RESUMEN
In the present study, the effects of levamlodipine benzenesulfonate on the development of fertile Sprague-Dawley (SD) rats, their embryos, and littermates were assessed using an embryo-fetal developmental toxicity test. Maternal body weight reduction was observed at a dose of 20â¯mg/kg, but it recovered after treatment cessation. The 20â¯mg/kg dose group showed a skewed sex ratio in fetal rats, with a higher proportion of males. While some effects on fetal sternum development were observed at 20â¯mg/kg, no skeletal malformations were observed. No significant gross morphological abnormalities were detected in the dams (mothers), no significant embryotoxicity or foetotoxicity in fetal rats and no significant effects on fetal length and weight development at doses of 5 and 10â¯mg/kg. Genotoxicity was evaluated using a combination of the Ames test, the Chinese hamster ovary (CHO) cell chromosome aberration assay, and the ICR mouse bone marrow micronucleus test. The Ames test results indicated substantial bacteriostatic effects at doses of 500 and 5000â¯mg/dish, with no mutagenicity observed at doses of 0.5, 5, and 50â¯mg/dish. No significant effect on the aberration rate of CHO cell chromosomes was found at doses of 2.8, 5.6, and 11.2â¯mg/mL. In the ICR mouse micronucleus test, no micronucleus-inducing effect was observed at doses of 3.125, 6.25, and 12.5â¯mg/kg in each treatment group. In conclusion, under the conditions of this experiment, the no-observed-adverse-effect level (NOAEL) for developmental toxicity of levamlodipine benzenesulfonate in fertile SD rats, their embryos, and littermates was established to be 10â¯mg/kg/day. Levamlodipine benzenesulfonate did not exhibit significant genotoxicity.
RESUMEN
Bedaquiline (BDQ), a first-in-class diarylquinoline anti-tuberculosis drug, and its analogue, TBAJ-587, prevent the growth and proliferation of Mycobacterium tuberculosis by inhibiting ATP synthase1,2. However, BDQ also inhibits human ATP synthase3. At present, how these compounds interact with either M. tuberculosis ATP synthase or human ATP synthase is unclear. Here we present cryogenic electron microscopy structures of M. tuberculosis ATP synthase with and without BDQ and TBAJ-587 bound, and human ATP synthase bound to BDQ. The two inhibitors interact with subunit a and the c-ring at the leading site, c-only sites and lagging site in M. tuberculosis ATP synthase, showing that BDQ and TBAJ-587 have similar modes of action. The quinolinyl and dimethylamino units of the compounds make extensive contacts with the protein. The structure of human ATP synthase in complex with BDQ reveals that the BDQ-binding site is similar to that observed for the leading site in M. tuberculosis ATP synthase, and that the quinolinyl unit also interacts extensively with the human enzyme. This study will improve researchers' understanding of the similarities and differences between human ATP synthase and M. tuberculosis ATP synthase in terms of the mode of BDQ binding, and will allow the rational design of novel diarylquinolines as anti-tuberculosis drugs.
Asunto(s)
Antituberculosos , Diarilquinolinas , Imidazoles , ATPasas de Translocación de Protón Mitocondriales , Mycobacterium tuberculosis , Piperidinas , Piridinas , Humanos , Antituberculosos/farmacología , Antituberculosos/química , Sitios de Unión , Microscopía por Crioelectrón , Diarilquinolinas/química , Diarilquinolinas/farmacología , Imidazoles/química , Imidazoles/farmacología , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , ATPasas de Translocación de Protón Mitocondriales/química , ATPasas de Translocación de Protón Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/ultraestructura , Modelos Moleculares , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/efectos de los fármacos , Piperidinas/química , Piperidinas/farmacología , Subunidades de Proteína/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/antagonistas & inhibidores , Piridinas/química , Piridinas/farmacologíaRESUMEN
Respiratory syncytial virus (RSV) is the major cause of bronchiolitis and pneumonia in young children and the elderly. There are currently no approved RSV-specific therapeutic small molecules available. Using high-throughput antiviral screening, we identified an oral drug, the prenylation inhibitor lonafarnib, which showed potent inhibition of the RSV fusion process. Lonafarnib exhibited antiviral activity against both the RSV A and B genotypes and showed low cytotoxicity in HEp-2 and human primary bronchial epithelial cells (HBEC). Time-of-addition and pseudovirus assays demonstrated that lonafarnib inhibits RSV entry, but has farnesyltransferase-independent antiviral efficacy. Cryo-electron microscopy revealed that lonafarnib binds to a triple-symmetric pocket within the central cavity of the RSV F metastable pre-fusion conformation. Mutants at the RSV F sites interacting with lonafarnib showed resistance to lonafarnib but remained fully sensitive to the neutralizing monoclonal antibody palivizumab. Furthermore, lonafarnib dose-dependently reduced the replication of RSV in BALB/c mice. Collectively, lonafarnib could be a potential fusion inhibitor for RSV infection.
