Insight into the Recognition, Binding, and Reactivity of Catalytic Metallodrugs Targeting Stem Loop IIb of Hepatitis C IRES RNA

Abstract The complex Cu‐GGHYrFK‐amide ( 1 ‐Cu) was previously reported as a novel metallotherapeutic that catalytically inactivates stem loop IIb (SLIIb) of the hepatitis C virus (HCV) internal ribosomal entry site (IRES) RNA and demonstrates significant antiviral activity in a cellular HCV replicon assay. Herein we describe additional studies focused on understanding the cleavage mechanism as well as the relationship of catalyst configuration to structural recognition and site‐selective cleavage of the structured RNA motif. These are advanced by use of a combination of MALDI‐TOF mass spectrometry, melting temperature determinations, and computational analysis to develop a structural model for binding and reactivity toward SLIIb of the IRES RNA. In addition, the binding, reactivity, and structural chemistry of the all‐ D ‐amino acid form of this metallopeptide, complex 2 ‐Cu, are reported and compared with those of complex 1 ‐Cu. In vitro RNA binding and cleavage assays for complex 2 ‐Cu show a K D value of 76±3 n M , and Michaelis–Menten parameters of k cat =0.14±0.01 min −1 and K M =7.9±1.2 μ M , with a turnover number exceeding 40. In a luciferase‐based cellular replicon assay Cu‐GGhyrfk‐amide shows activity similar to that of the 1 ‐Cu parent peptide, with an IC 50 value of 1.9±0.4 μ M and cytotoxicity exceeding 100 μ M . RT‐PCR experiments confirm a significant decrease in HCV RNA levels in replicon assays for up to nine days when treated with complex 1 ‐Cu in three‐day dosing increments. This study shows the influence that the α‐carbon stereocenter has for this new class of compounds, while detailed mass spectrometry and computational analyses provide new insight into the mechanisms of recognition, binding, and reactivity.