Hydroxychloroquine (HCQ), a traditional clinical anti-malarial medicine, has shown broad-spectrum activities, including anticancer, anti-inflammatory, and antiviral effects. Since the outbreak of coronavirus disease 2019 (COVID-19), HCQ has been extensively investigated due to its antiviral properties. However, the therapeutic effect and mechanism of how HCQ inhibits coronavirus are unclear, which limits clinical applications of HCQ and poses risks to its ongoing clinical trials.
In this study, we utilized chemical biology and proteomics approaches to reveal a lysosomal acid protease as a binding partner of HCQ and related antiviral mechanisms. A photo-crosslinking probe mimicking HCQ was synthesized to purify proteins that physically bind to HCQ. Next, multiple mass-spectrometry-based analyses were conducted to validate the interactions. Furthermore, HCQ significantly inhibited the enzymatic activity of the target protein and, thus, the endosomal entry pathway of coronavirus, as shown in a pseudovirus assay.
Finally, by comparing HCQ"s inhibitory effects on different SARS-CoV-2 variants with or without gene knockdown, we demonstrated that the anti-COVID-19 mechanism of HCQ was based on the inhibition of the target protein. Our study not only revealed a novel binding target of HCQ but also provided a promising antiviral therapy agent against endosomal pathway-dependent viruses.