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

Emma Forest, Jordan Lehoux, Alexandre Guy, Thierry Durand, Stéphane Audebert, Luc Camoin, Christopher D Spilling, Céline Crauste, Stéphane Canaan, Jean Michel Brunel, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Emma Forest, Jordan Lehoux, Alexandre Guy, Thierry Durand, Stéphane Audebert, et al. The polyamino-isoprenyl enhancer NV716 enables the antibacterial activity of two families of multi-target inhibitors against the ESKAPEE bacterium Enterobacter cloacae. mLife, 2025, 4 (3), pp.259-274. 10.1002/mlf2.70014. hal-05131779

HAL Id: hal-05131779

https://hal.science/hal-05131779v1

Submitted on 26 Jun 2025

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
ORIGINAL RESEARCH

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

Emma Forest<fnref n="1" />,² ID, Jordan Lehoux³ ID, Alexandre Guy³ ID, Thierry Durand³ ID, Stéphane Audebert⁴ ID, Luc Camoin⁴ ID, Christopher D. Spilling⁵, Céline Crauste³ ID, Stéphane Canaan<fnref n="1" /> ID, Jean Michel Brunel² ID, Jean-Michel Bolla²,\* ID
and Jean-François Cavalier<fnref n="1" />,\* ID

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 demon-strated 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.

**Keywords:** activity based-protein profiling; antibiotic resistance; Cyclipostins and Cyclophostin analogs; drug enhancers; oxadiazolone derivatives

Impact statement

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.

<footnote n="1">¹Aix Marseille Univ., CNRS, LISM UMR7255, IMM FR3479, Marseille, France. ²Aix Marseille Univ, INSERM, SSA, MCT, Marseille, France. ³IBM, Univ Montpellier, CNRS, ENSCM, Montpellier, France. ⁴Aix Marseille Univ, CNRS, INSERM, Institut Paoli-Calmettes, CRCM, Marseille Protéomique, Marseille, France. ⁵Department of Chemistry and Biochemistry, University of Missouri-St. Louis, St. Louis, Missouri, USA.</footnote>

<footnote>* Correspondence: Jean-Michel Bolla, jean-michel.BOLLA@univ-amu.fr; Jean-François Cavalier, jfcavalier@imm.cnrs.fr
Received September 30, 2024; Accepted March 19, 2025; Published online xxx</footnote>

<footnote>DOI: 10.1002/mlf2.70014</footnote>

<footnote>This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</footnote>
## INTRODUCTION

The discovery and development of antimicrobials, such as penicillin, during the 20th century revolutionized chemotherapy to a point where the healthcare community believed that the war against microbes had been won¹,². In fact, between the 1940s and the 1960s, the scientific community experienced what is now known as the “Golden Era” of antibiotics, during which a large number of new drugs were developed, but since then, very few new classes have been discovered. However, while antibiotics have strongly transformed our relationship with disease-causing bacteria, empirical prescription in the case of infections contributed greatly to the emergence of antibiotic resistance among bacterial populations. The spread of antimicrobial resistance (AMR), coupled with the decline in antibiotic development, has become a major public health concern. Recent studies have estimated that infections caused by multidrug-resistant (MDR) bacteria are responsible for 700,000 deaths each year³. Another statistical model estimated that nearly 5 million deaths were associated with AMR in 2019⁴. To refocus the scientific community's efforts on this issue, in 2017, the WHO regrouped the highest-priority pathogens with respect to AMR into the ESKAPEE list⁵,⁶, which includes two Gram-positive bacteria: *Enterococcus faecium* and *Staphylococcus aureus*, and five Gram-negative bacteria, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Pseudomonas aeruginosa*, *Enterobacter* spp., and *Escherichia coli*. Among these, Gram-negative bacteria are particularly

overrepresented. This is due to their high propensity to develop many resistance mechanisms⁷,⁸, in addition to their intrinsic resistance to many antimicrobials resulting from their membrane composition⁹,¹⁰, and the expression of broad-spectrum efflux pumps¹¹. In fact, the efflux pumps may act synergistically with the impermeability of the outer membrane to increase antibiotic resistance. As a direct consequence, some Gram-negative antibiotics may become clinically obsolete.

With a significant slowdown in the development of new antimicrobials, largely due to a poor return on investment¹, new strategies to overcome MDR need to be explored. The concept of drug adjuvants, also called “adjuvants,” “chemosensitizers,” or “antibiotics enhancers,” able to (re) activate poorly active antibiotics against resistant strains, constitutes a real valuable strategy¹²,¹³. These adjuvants are chemical entities that have no intrinsic antimicrobial activity, but can potentiate/activate inefficient antibiotics, either by protecting the drug from enzymatic degradation (i.e., β-lactamase inhibitors¹⁴) or by enhancing its efficacy through membrane permeabilization or efflux pump blockade¹³,¹⁵,¹⁶. In this context, we have demonstrated that the polyamino-isoprenyl derivative NV716 (Figure 1A) was able to restore the activity of disused antibiotics, such as doxycycline, chloramphenicol, and rifampicin, against *P. aeruginosa* MDR strains²⁴, including clinical isolates from cystic fibrosis (CF) patients¹³,²⁵. Its enhancing properties resulted from an

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Chemical structures of compounds. (A) Polyamino-isoprenyl enhancer NV716¹⁷,¹⁸. (B) Oxadiazolone-core derivatives¹⁹,²⁰. (C) The Cyclipostins and Cyclophostin analogs²¹⁻²³.</figcaption></figure>
increase in the outer-membrane permeabilization as well as the inhibition of efflux mechanisms²⁴,²⁶.

Some antibiotics, such as polymyxin B, which permeabilizes both the outer and the inner membrane of Gram-negative bacteria, or ciprofloxacin, which inhibits topoisomerases II and IV, are known to exert multiple mechanisms of action²⁷, and their use in combination therapy is frequent when dealing with MDR bacteria²⁸,²⁹. Accordingly, another valuable strategy is the development of multi-target compounds to prevent the emergence of target-specific resistant strains²⁷, thus simplifying polytherapy treatment for the patient. In this challenging context, a few years ago, we have developed two families of multi-target inhibitors: the oxadiazolone-core derivatives (OX) and the Cyclipostins and Cyclophostin analogs (CyC) (Figure 1B,C)³⁰. These compounds bind covalently to catalytic serine or cysteine residues in the active site of target proteins, thereby inhibiting the enzymatic activity of corresponding (Ser/Cys)-based enzymes¹⁹,²¹. Nontoxic to mammalian host cells, both OX and CyC specifically impair the growth of all mycobacteria, including *Mycobacterium abscessus*²²,³¹ and *Mycobacterium tuberculosis*²⁰,³², in broth medium and/or within infected macrophages. Using activity-based protein profiling (ABPP) approaches on *M. abscessus*²²,³¹ and *M. tuberculosis*²⁰,³²,³³ cultures, we clearly demonstrated the multi-target inhibitory activity of these compounds by identifying all their target mycobacterial enzymes. Despite the huge number of (Ser/Cys)-based enzymes in all prokaryotic cells, the OX and CyC are, however, not active against Gram-negative bacteria³⁴. Using fluorescent CyC analogs, we unambiguously confirmed that this lack of antibacterial activity was not due to the absence of targets, but rather due to the inability of these molecules to cross the outer membrane of such bacteria³⁵. Indeed, when treating a total lysate of *P. aeruginosa* with the same fluorescent CyC inhibitors, distinct fluorescent bands were detected on SDS-PAGE gel corresponding to bacterial (Ser/Cys)-based enzymes that may have reacted with our inhibitors³⁵.

From these findings, it is thus tempting to assume that if OX and CyC could penetrate the outer membrane of Gram-negative bacteria, they would be able to block various metabolic pathways impacting their growth, as in the case of *M. abscessus* or *M. tuberculosis*. Therefore, in this study, we evaluated the efficiency of the OX and CyC in association with the polyamino-isoprenyl enhancer NV716, against a panel of four Gram-negative ESKAPEE bacteria: *P. aeruginosa*, *K. pneumoniae*, *E. coli*, and *Enterobacter cloacae*. Of the four bacteria tested, only *En. cloacae* was sensitive to OX/CyC–NV716 combinations, with significant MIC values obtained. A series of real-time assays (permeabilization and/or disruption of the outer membrane, depolarization of the inner membrane, inhibition of efflux pumps) combined with quantitative fluorescence microscopy experiments helped to elucidate the mechanism of action by which NV716 potentiates the activity of OX and CyC in *En. cloacae*. The potential targets of the two most potent OX and CyC inhibitors were further identified by click-chemistry ABPP (CC-ABPP) using

their corresponding click-ready affinity-based probes on a bacterial culture of *En. cloacae* in the presence of NV 716. The same approach conducted on a *P. aeruginosa* lysate revealed similar target enzymes, suggesting that, provided they can cross the outer membrane, the OX and CyC inhibitors could represent an interesting therapeutic alternative against resistant Gram-negative bacteria.

## RESULTS

### The polyamino-isoprenyl derivative NV716 enables the antibacterial activity of the OX and CyC compounds against *En. cloacae* ATCC 23355

The antibacterial properties of selected OX and CyC active on *M. abscessus* and *M. tuberculosis* growth (i.e., 7 OXs and 9 CyCs; Figure 1B,C and Table 1)³⁰ have been tested in the presence or absence of NV716 against four Gram-negative bacteria belonging to the ESKAPEE group: *P. aeruginosa* PAO1, *K. pneumonia* ATCC 13883, *E. coli* ATCC 25922, and *En. cloacae* ATCC 23355. Their corresponding minimal inhibitory concentrations leading to 90% of growth inhibition, hereinafter referred to as MIC₉₀, determined by the rapid p-iodonitrophenyltetrazolium violet (INT) colorimetric assay following absorbance reading at 470 nm³⁶,³⁷, are shown in Table 1. As expected, when tested alone, OX and CyC were fully inactive against the four Gram-negative bacteria (MIC₉₀ > 100 µg/ml). To exclude any indirect growth defect caused by the polyamino-isoprenyl derivative, NV716 was used at a fixed sub-MIC concentration of 4.1 µg/ml (i.e., 0.2× MIC₉₀ – MICₙV716 = 20.3 µg/ml) with *P. aeruginosa* PAO1 and *K. pneumonia* ATCC 13883²⁴, and at 1.2 µg/ml (i.e., 0.24× MIC₉₀ – MICₙV716 = 5.0 µg/ml) with *E. coli* ATCC 25922²⁴ and *En. cloacae* ATCC 23355 for the combination assays with the OX and CyC compounds (Figure S1). Such sub-MIC concentrations have previously been validated through synergistic assays and subsequent determination of the Fractional Inhibitory Concentration Index (FICI) of various antibiotics in combination with NV716 against these Gram-negative bacteria³⁸⁻⁴⁰.

