RESUMEN
Aneuploidy, an incorrect chromosome number, is a hallmark of cancer. Compounds that cause lethality in aneuploid, but not euploid, cells could therefore provide new cancer therapies. We have identified the energy stress-inducing agent AICAR, the protein folding inhibitor 17-AAG, and the autophagy inhibitor chloroquine as exhibiting this property. AICAR induces p53-mediated apoptosis in primary mouse embryonic fibroblasts (MEFs) trisomic for chromosome 1, 13, 16, or 19. AICAR and 17-AAG, especially when combined, also show efficacy against aneuploid human cancer cell lines. Our results suggest that compounds that interfere with pathways that are essential for the survival of aneuploid cells could serve as a new treatment strategy against a broad spectrum of human tumors.
Asunto(s)
Aneuploidia , Antineoplásicos/aislamiento & purificación , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis , Benzoquinonas/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Cloroquina/farmacología , Segregación Cromosómica , Embrión de Mamíferos/citología , Fibroblastos/efectos de los fármacos , Humanos , Lactamas Macrocíclicas/farmacología , Ratones , Neoplasias/tratamiento farmacológico , Ribonucleótidos/farmacología , TrisomíaRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been associated with more than 780,000 deaths worldwide (as of 20 August 2020). To develop antiviral interventions quickly, drugs used for the treatment of unrelated diseases are currently being repurposed to treat COVID-19. Chloroquine is an anti-malaria drug that is used for the treatment of COVID-19 as it inhibits the spread of SARS-CoV-2 in the African green monkey kidney-derived cell line Vero1-3. Here we show that engineered expression of TMPRSS2, a cellular protease that activates SARS-CoV-2 for entry into lung cells4, renders SARS-CoV-2 infection of Vero cells insensitive to chloroquine. Moreover, we report that chloroquine does not block infection with SARS-CoV-2 in the TMPRSS2-expressing human lung cell line Calu-3. These results indicate that chloroquine targets a pathway for viral activation that is not active in lung cells and is unlikely to protect against the spread of SARS-CoV-2 in and between patients.
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Cloroquina/farmacología , Cloroquina/uso terapéutico , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Pulmón/citología , Pulmón/efectos de los fármacos , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/virología , Animales , Betacoronavirus/efectos de los fármacos , COVID-19 , Línea Celular , Chlorocebus aethiops , Humanos , Técnicas In Vitro , Pulmón/virología , Pandemias , SARS-CoV-2 , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Insuficiencia del Tratamiento , Células Vero , Internalización del Virus , Tratamiento Farmacológico de COVID-19RESUMEN
Immune evasion is a major obstacle for cancer treatment. Common mechanisms of evasion include impaired antigen presentation caused by mutations or loss of heterozygosity of the major histocompatibility complex class I (MHC-I), which has been implicated in resistance to immune checkpoint blockade (ICB) therapy1-3. However, in pancreatic ductal adenocarcinoma (PDAC), which is resistant to most therapies including ICB4, mutations that cause loss of MHC-I are rarely found5 despite the frequent downregulation of MHC-I expression6-8. Here we show that, in PDAC, MHC-I molecules are selectively targeted for lysosomal degradation by an autophagy-dependent mechanism that involves the autophagy cargo receptor NBR1. PDAC cells display reduced expression of MHC-I at the cell surface and instead demonstrate predominant localization within autophagosomes and lysosomes. Notably, inhibition of autophagy restores surface levels of MHC-I and leads to improved antigen presentation, enhanced anti-tumour T cell responses and reduced tumour growth in syngeneic host mice. Accordingly, the anti-tumour effects of autophagy inhibition are reversed by depleting CD8+ T cells or reducing surface expression of MHC-I. Inhibition of autophagy, either genetically or pharmacologically with chloroquine, synergizes with dual ICB therapy (anti-PD1 and anti-CTLA4 antibodies), and leads to an enhanced anti-tumour immune response. Our findings demonstrate a role for enhanced autophagy or lysosome function in immune evasion by selective targeting of MHC-I molecules for degradation, and provide a rationale for the combination of autophagy inhibition and dual ICB therapy as a therapeutic strategy against PDAC.
