Small-Molecule Inhibitors of the NusB-NusE Protein-Protein Interaction with Antibiotic Activity

The NusB-NusE protein-protein interaction (PPI) is critical to the formation of stable antitermination complexes required for stable RNA transcription in all bacteria. This PPI is an emerging antibacterial drug target. Pharmaco-phore-based screening of the mini-Maybridge compound library (56 000 molecules) identified N, N'-[1,4-butanediylbis( oxy-4,1-phenylene)] bis(N-ethyl) urea 1 as a lead of interest. Competitive enzyme-linked immunosorbent assay screening validated 1 as a 20 mu M potent inhibitor of NusB-NusE. Four focused compound libraries based on 1, comprising 34 compounds in total were designed, synthesized, and evaluated as NusB-NusE PPI inhibitors. Ten analogues displayed NusB-NusE PPI inhibition >= 50% at 25 mu M concentration in vitro. In contrast to representative Gram-negative Escherichia coli and Gram-positive Bacillus subtilis species, these analogues showed up to 100% growth inhibition at 200 mu M. 2-((Z)-4-(((Z)-4-(4-((E)-(Carbamimidoylimino) methyl) phenoxy) but-2-en-1-yl) oxy)benzylidene) hydrazine-1-carboximidamide 22 showed excellent activity against important pathogens. With minimum inhibitory concentration values of <= 3 mu g/mL for Gram-positive Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus and <= 51 mu g/mL for Gram-negative Pseudomonas aeruginosa and Acinetobacter baumannii, 22 is a potent lead for a novel antibacterial target. Epifluorescence studies in live bacteria were consistent with 22, inhibiting the NusB-NusE PPI as proposed.