In these conditions, and among the four bacteria tested, NV716 was able to potentiate the activity of 3 OXs and 4 CyCs against *En. cloacae* ATCC 23355 only. If the OX–NV716 association resulted in weak antimicrobial activity (MIC₉₀ = 75–100 µg/ml), significant and promising MIC₉₀ values were obtained with the four CyC–NV716 associations (Table 1). Interestingly, CyC₁₇ and CyC₃₁, which showed the best antibacterial activity against mycobacterial growth, did not affect *En. cloacae* ATCC 23355 even in the presence of the drug enhancer NV716. Conversely, NV716-potentiated CyC₇α,β/25/32 showed MIC₉₀ values in the same range as the reference drugs doxycycline (7.5 ± 0.3 µg/ml) and chloramphenicol (11 ± 0.6 µg/ml) when tested alone.

We further evaluated the efficiency of the OX and CyC combination with NV716 in the context of antimicrobial resistance by testing the susceptibility of five clinical *En. cloacae* strains collected in three hospital centers located in the south of France between October 2017 and May 2020 and characterized
in a previous study (Table S1)$^{41}$. First, all five clinical *En. cloacae* strains were almost 16 times less susceptible to the effect of NV716 (MIC$_{90}$$_{Ecl}$$_{-isolate}$ = 80 µg/ml) than the reference strain ATCC 23355 (MIC$_{90}$ = 5 µg/ml). Remarkably, the presence of only 1.2 µg/ml (= 0.015 × MIC$_{90}$$_{Ecl}$$_{-isolate}$) NV716 was sufficient to potentiate the antibacterial activity of doxycycline and chloramphenicol by at least 2.4-fold and up to 18-fold in the case of *Ecl71* (Table S2). Interestingly, *Ecl136* remained insensitive to chloramphenicol (MIC$_{90}$ > 64 µg/ml) even in the presence of NV716.

Drug susceptibility testing of the most active compounds, iBPOX and CyC$_{32}$, against the five *En. cloacae* clinical isolates in

the presence or absence of NV716 was further investigated (Table 2). With iBPOX, although no MIC$_{90}$ was achieved for any clinical isolate despite the addition of NV716 up to 20 µg/ml (= 0.25 × MIC$_{90}$$_{Ecl}$$_{-isolate}$), the same iBPOX–NV716 association was able to inhibit 50% of the growth of most isolates at concentrations (MIC$_{50}$ = 37.8–92.8 µg/ml), similar to the one obtained for the reference strain ATCC 23355 (MIC$_{50}$ = 44.9 µg/ml) (Table 2). On the other hand, the CyC$_{32}$–NV716 association displayed good MIC$_{50}$ and MIC$_{90}$ (Table 2) values against *Ecl71* (MIC$_{50/90}$ = 10.0 ± 1.9/39.1 ± 14.5 µg/ml) and *Ecl93* (MIC$_{50/90}$ = 5.3 ± 1.6/9.7 ± 4.5 µg/ml), similar to those obtained with reference drugs (Table S2).

<table><caption>**Table 1.** Antibacterial activities of selected OX and CyC in the presence or absence of NV716 against four Gram-negative bacteria belonging to the ESKAPEE groupª.</caption><thead><tr><th rowspan="3">Cpds</th><th colspan="8">MIC<sub>90</sub> (µg/ml)</th></tr><tr><th colspan="2">M. tuberculosis</th><th colspan="2">En. cloacae ATCC 23355</th><th colspan="2">E. coli ATCC 25922</th><th colspan="2">P. aeruginosa PAO1</th><th colspan="2">K. pneumonia ATCC 13883</th></tr><tr><th>mc<sup>2</sup>6230<sup>b</sup></th><th>M. abscessus CIP 104536<sup>T</sup> S<sup>c</sup></th><th>- NV716</th><th>+ NV716 (1.2 µg/ml)<sup>d</sup></th><th>- NV716</th><th>+NV716 (1.2 µg/ml)<sup>d</sup></th><th>- NV716</th><th>+ NV716 (4.1 µg/ml)<sup>d</sup></th><th>- NV716</th><th>+ NV716 (4.1 µg/ml)<sup>d</sup></th></tr></thead><tbody><tr><td>iBPOX</td><td>&gt;100</td><td>16 ± 0.56</td><td>&gt;100</td><td>80 ± 14.2</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td></tr><tr><td>iBpPPOX</td><td>22 ± 0.70</td><td>28 ± 1.8</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td></tr><tr><td>HPOX</td><td>43 ± 0.80</td><td>26 ± 1.4</td><td>&gt;100</td><td>80 ± 16.3</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td></tr><tr><td>HpPPOX</td><td>20 ± 0.8</td><td>28 ± 1.2</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td></tr><tr><td>MmPPOX</td><td>68 ± 2.9</td><td>34 ± 1.2</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td></tr><tr><td>MpPPOX</td><td>&gt;100</td><td>45 ± 1.8</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td></tr><tr><td>MemPPOX</td><td>39 ± 1.1</td><td>15 ± 0.65</td><td>&gt;100</td><td>75 ± 20.4</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td></tr><tr><td>CyC<sub>6α</sub></td><td>53 ± 2.3</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td></tr><tr><td>CyC<sub>6β</sub></td><td>&gt;100</td><td>65 ± 1.7</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td></tr><tr><td>CyC<sub>7α</sub></td><td>&gt;100</td><td>79 ± 1.8</td><td>&gt;100</td><td>19 ± 6.2</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td></tr><tr><td>CyC<sub>7β</sub></td><td>14 ± 1.5</td><td>73 ± 1.8</td><td>&gt;100</td><td>19 ± 6.2</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td></tr><tr><td>CyC<sub>8β</sub></td><td>&gt;100</td><td>74 ± 1.7</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td></tr><tr><td>CyC<sub>17</sub></td><td>1.2 ± 0.03</td><td>6.5 ± 0.12</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td></tr><tr><td>CyC<sub>25</sub></td><td>42 ± 1.5</td><td>34 ± 0.31</td><td>&gt;100</td><td>19 ± 5.1</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td></tr><tr><td>CyC<sub>31</sub></td><td>2.0 ± 0.06</td><td>0.44 ± 0.11</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td></tr><tr><td>CyC<sub>32</sub></td><td>2.0 ± 0.26</td><td>63 ± 0.74</td><td>&gt;100</td><td>6.5 ± 2.0</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td></tr><tr><td>DOX<sup>e</sup></td><td>-</td><td>-</td><td>7.5 ± 0.3</td><td>0.16 ± 0.01</td><td>2</td><td>0.031</td><td>16</td><td>1</td><td>1.1 ± 0.9</td><td>1.2 ± 0.7</td></tr><tr><td>CHL<sup>e</sup></td><td>-</td><td>-</td><td>11 ± 0.6</td><td>1.7 ± 0.6</td><td>8</td><td>0.5</td><td>32</td><td>0.125</td><td>2.5 ± 1.5</td><td>2.0</td></tr></tbody></table>

<table><caption>**Table 2.** Antibacterial activities of selected OX and CyC in the presence or absence of NV716 against *En. cloacae* clinical isolatesª.</caption><thead><tr><th rowspan="3">En. cloacae strains</th><th colspan="4">MIC<sub>50</sub>/MIC<sub>90</sub> (µg/ml)</th></tr><tr><th colspan="2">iBPOX</th><th colspan="2">CyC<sub>32</sub></th></tr><tr><th>- NV716</th><th>+NV716 (20 µg/ml)</th><th>- NV716</th><th>+NV716 (20 µg/ml)</th></tr></thead><tbody><tr><td>ATCC 23355</td><td>&gt;100</td><td>44.9 ± 4.3/80.0 ± 14.2</td><td>&gt;100</td><td>3.7 ± 0.26/6.5 ± 2.0</td></tr><tr><td>Ecl53</td><td>&gt;100</td><td>92.8 ± 1.4/&gt;100</td><td>&gt;100</td><td>&gt;100</td></tr><tr><td>Ecl71</td><td>&gt;100</td><td>70.1 ± 14.3/&gt;100</td><td>&gt;100</td><td>10.0 ± 1.9/39.1 ± 14.5</td></tr><tr><td>Ecl80</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td></tr><tr><td>Ecl93</td><td>&gt;100</td><td>&gt;100</td><td>&gt;100</td><td>5.3 ± 1.6/9.7 ± 4.5</td></tr><tr><td>Ecl136</td><td>&gt;100</td><td>37.8 ± 17.9/&gt;100</td><td>&gt;100</td><td>&gt;100</td></tr></tbody></table>

<footnote>ªExperiments were performed as described in the Materials and Methods section. MIC$_{90}$, compound minimal concentration leading to 90% of growth inhibition, as determined by the p-iodonitrophenyltetrazolium violet (INT) assay following absorbance reading at 470 nm. Values are mean of at least three independent assays performed in triplicate. ᵇData from Refs.²⁰,³²⁻³⁴. ᶜData from Refs.²²,³¹,³⁴,³⁵. ᵈThe final concentration of the enhancer is indicated under parenthesis. ᵉData on *E. coli* ATCC 25922, *P. aeruginosa* PAO1, and *K. pneumoniae* ATCC 13883 are from Refs.²⁴,²⁶. CHL, chloramphenicol; DOX, doxycycline; INT, p-iodonitrophenyltetrazolium violet.</footnote>

<footnote>ªExperiments were performed as described in the Materials and Methods section. MIC$_{50}$/MIC$_{90}$, compound minimal concentration leading to 50% and 90% of growth inhibition, respectively, as determined by the INT assay following absorbance reading at 470 nm. Values are means of at least three independent assays.</footnote>
Taken together, these results underscore the potentiating effect of NV716 in activating the OX and CyC antibacterial activity against the *En. cloacae* ATCC 23355 reference strain as well as clinical isolates.

