In Vitro Preclinical Evaluation of BPP-PNA Antisense Antibiotics: A Protocol for Efficacy, Safety, and Resistance Analysis.

The global challenge of antibacterial resistance necessitates urgent efforts to develop novel antimicrobial agents with innovative chemical scaffolds. Bacterial-penetrating peptide-peptide nucleic acid (BPP-PNA) conjugates represent a promising class of antisense antibiotics (ASObiotics) capable of selectively targeting bacterial gene expression. This chapter provides a structured protocol for the preclinical evaluation of new BPP-PNA compounds, focusing on efficacy, resistance potential, safety, and mechanism of action. Efficacy assessment includes determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), followed by time-kill assays to evaluate bactericidal kinetics. The combination efficacy of multiple BPP-PNAs is investigated using checkerboard assays. Safety and biocompatibility studies include hemolysis assays on red blood cells and cytotoxicity assessments using cell lines relevant to the infection site and drug metabolism and clearance. To evaluate the propensity for resistance development (spontaneous mutation frequency), bacterial populations (109-1010 CFU) are exposed to increasing BPP-PNA concentrations (4-16× MIC) to isolate resistant mutants, which are then analyzed via whole-genome sequencing and compared with parental strains. Finally, mechanistic studies examine the impact of BPP-PNAs on bacterial membrane integrity using Sytox Green and N-phenyl-1-naphthylamine (NPN) assays to assess inner and outer membrane permeability, respectively. This comprehensive protocol facilitates the systematic preclinical characterization of BPP-PNA antibiotics, contributing to the development of innovative therapeutic strategies to combat antibiotic resistance.