1. A High-Throughput HIV-1 Drug Screening Platform, Based on Lentiviral Vectors and Compatible with Biosafety Level-1
- Author
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Carol Stocking, Felix M Jäkel, Jannis Woens, Jeanette Reinshagen, Daniel Pohlmann, Kristoffer Riecken, V.S. Prassolov, Bernhard Ellinger, Boris Fehse, and Publica
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0301 basic medicine ,Anti-HIV Agents ,High-throughput screening ,BSL-1 screening platform ,mCat1 expressing PM1 T cell line ,030106 microbiology ,Genetic Vectors ,lcsh:QR1-502 ,Drug Evaluation, Preclinical ,lentiviral vectors ,Computational biology ,Biology ,Genome ,high-throughput screening ,lcsh:Microbiology ,Article ,Cell Line ,03 medical and health sciences ,Mice ,Drug Development ,Viral Envelope Proteins ,Virology ,Biosafety level ,Drug Discovery ,Animals ,Humans ,Luciferase ,Throughput (business) ,Drug discovery ,Cellular Assay ,Lentivirus ,Virion ,Containment of Biohazards ,Reverse transcriptase ,High-Throughput Screening Assays ,030104 developmental biology ,Infectious Diseases ,HEK293 Cells ,LeGO vectors ,HIV-1 ,HIV-1 drug development - Abstract
HIV-1 infection is a complex, multi-step process involving not only viral, but also multiple cellular factors. To date, drug discovery methods have primarily focused on the inhibition of single viral proteins. We present an efficient and unbiased approach, compatible with biosafety level 1 (BSL-1) conditions, to identify inhibitors of HIV-1 reverse transcription, intracellular trafficking, nuclear entry and genome integration. Starting with a fluorescent assay setup, we systematically improved the screening methodology in terms of stability, efficiency and pharmacological relevance. Stability and throughput were optimized by switching to a luciferase-based readout. BSL-1 compliance was achieved without sacrificing pharmacological relevance by using lentiviral particles pseudo-typed with the mouse ecotropic envelope protein to transduce human PM1 T cells gene-modified to express the corresponding murine receptor. The cellular assay was used to screen 26,048 compounds selected for maximum diversity from a 200,640-compound in-house library. This yielded z’ values greater than 0.8 with a hit rate of 3.3% and a confirmation rate of 50%. We selected 93 hits and enriched the collection with 279 similar compounds from the in-house library to identify promising structural features. The most active compounds were validated using orthogonal assay formats. The similarity of the compound profiles across the different platforms demonstrated that the reported lentiviral assay system is a robust and versatile tool for the identification of novel HIV-1 inhibitors.
- Published
- 2020
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