**The CyC<sub>32-Dansyl</sub> is able to label *En. cloacae* ATCC 23355 only in the presence of NV716**

The ability of NV716 to potentiate the iBPOX and CyC₃₂ compounds may result from better penetration of these two inhibitors into the bacteria following disruption/ permeabilization of the *En. cloacae* outer membrane by the adjuvant. To confirm this hypothesis, *En. cloacae* ATCC 23355 was preincubated with the CyC₃₂-Dansyl fluorescent probe in the presence or absence of NV716 and the obtained CyC₃₂-Dansyl-treated bacteria were further processed for fluorescent microscopy, as previously reported³⁵. In the absence of NV716, only a slight and diffuse fluorescence was detected, suggesting that the CyC₃₂-Dansyl alone was not able to cross the *En. cloacae* membrane. On the contrary, when

incubated in the presence of NV716, this fluorescent CyC probe was found to accumulate inside the bacteria. Interestingly, this compound seems to localize as foci closed to the bacterial pole (Figure 2A—inserted zoom). Quantitative analysis of CyC₃₂-Dansyl accumulation in *En. cloacae* ATCC 23355 was next performed (Figure 2B). In the presence of NV716, a significantly higher level of CyC₃₂-Dansyl associated with *En. cloacae* was reached rather than in the absence of the polyamino-isoprenyl adjuvant (average mean pixel intensity [a.u.] of 47.9 ± 1.5 vs. 17.6 ± 0.8, for CyC₃₂-Dansyl + NV716 positive bacteria vs. CyC₃₂-Dansyl positive bacteria, respectively, *p*-value < 0.0001) (Figure 2B). Conversely, incubation of *P. aeruginosa* PAO1 with CyC₃₂-Dansyl in the presence of NV716 did not result in any fluorescence uptake (Figure S2A). Indeed, no significant differences in the quantitative fluorescence signal were observed (average mean pixel intensity [a.u.] of 26.3 ± 1.4 vs. 24.3 ± 2.4, for CyC₃₂-Dansyl + NV716 vs. DMSO, respectively) (Figure S2B), indicating that NV716 is not able to potentiate CyC₃₂-Dansyl penetration inside *P. aeruginosa*.

<figure><img src="image_4.png" /><figcaption>**Figure 2.** NV716 allows fluorescent compound CyC₃₂-Dansyl to accumulate in *Enterobacter cloacae* ATCC 23355. (A) Representative fluorescence images of the *En. cloacae* ATCC 23355 strain in the presence of fluorescent CyC₃₂-Dansyl. The bacteria were exposed to 124 µg/ml CyC₃₂-Dansyl or DMSO (vehicle, negative control) in the presence or absence of 40.6 µg/ml NV716, washed, and fixed with 4% paraformaldehyde. Fixed bacteria were imaged using an Olympus IX81 confocal microscope³⁵. Scale bars: 10 µm. (B) Quantitative analysis of the Dansyl fluorescence signal per bacterium shown as a violin plot and expressed as pixel intensity arbitrary units (a.u.). To eliminate any false-positive hits due to bacterial autofluorescence, the maximum fluorescence intensity recovered with the vehicle (DMSO only) was used as the threshold. All CyC₃₂-Dansyl-treated bacteria with a fluorescence signal above this value (i.e., values above the dotted line) were therefore considered probe-positive. The number under each violin represents the number of “positive” bacteria counted above this threshold in each condition. Results are from three biologically independent experiments, *n* = 150, for each condition. The *p*-values were calculated using a two-tailed *t*-statistic test with Prism 8.0 (Graphpad, Inc). \*\*\*\* *p*-value < 0.0001; ns, not significant (*p*-value > 0.05).</figcaption></figure>
## NV716 acts through permeabilization of the outer membrane and depolarization of the inner membrane of *En. cloacae* ATCC 23355

A series of real-time assays have been previously reported<sup>13,17,24,40</sup> and used to determine the capacity of NV716, tested at two working concentrations of 6 and 50 µg/ml (i.e., 15 and 125 µM), to (i) permeabilize the outer membrane; (ii) both the outer and the inner membranes; and (iii) depolarize the inner membrane of Gram-negative bacteria, including the *En. cloacae* DSM 129 strain (MIC<sub>90_NV716</sub> = 20 µg/ml)<sup>39</sup>. The results obtained suggest that NV716 is capable of severely disrupting the integrity of the outer membrane of all Gram-negative pathogens, with only a poor impact on the permeabilization of the inner membrane, as evidenced by low levels of ATP efflux<sup>39</sup>. In order to investigate more in depth the discrepancy observed with the potentiation of OX and CyC only on *En. cloacae* ATCC 23355, we decided to perform the same real-time assays, but with increasing concentrations of NV716 (Figure S3), to evaluate the dose-response effect of this enhancer. For the tests focused on membrane integrity, Polymyxin B was used as the positive control due to its well-known pore-forming abilities<sup>42</sup>.

The impact of NV716 on outer-membrane permeability was first assessed by monitoring the absorbance at 490 nm of the hydrolysis product nitrocefin. This non-permanent yellow-colored cephalosporin is indeed degraded by periplasmic β-lactamases when the outer membrane has been permeabilized<sup>24</sup>. In the presence of Polymyxin B, the permeabilization was found to increase rapidly up to a plateau value reached at 10–1000 µg/ml (i.e., 100% permeabilization) (Figure S3A). Conversely, a classic sigmoidal dose-response curve was obtained with NV716, leading to significant ~155% membrane permeabilization (p-value < 0.0001) at the highest concentration used as compared to Polymyxin B (Figure S3A). Inner-membrane permeabilization was assessed by measuring the fluorescent signal generated when propidium iodide (PI) intercalates into bacterial DNA after permeabilization of both the outer and inner membranes<sup>43</sup>. Similarly, Polymyxin B increased PI fluorescence drastically, with an optimum reached at 1000 µg/ml, whereas NV716 failed to permeate the inner membrane irrespective of the concentration used (Figure S3B).

Finally, the capacity of NV716 to depolarize *En. cloacae* ATCC 23355 inner membrane was evaluated using the DiSC<sub>3</sub>(5) membrane potential-sensitive dye that accumulates on polarized membranes, causing fluorescence to quench, but that is released when the membrane is depolarized, restoring the fluorescence signal<sup>44</sup>. In both cases, Polymyxin B and NV716 were found to depolarize the inner membrane of *En. cloacae* ATCC 23355 in a dose-dependent manner (Figure S3C). However, at concentrations above 20 µg/ml, NV716 induced a stronger release of DiSC<sub>3</sub>(5) than Polymyxin B (Figure S3C). To confirm that the phenotypes observed in the presence of NV716 were the result of its effect on *En. cloacae* ATCC 23355 membranes rather than cell lysis, the survival of this bacterium under the operating conditions used in these real-time assay series was then checked. Regardless of the concentration of NV716 tested, no

significant differences in CFU counts were achieved compared to the positive control (Figure S3D). Taken together, the results of this dose-dependent mechanistic study conducted on *En. cloacae* ATCC 23355 confirmed a mode of action of NV716 similar to that previously reported on several other Gram-negative bacteria<sup>38-40,45</sup>, i.e., permeabilization of the outer membrane and depolarization of the inner membrane.

The fact that NV716 has been shown to efficiently inhibit active efflux pumps of *P. aeruginosa*, *E. coli*, and *K. pneumoniae*<sup>24,40,45</sup>, prompted us to investigate its effect on the active efflux of the 1,2'-dinaphtylamine (DiNA) fluorescent probe<sup>46,47</sup> in *En. cloacae* ATCC 23355. First, the resistance/nodulation/division (RND) superfamily of efflux pumps<sup>48</sup> was blocked by the addition of carbonyl cyanide-m-chlorophenylhydrazone (CCCP), an inhibitor of the proton motive force (PMF), and the bacteria were loaded with DiNA before re-energizing the RND pumps with glucose<sup>24</sup>. When NV716 was added before glucose addition, a reduction of the fluorescence linked to DiNA efflux was observed (Figure S3F). However, the fact that, despite our best efforts, *En. cloacae* ATCC 23355 was only able to efflux 25% of the preloaded DiNA after the addition of glucose indicates a very weak efflux activity of this strain, thus limiting the potential impact of NV716 (Figure S3E).

## Synthesis and antibacterial activity of the CyC<sub>32yne</sub> and the iBP<sub>yne</sub>OX “click-ready” probes

We recently validated the use of new efficient CyC<sub>yne</sub> activity-based probes (ABPs), obtained by the direct introduction of a terminal alkyne function, as a means to capture the target proteins of these probes through direct CC-ABPP<sup>33</sup>. Given the similar phenotypic mechanisms of action of NV716 in *En. cloacae* ATCC 23355 and *P. aeruginosa* PAO1, we decided to follow the same chemoproteomic CC-ABPP approach to determine whether the specific antimicrobial activity observed against *En. cloacae* only was the consequence of unique and/or multiple targets in this Gram-negative bacterium. To achieve this goal, click-ready ABPs of the most potent inhibitors of *En. cloacae* ATCC 23355 growth, that is, iBP<sub>yne</sub>OX and CyC<sub>32yne</sub><sup>33</sup>, respectively, were first synthesized (Figure 3).

