RESUMEN
The global health emergency posed by the outbreak of Zika virus (ZIKV), an arthropod-borne flavivirus causing severe neonatal neurological conditions, has subsided, but there continues to be transmission of ZIKV in endemic regions. As such, there is still a medical need for discovering and developing therapeutical interventions against ZIKV. To identify small-molecule compounds that inhibit ZIKV disease and transmission, we screened multiple small-molecule collections, mostly derived from natural products, for their ability to inhibit wild-type ZIKV. As a primary high-throughput screen, we used a viral cytopathic effect (CPE) inhibition assay conducted in Vero cells that was optimized and miniaturized to a 1536-well format. Suitably active compounds identified from the primary screen were tested in a panel of orthogonal assays using recombinant Zika viruses, including a ZIKV Renilla luciferase reporter assay and a ZIKV mCherry reporter system. Compounds that were active in the wild-type ZIKV inhibition and ZIKV reporter assays were further evaluated for their inhibitory effects against other flaviviruses. Lastly, we demonstrated that wild-type ZIKV is able to infect a 3D-bioprinted outer-blood-retina barrier tissue model and disrupt its barrier function, as measured by electrical resistance. One of the identified compounds (3-Acetyl-13-deoxyphomenone, NCGC00380955) was able to prevent the pathological effects of the viral infection on this clinically relevant ZIKV infection model.
Asunto(s)
Antivirales/farmacología , Modelos Biológicos , Impresión Tridimensional , Retina , Replicación Viral/efectos de los fármacos , Infección por el Virus Zika , Virus Zika/fisiología , Animales , Antivirales/química , Chlorocebus aethiops , Evaluación Preclínica de Medicamentos , Células Hep G2 , Humanos , Retina/metabolismo , Retina/virología , Células Vero , Replicación Viral/genética , Infección por el Virus Zika/tratamiento farmacológico , Infección por el Virus Zika/genética , Infección por el Virus Zika/metabolismoRESUMEN
Age-related Macular Degeneration (AMD), a blinding eye disease, is characterized by pathological protein- and lipid-rich drusen deposits underneath the retinal pigment epithelium (RPE) and atrophy of the RPE monolayer in advanced disease stages - leading to photoreceptor cell death and vision loss. Currently, there are no drugs that stop drusen formation or RPE atrophy in AMD. Here we provide an iPSC-RPE AMD model that recapitulates drusen and RPE atrophy. Drusen deposition is dependent on AMD-risk-allele CFH(H/H) and anaphylatoxin triggered alternate complement signaling via the activation of NF-κB and downregulation of autophagy pathways. Through high-throughput screening we identify two drugs, L-745,870, a dopamine receptor antagonist, and aminocaproic acid, a protease inhibitor that reduce drusen deposits and restore RPE epithelial phenotype in anaphylatoxin challenged iPSC-RPE with or without the CFH(H/H) genotype. This comprehensive iPSC-RPE model replicates key AMD phenotypes, provides molecular insight into the role of CFH(H/H) risk-allele in AMD, and discovers two candidate drugs to treat AMD.
Asunto(s)
Ácido Aminocaproico/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Degeneración Macular/tratamiento farmacológico , Piridinas/farmacología , Pirroles/farmacología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Alelos , Factor H de Complemento/genética , Factor H de Complemento/metabolismo , Evaluación Preclínica de Medicamentos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Degeneración Macular/genética , Degeneración Macular/metabolismo , Modelos Biológicos , Fenotipo , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
The brain's primary circadian pacemaker, the suprachiasmatic nucleus (SCN), is required to translate day-length and circadian rhythms into neuronal, hormonal, and behavioral rhythms. Here, we identify the homeodomain transcription factor ventral anterior homeobox 1 (Vax1) as required for SCN development, vasoactive intestinal peptide expression, and SCN output. Previous work has shown that VAX1 is required for gonadotropin-releasing hormone (GnRH/LHRH) neuron development, a neuronal population controlling reproductive status. Surprisingly, the ectopic expression of a Gnrh-Cre allele (Gnrhcre) in the SCN confirmed the requirement of both VAX1 (Vax1flox/flox:Gnrhcre, Vax1Gnrh-cre) and sine oculis homeobox protein 6 (Six6flox/flox:Gnrhcre, Six6Gnrh-cre) in SCN function in adulthood. To dissociate the role of Vax1 and Six6 in GnRH neuron and SCN function, we used another Gnrh-cre allele that targets GnRH neurons, but not the SCN (Lhrhcre). Both Six6Lhrh-cre and Vax1Lhrh-cre were infertile, and in contrast to Vax1Gnrh-cre and Six6Gnrh-cre mice, Six6Lhrh-cre and Vax1Lhrh-cre had normal circadian behavior. Unexpectedly, ~ 1/4 of the Six6Gnrh-cre mice were unable to entrain to light, showing that ectopic expression of Gnrhcre impaired function of the retino-hypothalamic tract that relays light information to the brain. This study identifies VAX1, and confirms SIX6, as transcription factors required for SCN development and function and demonstrates the importance of understanding how ectopic CRE expression can impact the results.