Abstract Affordable and effective antiviral therapies are needed worldwide, especially against agents such as dengue virus that are endemic in underserved regions. Many antiviral compounds have been studied in cultured cells but are unsuitable for clinical applications due to pharmacokinetic profiles, side effects, or inconsistent efficacy across dengue serotypes. Such tool compounds can, however, aid in identifying clinically useful treatments. Here, computational screening (Rapid Overlay of Chemical Structures) was used to identify entries in an in silico database of safe-in-human compounds (SWEETLEAD) that display high chemical similarities to known inhibitors of dengue virus. Molecules known to inhibit dengue proteinase NS2B/3, dengue capsid, and the host autophagy pathway were used as query compounds. Following computational and initial virological screening, three FDA-approved compounds that resemble the tool molecules structurally, cause little toxicity and display strong antiviral activity in cultured cells were selected for further analysis. Pyrimethamine (IC 50 = 1.2 µM), like the dengue proteinase inhibitor ARDP0006 to which it shows structural similarity, inhibited intramolecular NS2B/3 cleavage. Lack of toxicity allowed testing in mice, in which pyrimethamine also reduced viral loads. Niclosamide (IC 50 = 0.28 µM), like dengue core inhibitor ST-148, affected structural components of the virion and inhibited early processes during infection. Vandetanib (IC 50 = 1.6 µM), like cellular autophagy inhibitor spautin-1, blocked viral exit from cells and could further be shown to extend survival in vivo . Our approach confirmed previous studies which used extensive high-throughput screening to identify niclosamide and pyrimethamine as antivirals, and it also revealed their likely molecular targets. Thus, three FDA-approved compounds with promising utility for repurposing to treat dengue virus infections and their potential mechanisms were identified using computational tools and minimal phenotypic screening. Author Summary No antiviral therapeutics are currently available for dengue virus infections. By computationally overlaying the 3D chemical structures of compounds known to inhibit dengue virus with those of compounds known to be safe in humans, we identified three FDA-approved compounds that are attractive candidates for drug repurposing towards treatment of dengue virus infections. We identified potential targets of two previously identified antiviral compounds and revealed a previously unknown potential anti-dengue drug, vandetanib. This computational approach to analyze a highly curated library of structures has the benefits of speed and cost efficiency. It also leverages mechanistic work with query compounds used in biomedical research to provide strong hypotheses for the antiviral mechanisms of the safer hit compounds. This workflow to identify compounds with known safety profiles in humans can be expanded to any biological activity for which a small-molecule query compound has been identified, potentially expediting the translation of basic research to clinical interventions.
