Analysis of Structure–Activity Relationships Based on the Hepatitis C Virus SLIIb Internal Ribosomal Entry Sequence RNA‐Targeting GGHYRFK⋅Cu Complex

New therapeutics for targeting the hepatitis C virus (HCV) have been released in recent years. Although they are less prone to resistance, they are still administered in cocktails as a combination of drugs targeting various aspects of the viral life cycle. Herein, we aim to contribute to an arsenal of new HCV therapeutics by targeting the HCV internal ribosomal entry sequence (IRES) RNA through the development of catalytic metallodrugs that function to degrade rather than inhibit the target molecule. Based on a previously characterized HCV IRES stem‐loop IIb RNA‐targeting metallopeptide Cu‐GGHYrFK ( 1? Cu), an all‐l analogue ( 3? Cu) and a series of additional complexes with single alanine substitutions in the targeting domain were prepared and screened to determine the influence each amino acid side chain on RNA localization and recognition, and catalytic reactivity toward the RNA. Additional substitutions of the tyrosine position in complex 3? Cu were also investigated. Good agreement between calculated and measured binding affinities provided support for in silico modeling of the SLIIb RNA binding site and correlations with RNA cleavage sites. Examination of the cleavage products from reaction of the Cu complexes with SLIIb provided mechanistic insights, with the first observation of the 5′‐geminal diol and 5′‐phosphopropenal as products through the use of a Cu ? ATCUN catalytic motif. Together, the data yielded insights into structure–function relationships that will guide future optimization efforts.