# The polyamino‐isoprenyl enhancer NV716 enables the antibacterial activity of two families of multi‐target inhibitors against the ESKAPEE bacterium Enterobacter cloacae

## TL;DR

A new compound, NV716, helps two types of inactive antibiotics work against a drug-resistant bacterium by making the bacteria's outer membrane more permeable.

## Contribution

NV716 is shown to enhance the antibacterial activity of previously ineffective inhibitors against Gram-negative ESKAPEE bacteria.

## Key findings

- NV716 increases antibiotic activity by permeabilizing the outer membrane of Enterobacter cloacae.
- OX and CyC inhibitors target multiple conserved proteins essential for bacterial survival.
- Some target proteins are shared across multiple Gram-negative bacteria like Pseudomonas aeruginosa.

## Abstract

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.

The increasing incidence of resistant Gram‐negative bacteria and the lack of new drugs underscore the urgent need for new antimicrobial agents. A promising strategy to overcome this problem is the use of antibiotic adjuvants, which can revive poorly active antibiotics against resistant strains of interest. Here, we report the potentiating effect of the polyamino‐isoprenyl adjuvant NV716 in activating the antimicrobial activities of two families of multi‐target inhibitors, initially ineffective on Gram‐negative bacteria, against the ESKAPEE bacterium Enterobacter cloacae. The mechanism of action of NV716 and the potential target enzymes of our inhibitors in En. cloacae were also elucidated, highlighting the antibacterial potential of our inhibitors against bacterial pathogens.