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Adenocarcinoma/inmunología , Autofagia/inmunología , Carcinoma Ductal Pancreático/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Neoplasias Pancreáticas/inmunología , Escape del Tumor/inmunología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/inmunología , Autofagia/efectos de los fármacos , Autofagia/genética , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/inmunología , Línea Celular Tumoral , Cloroquina/farmacología , Femenino , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Masculino , Ratones , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Escape del Tumor/efectos de los fármacosRESUMEN
Glycinergic neurons regulate nociceptive and pruriceptive signaling in the spinal cord, but the identity and role of the glycine-regulated neurons are not fully known. Herein, we have characterized spinal glycine receptor alpha 3 (Glra3) subunit-expressing neurons in Glra3-Cre female and male mice. Glra3-Cre(+) neurons express Glra3, are located mainly in laminae III-VI, and respond to glycine. Chemogenetic activation of spinal Glra3-Cre(+) neurons induced biting/licking, stomping, and guarding behaviors, indicative of both a nociceptive and pruriceptive role for this population. Chemogenetic inhibition did not affect mechanical or thermal responses but reduced behaviors evoked by compound 48/80 and chloroquine, revealing a pruriceptive role for these neurons. Spinal cells activated by compound 48/80 or chloroquine express Glra3, further supporting the phenotype. Retrograde tracing revealed that spinal Glra3-Cre(+) neurons receive input from afferents associated with pain and itch, and dorsal root stimulation validated the monosynaptic input. In conclusion, these results show that spinal Glra3(+) neurons contribute to acute communication of compound 48/80- and chloroquine-induced itch in hairy skin.
Asunto(s)
Prurito , Receptores de Glicina , Médula Espinal , Animales , Prurito/inducido químicamente , Prurito/metabolismo , Ratones , Receptores de Glicina/metabolismo , Masculino , Femenino , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Cloroquina/farmacología , Ratones Transgénicos , Piel/inervación , Ratones Endogámicos C57BL , p-Metoxi-N-metilfenetilamina/farmacología , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/fisiologíaRESUMEN
BACKGROUND: In most of the Americas, the recommended treatment to prevent relapse of Plasmodium vivax malaria is primaquine at a total dose of 3.5 mg per kilogram of body weight, despite evidence of only moderate efficacy. METHODS: In this trial conducted in Brazil, we evaluated three primaquine regimens to prevent relapse of P. vivax malaria in children at least 5 years of age and in adults with microscopy-confirmed P. vivax monoinfection. All the patients received directly observed chloroquine for 3 days (total dose, 25 mg per kilogram). Group 1 received a total primaquine dose of 3.5 mg per kilogram (0.5 mg per kilogram per day) over 7 days with unobserved administration; group 2 received the same regimen as group 1 but with observed administration; and group 3 received a total primaquine dose of 7.0 mg per kilogram over 14 days (also 0.5 mg per kilogram per day) with observed administration. We monitored the patients for 168 days. RESULTS: We enrolled 63 patients in group 1, 96 in group 2, and 95 in group 3. The median age of the patients was 22.4 years (range, 5.4 to 79.8). By day 28, three P. vivax recurrences were observed: 2 in group 1 and 1 in group 2. By day 168, a total of 70 recurrences had occurred: 24 in group 1, 34 in group 2, and 12 in group 3. No serious adverse events were noted. On day 168, the percentage of patients without recurrence was 58% (95% confidence interval [CI], 44 to 70) in group 1, 59% (95% CI, 47 to 69) in group 2, and 86% (95% CI, 76 to 92) in group 3. Survival analysis showed a difference in the day 168 recurrence-free percentage of 27 percentage points (97.5% CI, 10 to 44; P<0.001) between group 1 and group 3 and a difference of 27 percentage points (97.5% CI, 12 to 42; P<0.001) between group 2 and group 3. CONCLUSIONS: The administration of primaquine at a total dose of 7.0 mg per kilogram had higher efficacy in preventing relapse of P. vivax malaria than a total dose of 3.5 mg per kilogram through day 168. (Supported by the U.S. Agency for International Development; ClinicalTrials.gov number, NCT03610399.).
