Over the past decades, an increasing number of viruses has emerged or re-emerged in humans. Unfortunately, currently approved antiviral drugs target a small set of viruses. Thus, there is an urgent need for the development of broad-spectrum antiviral drugs.Cyclophilins are cellular proteins involved in a large number of biological processes, and in different viral lifecycles from unrelated families. They appear as a potential target for the development of broad-spectrum antiviral approaches. However, currently available cyclophilin inhibitors have drawbacks which limit their clinical use.By means of "fragment-based drug design", we generated a new class of small-molecule cyclophilin inhibitors (SMCypI), unrelated with those already available. Cristallographic studies revealed that the SMCypIs bind to two close pockets of the active site and inhibit cyclophilin PPIase activity. These compounds do not bear immunosuppressive properties and inhibit the replication of HIV, HCV and coronaviruses in vitro.We characterized the anti-HCV activity of C31, the most potent inhibitor of cyclophilin PPIase activity. C31 had pan-genotypic HCV inhibitor properties, with a high barrier to resistance and additive effects with currently approved anti-HCV agents. C31 blocked HCV replication by disrupting the interaction between the nonstructural viral protein NS5A and cyclophilin A in a PPIase-dependent manner. Finally, C31 was active on zika, yellow fever, dengue and West-Nile virus infections.The antiviral activity of the SMCypIs has then been characterized on HCoV-229E infection. Interestingly, PPIase inhibition was necessary, but not sufficient for antiviral effect. A structure-activity relationship study identified a key moiety in the SMCypIs at the interface between the two cyclophilin pockets. F836 has been identified as the most potent compound which inhibited both the cytopathic effect and the intracellular RNA of HCoV-229E without associated cytotoxicity and as potently as alisporivir. This compound targeted HCoV-229E entry at a post-attachment step and was also active on HCoV-OC43 and MERS-CoV strains. We then demonstrated that cyclophilin A was associated with viral particles. By means of CRISPR-Cas9, cell lines depleted for cyclophilin A were generated. Cyclophilin A was identified as a proviral factor for HCoV-229E and was partially involved in F836 antiviral effect. Cyclophilin A expression level was drastically decreased by infection.SMCypIs represent a unique tool to decipher the cellular and molecular mechanisms by which cyclophilins interfere with viral lifecycles, as well as drugable compounds that could find an indication as broad-spectrum antiviral drugs.