Gene Expression
Nebulas
Gene Expression NebulasSummary: The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Here we describe a screening pipeline for the discovery of efficacious SARS-CoV-2 inhibitors. Two high-throughput, high-content imaging infection assays (one using HeLa cells expressing SARS-CoV-2 receptor ACE2 and the other using lung epithelial Calu-3 cells) were developed and used to screen ReFRAME, a best-in-class drug repurposing library. Among the promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 had most favorable in vitro activity, pharmacokinetic and human safety profiles, and both reduced SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. However, only MK-4482 effectively blocked SARS-CoV-2 infection in a hamster model, likely due to inadequate plasma exposure of nelfinavir.
Overall Design: Hamster lung from uninfected (U, n=2), vehicle treated (V, n=4) and MK-4482 (EIDD-2801) treated (T, n=4) samples were analyzed using RNASeq platform.
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| Growth Protocol: | Hamster lung samples were collected after two different treatment regimens. |
| Treatment Protocol: | Hamster were infected with SARS-CoV-2 using a BSL-3 facility. |
| Extract Protocol: | The lungs were harvested from the hamsters and homogenized in TRI-Reagent. RNA were extracted after infection using the Quick-RNA MicroPrep Kit (Zymo Research, USA) according to the manufacture’s instruction. RNA was converted into cDNA using the qScript™ cDNA SuperMix (Quantabio). |
| Library Construction Protocol: | RNA sequencing libraries were generated using the Illumina TruSeq Stranded Total RNA Library Prep Gold with TruSeq Unique Dual Indexes (Illumina, San Diego, CA). Samples were processed following manufacturer’s instructions, except modifying RNA shear time to five minutes. Resulting libraries were multiplexed and sequenced with 100 basepair (bp) Paired End (PE100) to a depth of approximately 25-40 million reads per sample on an Illumina NovaSeq 6000. Samples were demuxltiplexed using bcl2fastq v2.20 Conversion Software (Illumina, San Diego, CA). |
| Molecule Type: | polyA(+) RNA |
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| Library Layout: | PAIRED |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NovaSeq 6000 |
| Strand-Specific: | specific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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