The synthesis of CyC<sub>32yne</sub> has been described in a previous study<sup>33</sup>. Regarding the alkyne derivative of iBPOX (i.e., iBP<sub>yne</sub>OX, Figure 3), we deliberately chose to position the alkyne function on the phenyl group rather than on the iso-butyl motif, in order to avoid any steric hindrance near the reactive oxadiazolone core. The synthesis was first envisaged through the formation of the oxadiazolone ring using an alkyne phenyl-hydrazine derivative. Unfortunately, we failed to synthesize the starting hydrazine from ethynyl-aniline. The alkyne residue was thus introduced after the cyclization process<sup>20</sup>: the oxadiazolone ring was easily obtained starting from the commercial iodo-phenylhydrazine **1** and isobutyl chloroformate (iBCF). Following the one-pot (2 steps) cyclization process involving diphosgene, the corresponding iodo-oxadiazolone **2** was used in Sonogashira coupling for alkyne introduction<sup>49</sup>. The final trimethylsilyl group de-protection of **3** was achieved using a catalytic amount of
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<table><caption>**Figure 3.** Synthesis and antibacterial activity of click-ready activity-based probes (ABPs). (A) Synthesis of the alkyne derivative of iBPOX bearing an alkyne group on the phenyl moiety, that is, iBP<sub>yne</sub>OX. (B) Structure of the two alkyne click-ready probes used in this study, together with their parent molecules. The inset table shows the antibacterial activity of the four derivatives against *En. cloacae* ATCC 23355, reported as MIC<sub>90</sub>, that is, compound minimal concentration leading to 90% of growth inhibition as determined by the p-iodonitrophenyltetrazolium violet (INT) assay following absorbance reading at 470 nm. Values are means of three independent assays performed in triplicate. DOX, doxycycline used as a control drug. INT, p-iodonitrophenyltetrazolium violet.</caption><thead><tr><th rowspan="2"></th><th colspan="2">MIC<sub>90</sub> against <i>En. cloacae</i> (µg/ml)</th><th rowspan="2">Gain in MIC</th></tr><tr><th>- NV716</th><th>+ NV716 (1.2 µg/ml)</th></tr></thead><tbody><tr><td>iBPOX</td><td>&gt; 100</td><td>80 ± 14.2</td><td>&gt; ×1.2</td></tr><tr><td>iBP<sub>yne</sub>OX</td><td>&gt; 100</td><td>59 ± 8.9</td><td>&gt; ×1.7</td></tr><tr><td>CyC<sub>32</sub></td><td>&gt; 100</td><td>6.5 ± 2.0</td><td>&gt; ×15</td></tr><tr><td>CyC<sub>32yne</sub></td><td>&gt; 100</td><td>22 ± 1.5</td><td>&gt; ×4.5</td></tr><tr><td>DOX</td><td>7.5 ± 0.3</td><td>0.16 ± 0.01</td><td>×47</td></tr></tbody></table>

tetra-n-butylammonium fluoride (TBAF) to avoid ring opening<sup>50</sup>. This resulted in the synthesis of the desired iB-P<sub>yne</sub>OX click-ready ABP with a 39% overall yield (Figure 3A).

The antimicrobial potency of the two alkyne derivatives in the presence or absence of NV716 was evaluated against *En. cloacae* ATCC 23355 and compared with that of their respective parent molecules (Figure 3B). Although no antimicrobial activity was observed in the absence of NV716, comparable antibacterial activities against *En. cloacae* ATCC 23355 were obtained in the presence of NV716 for each inhibitor/probe pair, that is, iBPOX/iBP<sub>yne</sub>OX (80/59 µg/ml) and CyC<sub>32</sub>/CyC<sub>32yne</sub> (6.5/22 µg/ml), respectively (Figure 3B). Following this prerequisite, iBP<sub>yne</sub>OX and CyC<sub>32yne</sub> were used as affinity-based probes in a CC-ABPP strategy (Figure 4A).

## En. cloacae ATCC 23355 target proteins identified by CC-ABPP using the CyC<sub>32yne</sub> and iBP<sub>yne</sub>OX affinity-based probes

To take into account the critical issue of penetration of our probes inside *En. cloacae* ATCC 23355, each OX<sub>yne</sub> and CyC<sub>yne</sub> molecule was incubated with a bacterial culture of this Gram-negative bacterium in the presence of NV716. Accordingly, mid-log-phase *En. cloacae* ATCC 23355 cells were incubated with each inhibitor/enhancer pair: iBP<sub>yne</sub>OX-NV716 or CyC<sub>32yne</sub>-NV716 (or DMSO-NV716 as a control for the nonspecific condition). After cell lysis, each total lysate was subjected to a click-chemistry reaction using the copper(I)-catalyzed Huisgen's 1,3-dipolar cycloaddition reaction<sup>53</sup> to form a triazole cycle between the probe-enzyme complex and the Desthiobiotin-PEG<sub>3</sub>-N<sub>3</sub> reporter. The samples were enriched with streptavidin-agarose beads, then tryptic-digested, and the obtained peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by label-free

quantification analysis (Figure 4A). Comparative analysis between the proteomes of the DMSO control sample and each of the OX<sub>yne</sub>- & CyC<sub>yne</sub>-treated samples enabled a volcano plot representation (Figure 4B-a). Only proteins identified after applying *p*-value ≤ 0.01 and log<sub>2</sub>(fold change) ≥ 1.2 thresholds on the proteomics analysis results were selected, therefore leading to a panel of 143 and 48 enzyme candidates for the iBP<sub>yne</sub>OX-NV716 and the CyC<sub>32yne</sub>-NV716 combinations, respectively (Figure 4B, Tables S3 and S4).

As shown in the Venn diagram (Figure 4B-b), and despite their closely related mechanism of action<sup>30</sup>, both inhibitor probes share only 15 common targets (Table S5). It is noteworthy that, as depicted in the functional categorization profile of the captured proteins (Figure 4B-c), similar biological processes were, however, targeted by the two inhibitors. This result reflects the complementary selectivity of the two reactive warheads, that is, the oxadiazolone ring and the enol-phosphonate cycle, which in fact differ in their electrophilicity and enzyme specificity, as already observed in previous studies<sup>20,22,31,32</sup>. With the iBP<sub>yne</sub>OX, a significant number of ribosomal proteins involved in “Genetic information processing” were observed, suggesting stronger and enhanced activity in protein synthesis following high stress treatment with this OX-NV716 association. Such a feature is also observed for the proteins belonging to the “Signaling and cellular processes” category, which represent 21.7% and 52.1% of the total proteins captured with iBP<sub>yne</sub>OX and CyC<sub>32yne</sub> in bacterial culture, respectively. Notably, numerous transporters and stress response proteins were found, such as the Phage shock protein C (ECL_01778), which is produced when bacterial membranes are damaged.

To further analyze the results of these CC-ABPP experiments and compare them with previous work on the capture of target enzymes in mycobacteria<sup>30,33</sup>, the corresponding
<figure><img src="image_7.png" /><figcaption>**Figure 4.** Click-chemistry activity-based profiling (CC-ABPP) on *En. cloacae* ATCC 23355 culture. (A) CC-ABPP typical workflow for the identification of proteins covalently bound to OX~yne~/CyC~yne~ inhibitors. *En. cloacae* ATCC 23355 culture was pretreated with either the iBP~yne~ OX–NV716 or the CyC~32yne~–NV716 combination before cell lysis and click reaction with the Desthiobiotin-PEG~3–N~~3 reporter. Samples were then treated with streptavidin–agarose beads for the capture and enrichment of labeled proteins, followed by tryptic digestion. Tandem mass spectrometry analyses and subsequent differential peptides analysis led to the identification of the OX and CyC target enzymes. Created in BioRender (https://BioRender.com/q94r710). (B) Proteomics analysis of CC-ABPP applied to *En. cloacae* ATCC 23355 culture. (a) Volcano plot of the proteomics analysis of CyC~32yne~ showing the significance of the two-sample *t*-test (~log~₁₀(*p*-value)) vs. fold change (~log~₂(LFQ normalized intensity in CyC~32yne~ vs. nonspecific conditions)) on the *y* and *x* axes, respectively. Only the proteins with a positive fold-change are plotted here. The dashed lines indicate the threshold of *p*-value ≤ 0.01 and a fold change ≥ 1.2. (b) Venn diagram showing the total number of proteins differentially captured with iBP~yne~OX or CyC~32yne~ at a *p*-value ≤ 0.01 and a ~log~₂(FC) ≥ 1.2. (c) Functional categories of *En. cloacae* ATCC 23355 proteins identified following treatment with the iBP~yne~OX–NV716 or CyC~32yne~–NV716 combination according to the functional classification system of the KEGG database⁵¹,⁵². The numbers correspond to the total number of proteins identified in each category, with the corresponding percentage in parentheses based on either the 143 or 48 proteins identified with iBP~yne~OX or CyC~32yne~, respectively. Proteins without annotation are denoted as uncharacterized proteins.</figcaption></figure>

orthologs in *M. tuberculosis* H37Rv and/or *M. abscessus* ATCC 19977 were also shown using the KEGG database⁵¹,⁵² and then cross-referenced with the OrtholugeDB online database⁵⁴ (Table S5). Interestingly, 125 out of the 176 proteins identified from *En. cloacae* ATCC 23355 culture have orthologs in the *M. tuberculosis* and/or *M. abscessus* genome. Of these, 63 proteins had already been identified as potential targets of the OX/CyC probes through ABPP on culture and/or total lysate of *M. abscessus*²²,³¹ and *M. tuberculosis*²⁰,³²,³³ (Tables 3 and S5), 5 of them being annotated as essential genes according to the essential genes database (DEG)⁵⁵. These included three ribosomal proteins (ECL_04612/MAB_3752c; ECL_04692/Rv0706/MAB_3815c; ECL_04698/Rv0700/MAB_3821c), the cell division protein FtsZ (ECL_

00892/Rv2150c/MAB_2009), and the 3-oxoacyl-[acyl-carrier-protein] reductase FabG (ECL_02543/Rv1483/MAB_2723c), which are highly conserved both in mycobacteria and in Gram-negative ESKAPEE bacteria. In terms of subcellular localization, the fact that these target proteins originate from the cell envelope and the cytoplasm of this Gram-negative bacterium is consistent with all our previous studies, where identified protein targets of the OX and CyC in *M. tuberculosis*²⁰,³²,³³ and *M. abscessus*²²,³¹ were localized in both cellular compartments. Taken together, these findings clearly support the proof of concept that the polyamino-isoprenyl enhancer NV716 has the ability to render the OX and CyC inhibitors active against *En. cloacae* ATCC 23355, while allowing them to retain their multi-target properties.

