Targeted cleavage of HIV RRE RNA by Rev-coupled transition metal chelates.

Evans Laboratory of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
Journal of the American Chemical Society (Impact Factor: 11.44). 06/2011; 133(25):9912-22. DOI: 10.1021/ja203057z
Source: PubMed

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-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were linked to a lysine side chain 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 Fe(2+), Co(2+), Ni(2+), and Cu(2+) 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 K(D) values ranging from ~0.2 to 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 to 4.9 nM/min but were optimal for Cu(2+)-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 those of the two principal co-reagents for metal-promoted formation of reactive oxygen species: E°(ascorbate/ascorbyl radical) = -66 mV and E°(H(2)O(2)/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 RRE mRNA.

  • [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    ChemMedChem 04/2014; · 3.05 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The human immunodeficiency virus 1 (HIV-1) replication cycle is finely tuned with many important steps involving RNA–RNA or protein–RNA interactions, all of them being potential targets for the development of new antiviral compounds. This cycle can also be considered as a good benchmark for the evaluation of early-stage strategies aiming at designing drugs that bind to RNA, with the possibility to correlate in vitro activities with antiviral properties. In this review, we highlight different approaches developed to interfere with four important steps of the HIV-1 replication cycle: the early stage of reverse transcription, the transactivation of viral transcription, the nuclear export of partially spliced transcripts and the dimerization step.
    ChemMedChem 08/2014; · 3.05 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Metallo-biopolymers are an exciting class of materials that integrate the optical, electronic, magnetic, and chemical properties of metal complexes with the specificity and highly ordered structures of biopolymers. Metal complexes can be incorporated into the biopolymers using a multitude of synthetic methodologies. Developments in the fields of synthetic organic/organometallic chemistry and biological chemistry have enabled new conjugation methods that can expand the diversity and scope of such integrated metallo-biopolymer assemblies. The conjugation of metal complexes and biopolymers leads to new classes of hybrid materials for applications spanning biomaterials, drug delivery, imaging, sensing, photodynamic therapy, catalysis, optoelectronics, and energy conversion. This review article summarizes the synthetic strategies that have been adopted for the conjugation between biopolymers and metal complexes, as well as some of the various applications of these metallo-biopolymers.
    Polymer Reviews 10/2014; 54(4). · 6.59 Impact Factor


Available from