Asunto(s)
Antimaláricos , Cloroquina , Malaria Vivax , Primaquina , Adolescente , Adulto , Anciano , Antimaláricos/administración & dosificación , Antimaláricos/efectos adversos , Antimaláricos/uso terapéutico , Brasil , Niño , Preescolar , Cloroquina/administración & dosificación , Cloroquina/efectos adversos , Cloroquina/uso terapéutico , Terapia por Observación Directa , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Humanos , Malaria Vivax/tratamiento farmacológico , Malaria Vivax/prevención & control , Persona de Mediana Edad , Primaquina/administración & dosificación , Primaquina/efectos adversos , Primaquina/uso terapéutico , Recurrencia , Prevención Secundaria , Adulto JovenRESUMEN
The chloroquine resistance transporter (PfCRT) confers resistance to a wide range of quinoline and quinoline-like antimalarial drugs in Plasmodium falciparum, with local drug histories driving its evolution and, hence, the drug transport specificities. For example, the change in prescription practice from chloroquine (CQ) to piperaquine (PPQ) in Southeast Asia has resulted in PfCRT variants that carry an additional mutation, leading to PPQ resistance and, concomitantly, to CQ re-sensitization. How this additional amino acid substitution guides such opposing changes in drug susceptibility is largely unclear. Here, we show by detailed kinetic analyses that both the CQ- and the PPQ-resistance conferring PfCRT variants can bind and transport both drugs. Surprisingly, the kinetic profiles revealed subtle yet significant differences, defining a threshold for in vivo CQ and PPQ resistance. Competition kinetics, together with docking and molecular dynamics simulations, show that the PfCRT variant from the Southeast Asian P. falciparum strain Dd2 can accept simultaneously both CQ and PPQ at distinct but allosterically interacting sites. Furthermore, combining existing mutations associated with PPQ resistance created a PfCRT isoform with unprecedented non-Michaelis-Menten kinetics and superior transport efficiency for both CQ and PPQ. Our study provides additional insights into the organization of the substrate binding cavity of PfCRT and, in addition, reveals perspectives for PfCRT variants with equal transport efficiencies for both PPQ and CQ.
Asunto(s)
Antimaláricos , Malaria Falciparum , Plasmodium falciparum , Quinolinas , Humanos , Antimaláricos/química , Cloroquina/farmacología , Cloroquina/uso terapéutico , Resistencia a Medicamentos/genética , Cinética , Malaria Falciparum/tratamiento farmacológico , Mutación , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Quinolinas/farmacología , Quinolinas/uso terapéuticoRESUMEN
Understanding and controlling the spread of antimalarial resistance, particularly to artemisinin and its partner drugs, is a top priority. Plasmodium falciparum parasites resistant to chloroquine, amodiaquine, or piperaquine harbor mutations in the P. falciparum chloroquine resistance transporter (PfCRT), a transporter resident on the digestive vacuole membrane that in its variant forms can transport these weak-base 4-aminoquinoline drugs out of this acidic organelle, thus preventing these drugs from binding heme and inhibiting its detoxification. The structure of PfCRT, solved by cryogenic electron microscopy, shows mutations surrounding an electronegative central drug-binding cavity where they presumably interact with drugs and natural substrates to control transport. P. falciparum susceptibility to heme-binding antimalarials is also modulated by overexpression or mutations in the digestive vacuole membrane-bound ABC transporter PfMDR1 (P. falciparum multidrug resistance 1 transporter). Artemisinin resistance is primarily mediated by mutations in P. falciparum Kelch13 protein (K13), a protein involved in multiple intracellular processes including endocytosis of hemoglobin, which is required for parasite growth and artemisinin activation. Combating drug-resistant malaria urgently requires the development of new antimalarial drugs with novel modes of action.
