A catalytic metallodrug that targets stem-loop IIb of the internal ribosomal entry site (IRES) RNA of hepatitis C virus (HCV) demonstrates enzyme-like turnover with K(M) of 0.85 μM, k(cat) of 0.53 min(-1), and a turnover number of 31.9 for Cu·GGHYrFK-amide (1-Cu), and yielded an antiviral activity (IC(50)) of 0.58 μM in an HCV cellular replicon assay.

Complex Cu-GGHYrFK-amide (1-Cu) was previously reported as a novel metallotherapeutic that catalytically inactivates stem loop IIb of the Hepatitis C Virus (HCV) Internal Ribosomal Entry Site (IRES) RNA and demonstrates significant antiviral activity in a cellular HCV replicon assay. Herein are described 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 determination, 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, is reported and compared to complex 1-Cu. In vitro RNA binding and cleavage assays for complex 2-Cu show a KD of 76 ± 3 nM, and Michaelis-Menten parameters of kcat of 0.14 ± 0.01 min−1 and KM of 7.9 ± 1.2 µM, with a turnover number exceeding 40. In a luciferase-based cellular replicon assay Cu-GGhyrfk-amide shows activity similar to the parent peptide, complex 1-Cu, with IC50 of 1.9 ± 0.4 µM and cytotoxicity exceeding 100 µM. RT-PCR experiments confirm a significant reduction 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 the new class of compounds, while detailed mass spectrometry and computational analysis provide new insights into the mechanisms of recognition, binding, and reactivity.

No abstract

Prior work has demonstrated the potential effectiveness of a new class of metallopeptides as catalytic metallodrugs that target HCV IRES SLIIb RNA (Cu-GGHYrFK, 1). Herein new catalytic metallodrugs (GGHKYKETDLLILFKDDYFAKKNEERK, 2; and GGHKYKETDL, 3) are described based on the LaR2C peptide that has been shown to bind to the SLIV HCV IRES domain. In vitro fluorescence assays yielded KD values ~10 μM for both peptides and reaction of the copper derivatives with SLIV RNA demonstrated initial rates comparable across different assays as well as displaying pseudo-Michaelis-Menten behavior. The sites of reaction and cleavage mechanisms were determined by MALDI-TOF mass spectrometry. The primary site of copper-promoted SLIV cleavage is shown to occur in the vicinity of the 5’-G17C18A19C20-3’ sequence that corresponds to a known binding site of the RM2 motif of the human La protein and has previously been reported to be important for viral translation. This domain also flanks the internal start codon (AUG). Both copper complexes also showed efficacy in an HCV replicon assay (IC50 = 0.75 μM for 2-Cu, and 2.17 μM for 3-Cu) and show potential for treatment of hepatitis C, complementing other marketed drugs by acting on a distinct therapeutic target by a novel mechanism of action.