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

Abstract

New therapeutics for the targeting of the hepatitis C virus have been released in recent years. Although these therapies 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 HCV internal ribosomal entry sequence (IRES) RNA via 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 of complex 3-Cu were also investigated. Good agreement of 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 cleavage productions from reaction of the Cu-complexes with the SLIIb provided mechanistic insights with the first observation of the 5′-geminal diol and 5′-phosphopropenal as products through use of a Cu-ATCUN catalytic motif. Together, the data yielded insights on structure-function relationships that will guide future optimization efforts.

The effects of nucleotide usage in key nucleotide positions +4 and -3 flanking start codon on translation levels mediated by IRES of hepatitis C virus

“…According to tRNA copy numbers for the tRNA abundance pool in CHO cell line (Genomic tRNA Database), when the position +4 mutates different nucleotide types in bicistronic reporter plasmids, the tRNA abundances for the second codon (+4XTG+6, X means any nucleotide, tRNA copy number for GTG is 9, tRNA copy number for CTG is 10, tRNA copy number for TTG is 4, tRNA copy number for ATG is 16) following the start codon have no obvious effect on mediating the EGFP gene expression levels. This result implies that nucleotide context at +4 nucleotide position might influence the local structural formation rather than tRNA abundances, leading to the changes of gene expression mediated by HCV IRES (Jaafar et al, 2016;Bugaud et al, 2017;Ma et al, 2018;Mengardi et al, 2017;Ross et al, 2017;Zhu et al, 2017). Of note, the results show that the positions -3A and +4G had a better performance in improving the translation efficiency mediated by HCV IRES than the preferred nucleotide usage bias (-3A, +4A) in the positions -3 & +4 of the translation initiation region of HCV, implying that the Kozak-like nucleotide context (-3A, +4G) probably impacts the ribosome binding to the translation initiation region of HCV.…”

Designed Metal-ATCUN Derivatives: Redox- and Non-redox-Based Applications Relevant for Chemistry, Biology, and Medicine

“…Interestingly, Cu-GGh-yrfk, exhibits major cleavage sites, U 14 and A 15 , whereas Cu-GGH-YrFK exhibits a wider range of cleavage sites A 6 -G 9 of HCV SLIIb RNA ( Figure 13 ). This concept is expanded to design a series of L-analog of Cu-GGH-YrFK derivative where recognition domain, YrFK is shuffled with existing amino acid or substituted other amino acids like A, D, N, and K (YRFK, ARFK, YAFK, YRAK, YRFA, KFRY, DRFK, NRFK, KRFK, and FRFK) in order to understand the impact of each amino acid residue toward SLIIb RNA binding and cleavage efficiency ( Ross et al., 2017 ). Binding affinity and reactivity patterns of each metallo-peptide are modulated by stereochemistry, charge, size and position of amino acids in the targeting domain.…”

“…Binding affinity and reactivity patterns of each metallo-peptide are modulated by stereochemistry, charge, size and position of amino acids in the targeting domain. For instance, Cu-GGh-YRFK and Cu-GGH-FRFK show the low catalytic efficiency compared to Cu-GGH-YrFK and Cu-GGh-yrfk ( Ross et al., 2017 ).

Figure 13 Selective Cleavage of HCV RNA by M-ATCUN Derivatives (A and B) (A) Two-dimensional representation of subdomain SL-IIb RNA of HCV, and (B) crystal structure of HCV SL-IIb RNA (PDB: 1P5N ) showing the main cleavage sites (highlighted blue and orange color) by CuGGHYrFK or CuGGhyrfk respectively.

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Figure 13 Selective Cleavage of HCV RNA by M-ATCUN Derivatives (A and B) (A) Two-dimensional representation of subdomain SL-IIb RNA of HCV, and (B) crystal structure of HCV SL-IIb RNA (PDB: 1P5N ) showing the main cleavage sites (highlighted blue and orange color) by CuGGHYrFK or CuGGhyrfk respectively. Modified from Ross et al. (2017) .

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“…Over the last few decades, the research on designed metal-ATCUN derivatives (ATCUN; amino terminal copper and nickel binding motif possessing H 2 N–X-X-His sequence; X = any amino acid) has greatly progressed due to the wide range of applications envisaged, relevant for biology and chemistry, which include DNA ( Harford and Sarkar, 1997 ; Mack and Dervan, 1992 ; Jin and Cowan, 2005 , 2007 ; Agbale et al., 2016 ; Yu and Cowan, 2017a , 2017b ; Gonzalez et al., 2018 ), RNA ( Yu and Cowan, 2017a , 2017b ; Gonzalez et al., 2018 ; Ross et al., 2017 ), proteins ( Agbale et al., 2016 ; Yu and Cowan, 2017a , 2017b ; Gonzalez et al., 2018 ; Pinkham et al., 2018a , 2018b ), sugar ( Yu and Cowan, 2017a , 2017b ), and lipids cleavage ( Alexander et al., 2017 ), antitumor ( Kimoto et al., 1983 ), and antimicrobial activity ( Alexander et al., 2018 , 2019 ), inhibition of enzyme activity ( Gokhale et al., 2008 ), telomere shortening ( Yu et al., 2015 ), aggregation of amyloid-β peptides ( Zhang et al., 2016 ), nitration of tyrosine ( Maiti et al., 2019 ), protein-protein cross-linking ( Horowitz et al., 2012 ; Brown et al., 1998 ), water oxidation ( Deng et al., 2018 ), hydrogen evolution ( Kandemir et al., 2016 ), nitrite to NH 3 ( Guo et al., 2018 ), useful in spectroscopic probes ( Donaldson et al., 2001 ; Maiti et al, 2017b ; Deng et al., 2019a , 2019b ; Torrado et al., 1998 ), and imaging ( Miyamoto et al., 2016 ).…”