Abstract

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.

γ-Secretase is an aspartyl protease that cleaves multiple substrates including the amyloid precursor protein (APP) and the Notch proteins. Abnormal proteolysis of APP is involved in the pathogenesis of Alzheimer’s disease (AD) and overactive Notch signaling plays an oncogenic role in a variety of cancers. γ-Secretase has emerged as a promising target for drug development in the treatment of AD and cancer. Assays with increased capacity for high-throughput screening would allow for quicker screening of chemical libraries and facilitate inhibitor development. We have developed a homogeneous time-resolved fluorescence (HTRF)-based assay that makes use of a novel biotinylated recombinant APP substrate and solubilized membrane preparation as the source of the γ-secretase enzyme. The assay was miniaturized to a 1536-well format and validated in a pilot screen against a library of ∼3,000 compounds. The overall assay performance was robust due to a calculated Z′ factor of 0.74 and its demonstrated ability to identify known γ-secretase inhibitors such as pepstatin A. This validated assay can readily be used for primary screening against large chemical libraries searching for novel inhibitors of γ-secretase activity that may represent potential therapeutics for AD and a variety of neoplasms.