Design, synthesis and in vitro validation of bivalent binders of SARS-CoV-2 spike protein: Obeticholic, betulinic and glycyrrhetinic acids as building blocks

: SARS-CoV-2 is the virus responsible for the COVID-19 pandemic, which caused over 6.7 million deaths worldwide. The Spike protein plays a crucial role in the infection process, mediating the binding of the virus to its cellular receptor, angiotensin-converting enzyme 2 (ACE2), and its subsequent entry into target cells. Previous studies identified, through virtual screening, several natural products capable of binding to two distinct pockets of the Spike protein: triterpenoids binding to pocket 1 and bile acid derivatives binding to pocket 5. Building on these findings, our study advances the field by developing bivalent compounds 1-4 that through a spacer combine a triterpenoid (betulinic acid or glycyrrhetinic acid) with a semisynthetic bile acid derivative (obeticholic acid). These bivalent compounds are designed to simultaneously bind both pockets of the Spike protein, offering significant advantages over single molecules or the combination of the two natural products. In vitro cell assays using pseudotyped recombinant lentiviral particles with selected SARS-CoV-2 Spike proteins demonstrated that 1 and 2 exhibit enhanced activity in reducing viral entry into target cells compared to individual natural products, thus highlighting their potential as superior antiviral agents with reduced side effects.