A platform of assays for the discovery of anti-Zika small-molecules with activity in a 3D-bioprinted outer-blood-retina model

Abstract

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.

High throughput 3D gel-based neural organotypic model for cellular assays using fluorescence biosensors

“…It has also been shown that addition or enhancement of ECM deposition in neural 2D co-cultures accelerated the formation of neural networks, and in one example, the drug responses were more similar to those obtained in vivo 11,12 , thus reproducing evidence from in vivo experiments that ECM plays an important role in the development and function of the neural circuitry in the brain. Similar data has now also been shown for many non-neuronal 3D tissue models [13][14][15] .…”

Advances in the engineering of the outer blood-retina barrier: From in-vitro modelling to cellular therapy

“…Since these early developments, multiple oBRB models have used hiPSCs, most frequently derived from dermal fibroblasts [ 65 , 98 , [111] , [112] , [113] , [137] , [138] , [139] ]. The use of hiPSCs has notably enabled the development of multiple disease models by deriving hiPSCs-RPE cells from fibroblasts from patients with retinal diseases [ 58 , 65 , 66 , 98 , 112 , 140 ].…”

“…The advantage of using iPSCs has already been mentioned before (section 4.1.1 ). In the case of co-culture models, the ability to obtain both iPSCs derived RPE cells and ECs from the same patient offers the possibility to create disease models for personalized medicine [ 112 , 113 , 139 ]. To our knowledge, only three groups have used hiPSCs-derived ECs, characterized by the expression of CD31, ETV2 and vWF markers [ 112 , 113 , 139 ].…”

“…In the case of co-culture models, the ability to obtain both iPSCs derived RPE cells and ECs from the same patient offers the possibility to create disease models for personalized medicine [ 112 , 113 , 139 ]. To our knowledge, only three groups have used hiPSCs-derived ECs, characterized by the expression of CD31, ETV2 and vWF markers [ 112 , 113 , 139 ]. Cell phenotype of differentiated cells differed from choroidal cells, with lack of fenestra.…”

Generation of Reporter-Expressing New World Arenaviruses: A Systematic Comparison

“…Unfortunately, there are currently few resources available for such work and, in particular, traditional virus titration-based analysis of virus infection is cumbersome and presents a challenge to the use of these viruses in larger-scale applications. For other virus families, the development of reporter-protein-expressing viruses has proven valuable for antiviral drug discovery efforts [ 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ], as well as downstream mechanism of action studies [ 29 , 30 ], and even the identification of host factors required for the viral lifecycle [ 31 , 32 , 33 , 34 ], which can themselves then be leveraged as targets for treatment. Further, for arenaviruses, recent work has shown that the development of reporter protein-expressing arenaviruses is possible [ 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 ], and some have even been successfully used for high-throughput screening [ 35 , 36 , 44 ].…”