title: Antimicrobial activity and mechanism of action of Nu-3, a protonated modified nucleotide.
abstract: 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™ 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™ 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.
authors.affiliation: College of Veterinary Medicine, China Agricultural University No, 2 Yuanmingyuan West Road, Beijing, 100193, China.
authors.forename: Shanping
authors.initials: S
authors.lastname: Cao
authors.forename: Lun-Quan
authors.initials: LQ
authors.lastname: Sun
authors.forename: Ming
authors.initials: M
authors.lastname: Wang
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D001419:Bacteria / Q000187:drug effects / Q000235:genetics; D001424:Bacterial Infections / Q000188:drug therapy* / Q000382:microbiology; D004261:DNA Replication / Q000187:drug effects; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D009711:Nucleotides / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D014946:Wound Infection / Q000188:drug therapy* / Q000382:microbiology
chemical_list: D000900:Anti-Bacterial Agents; D009711:Nucleotides