Targeted Cleavage of HIV RRE RNA by Rev-Coupled Transition Metal Chelates

Abstract

A series of compounds that target reactive metal-chelates to the HIV-1 Rev Response Element (RRE) mRNA have been synthesized. Dissociation constants and chemical reactivity toward HIV RRE RNA have been determined and evaluated in terms of reduction potential, coordination unsaturation, and overall charge associated with the metal-chelate-rev complex. Ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), and 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA) were linked to a lysine sidechain of a Rev-derived peptide by either EDC/NHS or isothiocyanate coupling. The resulting chelate-Rev (EDTA-Rev, DTPA-Rev, NTA-Rev, and DOTA-Rev) conjugates were used to form coordination complexes with Fe2+, Co2+, Ni2+, and Cu2+ such that the arginine-rich Rev peptide could mediate localization of the metal-chelates to the Rev peptide’s high-affinity mRNA binding partner, RRE stem loop IIB. Metal complexes of the extended peptides GGH-Rev and KGHK-Rev, which also contain N-terminal peptidic chelators (ATCUN motifs), were studied for comparison. A fluorescence titration assay revealed high affinity RRE RNA binding by all 22 metal-chelate-Rev species, with KD values ranging from ~ 0.2 – 16 nM, indicating little to no loss of RNA affinity due to the coupling of the metal-chelates to the Rev peptide. Dissociation constants for binding at a previously unobserved low-affinity site are also reported. Rates of RNA modification by each metal-chelate-Rev species were determined and varied from ~ 0.28 – 4.9 nM/min, but were optimal for Cu2+-NTA-Rev. Metal-chelate reduction potentials were determined and varied from −228 to +1111 mV vs. NHE under similar solution conditions, allowing direct comparison of reactivity with redox thermodynamics. Optimal activity was observed when the reduction potential for the metal center was poised between that of the two principal coreagents for metal-promoted formation of reactive oxygen species: E°ascorbate/ascorbyl radical = −66 mV; E°H2O2/hydroxyl radical = 380 mV. Given the variety of oxidative activities of these metal-complexes and their high-affinity binding to the targeted RRE mRNA following coupling to the Rev peptide, this class of metal-chelate-Rev derivatives constitutes a promising step toward development of multiple-turnover reagents for selective eradication of HIV-1 Rev response element mRNA.

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

“…Oxidation of RNA is likely facilitated by the high oxidation state Cu 3+ and formation of intermediate ROS, a characteristic of Cu-ATCUN complexes. [2, 19–24] …”

“…[1–2] Incorporation of a reactive metal-binding domain allows for the irreversible inactivation of a target protein or RNA and the potential for multiple turnover to irreversibly modify and inactivate many copies of a target, resulting in a therapeutic effect at lower dosage. [3] Previous work has shown that the tetrapeptide sequence YrFK-amide (where the arginine is in the d -conformation) binds to stem loop IIb (SLIIb) of the Internal Ribosomal Entry Site (IRES) of Hepatitis C Virus (HCV) RNA [4] and that incorporation of a metal-binding ATCUN [5] (amino terminal copper and nickel binding, XXH, where X ≠ P) motif results in catalytic degradation of the target RNA under oxidative conditions.…”

The reactivity of macrocyclic Fe(II) paraCEST MRI contrast agents towards biologically relevant anions, cations, oxygen or peroxide

“…To gauge the reactivity of the Fe(II) complexes in air under reducing conditions analogous to those in blood, we used ascorbate as a biologically relevant reductant (E o = − 66 mV [16,40]. The fact that [Fe(L4)] 2+ has the lowest ascorbate consumption suggests that the eight donor groups protect the Fe(II) center and inhibit redox cycling.…”

The Design of RNA Binders: Targeting the HIV Replication Cycle as a Case Study

“…[72] The targeted cleavage of RRE by a metallopeptide bearing an amino-terminal copper binding motif connected the binding domain of Rev has also been reported. [73,74] Boronic acids are known to covalently link to the 2'-and/or 3'-hydroxyl group of ribose in a reversible manner, under physiological conditions. [75] As a consequence, it can be expected that compounds bearing boronic acid moieties could bind to RNA with improved selectivity over DNA thanks to an additional interaction with the 2'-OH of ribonucleic acids.…”