Insight into the recognition, binding, and reactivity of catalytic metallodrugs targeting stem loop IIb of hepatitis C IRES RNA.
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 KD value of 76 ± 3 nM, and Michaelis-Menten parameters of kcat =0.14 ± 0.01 min(-1) and KM =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 that of the 1-Cu parent peptide, with an IC50 value of 1.9 ± 0.4 μM and cytotoxicity exceeding 100 μM. 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.
Evans Laboratory of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, OH 43210 (USA), Fax: (+1) 614-292-1685.
Seth S
SS
Bradford
Martin James
MJ
Ross
Insiya
I
Fidai
James A
JA
Cowan
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000737:chemistry* / Q000633:toxicity; D001665:Binding Sites; D002384:Catalysis; D056831:Coordination Complexes / Q000737:chemistry* / Q000633:toxicity; D003300:Copper / Q000737:chemistry*; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism; D062105:Molecular Docking Simulation; D009690:Nucleic Acid Conformation; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D044366:Transition Temperature; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; D056831:Coordination Complexes; D010455:Peptides; D012367:RNA, Viral; D003300:Copper
HCV IRES RNA; antiviral agents; catalysis; copper; metallodrugs