Asunto(s)
Piridinas , Infecciones por Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Proteínas Virales de Fusión , Humanos , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Infecciones por Virus Sincitial Respiratorio/genética , Piridinas/farmacología , Ratones , Animales , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Virus Sincitial Respiratorio Humano/genética , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/antagonistas & inhibidores , Farnesiltransferasa/antagonistas & inhibidores , Farnesiltransferasa/genética , Antivirales/farmacología , Antivirales/química , Piperidinas/farmacología , Piperidinas/química , Ratones Endogámicos BALB C , Conformación Proteica , DibenzocicloheptenosRESUMEN
Recombinant adeno-associated viruses (rAAVs) have emerged as promising gene therapy vectors due to their proven efficacy and safety in clinical applications. In non-human primates (NHPs), rAAVs are administered via suprachoroidal injection at a higher dose. However, high doses of rAAVs tend to increase additional safety risks. Here, we present a novel AAV capsid (AAVv128), which exhibits significantly enhanced transduction efficiency for photoreceptors and retinal pigment epithelial (RPE) cells, along with a broader distribution across the layers of retinal tissues in different animal models (mice, rabbits, and NHPs) following intraocular injection. Notably, the suprachoroidal delivery of AAVv128-anti-VEGF vector completely suppresses the Grade IV lesions in a laser-induced choroidal neovascularization (CNV) NHP model for neovascular age-related macular degeneration (nAMD). Furthermore, cryo-EM analysis at 2.1 Å resolution reveals that the critical residues of AAVv128 exhibit a more robust advantage in AAV binding, the nuclear uptake and endosome escaping. Collectively, our findings highlight the potential of AAVv128 as a next generation ocular gene therapy vector, particularly using the suprachoroidal delivery route.
Asunto(s)
Neovascularización Coroidal , Dependovirus , Terapia Genética , Vectores Genéticos , Epitelio Pigmentado de la Retina , Animales , Dependovirus/genética , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificación , Terapia Genética/métodos , Ratones , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/virología , Neovascularización Coroidal/terapia , Neovascularización Coroidal/genética , Conejos , Humanos , Técnicas de Transferencia de Gen , Degeneración Macular/terapia , Degeneración Macular/genética , Degeneración Macular/patología , Modelos Animales de Enfermedad , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Transducción Genética , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ratones Endogámicos C57BL , Retina/metabolismo , Retina/virología , Masculino , Células HEK293RESUMEN
The type I adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter DppABCD is believed to be responsible for the import of exogenous heme as an iron source into the cytoplasm of the human pathogen Mycobacterium tuberculosis (Mtb). Additionally, this system is also known to be involved in the acquisition of tri- or tetra-peptides. Here, we report the cryo-electron microscopy structures of the dual-function Mtb DppABCD transporter in three forms, namely, the apo, substrate-bound, and ATP-bound states. The apo structure reveals an unexpected and previously uncharacterized assembly mode for ABC importers, where the lipoprotein DppA, a cluster C substrate-binding protein (SBP), stands upright on the translocator DppBCD primarily through its hinge region and N-lobe. These structural data, along with biochemical studies, reveal the assembly of DppABCD complex and the detailed mechanism of DppABCD-mediated transport. Together, these findings provide a molecular roadmap for understanding the transport mechanism of a cluster C SBP and its translocator.