through alternative mitophagy. Conclusion: Our results highlight the therapeutic potential of ALT001 for AD via alleviation of mitochondrial dysfunction and indicate the usefulness of the ULK1-Rab9 alternative mitophagy pathway as a therapeutic target.
Asunto(s)
Enfermedad de Alzheimer , Enfermedades Mitocondriales , Neuroblastoma , Humanos , Ratones , Animales , Enfermedad de Alzheimer/metabolismo , Mitofagia , Modelos Animales de Enfermedad , Isoquinolinas/farmacología , CogniciónRESUMEN
An efficient synthetic route to biologically relevant (-)-5-fluorocarbodine 6 was developed. Direct coupling of N(6)-protected 5-fluorouracil 15 with cyclopentenyl intermediate 13, followed by formation of a macrocycle between the base and the carbocyclic sugar moiety, via ring-closing metathesis, allowed for a facial selective hydrogenation of the sugar double bond to give, exclusively, the desired 4'-ß stereoisomer.