<figure><img src="image_8.png" /></figure>
<table><caption>**Table 3.** En. cloacae ATCC 23555 target proteins identified by CC-ABPP using the iBP<sub>yne</sub>OX and CyC<sub>32yne</sub> affinity-based probes, together with their respective *Mycobacterium tuberculosis* and/or *Mycobacterium abscessus* orthologs<sup>a</sup>.</caption><thead><tr><th rowspan="2">iBP<sub>yne</sub>OX</th><th rowspan="2">CyC<sub>32yne</sub></th><th rowspan="2">Gene name</th><th colspan="3">Ortholog</th><th rowspan="2">Protein name</th><th rowspan="2">Pathway</th></tr><tr><th>M. tuberculosis</th><th>M. abscessus</th><th>M. abscessus</th></tr></thead><tbody><tr><td>+</td><td>ECL_00157</td><td>Rv2335</td><td>MAB_1148<sup>b</sup></td><td>Serine acetyl/transferase</td><td></td><td>Amino acid metabolism</td></tr><tr><td>+</td><td>ECL_01853</td><td>Rv2858<sup>b</sup></td><td>MAB_4203<sup>b</sup></td><td>Gamma-aminobutyraldehyde dehydrogenase</td><td></td><td>Amino acid metabolism</td></tr><tr><td>+</td><td>ECL_03989</td><td>Rv3565<sup>b</sup></td><td>MAB_3582</td><td>GntR family transcriptional regulator</td><td></td><td>Amino acid metabolism</td></tr><tr><td>+</td><td>ECL_05036</td><td>Rv1908<sup>b</sup></td><td>MAB_2470<sup>b</sup></td><td>Catalase-peroxidase</td><td></td><td>Amino acid metabolism</td></tr><tr><td>+</td><td>ECL_03341</td><td>Rv1600</td><td>MAB_2669<sup>b</sup></td><td>Histidinol-phosphate aminotransferase</td><td></td><td>Amino acid metabolism</td></tr><tr><td>+</td><td>ECL_05014</td><td>Rv3003<sup>c</sup></td><td>MAB_3323<sup>b</sup></td><td>Acetolactate synthase</td><td></td><td>Amino acid metabolism</td></tr><tr><td>+</td><td>ECL_04055</td><td>Rv0533<sup>c</sup></td><td>MAB_3192<sup>c</sup></td><td>Cryptic 6-phospho-beta-glucosidase</td><td></td><td>Carbohydrate metabolism</td></tr><tr><td>+</td><td>ECL_01284</td><td>Rv0844<sup>c</sup></td><td>MAB_4519<sup>c</sup></td><td>Two-component system, NaL family, response regulator, fimbrial Z</td><td></td><td>Environmental information processing</td></tr><tr><td>+</td><td>ECL_01619</td><td>Rv0844<sup>c</sup></td><td>MAB_0060<sup>b</sup></td><td>Nitrate/nitrite response regulator</td><td></td><td>Environmental information processing</td></tr><tr><td>+</td><td>ECL_04457</td><td>Rv3565<sup>b</sup></td><td>MAB_3387<sup>b</sup></td><td>Methyl-accepting chemotaxis sensory transducer</td><td></td><td>Environmental information processing</td></tr><tr><td>+</td><td>ECL_01778</td><td>Rv2027<sup>c</sup></td><td>MAB_3715<sup>c</sup></td><td>Phage shock protein C</td><td></td><td>Genetic information processing</td></tr><tr><td>+</td><td>ECL_00317</td><td>Rv0054<sup>b</sup></td><td>MAB_4989<sup>c</sup></td><td>Single-stranded DNA-binding protein</td><td></td><td>Genetic information processing</td></tr><tr><td>+</td><td>ECL_00242</td><td>Rv0641<sup>b</sup></td><td>MAB_3892<sup>c</sup></td><td>Large ribosomal subunit protein uL1</td><td></td><td>Genetic information processing</td></tr><tr><td>+</td><td>ECL_00597</td><td>Rv0056<sup>b</sup></td><td>MAB_4896<sup>c</sup></td><td>Large ribosomal subunit protein bL9</td><td></td><td>Genetic information processing</td></tr><tr><td>+</td><td>ECL_00594</td><td>Rv0053<sup>b</sup></td><td>MAB_4899<sup>c</sup></td><td>Small ribosomal subunit protein bS6</td><td></td><td>Genetic information processing</td></tr><tr><td>+</td><td>ECL_00109</td><td>Rv1644</td><td>MAB_0572<sup>b</sup></td><td>tRNA (guanosine(18)-2'-O)-methyltransferase</td><td></td><td>Genetic information processing</td></tr><tr><td>+</td><td>ECL_00240</td><td>Rv0639<sup>b</sup></td><td>MAB_3894<sup>c</sup></td><td>Transcription termination/antitermination protein NusG</td><td></td><td>Genetic information processing</td></tr><tr><td>+</td><td>ECL_03489</td><td>Rv2534<sup>c</sup></td><td>MAB_2837<sup>c</sup></td><td>Elongation factor P-like protein</td><td></td><td>Genetic information processing</td></tr><tr><td>+</td><td>ECL_04612</td><td>Rv3443<sup>c</sup></td><td>MAB_3752<sup>c</sup></td><td>Large ribosomal subunit protein uL13</td><td></td><td>Genetic information processing</td></tr><tr><td>+</td><td>ECL_04698</td><td>Rv0700<sup>b</sup></td><td>MAB_3821<sup>b</sup></td><td>Small ribosomal subunit protein uS10</td><td></td><td>Genetic information processing</td></tr><tr><td>+</td><td>ECL_05119</td><td>Rv1629<sup>c</sup></td><td>MAB_2615<sup>c</sup></td><td>DNA polymerase I</td><td></td><td>Genetic information processing</td></tr><tr><td>+</td><td>ECL_04695</td><td>Rv0703</td><td>MAB_3818<sup>c</sup></td><td>Large ribosomal subunit protein uL23</td><td></td><td>Genetic information processing</td></tr><tr><td>+</td><td>ECL_04706</td><td>Rv0683</td><td>MAB_3850<sup>c</sup></td><td>Small ribosomal subunit protein uS7</td><td></td><td>Genetic information processing</td></tr><tr><td>+</td><td>ECL_04696</td><td>Rv0702<sup>b</sup></td><td>MAB_3819<sup>c</sup></td><td>Large ribosomal subunit protein uL4</td><td></td><td>Genetic information processing</td></tr><tr><td>+</td><td>ECL_04611</td><td>Rv3442<sup>c</sup></td><td>MAB_3751<sup>c</sup></td><td>Small ribosomal subunit protein uS9</td><td></td><td>Genetic information processing</td></tr><tr><td>+</td><td>ECL_04692</td><td>Rv0706</td><td>MAB_3815<sup>c</sup></td><td>Large ribosomal subunit protein uL22</td><td></td><td>Genetic information processing</td></tr><tr><td>+</td><td>ECL_04697</td><td>Rv0701</td><td>MAB_3820<sup>c</sup></td><td>Large ribosomal subunit protein uL3</td><td></td><td>Genetic information processing</td></tr><tr><td>+</td><td>ECL_03742</td><td>Rv2587<sup>c</sup></td><td>MAB_0803</td><td>Cell division protein ZipA</td><td></td><td>Genetic information processing</td></tr><tr><td>+</td><td>ECL_01194</td><td>Rv2462<sup>c</sup></td><td>MAB_1580<sup>b</sup></td><td>Trigger factor Tig</td><td></td><td>Genetic information processing</td></tr><tr><td>+</td><td>ECL_02592</td><td>Rv0074<sup>b</sup></td><td>MAB_3236<sup>c</sup></td><td>Glucans biosynthesis glucosyltransferase H</td><td></td><td>Glycan biosynthesis and metabolism</td></tr><tr><td>+</td><td>ECL_04529</td><td>Rv2587<sup>c</sup></td><td>MAB_0108<sup>c</sup></td><td>Penicillin-binding protein activator LpoA</td><td></td><td>Glycan biosynthesis and metabolism</td></tr><tr><td>+</td><td>ECL_02543</td><td>Rv1483<sup>b</sup></td><td>MAB_2723<sup>c</sup></td><td>3-oxoacyl-[acyl-carrier-protein] reductase FabG</td><td></td><td>Lipid metabolism</td></tr><tr><td>+</td><td>ECL_03683</td><td>Rv0243<sup>b</sup></td><td>MAB_1002<sup>b</sup></td><td>3-ketoacyl-CoA thiolase</td><td></td><td>Lipid metabolism</td></tr><tr><td>+</td><td>ECL_01173</td><td>Rv1416<sup>b</sup></td><td>MAB_2795<sup>c</sup></td><td>6,7-dimethyl-8-ribityllumazine synthase</td><td></td><td>Metabolism of cofactors and vitamins</td></tr><tr><td>+</td><td>ECL_02952</td><td>Rv0984<sup>b</sup></td><td>MAB_1079</td><td>Molybdenum cofactor biosynthesis protein B</td><td></td><td>Metabolism of cofactors and vitamins</td></tr><tr><td>+</td><td>ECL_00113</td><td>Rv1389</td><td>MAB_2823<sup>c</sup></td><td>Guanylate kinase</td><td></td><td>Nucleotide metabolism</td></tr><tr><td>+</td><td>ECL_02171</td><td>Rv0484<sup>c</sup></td><td>MAB_4060<sup>b</sup></td><td>NADP-dependent 3-hydroxy acid dehydrogenase YdfG</td><td></td><td>Nucleotide metabolism</td></tr><tr><td>+</td><td>ECL_02486</td><td>Rv1699<sup>b</sup></td><td>MAB_2364</td><td>CTP synthase (glutamine hydrolyzing)</td><td></td><td>Nucleotide metabolism</td></tr><tr><td>+</td><td>ECL_02744</td><td>Rv1712<sup>b</sup></td><td>MAB_2371<sup>*</sup></td><td>Cytidylate kinase (CK)</td><td></td><td>Nucleotide metabolism</td></tr><tr><td>+</td><td>ECL_03774</td><td>Rv0780</td><td>MAB_0689<sup>b</sup></td><td>Phosphoribosylaminoimidazole-succinocarboxamide synthase</td><td></td><td>Nucleotide metabolism</td></tr></tbody></table>