Asunto(s)
Antimaláricos/farmacología , Resistencia a Medicamentos/genética , Proteínas de Transporte de Membrana/genética , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Antimaláricos/uso terapéutico , Artemisininas/farmacología , Artemisininas/uso terapéutico , Cloroquina/farmacología , Cloroquina/uso terapéutico , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Mutación , Quinolinas/farmacología , Quinolinas/uso terapéuticoRESUMEN
Polymorphisms in the Plasmodium falciparum multidrug resistance protein 1 (pfmdr1) gene and the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene alter the malaria parasite's susceptibility to most of the current antimalarial drugs. However, the precise mechanisms by which PfMDR1 contributes to multidrug resistance have not yet been fully elucidated, nor is it understood why polymorphisms in pfmdr1 and pfcrt that cause chloroquine resistance simultaneously increase the parasite's susceptibility to lumefantrine and mefloquine-a phenomenon known as collateral drug sensitivity. Here, we present a robust expression system for PfMDR1 in Xenopus oocytes that enables direct and high-resolution biochemical characterizations of the protein. We show that wild-type PfMDR1 transports diverse pharmacons, including lumefantrine, mefloquine, dihydroartemisinin, piperaquine, amodiaquine, methylene blue, and chloroquine (but not the antiviral drug amantadine). Field-derived mutant isoforms of PfMDR1 differ from the wild-type protein, and each other, in their capacities to transport these drugs, indicating that PfMDR1-induced changes in the distribution of drugs between the parasite's digestive vacuole (DV) and the cytosol are a key driver of both antimalarial resistance and the variability between multidrug resistance phenotypes. Of note, the PfMDR1 isoforms prevalent in chloroquine-resistant isolates exhibit reduced capacities for chloroquine, lumefantrine, and mefloquine transport. We observe the opposite relationship between chloroquine resistance-conferring mutations in PfCRT and drug transport activity. Using our established assays for characterizing PfCRT in the Xenopus oocyte system and in live parasite assays, we demonstrate that these PfCRT isoforms transport all 3 drugs, whereas wild-type PfCRT does not. We present a mechanistic model for collateral drug sensitivity in which mutant isoforms of PfMDR1 and PfCRT cause chloroquine, lumefantrine, and mefloquine to remain in the cytosol instead of sequestering within the DV. This change in drug distribution increases the access of lumefantrine and mefloquine to their primary targets (thought to be located outside of the DV), while simultaneously decreasing chloroquine's access to its target within the DV. The mechanistic insights presented here provide a basis for developing approaches that extend the useful life span of antimalarials by exploiting the opposing selection forces they exert upon PfCRT and PfMDR1.
Asunto(s)
Antimaláricos , Malaria Falciparum , Parásitos , Animales , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Cloroquina/metabolismo , Cloroquina/farmacología , Cloroquina/uso terapéutico , Resistencia a Medicamentos/genética , Resistencia a Múltiples Medicamentos , Lumefantrina/farmacología , Lumefantrina/uso terapéutico , Malaria Falciparum/parasitología , Mefloquina/metabolismo , Mefloquina/farmacología , Mefloquina/uso terapéutico , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/uso terapéutico , Parásitos/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Triple-negative breast cancer is more common among younger than older women and is associated with the poorest survival outcomes of all breast cancer types. Fluvastatin inhibits tumour progression and induces the autophagy of breast cancer cells; however, the role of autophagy in fluvastatin-induced inhibition of breast cancer metastasis is unknown. Therefore, this study aimed to determine this mechanism. The effect of fluvastatin on human hormone receptor-negative breast cancer cells was evaluated in vitro via migration and wound healing assays, western blotting, and morphological measurements, as well as in vivo using a mouse xenograft model. Chloroquine, a prophylactic medication used to prevent malaria in humans was used as an autophagy inhibitor. We found that fluvastatin administration effectively prevented the migration/invasion of triple-negative breast cancer cells, an effect that was largely dependent on the induction of autophagy. Administration of the autophagy inhibitor chloroquine prevented the fluvastatin-induced suppression of lung metastasis in the nude mouse model. Furthermore, fluvastatin increased Ras homolog family member B (RhoB) expression, and the autophagy and anti-metastatic activity induced by fluvastatin were predominantly dependent on the regulation of RhoB through the protein kinase B-mammalian target of rapamycin (Akt-mTOR) signaling pathway. These results suggest that fluvastatin inhibits the metastasis of triple-negative breast cancer cells by modulating autophagy via the up regulation of RhoB through the AKT-mTOR signaling pathway. Fluvastatin may be a promising therapeutic option for patients with triple-negative breast cancer.
Asunto(s)
Neoplasias Pulmonares , Neoplasias de la Mama Triple Negativas , Animales , Femenino , Humanos , Ratones , Autofagia , Línea Celular Tumoral , Proliferación Celular , Cloroquina/farmacología , Cloroquina/uso terapéutico , Fluvastatina/farmacología , Fluvastatina/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/prevención & control , Mamíferos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide1. Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy2. Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite3-9. Here we present the structure, at 3.2 Å resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance5, but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively6,9, reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures.
