Agradecimentos: We would like to thank Mr. Valber Ferreira and Ms. Tereza Silva for all technical support. We thank Dr. Celso Benedetti for critically reviewing our manuscript. Graphical abstract was in created BioRender.com. Finally, we thank MMV for providing the Pathogen box. We thank RedeVirus MCTI (Brazilian Ministry of Sciences, Technology and Innovation), CNPq: Brazilian National Council for Scientific and Technological Development (grant #403709/2020-2), Finep (Brazilian Funding Authority for Studies and Projects) and FNDCT (Brazilian National Fund for Scientific and Technological Development) for the financial support (grant #01.20.0003.00). We also thank FAPESP (#2016/00194-8, #2020/04558-0, #2019/06459-1, #2020/02159-0) and FAEPEX-UNICAMP (#2266/20) for supporting this study Ver menos

Abstract: Until December 2021, the COVID-19 pandemic has caused more than 5.5 million deaths. Vaccines are being deployed worldwide to mitigate severe disease and death, but continued transmission and the emergence of SARS-CoV-2 variants indicate that specific treatments against COVID-19 are still necessary. We screened 400 compounds from the Medicines for Malaria Venture (MMV) Pathogen Box seeking for molecules with antiviral activity against SARS-CoV-2 by using a high-throughput screening (HTS) infection assay in Vero CCL81 cells. On resupply of 15 selected hit compounds, we confirmed that 7 of them presented a dose-dependent cytoprotective activity against SARS-CoV-2-induced cytopathic effect (CPE) in the micromolar range. They were validated in low-throughput infection assays using four different cell lines, including the human lung Calu-3 cell line. MMV000063, MMV024937, MMV688279, and MMV688991 reduced viral load in cell culture, assessed by RT-qPCR and viral plaque assay, while MMV688279 and MMV688991 (also known as nitazoxanide) were the most promising, reducing SARS-CoV-2 load by at least 100-fold at 20 µM in almost all cell types tested. Our results indicate that active anti-SARS-CoV-2 molecules exist within the repertoire of antiviral, antiparasitic and antimicrobial compounds available to date. Although the mode of action by which MMV688279 and MMV688991 reduce SARS-CoV-2 replication is yet unknown, the fact that they were active in different cell types holds promise not only for the discovery of new therapeutic targets, but also for the development of novel antiviral medicines against COVID-19 Ver menos