High-content assay to identify inhibitors of dengue virus infection.
Dengue virus (DENV) infections are vectored by mosquitoes and constitute one of the most prevalent infectious diseases in many parts of the world, affecting millions of people annually. Current treatments for DENV infections are nonspecific and largely ineffective. In this study, we describe the adaptation of a high-content cell-based assay for screening against DENV-infected cells to identify inhibitors and modulators of DENV infection. Using this high-content approach, we monitored the inhibition of test compounds on DENV protein production by means of immunofluorescence staining of DENV glycoprotein envelope, simultaneously evaluating cytotoxicity in HEK293 cells. The adapted 384-well microtiter-based assay was validated using a small panel of compounds previously reported as having inhibitory activity against DENV infections of cell cultures, including compounds with antiviral activity (ribavirin), inhibitors of cellular signaling pathways (U0126), and polysaccharides that are presumed to interfere with virus attachment (carrageenan). A screen was performed against a collection of 5,632 well-characterized bioactives, including U.S. Food and Drug Administration-approved drugs. Assay control statistics show an average Z' of 0.63, indicative of a robust assay in this cell-based format. Using a threshold of >80% DENV inhibition with <20% cellular cytotoxicity, 79 compounds were initially scored as positive hits. A follow-up screen confirmed 73 compounds with IC₅₀ potencies ranging from 60 nM to 9 μM and yielding a hit rate of 1.3%. Over half of the confirmed hits are known to target transporters, receptors, and protein kinases, providing potential opportunity for drug repurposing to treat DENV infections. In summary, this assay offers the opportunity to screen libraries of chemical compounds, in an effort to identify and develop novel drug candidates against DENV infections.
HTS Core Facility, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.
David
D
Shum
Jessica L
JL
Smith
Alec J
AJ
Hirsch
Bhavneet
B
Bhinder
Constantin
C
Radu
David A
DA
Stein
Jay A
JA
Nelson
Klaus
K
Früh
Hakim
H
Djaballah
D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology* / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects; D003715:Dengue / Q000188:drug therapy*; D003716:Dengue Virus / Q000187:drug effects* / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D008826:Microbial Sensitivity Tests; D018613:Microscopy, Confocal; D008904:Miniaturization; D012984:Software; D014764:Viral Proteins / Q000378:metabolism; D053585:Virus Attachment / Q000187:drug effects
D000998:Antiviral Agents; D014764:Viral Proteins