Targeting oxysterol-binding protein (OSBP) as a novel strategy to inhibit herpes simplex virus 2 (HSV-2) replication: First structure-activity relationship study of antiviral cholenamide derivatives

25-Hydroxycholesterol (25-HC) and 25R,26-hydroxycholesterol (25R,26-HC) are two endogenous oxysterols endowed with broad-spectrum antiviral activity. They hamper viral replication by targeting oxysterol-binding protein (OSBP), a host lipid transporter which plays a critical role in the replicative cycle of several viruses. Recently, we have reported the remarkable antiviral activity against herpes simplex virus 2 (HSV-2) of N,N- dimethyl-3β-hydroxychol-5-en-24-amide (PFM067, 3), identified by the screening of our synthetic oxysterol li brary. Further development of antiviral cholenamide-based fluorescent probes allowed us to support the hy pothesis that OSBP was involved in the antiviral mechanism of action of the parent PFM067. Herein, we report the first study aimed at the definition of structure-antiviral activity relationships for PFM067 (3); successfully, we identified 24-(morpholin-4-yl)-3β-hydroxychol-5-en-24-one (8d), as highly potent, nanomolar inhibitor of HSV-2 replication, endowed with promising selectivity index. Moreover, we demonstrated that OSBP was the molecular target underlying the anti-HSV-2 activity of PFM067 (3) and its analogues by the development of a microscale thermophoresis binding assay. In-depth experiments performed using an OSBP-silenced cell line allowed us to disclose the role played by OSBP in the replicative cycle of HSV-2. Finally, molecular modelling studies evi denced a peculiar orientation of the active compound 8d within the binding site of OSBP respect to that stroked by either the endogenous ligand or an inactive compound.