(Continues)
<table><caption>**Table 3. (Continued)**</caption><thead><tr><th rowspan="2">iBP<sub>ox</sub></th><th rowspan="2">CyC<sub>32</sub>yne</th><th colspan="3">Ortholog</th><th rowspan="2">Pathway</th></tr><tr><th>Gene name</th><th>M. tuberculosis</th><th>M. abscessus</th><th>Protein name</th></tr></thead><tbody><tr><td>+</td><td>ECL_03860</td><td>Rv2445c<sup>b</sup></td><td>MAB_1606<sup>b</sup></td><td>Nucleoside diphosphate kinase</td><td>Nucleotide metabolism</td></tr><tr><td>+</td><td>ECL_00721</td><td>Rv2461c<sup>b</sup></td><td>MAB_1581<sup>*</sup></td><td>ATP-dependent Clp protease proteolytic subunit</td><td>Peptidases and inhibitors</td></tr><tr><td>+</td><td>ECL_04557</td><td>Rv3610c</td><td>MAB_0533<sup>b</sup></td><td>ATP-dependent zinc metalloprotease FtsH</td><td>Peptidases and inhibitors</td></tr><tr><td>+</td><td>ECL_05045</td><td>Rv2110c<sup>b</sup></td><td>MAB_2172</td><td>ATP-dependent protease subunit HslV</td><td>Peptidases and inhibitors</td></tr><tr><td>+</td><td>ECL_02464</td><td>Rv0724<sup>b</sup></td><td>MAB_3789c</td><td>Protease 4</td><td>Peptidases and inhibitors</td></tr><tr><td>+</td><td>ECL_02504</td><td>Rv1027c</td><td>MAB_3250c<sup>b</sup></td><td>DNA-binding transcriptional regulator PhoP</td><td>Signaling and cellular processes</td></tr><tr><td>+</td><td>ECL_00892</td><td>Rv2150c<sup>b</sup></td><td>MAB_2009</td><td>Cell division protein FtsZ</td><td>Signaling and cellular processes</td></tr><tr><td>+</td><td>ECL_01160</td><td>Rv2428<sup>b</sup></td><td>MAB_4408c<sup>*</sup></td><td>Alkyl hydroperoxide reductase C</td><td>Signaling and cellular processes</td></tr><tr><td>+</td><td>ECL_03521</td><td>Rv0844c<sup>b</sup></td><td>MAB_1522<sup>b</sup></td><td>Transcriptional regulatory protein RcsB</td><td>Signaling and cellular processes</td></tr><tr><td>+</td><td>ECL_03943</td><td>Rv2916c</td><td>MAB_3237c<sup>b</sup></td><td>Signal recognition particle protein</td><td>Signaling and cellular processes</td></tr><tr><td>+</td><td>ECL_00368</td><td>Rv1200</td><td>MAB_2263c<sup>b</sup></td><td>Proline/glycine betaine transporter</td><td>Signaling and cellular processes</td></tr><tr><td>+</td><td>ECL_01828</td><td>Rv1239c<sup>b</sup></td><td>MAB_4693</td><td>Zinc transport protein ZntB</td><td>Signaling and cellular processes</td></tr><tr><td>+</td><td>ECL_03532</td><td>Rv3476c</td><td>MAB_2263c<sup>b</sup></td><td>sn-glycerol-3-phosphate transporter</td><td>Signaling and cellular processes</td></tr><tr><td>+</td><td>ECL_04977</td><td>Rv1239c<sup>b</sup></td><td>MAB_1378c</td><td>Magnesium transport protein CorA</td><td>Signaling and cellular processes</td></tr><tr><td>+</td><td>ECL_00740</td><td>Rv0234c<sup>b</sup></td><td>MAB_4322</td><td>Putative aldehyde dehydrogenase</td><td>Uncharacterized protein</td></tr><tr><td>+</td><td>ECL_01005</td><td>Rv2971<sup>b</sup></td><td>MAB_1528c</td><td>NADP-dependent oxidoreductase domain-containing protein</td><td>Uncharacterized protein</td></tr><tr><td>+</td><td>ECL_03029</td><td>Rv0045c<sup>b</sup></td><td>MAB_1169</td><td>AB hydrolase-1 domain-containing protein</td><td>Uncharacterized protein</td></tr><tr><td>+</td><td>ECL_02982</td><td>Rv0489<sup>b</sup></td><td>MAB_4049c</td><td>2,3-bisphosphoglycerate-dependent phosphoglycerate mutase</td><td>Uncharacterized protein</td></tr><tr><td>+</td><td>ECL_04029</td><td>Rv3400<sup>b</sup></td><td>MAB_2130</td><td>Fructose-1-phosphatase</td><td>Uncharacterized protein</td></tr><tr><td>+</td><td>ECL_04104</td><td>Rv2392<sup>b</sup></td><td>MAB_1661c</td><td>Phosphoadenosine 5'-phosphosulfate reductase</td><td>Uncharacterized protein</td></tr><tr><td>+</td><td>ECL_03668</td><td>Rv3708c<sup>b</sup></td><td>MAB_0344</td><td>Aspartate-semialdehyde dehydrogenase</td><td>Uncharacterized protein</td></tr><tr><td>+</td><td>ECL_03953</td><td>Rv3100c</td><td>MAB_3473c<sup>b</sup></td><td>SsrA-binding protein</td><td>Uncharacterized protein</td></tr><tr><td>+</td><td>ECL_04636</td><td>Rv0149<sup>b</sup></td><td>MAB_4603c<sup>b</sup></td><td>Alcohol dehydrogenase</td><td>Uncharacterized protein</td></tr><tr><td>+</td><td>ECL_00728</td><td>Rv0223c<sup>b</sup></td><td>MAB_4484<sup>b</sup></td><td>Aldehyde dehydrogenase</td><td>Uncharacterized protein</td></tr></tbody></table>

<footnote><sup>a</sup>En. cloacae ATCC 23355 orthologs were retrieved using the KEGG database<sup>51,52</sup> and then cross-referenced with the OrtholugeDB online database<sup>54</sup>. <sup>b</sup> Proteins previously identified from ABPP experiments on culture or total lysates of *M. abscessus*<sup>22,31</sup> or *M. tuberculosis*<sup>20,32,33</sup> with the OX and CyC.</footnote>
## Identification of potential target proteins from a total lysate of *P. aeruginosa* PAO1 by CC-ABPP using the CyC₃₂yne affinity-based probe

In a previous work, we have shown that the CyC₃₂-Dansyl fluorescent probe was able to efficiently label some bacterial enzymes present in a total lysate of *P. aeruginosa* PAO1³⁵. In this context, we decided to apply the same CC-ABPP approach on a CyC₃₂yne-treated total lysate of this Gram-negative bacterium. Following proteomic analysis, 36 potential target enzymes have been identified, of which 10 are annotated as essential proteins for bacterial growth⁵⁵ (Table S6). Interestingly, 30 of these 36 targets have *En. cloacae* genomic orthologs (Table S6), including 5 proteins that have also been captured from the *En. cloacae* ATCC 23355 culture (Table 4). These included the nucleoside diphosphate kinase Ndk (PA3807/ECL_03860/Rv2445c/MAB_1606) involved in Nucleotide metabolism; the outer membrane LPS-assembly protein LptD (PA0595/ECL_00852) involved in the assembly of LPS; the essential Trigger factor Tig (PA1800/ECL_01194/Rv2462c/MAB_1580), which functions as a peptidyl-prolyl cis-trans isomerase and is involved in the genetic information processing pathway; and finally, an acyl-CoA thioesterase (PA2856/ECL_01261) and the essential 3-oxoacyl-[acyl-carrier-protein] reductase FabG (PA2967/ECL_02543/Rv1483/MAB_2723c), both involved in the lipid metabolism pathway (Table 4). Notably, Rv2445c/MAB_1606, Rv2462c/MAB_1580, and Rv1483/MAB_2723c are part of the set of previously identified target proteins of the OX/CyC from *M. tuberculosis*²⁰,³²,³³ and *M. abscessus*²²,³¹. In particular, based on the fact that the latter five proteins, including two essential enzymes, are highly conserved in all Gram-negative ESKAPEE bacteria, we can hypothesize that the OX and CyC inhibitors should be able to block the growth of these pathogens, provided that they penetrate and enter the bacteria, as in the case of *En. cloacae*.

## DISCUSSION

As the race for efficient, broad-spectrum antibiotics is slowing down, new strategies to eradicate drug-resistant

bacteria are thus needed. In this context, “adjuvant therapy” represents a promising but still underexplored area. Antibiotic adjuvants are molecules with little or no antibacterial activity that can enhance the activity of existing antibiotics by minimizing, bypassing, or directly blocking the resistance mechanisms associated with them¹². The two main classes of adjuvants currently in development are β-lactamase inhibitors, the best example of which is the clinically approved and widely prescribed Augmentin™, a combination of the β-lactam antibiotic amoxicillin and the β-lactam-based inhibitor clavulanic acid⁵⁶, and membrane disruptors¹⁵.

Membrane-targeting compounds usually bypass passive resistance mechanisms such as the permeability barrier of Gram-negative bacteria and can also block efflux mechanisms¹². Among the reported membrane-disrupting adjuvants, pentamidine, an antiprotozoal drug, was able to potentiate novobiocin against colistin-resistant strains of *A. baumannii*, and interestingly, a combination of pentamidine (10 mg/kg) and novobiocin (50 mg/kg) significantly reduced splenic bacterial load in an in vivo model of infected mice⁵⁷. Recently, P35, a new synthetic analog of pentamidine, demonstrated increased in vivo efficacy and reduced toxicity in a mouse model of *A. baumannii* infection when combined with novobiocin⁵⁸. Similar extensive structure activity relationship (SAR) studies conducted on polymyxin B-derived molecules led to the identification of SPR741⁵⁹. This molecule, which showed synergistic effects with various antibiotics, notably by significantly reducing bacterial burden and promoting animal survival in a murine pulmonary model of *A. baumannii* infection, has completed Phase I clinical trials⁵⁹⁻⁶¹. Apart from these successful examples, few membrane-disrupting adjuvants have been the subject of in vivo studies¹⁵.

