Repurposing farnesyltransferase inhibitors tipifarnib and lonafarnib as antibacterial agents: proteomic and...

Farnesyltransferase inhibitors (FTIs) like tipifarnib and lonafarnib, originally developed as anticancer agents, show antibacterial activity against Gram-positive pathogens through an unknown mechanism of action. Here, we investigated the proteomic response of Bacillus subtilis 168 (Gram-positive) and an efflux-deficient Escherichia coli ΔtolC mutant (Gram-negative) to sublethal concentrations of FTIs. Concentrations resulting in approximately 50% growth inhibition were applied, followed by two complementary proteomic approaches: gel-free quantitative LC-HDMS^E (both organisms) and 2D-PAGE with [³⁵S]-methionine pulse labelling of newly synthesized proteins (B. subtilis). Marker proteins from the B. subtilis 2D-PAGE approach were used for comparative proteomic response profiling against a reference library of 96 antibacterial agents. Bacillus subtilis was more susceptible to both FTIs than E. coli ΔtolC, with tipifarnib being more potent. LC-HDMS^E analysis revealed a pronounced cell envelope stress signature in B. subtilis, including regulation of YocH, YabE, and YjhA, along with several σ^B, σ^W, and σ^M-regulated stress proteins. In the 2D-PAGE analysis, reflecting acute cellular responses by monitoring newly synthesized proteins, both FTIs induced proteins such as ClpC, PspA, and LiaH. Comparative proteomic response analysis in B. subtilis showed that both FTIs cluster with membrane-active antibiotics, including xanthoangelol, MP196, gramicidin S, MP66, and nisin, which binds the cell wall precursor lipid II and forms pores in the bacterial membrane. Similarities were also observed with pseudopterosin G and pseudopteroxazole, which target the bacterial cell envelope by integration into the membrane and interfering with membrane-bound steps of cell wall biosynthesis. Yet, the similarity of the proteomic profiles does not indicate a perfect match or an identical molecular mode of action within the reference library. In E. coli ΔtolC, the proteomic response was less pronounced, but for treatment with tipifarnib, regulated proteins of the category "membrane-bounded periplasmic space" were enriched. Proteomic profiling, combined with functional validation via membrane integrity and membrane potential assays, indicates that FTIs compromise the bacterial membrane and activate stress and repair pathways. The FTIs may also indirectly affect cell wall biosynthesis. The lack of a perfect match with any of the 96 compounds in the proteomic reference library highlights FTIs as promising lead structures for the development of novel antibiotics.