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Joyner J.C.,Ohio State University | Reichfield J.,Ohio State University | Cowan J.A.,Ohio State University | Cowan J.A.,Metallopharm, Llc
Journal of the American Chemical Society | Year: 2011

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 co-reactants were quantified by gel electrophoresis, yielding second-order rate constants for DNA supercoiled to DNA nicked conversion up to 2.5 × 10 6 M -1 min -1, and for DNA nicked to DNA linear up to 7 × 10 5 M -1 min -1. Relative rates of radical generation and characterization of radical species were determined by reaction with the fluorescent radical probes TEMPO-9-AC and rhodamine B. Ascorbate turnover rate constants ranging from 3 × 10 -4 to 0.13 min -1 were determined, although many complexes demonstrated no measurable activity. Inhibition and Freifelder-Trumbo analysis of DNA cleavage supported concerted cleavage of dsDNA by a metal-associated reactive oxygen species (ROS) in the case of Cu 2+(aq), Cu-KGHK, Co-KGHK, and Cu-NTA and stepwise cleavage for Fe 2+(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 to +1111 mV versus NHE, and complexes demonstrated optimal catalytic activity in the range of the physiological redox co-reactants ascorbate and peroxide (-66 to +380 mV). © 2011 American Chemical Society.

Joyner J.C.,Ohio State University | Cowan J.A.,Ohio State University | Cowan J.A.,Metallopharm, Llc
Journal of the American Chemical Society | Year: 2011

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+, 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 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 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 those of the two principal co-reagents for metal-promoted formation of reactive oxygen species: E°ascorbate/ascorbyl-radical = -66 mV and E°H 2O2/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. © 2011 American Chemical Society.

Bradford S.,Ohio State University | Cowan J.A.,Ohio State University | Cowan J.A.,Metallopharm, Llc
Chemical Communications | Year: 2012

A catalytic metallodrug that targets stem-loop IIb of the internal ribosomal entry site (IRES) RNA of hepatitis C virus (HCV) demonstrates enzyme-like turnover with K M of 0.85 μM, k cat of 0.53 min -1, and a turnover number of 31.9 for Cu·GGHYrFK-amide (1-Cu), and yielded an antiviral activity (IC 50) of 0.58 μM in an HCV cellular replicon assay. © The Royal Society of Chemistry 2012.

Agency: NSF | Branch: Standard Grant | Program: | Phase: | Award Amount: 150.00K | Year: 2014

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) Phase I project is to develop a simple general test strip that can be broadly used for the detection and characterization of bacteria such as pathogens and drug-resistant organisms. The project is expected to result in a prototype low-cost device for rapid and specific characterization of bacteria. The product will take the form similar to that of a home pregnancy test and will be ideal for immediate point-of-care diagnoses. There is no such general product currently on the market and commercial potential is deemed high. The proposed diagnostic platform will be versatile and broadly applicable for different applications.

The proposed project is to deliver a low-cost, simple, handheld product that gives a rapid and accurate visible colorimetric indication (Yes or NO answer similar to a pregnancy home test strip) of bacteria relevant to disease, biowarfare concerns, environmental contamination, and the food chain. There is a need for a rapid, simple and inexpensive point-of-care test, especially in hospital ICU?s where there is a high risk and frequency of getting infection from sick patients who have compromised immune systems. The detection platform is versatile and applicable to a large array of microbial infections, and has multiple applications in clinical and non-clinical setting. It is intended to transform the way pathogens are detected in the professional and consumer markets. The proposal seeks to develop the design principles and fashioning of practical devices to achieve a rapid diagnostic kit for identification of bacteria using human samples.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 2.00M | Year: 2011

DESCRIPTION (provided by applicant): HCV is responsible for 60% of the cases of chronic hepatitis and 50% of cases of cirrhosis, end- stage liver disease, and liver cancer. An effective vaccine has proved elusive and the preferred therapy with pegylated interferon is effective in less than 50% of patients with genotype 1 and 75% of patients with genotypes 2 or 3. Clearly, new treatment alternatives are needed. Interest in HCV IRES RNA as a drug target is reflected by the increasing number of small and largepharma companies pursuing that goal. MetalloPharm has created a novel platform technology (metallodrugs) that has the potential to irreversibly destroy the HCV IRES RNA. The specific aims are directed toward selection of a lead and back-up drug candidatefor IND-enabling pre- clinical testing following validation of cellular mode of action against IRES RNA and uptake mechanisms; assessment of PK, toxicity and efficacy data; and exploration of methods to improve serum half life. PUBLIC HEALTH RELEVANCE: Millions of people worldwide are infected with HCV, including a significant portion of the US population. HCV is responsible for 60% of the cases of chronic hepatitis and 50% of cases of cirrhosis, end-stage liver disease, and liver cancer. An effective vaccine has proved elusive and current therapy is effective in less than 50% of patients. Clearly, new treatment alternatives are needed. MetalloPharm has created a novel class of therapeutics (metallodrugs) that function by destroying a key molecule inthe viral life cycle with the potential to halt the progression of, or completely eliminate the virus.

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