Antimicrobial activity and mechanism of action of Nu-3, a protonated modified nucleotide

Background: "Nubiotics" are synthetic oligonucleotides and nucleotides with nuclease-resistant backbones, and are fully protonated for enhanced ability to be taken up by bacterial cells. Nu-3 [butyl-phosphate-5'-thymidine-3'-phosphate-butyl], one of the family members of Nubiotics was efficacious in the treatment of burn-wound infections by Pseudomonas aeruginosa in mice. Subsequent studies revealed that Nu-3 had a favorable toxicological profile for use as a pharmaceutical agent. This study evaluated the antibacterial activity of Nu-3 in vitro and its efficacy as a topical antibiotic. In addition, we investigated the possible mechanisms of Nu-3 action at the levels of DNA synthesis and bacterial membrane changes. Methods: Antimicrobial minimum inhibitory concentrations (MIC) experiments with Nu-3 and controls were measured against a range of Gram-positive and Gram-negative bacteria, including some hospital isolates according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Analysis of the killing kinetics of Nu-3 was also performed against two strains (Staphylococcus aureus cvcc 2248 and Pseudomonas aeruginosa cvcc 5668). The mouse skin suture-wound infection model was used to evaluate the antibacterial activity of Nu-3. We used a 5-Bromo-2'-deoxy-uridine Labeling and Detection Kit III (Roche, Switzerland) to analyze DNA replication in bacteria according to the manufacturer's instruction. The BacLight (TM) Bacterial Membrane Potential Kit (Invitrogen) was used to measure the bacterial membrane potential in S. aureus. Results: Nu-3 had a wide antibacterial spectrum to Gram-positive, Gram-negative and some resistant bacteria. The MIC values of Nu-3 against all tested MRSA and MSSA were roughly in a same range while MICs of Oxacillin and Vancomycin varied between the bacteria tested. In the mouse model of skin wound infection study, the treatment with 5% Nu-3 glycerine solution also showed comparable therapeutic effects to Ciprofloxacin Hydrochloride Ointment. While Nu-3 had no effect on DNA synthesis of the tested bacteria as demonstrated in a BrdU assay, it could cause bacterial cell membrane depolarization, as measured using a BacLight T Bacterial Membrane Potential Kit. Conclusions: These results provide additional experimental data that are consistent with the hypothesis that Nu-3 represents a new class of antibacterial agents for treating topical infections and acts via a different mechanism from conventional antibiotics.