An innovative approach for anti-respiratory syncytial virus compound discovery from plant extracts, combining high-throughput screening and metabolomics

The respiratory syncytial virus (RSV) has been identified as a major causative agent of bronchiolitis in children; nevertheless, available therapeutic options remain limited. Natural products (NPs) have been valued in the field of drug discovery and hold great promise for addressing this gap. However, there is a lack of methodologies that combine high-throughput screening (HTS) of natural extracts (NEs) with parallel metabolomic profiling to target the isolation of potential antivirals. This study presents an antiviral discovery approach that integrates HTS of 192 NEs against RSV in human lung adenocarcinoma cells (A549) with their systematic metabolite profiling. To optimize the identification of compounds with antiviral activity, we developed a scoring system, referred to as the “bioscore”. This score correlates the antiviral activity of each NE with the normalized intensities of its constituents across the 192 NEs. Using this approach, triterpenes, including betulinic acid (BA), were frequently identified in active NEs, contributing to a high hit rate of 38 %. After isolating BA from Pilea plataniflora , we confirmed its antiviral activity in A549 cells, as well as in physiologically relevant models such as airway organoids and human airway epithelia cultured at the air-liquid interface. We further investigated its mechanism of action and found an effect at the early stage of viral replication. Protoapigenone, a species-specific flavonoid from this set of 192 NEs, was also identified as a potential antiviral NP, though limited by its cytotoxicity. This integrative workflow enables scalable antiviral screening of NEs by directly linking metabolomics with bioactivity through bioscore-driven prioritization. • A 384-well HTS assay on A549 cells assessed the anti-RSV activity of NEs. • A “bioscore” strategy was developed to prioritize antiviral molecules from NEs. • Betulinic acid was a high-bioscore antiviral, active in A549 and relevant models. • Its mode of action likely occurs at an early step of the RSV replication cycle. • Protoapigenone also scored high and showed activity, but caused cytotoxic effects.