Bacterial infections and the rise of antibiotic resistance, especially multidrug resistance, have generated a clear need for discovery of novel therapeutics. We demonstrated that a small molecule drug PKZ18 targets the T-box mechanism and inhibits bacterial growth. The T-box is a structurally conserved riboswitch-like gene regulator in the 5′-untranslated region of numerous essential genes of Gram-positive bacteria. T-boxes are stabilized by cognate, unacylated tRNA ligands, allowing the formation of an anti-terminator hairpin in the mRNA that enables transcription of the gene. In the absence of an unacylated cognate tRNA, transcription is halted due to the formation of a thermodynamically more stable terminator hairpin. PKZ18 targets the site of the codon/anticodon interaction of the conserved Stem I and reduces T-box controlled gene expression. Here we show that novel analogs of PKZ18 have improved minimum inhibitory concentrations, bactericidal effects against methicillin resistant Staphylococcus aureus (MRSA), and increased efficacy in nutrient limiting conditions. The analogs have reduced cytotoxicity against eukaryotic cells compared to PKZ18. The PKZ18 analogs acted synergistically with aminoglycosides to significantly enhance the efficacy of the analogs and aminoglycosides, further increasing their therapeutic windows. RNA sequencing showed that the analog PKZ18-22 affects expression of 8 of 12 T-box controlled genes in a statistically significant manner, but not other 5′UTR regulated genes in MRSA. Very low levels of resistance further support the existence of multiple T-box targets for PKZ18 analogs in the cell. Together the multiple targets, low resistance, and synergy make PKZ18 analogs promising drugs for development and future clinical applications.

Combating single and multi-drug-resistant infections in the form of biofilms is an immediate challenge. The challenge is to discover innovative targets and develop novel chemistries that combat biofilms and drug-resistant organisms, and thwart emergence of future resistant strains. An ideal novel target would control multiple genes, and can be inhibited by a single compound. We previously demonstrated success against Staphylococcus aureus biofilms by targeting the tRNA-dependent regulated T-box genes, not present in the human host. Present in Gram-positive bacteria, T-box genes attenuate transcription with a riboswitch-like element that regulates the expression of aminoacyl-tRNA synthetases and amino acid metabolism genes required for cell viability. PKZ18, the parent of a family of compounds selected in silico from 305,000 molecules, inhibits the function of the conserved T-box regulatory element and thus blocks growth of antibiotic-resistant S. aureus in biofilms. The PKZ18 analog PKZ18-22 was 10-fold more potent than vancomycin in inhibiting growth of S. aureus in biofilms. In addition, PKZ18-22 has a synergistic effect with existing antibiotics, e.g., gentamicin and rifampin. PKZ18-22 inhibits the T-box regulatory mechanism, halts the transcription of vital genes, and results in cell death. These effects are independent of the growth state, planktonic or biofilm, of the bacteria, and could inhibit emergent strains.