A library of complexes that included iron, cobalt, nickel, and copper chelates of cyclam, cyclen, DOTA, DTPA, EDTA, tripeptide GGH, tetrapeptide KGHK, NTA, and TACN was evaluated for DNA nuclease activity, ascorbate consumption, superoxide and hydroxyl radical generation, and reduction potential under physiologically relevant conditions. Plasmid DNA cleavage rates demonstrated by combinations of each complex and biological coreactants were quantified by gel electrophoresis, yielding second-order rate constants for DNAsupercoiled to DNAnicked conversion up to 2.5 ×106 M-1min-1, and for DNAnicked to DNAlinear up to 7 ×105 M-1min-1. Relative rates of radical generation and characterization of radical species were determined by reaction with the fluorescent radical probe TEMPO-9-AC and rhodamine B. Ascorbate turnover rate constants ranging from 9.1×10-3 to 8.2 min-1 were determined, although many complexes demonstrated no measureable activity. Inhibition and Freifelder-Trumbo analysis of DNA cleavage supported concerted cleavage of dsDNA by a metal associated ROS in the case of Cu2+(aq), Cu-KGHK, Co-KGHK, and Cu-NTA and stepwise cleavage for Fe2+(aq), Cu-cyclam, Cu-cyclen, Co-cyclen, Cu-EDTA, Ni-EDTA, Co-EDTA, Cu-GGH, and Co-NTA. Reduction potentials varied over the range from -362 mV to +1111 mV versus NHE, and complexes demonstrated optimal catalytic activity in the range of the physiological redox coreactants ascorbate and peroxide (-66 to +380 mV).

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.

A series of compounds that target reactive transition metal chelates to somatic Angiotensin Converting Enzyme (sACE-1) have been synthesized. Half maximal inhibitory concentrations (IC50) and rate constants for both inactivation and cleavage of full length sACE-1 have been determined and evaluated in terms of metal-chelate size, charge, reduction potential, coordination unsaturation, and coreactant selectivity. Ethylenediamine-tetraacetic acid (EDTA), nitrilotriacetic acid (NTA), 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA), and tripeptide GGH were linked to the lysine sidechain of lisinopril by EDC/NHS coupling. The resulting amide-linked chelate-lisinopril (EDTA-lisinopril, NTA-lisinopril, DOTA-lisinopril, and GGH-lisinopril) conjugates were used to form coordination complexes with iron, cobalt, nickel and copper, such that lisinopril could mediate localization of the reactive metal chelates to sACE-1. ACE activity was assayed by monitoring cleavage of the fluorogenic substrate Mca-RPPGFSAFK(Dnp)-OH, a derivative of bradykinin, following pre-incubation with metal-chelate-lisinopril compounds. Concentration-dependent inhibition of sACE-1 by metal-chelate-lisinopril complexes revealed IC50 values ranging from 44 nM to 4,500 nM for Ni-NTA-lisinopril and Ni-DOTA-lisinopril, respectively, versus 1.9 nM for lisinopril. Stronger inhibition was correlated with smaller size and lower negative charge of the attached metal chelates. Time-dependent inactivation of sACE-1 by metal-chelate-lisinopril complexes revealed a remarkable range of catalytic activities, with second order rate constants as high as 150,000 M−1min−1 (Cu-GGH-lisinopril), while catalyst-mediated cleavage of sACE-1 typically occurred at much lower rates, indicating that inactivation arose primary from sidechain modification. Optimal inactivation of sACE-1 was observed when the reduction potential for the metal center was poised near 1000 mV, reflecting the difficulty of protein oxidation. This class of metal-chelate-lisinopril complexes possesses a range of high-affinity binding to ACE, introduces the advantage of irreversible catalytic turnover, and marks an important step toward the development of multiple-turnover drugs for selective inactivation of sACE-1.

Metallopeptides containing both the complex Cu2+-glycyl-glycyl-histidine (Cu-GGH) and the sequence WRWYCR were shown to possess antimicrobial activity against a variety of pathogenic bacteria, as well as bind to and cleave a variety of nucleic acids, suggesting potential mechanisms for antimicrobial activity that involve binding and/or irreversible cleavage of bacterial nucleic acids.

A method of analysis is presented that utilizes matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to monitor the kinetics and products of RNA cleavage, by use of a program designed to mass-match observed MS peaks with predicted RNA cleavage products. The method is illustrated through application to the study of targeted oxidation of RNA stem loops from HIV-1 Rev Response Element mRNA (RRE RNA) and ribosomal 16S A-site RNA (16S RNA) by metallonucleases. Following incubation of each RNA with catalysts and/or redox co-reactants, reaction mixtures were desalted, and MALDI-TOF MS was used to monitor both time-resolved formation of cleavage products and disappearance of full-length RNA. For each RNA, a unique list was generated that contained the predicted masses of both the full-length, and all of the possible RNA cleavage fragments that resulted from the combination of all possible cleavage sites and each of the six expected overhangs formed at nascent termini adjacent to the cleavage sites. The overhangs corresponded to 2′,3′-cyclic phosphate, 3′-phosphate, 3′-phosphoglycolate, 5′- hydroxyl and 5′- phosphate, which corresponded to differing oxidative, hydrolytic, and/or 2′-OH-mediated-endonucleolytic modes of scission. Each mass spectrum was compared with a corresponding list of predicted masses, and peaks were rapidly assigned by use of a Perl script, with a mass-matching tolerance of 200 ppm. Both time-dependent cleavage mediated by metallonucleases and MALDI-TOF-induced fragmentation were observed, and these were distinguished by time-dependent experiments. The resulting data allowed a semi-quantitative assessment of the rate of formation of each overhang at each nucleotide position. Limitations included artifactual skewing of quantification by mass bias, a limited mass range for quantification, and a lack of detection of secondary cleavage products. Nevertheless, the method presented herein provides a rapid, accurate, highly-detailed and semi-quantitative analysis of RNA cleavage that should be widely applicable.