DNA to RNA Tuning of a Pyrrolidine-Derived Quinoxaline Scaffold: Magnesium Induces Binding and Inhibition of Internal Ribosomal Entry Site RNA-Mediated Translational of Hepatitis C Virus

Selective targeting of disease-relevant RNA with small molecules remains challenging due to the dynamic conformation of RNA and the nonspecific binding of ligands toward DNA. The internal ribosome entry site (IRES) RNA of the hepatitis C virus (HCV) is a critical antiviral drug target for structure-specific therapeutics. Small molecule inhibitors capable of altering the Mg²⁺ ion-induced L-shaped conformation of IRES subdomain IIa can effectively suppress the IRES-driven translation and HCV RNA replication. Here, we report the development of quinoxaline analogues designed to shift binding selectivity from DNA to HCV-IRES RNA by modifying a known DNA intercalator and describe their structure-activity relationship (SAR). Lead compound <b>8e</b>, featuring a pyrrolidine ring with an ether modification, exhibited Mg²⁺-dependent binding to HCV-IRES subdomain IIa RNA, evading DNA interactions and inhibiting HCV-IRES-mediated translation. Biophysical studies (CD, NMR, and FRET) revealed that <b>8e</b> undergoes a conformational change in the presence of Mg²⁺, favoring its binding to HCV-IRES RNA and altering its subdomain IIa structure, preventing the binding of the 40S ribosome. Moreover, <b>8e</b> significantly reduced the viral load in HCV-infected cells without observable cytotoxicity. Overall, our study highlights a strategy for fine-tuning quinoxaline-based small molecules to enhance RNA selectivity over DNA, and we present <b>8e</b> as a promising lead for HCV therapeutics.