Antimicrobial resistance poses a growing global challenge, necessitating the development of novel antibacterial strategies. One promising approach is based on short amino acid motifs that are significantly underrepresented or absent in bacterial proteomes (URS).

Using a computational strategy developed in our laboratory, Navon et al. (PNAS 2016) identified URSs by detecting patterns missing from proteomic datasets. These sequences are often species-specific and, in some cases, completely absent from bacterial proteomes. Introduction of a URSs can lead to translational arrest and even cell death. These sequences are not URSs in the human proteome, suggesting potential use as antibiotics.

Two URSs shown to induce translational inhibition in E. coli are CMY and CMYW. Based on these findings, we proposed two implementation strategies: delivery of synthetic URS-containing peptides or expression of URS-encoding sequences.

Chemically synthesized peptides, including CMY- and CMYW-based sequences, were applied to Escherichia coli and Salmonella cultures. These peptides exhibited limited antibacterial activity, likely due to restricted membrane permeability associated with their hydrophobic nature. To overcome this limitation, the peptides were conjugated to cell-penetrating peptides (CPPs), resulting in a substantial improvement in antibacterial activity. Optimized peptides, including RLLRRCMYW, exhibited MIC values in the range of 8–16 µM, comparable to those of conventional antibiotics, due to improved cellular uptake and stability. Notably, CMYW-based peptides showed enhanced activity relative to CMY, consistent with stronger ribosomal stalling and their absence from bacterial proteomes.

In addition, heterologous expression of URS-encoding sequences inhibited bacterial growth, supporting that URS motifs impair growth through disruption of translation.

Structural determination of ribosome–URSpep complexes (Tarabeh et al., in preparation) provides insight into their molecular interactions with the ribosome.

Our current work focuses on further optimizing peptide permeability and stability to enhance intracellular targeting of the ribosome and maximize antibacterial activity.