Asunto(s)
Microscopía por Crioelectrón , Resistencia a Medicamentos/efectos de los fármacos , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/ultraestructura , Plasmodium falciparum/química , Proteínas Protozoarias/química , Proteínas Protozoarias/ultraestructura , Cloroquina/metabolismo , Cloroquina/farmacología , Resistencia a Medicamentos/genética , Concentración de Iones de Hidrógeno , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Modelos Moleculares , Mutación , Plasmodium falciparum/genética , Plasmodium falciparum/ultraestructura , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Quinolinas/metabolismo , Quinolinas/farmacologíaRESUMEN
The amygdala plays a key role in the processing of itch and pain signals as well as emotion. A previous study revealed that the central nucleus of the amygdala (CeA)-parabrachial nucleus (PBN) pathway is involved in pain regulation. The same pathway might also control itch. To test this possibility, prodynorphin (Pdyn)-Cre mice were used to optogenetically manipulate Pdyn+ CeA-to-PBN projections. We found that optogenetic stimulation of Pdyn+ amygdala neurons or Pdyn+ CeA-to-PBN projections inhibited histamine-evoked and chloroquine-evoked scratching. The number of Fos-positive neurons in the PBN increased following intradermal injection of chloroquine. Optogenetic stimulation of Pdyn+ CeA-to-PBN projections suppressed the increase in Fos expression in the PBN. Optogenetic stimulation of Pdyn+ CeA-to-PBN projections increased thermal and mechanical thresholds without affecting anxiety-like behavior. These results highlight the importance of dynorphinergic projections from the central amygdala to the parabrachial nucleus in the regulation of itch signaling.SIGNIFICANCE STATEMENT The central nucleus of the amygdala (CeA)-parabrachial nucleus (PBN) pathway regulates pain signaling. Using prodynorphin (Pdyn)-cre mice, we investigated the role of Pdyn+ CeA-to-PBN projections in itch. Optogenetic stimulation of Pdyn+ CeA-to-PBN projections inhibited pruritogen-evoked scratching and neuronal activity (c-Fos expression) in the PBN. Together, dynorphinergic projections from the central amygdala to the parabrachial nucleus are important for regulating itch information.
Asunto(s)
Núcleo Amigdalino Central , Núcleos Parabraquiales , Ratones , Animales , Dolor , Neuronas/fisiología , Prurito/inducido químicamente , CloroquinaRESUMEN
The HSP70 co-chaperone BAG3 targets unfolded proteins to degradation via chaperone assisted selective autophagy (CASA), thereby playing pivotal roles in the proteostasis of adult cardiomyocytes (CMs). However, the complex functions of BAG3 for regulating autophagy in cardiac disease are not completely understood. Here, we demonstrate that conditional inactivation of Bag3 in murine CMs leads to age-dependent dysregulation of autophagy, associated with progressive cardiomyopathy. Surprisingly, Bag3-deficient CMs show increased canonical and non-canonical autophagic flux in the juvenile period when first signs of cardiac dysfunction appear, but reduced autophagy during later stages of the disease. Juvenile Bag3-deficient CMs are characterized by decreased levels of soluble proteins involved in synchronous contraction of the heart, including the gap junction protein Connexin 43 (CX43). Reiterative administration of chloroquine (CQ), an inhibitor of canonical and non-canonical autophagy, but not inactivation of Atg5, restores normal concentrations of soluble cardiac proteins in juvenile Bag3-deficient CMs without an increase of detergent-insoluble proteins, leading to complete recovery of early-stage cardiac dysfunction in Bag3-deficient mice. We conclude that loss of Bag3 in CMs leads to age-dependent differences in autophagy and cardiac dysfunction. Increased non-canonical autophagic flux in the juvenile period removes soluble proteins involved in cardiac contraction, leading to early-stage cardiomyopathy, which is prevented by reiterative CQ treatment.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la Apoptosis , Autofagia , Cardiomiopatías , Miocitos Cardíacos , Animales , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Cardiomiopatías/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/deficiencia , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Ratones , Miocardio/metabolismo , Miocardio/patología , Cloroquina/farmacología , Ratones NoqueadosRESUMEN
The molecular basis of renal interstitial fibrosis, a major pathological feature of progressive kidney diseases, remains poorly understood. Autophagy has been implicated in renal fibrosis, but whether it promotes or inhibits fibrosis remains controversial. Moreover, it is unclear how autophagy is activated and sustained in renal fibrosis. The present study was designed to address these questions using the in vivo mouse model of unilateral ureteral obstruction and the in vitro model of hypoxia in renal tubular cells. Both models showed the activation of hypoxia-inducible factor-1 (HIF-1) and autophagy along with fibrotic changes. Inhibition of autophagy with chloroquine reduced renal fibrosis in unilateral ureteral obstruction model, whereas chloroquine and autophagy-related gene 7 knockdown decreased fibrotic changes in cultured renal proximal tubular cells, supporting a profibrotic role of autophagy. Notably, pharmacological and genetic inhibition of HIF-1 led to the suppression of autophagy and renal fibrosis in these models. Mechanistically, knock down of BCL2 and adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), a downstream target gene of HIF, decreased autophagy and fibrotic changes during hypoxia in BUMPT cells. Together, these results suggest that HIF-1 may activate autophagy via BNIP3 in renal tubular cells to facilitate the development of renal interstitial fibrosis.NEW & NOTEWORTHY Autophagy has been reported to participate in renal fibrosis, but its role and underlying activation mechanism is unclear. In this study, we report the role of HIF-1 in autophagy activation in models of renal fibrosis and further investigate the underlying mechanism.
