Cholenamide-based, antiviral fluorescent probes targeting oxysterol-binding protein

Oxysterols (OSs) represent a large family of cholesterol-derived molecules, involved in several physiological and pathological processes. Recently, we reported the remarkable antiviral activity against herpes simplex virus 2 (HSV-2) infection of three cholenamide or homocholenamide derivatives, namely PFM067, PFM064, and PFM069, identified by the screening of an in-house library of OS derivatives. With the aim to shed light on the antiviral mechanism of action of this class of molecules, we assumed to exploit the use of cholenamide-based fluorescent probes. Herein, we report that PFM120 and PFM124, two fluorescent tagged version of PFM067 maintain the same antiviral properties against HSV-2 as the parent compound and localize intracellularly inside the endoplasmic reticulum and the cis-Golgi network. Moreover, we also demonstrate that both tagged molecules co-localize with oxysterol-binding protein (OSBP) and are able to induce its re-localization. Finally, we report that PFM120 and PFM124 are endowed with antiviral activity against another OSBP-dependent viral pathogen, i.e. the human rhinovirus (HRV), different in structure and replication strategy from HSV-2. Taken together, these results candidate PFM120 and PFM124 as useful tools to investigate the actual mechanism of action and molecular target(s) of cholenamide-based antivirals and provide a proof of principle to explore them as a promising broad-spectrum class of antiviral agents.