In this challenging context, here, we have tested the association of multi-target inhibitors of (Ser/Cys)-based enzymes, namely, OX and CyC, with a known antibiotic adjuvant: the polyamino-isoprenyl derivative NV716. Indeed, although OX and CyC displayed promising antibacterial activity against pathogenic mycobacterial species, such as *M. abscessus* and *M. tuberculosis*, with no toxicity to mammalian host cells, they are inactive against Gram-negative

<table><caption>**Table 4.** CyC₃₂yne target proteins identified in *Pseudomonas aeruginosa* PAO1 as well as in *En. cloacae* ATCC 23355 culture by LC-ESI-MS/MS analysisª.</caption><thead><tr><th colspan="7">En. cloacae ortholog</th></tr><tr><th>Gene name</th><th>Essentiality</th><th>Gene name</th><th>Sequence identity</th><th>Overlap</th><th>Protein name</th><th>Pathway</th></tr></thead><tbody><tr><td>PA1800</td><td>Essential</td><td>ECL_01194</td><td>0.507</td><td>426</td><td>Trigger factor Tig<sup>b</sup></td><td>Genetic information processing</td></tr><tr><td>PA2967</td><td>Essential</td><td>ECL_02543</td><td>0.642</td><td>246</td><td>3-oxoacyl-[acyl-carrier-protein] reductase FabG<sup>b</sup></td><td>Lipid metabolism</td></tr><tr><td>PA2856</td><td>Non-Essential</td><td>ECL_01261</td><td>0.477</td><td>193</td><td>acyl-CoA thioesterase</td><td>Lipid metabolism</td></tr><tr><td>PA3807</td><td>Non-Essential</td><td>ECL_03860</td><td>0.629</td><td>143</td><td>Nucleoside diphosphate kinase Ndk<sup>b</sup></td><td>Nucleotide metabolism</td></tr><tr><td>PA0595</td><td>Non-Essential</td><td>ECL_00852</td><td>0.306</td><td>797</td><td>Lipopolysaccharide-assembly protein LptD</td><td>Signaling and cellular processes</td></tr></tbody></table>

<footnote>ªEn. cloacae ATCC 23355 orthologs were retrieved using the KEGG database⁵¹,⁵² and then cross-referenced with the OrtholugeDB online database⁵⁴. The essentiality of each *P. aeruginosa* PAO1 gene was checked using the essential genes database (DEG)⁵⁵. ᵇProteins previously identified from ABPP experiments on culture or total lysates of *Mycobacterium abscessus*²²,³¹ or *Mycobacterium tuberculosis*²⁰,³²,³³ with OX and CyC.</footnote>
bacteria due to their incapacity to cross their outer membrane. In this study, we showed that NV716, which has been shown to efficiently potentiate disused antibiotics against Gram-negative bacteria, such as *P. aeruginosa*, *E. coli*, or *K. pneumoniae*<sup>24,26,40,45</sup>, was able to sensitize the ESKAPEE bacterium *En. cloacae* ATCC 23355 to some OX and CyC compounds. Mechanistically, we confirmed that the NV716 potentiation likely occurs through effective strategies to fight Gram-negative bacteria<sup>62,63</sup>: permeabilization of the outer membrane and depolarization of the inner membrane of *En. cloacae* ATCC 23355.

However, the MIC<sub>90</sub> of NV716 against the five clinical isolates of *En. cloacae* was found to be 16 times higher than that of the above reference strain. This finding suggests that the ability of this enhancer to potentiate the OX and CyC compounds may strongly depend on the isolate resistance and efflux phenotypes (Table S1)<sup>41</sup>, as shown with reference drugs (Table S2). Moreover, while the iBPOX–NV716 association showed only poor antibacterial activity against the five clinical strains, the CyC<sub>32</sub>–NV716 combination was able to efficiently inhibit the growth of *Ecl*71 and *Ecl*93 (Table 2). *Ecl*71 was characterized as a wild-type clinical strain, with a basal efflux profile. *Ecl*53 and *Ecl*136 are highly resistant isolates, with the same substitutions in AcrA and AcrR efflux pump components. Strains *Ecl*80 and *Ecl*93 showed the same substitutions in the efflux key gene *tolC*, but only strain *Ecl*80 showed mutations in the multidrug efflux pump component OqxA, known to confer resistance to multiple agents including fluoroquinolones<sup>64</sup>. Since NV716 has nearly no effect on the efflux in *En. cloacae* ATCC 23355 (Figure S3), we hypothesize that the observed resistance of *Ecl*53, *Ecl*136, and *Ecl*80 to iBPOX and CyC<sub>32</sub> in the presence of NV716 may result from the over-expressing efflux phenotypes of these three isolates, thereby preventing the accumulation of the two inhibitors within the bacteria. This hypothesis is supported by evidence that NV716, even at a concentration as low as 0.015× MIC<sub>90</sub>_Ecl-isolate, enhances the antibacterial activity of doxycycline against these three clinical isolates (Table S2) by permeabilizing their outer membrane.

However, the inability of NV716 to potentiate the OX and CyC antibacterial activity on Gram-negative ESKAPEE bacteria other than *En. cloacae* ATCC 23355 and some clinical isolates despite a similar mechanism of action<sup>40,45</sup> suggests that the permeation of the outer membrane caused by NV716 may not be sufficient to allow the penetration, accumulation, and subsequent antibacterial activity of the OX and CyC compounds.

The hydrophobic/hydrophilic balance of a drug candidate is indeed a critical issue that determines its physicochemical compatibility with the bacterial outer membrane and thus its ability to penetrate the bacteria. In this context, the distribution coefficient, LogD, is a widely used metric of the lipophilicity for a given molecule. Here, the calculated cLogD at pH 7.4 of our OX and CyC compounds using Marvin Suite (ChemAxon) shows that they all display a high lipophilic nature (2 < cLogD < 6). This property is in line with their very good penetration and

antibacterial activity on mycobacterial species, which have a very complex, hydrophobic, and thick lipid-rich membrane consisting of up to 60% cell wall lipids<sup>65,66</sup>. In comparison, the cell wall of Gram-negative bacteria contains no more than 20% of lipid content. A recent study conducted on cycline derivatives showed that their potentiation by NV716 against *P. aeruginosa* correlated with their hydrophobic/hydrophilic balance<sup>18</sup>, the most active derivatives having a cLogD < -1.8 (pH 7.4), indicative of very high hydrophilicity. Regarding the OX and CyC compounds, their high hydrophobicity might limit their compatibility with the Gram-negative outer membrane. Indeed, it is well acknowledged that variations in the chemical structure of LPS, for example, in the composition of the sugar head group, can strongly influence the activity of membrane-active compounds and so their antimicrobial activity<sup>10,67</sup>. Taken together, all these findings may provide some insights into the differential potentiation of OX and CyC compounds observed among the four bacterial species tested. This latter point is of particular interest, considering that the CC-ABPP approach on bacterial culture (Figure 4) revealed that OX and CyC compounds still retain their multi-target properties on Gram-negative bacteria (Tables 3, 4 and S3–S6). In addition, these proteomic data also indicate that the presence of NV716, which is required for the activity of the compounds, strongly alters the bacterial metabolism and may influence the nature of the proteins captured by the OX and CyC probes. Furthermore, the fact that several highly conserved proteins across all Gram-negative ESKAPEE bacteria (70-100% positive hits, at least 60% identities when BLASTed) have been identified is very encouraging and should provide good insights into the antibacterial potential of our OX and CyC compounds against bacterial pathogens.

In summary, this study points out the efficiency of NV716 as an antibiotic enhancer that enables the OX and CyC multi-target inhibitors to impair the bacterial growth of the Gram-negative *En. cloacae* ATCC 23355 bacterium. Furthermore, from all our data, we anticipate that the synthesis of novel OX and CyC inhibitors with enhanced hydrophilicity (i.e., negative cLogD) should facilitate their penetration, maximize the physicochemical compatibility, and thus boost their bactericidal activity against other clinically relevant Gram-negative ESKAPEE species. Such studies are currently ongoing and will be reported in due course.

## MATERIALS AND METHODS

All detailed protocols are provided in the Supporting Information.

### Chemistry

**Synthesis of OX and CyC activity-based probes.** The Cyclpostins analogs CyC<sub>32</sub>, CyC<sub>32yne</sub> and CyC<sub>32-Dansyl</sub>, as well as the oxadiazolone derivative HPOX, were synthesized as described previously<sup>20,33,35</sup>. Stock solutions (10 mM) of the OX and CyC molecules (purity of ≥95%) were prepared in
dimethyl sulfoxide (DMSO) and stored at 4°C. See Figure S4 for NMR spectra of the new compounds synthesized to access the iBP<sub>yne</sub>OX probe and for additional details.

**3-(4-iodophenyl)-5-isobutoxy-1,3,4-oxadiazol-2(3H)-one (2).** A solution of *p*-iodophenylhydrazine hydrochloride (2.3 g, 8.50 mmol, 1 equiv.) in dry pyridine (18 ml) was stirred at room temperature for 15 min. The mixture was then cooled to 0°C and a solution of isobutyl chloroformate (1.12 ml, 8.50 mmol, 1 equiv.) in dry CH<sub>2</sub>Cl<sub>2</sub> (6 ml) was slowly added. The mixture was stirred at 0°C for 30 min, followed by 1 h at room temperature. The mixture was then diluted with dry CH<sub>2</sub>Cl<sub>2</sub> (36 ml) and dry pyridine (9 ml) and cooled to 0°C. A solution of diphosgene (1.03 ml, 8.50 mmol, 1 equiv.) in dry CH<sub>2</sub>Cl<sub>2</sub> (12 ml) was added dropwise. The mixture was stirred at 0°C for 30 min, followed by 2 h at room temperature. The mixture was then degassed with N<sub>2</sub> to eliminate the remaining phosgene, treated with 1 M HCl (60 ml), and extracted with Et<sub>2</sub>O (2 × 100 ml). Organic layers were washed with a Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub> 10% solution (60 ml) and brine, dried over MgSO<sub>4</sub>, and solvents were removed under reduced pressure. The crude product was then adsorbed on silica and purified through silica gel chromatography with a gradient pentane/Et<sub>2</sub>O 100/0 to 95/5 to yield oxadiazolone **2** (2.49 g, 7.50 mmol, 81% over 2 steps) as a yellow solid. Analytical data for **(2)**: R<sub>f</sub> (cyclohexane/EtOAc 90:10, v/v) 0.55; <sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>): δ 7.71 (d,<sup>3</sup>J<sub>H,H</sub> = 9.0 Hz, 2H, H<sub>Ar</sub>), 7.57 (d,<sup>3</sup>J<sub>H,H</sub> = 9.0 Hz, 2H, H<sub>Ar</sub>), 4.16 (d,<sup>3</sup>J<sub>H,H</sub> = 6.6 Hz, 2H, CH<sub>2</sub>-O), 2.15 (non,<sup>3</sup>J<sub>H,H</sub> = 6.7 Hz, 1H, CH-(CH<sub>3</sub>)<sub>2</sub>), 1.04 (d,<sup>3</sup>J<sub>H,H</sub> = 6.7 Hz, 6H, CH-(CH<sub>3</sub>)<sub>2</sub>); <sup>13</sup>C NMR (75 MHz, CDCl<sub>3</sub>): δ 155.6, 148.2, 138.2 (2C), 136.2, 119.7 (2C), 89.4, 77.7, 27.9, 18.8 (2C).

