Discovery of Novel SARS-CoV-2 Fusion Inhibitors—Posaconazole-Polyarginine Conjugates

Objectives: The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the current treatment limitations—particularly the emergence of drug resistance and the reduced efficacy of some existing drugs against new variants—highlight the need for novel antiviral strategies with novel action mechanisms. Fusion inhibitors that disrupt six-helix bundle (6-HB) formation during viral entry represent a promising approach. Posaconazole, an antifungal agent, has been identified as a weak fusion inhibitor, but suffers from poor membrane permeability and modest activity. This study aimed to enhance its antiviral potency by conjugating it with cell-penetrating polyarginine peptides and to investigate the mechanism of action. Methods: A series of posaconazole-polyarginine conjugates were synthesized via click chemistry. Antiviral activity was evaluated using pseudotyped SARS-CoV-2 Omicron XDV in HEK293T cells. Mechanisms were investigated by circular dichroism, native PAGE, size-exclusion HPLC, molecular docking, and isothermal titration calorimetry. Metabolic stability was assessed using hepatic microsomes. Results: Posa-R8 exhibited potent antiviral activity comparable to the clinical candidate EK1, with minimal cytotoxicity. Mechanistic studies confirmed that Posa-R8 binds the HR2 region of the spike protein, disrupts 6-HB formation, and inhibits membrane fusion. It also showed strong lipid bilayer affinity and improved phase I metabolic stability over EK1. Conclusions: Polyarginine conjugation enhances the membrane-binding affinity and antiviral efficacy of posaconazole. Posa-R8 represents a promising lead for developing next-generation SARS-CoV-2 fusion inhibitors.