Asunto(s)
Enfermedades Renales , Obstrucción Ureteral , Ratones , Animales , Obstrucción Ureteral/complicaciones , Obstrucción Ureteral/genética , Obstrucción Ureteral/metabolismo , Factor 1 Inducible por Hipoxia , Enfermedades Renales/patología , Hipoxia , Autofagia/genética , Fibrosis , Cloroquina/farmacologíaRESUMEN
Primaquine (PQ) is the main drug used to eliminate dormant liver stages and prevent relapses in Plasmodium vivax malaria. It also has an effect on the gametocytes of Plasmodium falciparum; however, it is unclear to what extent PQ affects P. vivax gametocytes. PQ metabolism involves multiple enzymes, including the highly polymorphic CYP2D6 and the cytochrome P450 reductase (CPR). Since genetic variability can impact drug metabolism, we conducted an evaluation of the effect of CYP2D6 and CPR variants on PQ gametocytocidal activity in 100 subjects with P. vivax malaria. To determine gametocyte density, we measured the levels of pvs25 transcripts in samples taken before treatment (D0) and 72 hours after treatment (D3). Generalized estimating equations (GEEs) were used to examine the effects of enzyme variants on gametocyte densities, adjusting for potential confounding factors. Linear regression models were adjusted to explore the predictors of PQ blood levels measured on D3. Individuals with the CPR mutation showed a smaller decrease in gametocyte transcript levels on D3 compared to those without the mutation (P = 0.02, by GEE). Consistent with this, higher PQ blood levels on D3 were associated with a lower reduction in pvs25 transcripts. Based on our findings, the CPR variant plays a role in the persistence of gametocyte density in P. vivax malaria. Conceptually, our work points to pharmacogenetics as a non-negligible factor to define potential host reservoirs with the propensity to contribute to transmission in the first days of CQ-PQ treatment, particularly in settings and seasons of high Anopheles human-biting rates.
Asunto(s)
Antimaláricos , Artemisininas , Malaria Falciparum , Malaria Vivax , Malaria , Humanos , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Malaria Vivax/tratamiento farmacológico , Malaria Falciparum/tratamiento farmacológico , NADPH-Ferrihemoproteína Reductasa , Cloroquina/farmacología , Citocromo P-450 CYP2D6/genética , Artemisininas/farmacología , Primaquina/farmacología , Primaquina/uso terapéutico , Malaria/tratamiento farmacológico , Plasmodium falciparum , Plasmodium vivax/genéticaRESUMEN
Malaria tropica, caused by the parasite Plasmodium falciparum (P. falciparum), remains one of the greatest public health burdens for humankind. Due to its pivotal role in parasite survival, the energy metabolism of P. falciparum is an interesting target for drug design. To this end, analysis of the central metabolite adenosine triphosphate (ATP) is of great interest. So far, only cell-disruptive or intensiometric ATP assays have been available in this system, with various drawbacks for mechanistic interpretation and partly inconsistent results. To address this, we have established fluorescent probes, based on Förster resonance energy transfer (FRET) and known as ATeam, for use in blood-stage parasites. ATeams are capable of measuring MgATP2- levels in a ratiometric manner, thereby facilitating in cellulo measurements of ATP dynamics in real-time using fluorescence microscopy and plate reader detection and overcoming many of the obstacles of established ATP analysis methods. Additionally, we established a superfolder variant of the ratiometric pH sensor pHluorin (sfpHluorin) in P. falciparum to monitor pH homeostasis and control for pH fluctuations, which may affect ATeam measurements. We characterized recombinant ATeam and sfpHluorin protein in vitro and stably integrated the sensors into the genome of the P. falciparum NF54attB cell line. Using these new tools, we found distinct sensor response patterns caused by several different drug classes. Arylamino alcohols increased and redox cyclers decreased ATP; doxycycline caused first-cycle cytosol alkalization; and 4-aminoquinolines caused aberrant proteolysis. Our results open up a completely new perspective on drugs' mode of action, with possible implications for target identification and drug development.
