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              <p>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 Enterobacter cloacae . Remarkably, a large set of these identified proteins had already been captured in previous ABPP experiments conducted on Mycobacterium tuberculosis and/or Mycobacterium abscessus culture. Furthermore, we showed that five of the identified target proteins were present in a total lysate of Pseudomonas aeruginosa . Importantly, these latter enzymes are highly conserved among Gram‐negative bacteria, with two of them annotated as essential for bacterial survival. These results provide proof of concept that both OX and CyC, if successfully potentiated, could be used against ESKAPEE Gram‐negative bacteria.</p>
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