In vitro broad-spectrum antiviral activity of MIT-001, a mitochondria-targeted reactive oxygen species scavenger, against severe acute respiratory syndrome coronavirus 2 and multiple zoonotic viruses

•MIT-001 demonstrates potent antiviral activity against SARS-CoV-2 and zoonotic viruses, offering a promising therapeutic approach for viral infections.•MIT-001 inhibits the replication of SARS-CoV-2 variants, ZIKV, SEOV, and VACV, indicating broad-spectrum antiviral efficacy.•Restoration of antioxidant gene expression highlights MIT-001 ability to counteract oxidative stress and enhance cellular defense mechanisms compromised by SARS-CoV-2 infection.•MIT-001 preserves mitochondrial function and cellular homeostasis by mitigating mitochondrial depolarization caused by SARS-CoV-2 infection. The COVID-19 pandemic caused by SARS-CoV-2 becomes a serious threat to global health and requires the development of effective antiviral therapies. Current therapies that target viral proteins have limited efficacy with side effects. In this study, we investigated the antiviral activity of MIT-001, a small molecule reactive oxygen species (ROS) scavenger targeting mitochondria, against SARS-CoV-2 and other zoonotic viruses in vitro. The antiviral activity of MIT-001 was quantified by RT-qPCR and plaque assay. We also evaluated the functional analysis of MIT-001 by JC-1 staining to measure mitochondrial depolarization, total RNA sequencing to investigate gene expression changes, and immunoblot to quantify protein expression levels. The results showed that MIT-001 effectively inhibited the replication of B.1.617.2 and BA.1 strains, Zika virus, Seoul virus, and Vaccinia virus. Treatment with MIT-001 restored the expression of heme oxygenase-1 (HMOX1) and NAD(P)H: quinone oxidoreductase 1 (NqO1) genes, anti-oxidant enzymes reduced by SARS-CoV-2, to normal levels. The presence of MIT-001 also alleviated mitochondrial depolarization caused by SARS-CoV-2 infection. These findings highlight the potential of MIT-001 as a broad-spectrum antiviral compound that targets for zoonotic RNA and DNA viruses, providing a promising therapeutic approach to combat viral infection.