Efficacy of topical cobalt chelate CTC-96 against adenovirus in a cell culture model and against adenovirus keratoconjunctivitis in a rabbit model

BACKGROUND: Adenovirus (Ad), associated with significant morbidity, has no topical treatment. A leading CTC compound (CTC-96), a Co(III )chelate, was found to have potent in vitro and in vivo antiviral efficacy against herpes viruses. In this study CTC-96 is being tested for possible anti-Adenovirus activity. METHODS: The biological anti-adenovirus activity of CTC-96 in concentrations from 5 to 250 ug/ml, was evaluated initially by viral inactivation (viral exposure to CTC-96 followed by dilution and inoculation of cells), virucidal (viral exposure to CTC-96 and inoculation of cells without dilution) and antiviral (effect of CTC-96 on previously adsorbed virus) plaque assays on HeLa (human cervical carcinoma), A549 (human lung carcinoma) and SIRC (rabbit corneal) cells. After verifying the antiviral activity, New Zealand White rabbits were infected with Ad-5 into: 1) the anterior cul-de-sac scarifying the conjunctiva (Group "C+"); 2) the anterior cul-de-sac scarifying the conjunctiva and cornea (Group "CC+"); 3) the stroma (Group "CI+"). Controls were sham-infected ("C-", "CC-", "CI-"). Other rabbits, after "CC", were treated for 21 days with: 1) placebo, 9x/day ("-"); 2) CTC-96, 50 ug/ml, 9x/day ("50/9"); CTC-96, 50 ug/ml, 6x/day ("50/6"); CTC-96, 25 ug/ml, 6x/day ("25/6"). All animals were monitored via examination and plaque assays. RESULTS: In vitro viral inactivation, virucidal and antiviral assays all demonstrated CTC-96 to be effective against Adenvirus type 5 (ad-5). The in vivo model of Ad keratoconjunctivitis most similar to human disease and producing highest viral yield was "CC". All eyes (6/6) developed acute conjunctivitis. "CI" yielded more stromal involvement (1/6) and iritis (5/6), but lower clinical scores (area × severity). Infection via "C" was inconsistent (4/6). Fifty (50) ug/ml was effective against Ad-5 at 6x, 9x dosings while 25 ug/ml (6x) was only marginally effective. CONCLUSION: CTC-96 demonstrated virucidal activity against Ad5 in tissue culture with HeLa, A549 and SIRC cell lines. Animal Model Development: 1) "CC" produced conjunctival infection with occasional keratitis similar to human disease; "CI" yielded primarily stromal involvement; 2) "C" consistently produced neither conjunctivitis nor keratitis. CTC Testing: 1) Conjunctivitis in all eyes; 2) Resolution fastest in "50/9" ("50/9". "50/6" > "25/6" > "-"); 3) Efficacy in "50/6" was not statistically different than "50/9"; 4) Conjunctival severity was lower in treatment groups then controls; 5) Little corneal or intra-ocular changes were noted

An Eye to a Kill: Using Predatory Bacteria to Control Gram-Negative Pathogens Associated with Ocular Infections

Ocular infections are a leading cause of vision loss. It has been previously suggested that predatory prokaryotes might be used as live antibiotics to control infections. In this study, Pseudomonas aeruginosa and Serratia marcescens ocular isolates were exposed to the predatory bacteria Micavibrio aeruginosavorus and Bdellovibrio bacteriovorus. All tested S. marcescens isolates were susceptible to predation by B. bacteriovorus strains 109J and HD100. Seven of the 10 P. aeruginosa isolates were susceptible to predation by B. bacteriovorus 109J with 80% being attacked by M. aeruginosavorus. All of the 19 tested isolates were found to be sensitive to at least one predator. To further investigate the effect of the predators on eukaryotic cells, human corneal-limbal epithelial (HCLE) cells were exposed to high concentrations of the predators. Cytotoxicity assays demonstrated that predatory bacteria do not damage ocular surface cells in vitro whereas the P. aeruginosa used as a positive control was highly toxic. Furthermore, no increase in the production of the proinflammatory cytokines IL-8 and TNF-alpha was measured in HCLE cells after exposure to the predators. Finally, injection of high concentration of predatory bacteria into the hemocoel of Galleria mellonella, an established model system used to study microbial pathogenesis, did not result in any measurable negative effect to the host. Our results suggest that predatory bacteria could be considered in the near future as a safe topical bio-control agent to treat ocular infections. © 2013 Shanks et al

Structures of Helicobacter pylori Shikimate Kinase Reveal a Selective Inhibitor-Induced-Fit Mechanism

Shikimate kinase (SK), which catalyzes the specific phosphorylation of the 3-hydroxyl group of shikimic acid in the presence of ATP, is the enzyme in the fifth step of the shikimate pathway for biosynthesis of aromatic amino acids. This pathway is present in bacteria, fungi, and plants but absent in mammals and therefore represents an attractive target pathway for the development of new antimicrobial agents, herbicides, and antiparasitic agents. Here we investigated the detailed structure–activity relationship of SK from Helicobacter pylori (HpSK). Site-directed mutagenesis and isothermal titration calorimetry studies revealed critical conserved residues (D33, F48, R57, R116, and R132) that interact with shikimate and are therefore involved in catalysis. Crystal structures of HpSK·SO4, R57A, and HpSK•shikimate-3-phosphate•ADP show a characteristic three-layer architecture and a conformationally elastic region consisting of F48, R57, R116, and R132, occupied by shikimate. The structure of the inhibitor complex, E114A•162535, was also determined, which revealed a dramatic shift in the elastic LID region and resulted in conformational locking into a distinctive form. These results reveal considerable insight into the active-site chemistry of SKs and a selective inhibitor-induced-fit mechanism

Exploiting bacterial DNA gyrase as a drug target: current state and perspectives

DNA gyrase is a type II topoisomerase that can introduce negative supercoils into DNA at the expense of ATP hydrolysis. It is essential in all bacteria but absent from higher eukaryotes, making it an attractive target for antibacterials. The fluoroquinolones are examples of very successful gyrase-targeted drugs, but the rise in bacterial resistance to these agents means that we not only need to seek new compounds, but also new modes of inhibition of this enzyme. We review known gyrase-specific drugs and toxins and assess the prospects for developing new antibacterials targeted to this enzyme