Asunto(s)
Adenosina Trifosfato , Antimaláricos , Transferencia Resonante de Energía de Fluorescencia , Plasmodium falciparum , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/metabolismo , Plasmodium falciparum/genética , Adenosina Trifosfato/metabolismo , Antimaláricos/farmacología , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Humanos , Quinina/farmacología , Doxiciclina/farmacología , Artemisininas/farmacología , Cloroquina/farmacología , Concentración de Iones de HidrógenoRESUMEN
Testing Plasmodium vivax antimicrobial sensitivity is limited to ex vivo schizont maturation assays, which preclude determining the IC50s of delayed action antimalarials such as doxycycline. Using Plasmodium cynomolgi as a model for P. vivax, we determined the physiologically significant delayed death effect induced by doxycycline [IC50(96 h), 1,401 ± 607 nM]. As expected, IC50(96 h) to chloroquine (20.4 nM), piperaquine (12.6 µM), and tafenoquine (1,424 nM) were not affected by extended exposure.
Asunto(s)
Aminoquinolinas , Antimaláricos , Doxiciclina , Piperazinas , Plasmodium cynomolgi , Plasmodium vivax , Doxiciclina/farmacología , Antimaláricos/farmacología , Aminoquinolinas/farmacología , Plasmodium vivax/efectos de los fármacos , Plasmodium cynomolgi/efectos de los fármacos , Cloroquina/farmacología , Animales , Malaria Vivax/tratamiento farmacológico , Malaria Vivax/parasitología , Quinolinas/farmacología , Concentración 50 Inhibidora , Humanos , Pruebas de Sensibilidad ParasitariaRESUMEN
The emergence of clinically drug-resistant malaria parasites requires the urgent development of new drugs. Mosquitoes are vectors of multiple pathogens and have developed resistance mechanisms against them, which often involve antimicrobial peptides (AMPs). An-cecB is an AMP of the malaria-transmitting mosquito genus Anopheles, and we herein report its antimalarial activity against Plasmodium falciparum 3D7, the artemisinin-resistant strain 803, and the chloroquine-resistant strain Dd2 in vitro. We also demonstrate its anti-parasite activity in vivo, using the rodent malaria parasite Plasmodium berghei (ANKA). We show that An-cecB displays potent antimalarial activity and that its mechanism of action may occur through direct killing of the parasite or through interaction with infected red blood cell membranes. Unfortunately, An-cecB was found to be cytotoxic to mammalian cells and had poor antimalarial activity in vivo. However, its truncated peptide An-cecB-1 retained most of its antimalarial activity and avoided its cytotoxicity in vitro. An-cecB-1 also showed better antimalarial activity in vivo. Mosquito-derived AMPs may provide new ideas for the development of antimalarial drugs against drug-resistant parasites, and An-cecB has potential use as a template for antimalarial peptides.
