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

Abstract

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.

From Traditional Drug Design to Catalytic Metallodrugs: A Brief History of the Use of Metals in Medicine

“…Site specific oxidative degradation of the target RNA was demonstrated and the most likely mechanism of action for these compounds is a mixed oxidative/ hydrolytic mechanism where generation of the copper (III) center is necessary to enhance the Lewis acidity of the metal ion. Real-time polymerase chain reaction (RT-PCR) experiments showed that the mechanism in cells is consistent with the proposed mechanism of HCV RNA cleavage (Figure 13, right) [88].…”

Catalytic metallodrugs based on the LaR2C peptide target HCV SLIV IRES RNA

“…2, 3 Time-dependence plots were found to display first order exponential behavior. These initial rates were then plotted as a function of catalyst concentration (Figure 5) and the pseudo Michaelis-Menten parameters are summarized in Table 2.…”

“…3 A sample plot is shown in Figure SM1 and the data are summarized in Table 1. Both 2 and 3 displayed similar K D values for binding to SLIV RNA.…”

“…Reactions for MALDI-TOF analysis were performed as described earlier 3 by use of 10 μM fluorescein-labeled IRES SLIIb with 10 μM copper-peptide, and incubated for up to 90 min. Reactions were then quenched by placing on ice and desalted by use of C 18 ZipTips from Millipore Co. prior to mass spectrometric analysis.…”

Toward the Design of a Catalytic Metallodrug: Selective Cleavage of G‐Quadruplex Telomeric DNA by an Anticancer Copper–Acridine–ATCUN Complex

“…Unlike the oxidative path initiated by hydrogen abstraction from 1′, 3′, and 4′ CH positions of the deoxyribose ring, [17] the 3′-hydroxy product should reflect hydrolysis of phosphate backbone by transient formation of the strong Lewis acid Cu 3+ species. [18] Since oxidative cleavage products are not generally accepted as typical substrates of telomerase, [19] the advantage of this cleavage pattern is that telomeric DNA will be more difficult to elongate by telomerase following cleavage, unless these DNA lesions are first repaired by other DNA repair mechanisms.…”