Abstract
Coronaviruses have caused three major endemics in the past two decades. Alarmingly, recent identification of novel zoonotic coronaviruses that caused human infections suggests the risk of future coronavirus outbreak caused by spillover infection from animal reservoirs remains high1,2. Therefore, development of novel therapeutic options with broad-spectrum anti-coronavirus activities are urgently needed. Here, we develop an orally-available bispecific inhibitor, TMP1, which simultaneously targets key coronavirus replication protease Mpro and the essential airway protease TMPRSS23,4. TMP1 shows broad-spectrum protection not only against different SARS-CoV-2 variants but also against multiple human-pathogenic coronaviruses in vitro. By using the K18-hACE2 transgenic mouse, hDPP4 knock-in mouse and golden Syrian hamster models, we demonstrate TMP1 cross-protects against highly-pathogenic coronaviruses (SARS-CoV-1, SARS-CoV-2 and MERS-CoV) in vivo and efficiently abrogates SARS-CoV-2 transmission. Through structural and mutagenesis studies, we confirmed the direct interaction of TMP1 with Mpro and TMPRSS2, and pinpoint the key sites of interactions. Importantly, TMP1 inhibits the infection of nirmatrelvir-resistant SARS-CoV-2 escape mutants. Together, our findings demonstrate the antiviral potential of the novel bispecific Mpro/TMPRSS2 antiviral design against human-pathogenic coronaviruses and other emerging coronaviruses.