Outbreaks of emerging Coronaviruses (CoVs) can provoke life-threatening diseases. The coronavirus disease 2019 (COVID-19) is the latest one, caused by SARS-CoV-2 virus. Like its predecessors SARS-CoV and MERS-CoV, SARS-CoV-2 is a betacoronavirus which is thought to have originated in bats, their natural host. Originally transmitted from animals to human, the virus transmission quickly shifted to human-to-human transmission, provoking an unprecedented spread into actual pandemic. No vaccines and treatments for COVID-19 are currently available and are thus urgently needed to address the outbreak. Drugs that target viral entry into the host cell have been proven effective against a wide range of viral diseases. In this proposal, we will apply the results of our fundamental research, and more particularly inhibition of the viral fusion, including that of the measles virus, recognized to be one of the most contagious viruses known so far, to the development of novel inhibitors of SARS-CoV-2 entry into target cells. The strategy is based on inhibiting viral entry, using peptides specific for the viral fusion protein. Infection by coronavirus requires membrane fusion between the viral envelope and the cell membrane. The fusion process is mediated by the viral envelope glycoprotein (S). Based on peptides reported to inhibit MERS-CoV that fuses in the endosome, we designed lipid-conjugated inhibitory with enhanced activities. In this application, we will similarly design SARS-CoV-2 derived peptides and improve their endosomal delivery. Peptides will be fused to the proven lipidation and pegylating strategies and to a cell penetrating peptide sequence to further promote their endocytosis and dimerized to improve the antiviral efficiency, according to the results from our previous work. We propose to design, synthesize and evaluate novel SARS-CoV-2 peptide analogs with enhanced efficacy, which could be administrated by various routes. The proposed work will address two specific aims: 1) Utilization of the protein engineering to optimize the antiviral potency and endosomal localization of SARS-CoV-2 peptide fusion inhibitors and analyze them in in vitro fusion assay and ex vivo models of coronavirus infection to evaluate of their potential to block fusion, virus entry and propagation; 2) Establishment of the transgenic murine model of SARS-CoV-2 infection and evaluation biodistribution and toxicity of improved SARS-CoV-2 peptides and their antiviral protection in vivo. The project will be achieved in the close collaboration between two groups, B. Horvat’s in International Center for Infectiology Research (CIRI) in Lyon and M. Porotto’s at Columbia University Medical Center (CUMC) in New York, with complementary skills and sharing the common goal is to develop new efficient anti-viral approaches. The partners share long-term collaboration experience and have the excellent expertise in the immunovirological studies (CIRI) and analysis of the viral fusion mechanism (CUMC, USA). Project should lead to the identification of the novel class of peptide fusion inhibitors targeted to endosomes, with potent antiviral activity against the COVID-19, and possibly against multiple coronavirus, as well as optimal pharmacological and chemical solubility. The obtained results will provide the critical set up of a novel prophylactic and therapeutic strategy against the COVID-19 and thus open up new prospects for preventing and treating this highly infectious disease. Successful development of these fusion inhibitors for human use will set the stage for a platform technology suitable not only for SARS-CoV-2, but for other coronaviruses which may emerge in the future, as well as other viruses using similar fusion mechanism for viral entry.