Asunto(s)
Anopheles , Antimaláricos , Plasmodium berghei , Plasmodium falciparum , Animales , Antimaláricos/farmacología , Anopheles/efectos de los fármacos , Anopheles/parasitología , Plasmodium falciparum/efectos de los fármacos , Plasmodium berghei/efectos de los fármacos , Ratones , Cecropinas/farmacología , Péptidos Antimicrobianos/farmacología , Péptidos Antimicrobianos/química , Malaria/tratamiento farmacológico , Malaria/parasitología , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Humanos , Mosquitos Vectores/efectos de los fármacos , Mosquitos Vectores/parasitología , Femenino , Proteínas de Insectos/farmacología , Resistencia a Medicamentos/efectos de los fármacos , Cloroquina/farmacología , Pruebas de Sensibilidad ParasitariaRESUMEN
Malaria is a severe disease caused by cytozoic parasites of the genus Plasmodium, which infiltrate and infect red blood cells. Several drugs have been developed to combat the devastating effects of malaria. Antimalarials based on quinolines inhibit the crystallization of hematin into hemozoin within the parasite, ultimately leading to its demise. Despite the frequent use of these agents, there are unanswered questions about their mechanisms of action. In the present study, the quinoline chloroquine and its interaction with the target structure hematin was investigated using an advanced, highly parallelized Raman difference spectroscopy (RDS) setup. Simultaneous recording of the spectra of hematin and chloroquine mixtures with varying compositions enabled the observation of changes in peak heights and positions based on the altered molecular structure resulting from their interaction. A shift of (-1.12 ± 0.05) cm-1 was observed in the core-size marker band ν(CαCm)asym peak position of the 1:1 chloroquine-hematin mixture compared to pure hematin. The oxidation-state marker band ν(pyrrole half-ring)sym exhibited a shift by (+0.93 ± 0.13) cm-1. These results were supported by density functional theory (DFT) calculations, indicating a hydrogen bond between the quinolinyl moiety of chloroquine and the oxygen atom of ferric protoporphyrin IX hydroxide (Fe(III)PPIX-OH). The consequence is a reduced electron density within the porphyrin moiety and an increase in its core size. This hypothesis provided further insights into the mechanism of hemozoin inhibition, suggesting chloroquine binding to the monomeric form of hematin, thereby preventing its further crystallization to hemozoin.
Asunto(s)
Antimaláricos , Hemoproteínas , Malaria , Humanos , Antimaláricos/farmacología , Cloroquina/farmacología , Cloroquina/química , Hemina/química , Hemoproteínas/química , Análisis Espectral , Plasmodium falciparumRESUMEN
Itch, a common somatic sensation, serves as a crucial protective system. Recent studies have unraveled the neural mechanisms of itch at peripheral, spinal cord as well as cerebral levels. However, a comprehensive understanding of the central mechanism governing itch transmission and regulation remains elusive. Here, we report the role of the medial septum (MS), an integral component of the basal forebrain, in modulating the acute itch processing. The increases in c-Fos+ neurons and calcium signals within the MS during acute itch processing were observed. Pharmacogenetic activation manipulation of global MS neurons suppressed the scratching behaviors induced by chloroquine or compound 48/80. Microinjection of GABA into the MS or pharmacogenetic inhibition of non-GABAergic neurons markedly suppressed chloroquine-induced scratching behaviors. Pharmacogenetic activation of the MS-ACC GABAergic pathway attenuated chloroquine-induced acute itch. Hence, our findings reveal that MS has a regulatory role in the chloroquine-induced acute itch through local increased GABA to inhibit non-GABAergic neurons and the activation of MS-ACC GABAergic pathway.
Asunto(s)
Cloroquina , Giro del Cíngulo , Prurito , Ácido gamma-Aminobutírico , Cloroquina/farmacología , Animales , Prurito/inducido químicamente , Prurito/metabolismo , Prurito/tratamiento farmacológico , Masculino , Ácido gamma-Aminobutírico/metabolismo , Giro del Cíngulo/metabolismo , Giro del Cíngulo/efectos de los fármacos , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones , Núcleos Septales/metabolismo , Núcleos Septales/efectos de los fármacosRESUMEN
Interfering with intratumoral metabolic processes is proven to effectively sensitize different antitumor treatments. Here, a tumor-targeting catalytic nanoplatform (CQ@MIL-GOX@PB) loading with autophagy inhibitor (chloroquine, CQ) and glucose oxidase (GOX) is fabricated to interfere with the metabolisms of tumor cells and tumor-associated macrophages (TAMs), then realizing effective antitumor chemodynamic therapy (CDT). Once accumulating in the tumor site with the navigation of external biotin, CQ@MIL-GOX@PB will release Fe ions and CQ in the acid lysosomes of tumor cells, the latter can sensitize Fe ions-involved antitumor CDT by blocking the autophagy-dependent cell repair. Meanwhile, the GOX component will consume glucose, which not only generates many H2O2 for CDT but also once again decelerates the tumor repair process by reducing energy metabolism. What is more, the release of CQ can also drive the NO anabolism of TAMs to further sensitize CDT. This strategy of multiple metabolic regulations is evidenced to significantly improve the antitumor effect of traditional CDT nanoagents and might provide a new sight to overcome the bottlenecks of different antitumor treatments.