The polyamino-isoprenyl enhancer NV716 enables the antibacterial activity of two families of multi-target inhibitors against the ESKAPEE bacterium

Gram-negative bacteria are particularly prone to developing antimicrobial resistance (AMR), as evidenced by the WHO's ESKAPEE list of high-priority pathogens. One strategy that has increased is the use of antibiotic enhancers, which can re-empower abandoned or poorly active antibiotics against the resistant strain of interest. In this study, the polyamino-isoprenyl antibiotic enhancer, NV716, was tested in combination with two families of multi-target Ser/Cys-based enzyme inhibitors, the oxadiazolone derivatives (OX) and the Cyclipostins and Cyclophostin analogs (CyC), which are inactive against Gram-negative ESKAPEE bacteria, to potentiate their antibacterial activity and thus make them active against these bacteria. We demonstrated that NV716 potentiates some OX and CyC compounds by permeabilizing the outer membrane and thus by increasing the inhibitor accumulation, as shown by fluorescence microscopy. By using the click-chemistry activity-based protein profiling (ABPP) approach coupled with proteomic analysis, we also confirmed the multi-target nature of the best OX and CyC inhibitors by identifying their target proteins on a bacterial culture of