**5-isobutoxy-3-(4-((trimethylsilyl)ethynyl)phenyl)-1,3,4-oxadiazol-2(3H)-one (3).** A solution of **2** (1 g, 2.99 mmol, 1.0 equiv.), trimethylsilylacetylene (634 µl, 4.49 mmol, 1.5 equiv.), and dry distilled Et<sub>3</sub>N (1.04 ml, 7.48 mmol, 2.5 equiv.) in dry MeCN (10 ml) was cooled to 0°C. Then, Pd(PPh<sub>3</sub>)<sub>4</sub> (243 mg, 0.21 mmol, 0.07 equiv.) and Cul (399 mg, 2.09 mmol, 0.7 equiv.) were added and the mixture was stirred at 0°C for 30 min, followed by 3 h at room temperature. The mixture was then filtered on Celite® and the solvent was removed under reduced pressure. The crude was then adsorbed on silica and purified through silica gel chromatography with a gradient pentane/Et<sub>2</sub>O 100/0 to 95/5 to yield oxadiazolone **3** (840 mg, 2.54 mmol, 85%) as an off-white solid. Analytical data for **(3)**: R<sub>f</sub> (cyclohexane/EtOAc 90:10 v/v) 0.55; <sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>): δ 7.75 (d,<sup>3</sup>J<sub>H,H</sub> = 9.0 Hz, 2H, H<sub>Ar</sub>), 7.50 (d,<sup>3</sup>J<sub>H,H</sub> = 9.0 Hz, 2H, H<sub>Ar</sub>), 4.17 (d,<sup>3</sup>J<sub>H,H</sub> = 6.6 Hz, 2H, CH<sub>2</sub>-O), 2.15 (non,<sup>3</sup>J<sub>H,H</sub> = 6.7 Hz, 1H, CH-(CH<sub>3</sub>)<sub>2</sub>), 1.04 (d,<sup>3</sup>J<sub>H,H</sub> = 6.8 Hz, 6H, CH-(CH<sub>3</sub>)<sub>2</sub>), 0.25 (s, 9H, (CH<sub>3</sub>)<sub>3</sub>-Si); <sup>13</sup>C NMR (75 MHz, CDCl<sub>3</sub>): δ 155.6, 148.2, 136.2, 133.0 (2C), 120.2, 117.5 (2C), 104.5, 94.7, 77.7, 27.9, 18.8 (2C), 0.1 (3C).

**3-(4-ethynylphenyl)-5-isobutoxy-1,3,4-oxadiazol-2(3H)-one or iBP<sub>yne</sub>OX.** A solution of **3** (828 mg, 2.51 mmol, 1 equiv.) in dry THF (25 ml) was cooled to 0°C. A solution of TBAF 1M (500 µl, 0.50 mmol, 0.20 equiv.) was slowly added. The mixture was stirred at 0°C for 2 h and was then quenched with a NaHCO<sub>3</sub> saturated solution (50 ml). The resulting solution was extracted with Et<sub>2</sub>O (2 × 50 ml), organic layers were gathered, washed with brine, dried over MgSO<sub>4</sub>,

and solvents were evaporated under reduced pressure. The crude was then adsorbed on silica and purified through silica gel chromatography with a gradient pentane/Et<sub>2</sub>O 100/0 to 90/10. The product was then further purified by re-crystallization in hot pentane to yield the desired iBP<sub>yne</sub>OX probe (360 mg, 1.39 mmol, 56%) as a yellow solid. Analytical data for iBP<sub>yne</sub>OX: R<sub>f</sub> (cyclohexane/EtOAc 90:10, v/v) 0.27; M<sub>p</sub> 71–72°C; IR (neat) ν 3301 (C≡C-H), 2968, 2878 (C-H), 1779 (C=O), 1606, 1509, 1470 (C=C) cm<sup>-1</sup>; <sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>): δ 7.78 (d,<sup>3</sup>J<sub>H,H</sub> = 8.9 Hz, 2H, H<sub>Ar</sub>), 7.53 (d,<sup>3</sup>J<sub>H,H</sub> = 8.9 Hz, 2H, H<sub>Ar</sub>), 4.17 (d,<sup>3</sup>J<sub>H,H</sub> = 6.6 Hz, 2H, CH<sub>2</sub>-O), 3.09 (s, 1H, C≡CH), 2.17 (non,<sup>3</sup>J<sub>H,H</sub> = 6.7 Hz, 1H, CH-(CH<sub>3</sub>)<sub>2</sub>), 1.04 (d,<sup>3</sup>J<sub>H,H</sub> = 6.7 Hz, 6H, CH-(CH<sub>3</sub>)<sub>2</sub>); <sup>13</sup>C NMR (75 MHz, CDCl<sub>3</sub>): δ 155.7, 148.2, 136.5, 133.1 (2C), 119.2, 117.7 (2C), 83.1, 77.7, 77.4, 27.9, 18.8 (2C); HRMS (+ESI) m/z [M+H]<sup>+</sup> calculated for C<sub>14</sub>H<sub>14</sub>N<sub>2</sub>O<sub>3</sub>: 259.1077, found: 259.1078.

## Biological evaluation

**Bacteria strains and growth conditions.** The following bacterial reference strains were used in this study: *P. aeruginosa* PAO1 strain, *E. coli* ATCC 25922, *K. pneumoniae* ATCC 13883, and *En. cloacae* ATCC 23355. Five clinical *En. cloacae* strains collected in three hospital centers located in the south of France between March 2017 and November 2020 and characterized in a previous study (Table S1)⁴¹ were also tested. All bacteria were stored at -80°C in 25% (v/v) glycerol for cryoprotection. Bacteria were routinely grown on cation-adjusted Mueller-Hinton (CA-MHB; Sigma-Aldrich, Saint-Quentin Fallavier, France) agar plates and grown in Mueller-Hinton II broth (MHIIIB; Sigma-Aldrich) at 37°C under agitation at 180 rpm.

**Antibiotics and adjuvant compounds.** Chloramphenicol (98%), doxycycline (98%), and ampicillin (96%) reference drugs as well as Polymyxin B [USP grade] were purchased from Sigma-Aldrich. NV716 was synthesized as previously reported¹⁷. Stock solutions of NV716 (10 mM) were prepared in sterile water and stored at -20°C until use.

**Susceptibility testing on *P. aeruginosa* PAO1, *E. coli* ATCC 25922, *K. pneumoniae* ATCC 13883, and *En. cloacae* ATCC 23355.** The concentrations of compound leading to 90% of bacterial growth (MIC<sub>90</sub>) were determined using the rapid INT colorimetric assay, as reported previously³⁶,³⁷. Consistent with previous experiments with NV716 on these Gram-negative bacteria¹³,¹⁵,¹⁶,²⁵,³⁸,³⁹, sub-MIC final concentrations of this adjuvant were used (i.e., 4.1 µg/ml = 0.2× MIC<sub>90</sub> for *P. aeruginosa* and *K. pneumoniae*; 1.2 µg/ml = 0.24× MIC<sub>90</sub> for *E. coli* and *En. cloacae*). All experiments were performed independently in triplicate.

## Effects of NV716 on *En. cloacae* membranes—adapted from Ref.³⁹,⁴⁰

**Outer membrane permeabilization assay.** The permeabilization of the outer membrane of *En. cloacae* was
assessed using a nitrocefin hydrolysis assay, as previously reported⁴⁰. Potassium Phosphate Buffer (PPB, pH 7.4) was used as a negative control and Polymyxin B²⁵,⁴⁵ was used as a positive control. Absorbance at 490 nm related to nitrocefin hydrolysis was monitored over 1 h using a Tecan Infinite® 200 Pro multimode microplate reader (Tecan Group Ltd). Experiments were performed in triplicate.

**Inner membrane permeability assay.** The inner-membrane permeabilization by NV716 was evaluated using propidium iodide (PI, Sigma-Aldrich), a cell-impermeable DNA/RNA fluorescent dye, as previously described⁴⁰. Polymyxin B²⁵,⁴⁵ was used as a positive control and PBS buffer was used as a negative control.

**Inner membrane depolarization assay.** The inner-membrane depolarization was evaluated using the DiSC₃(5) (3,3'-dipropylthiadicarbocyanine iodide, Sigma-Aldrich) assay, as described previously⁴⁵. Polymyxin B²⁵,⁴⁵ was used as a positive control and HEPES-Sucrose buffer was used as a negative control.

**Glucose-triggered 1,2'-DiNA real-time efflux assay.** The efflux activity in *En. cloacae* was assessed as reported previously²⁴. Maximum efflux activity (100%) was defined as the difference between the fluorescence value obtained after 620 s in the presence/absence of glucose, thereby corresponding to the efflux of 1,2'-DiNA (TCI-Europe SA).

**Bacteria labeling with CyC₃₂-Dansyl.** Bacterial cells (2.0 × 10¹⁰ cells/ml) were incubated overnight at 37°C under shaking at 180 rpm with 124 µg/ml (200 µM) of CyC₃₂-Dansyl or DMSO (negative control) and in the presence or absence of 40.6 µg/ml (100 µM) NV716, then washed, resuspended in 250 µl PBS, and fixed with 4% paraformaldehyde. Fluorescence microscopy imaging and the analysis of Dansyl mean fluorescence intensity were performed as previously reported³⁵.

## Activity-based protein profiling

The capture of *En. cloacae* ATCC23355 potential target proteins from CyC₃₂yne⁻ and iBPyneOX-treated culture via ABPP experiments, the mass spectrometry analysis of resulting proteomes, and the subsequent proteins identification and quantification were performed as previously described³³.

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (www.proteomexchange.org)⁶⁸ via the PRIDE partner repository⁶⁹ (https://www.ebi.ac.uk/pride/login) with the dataset identifiers PXD053955 for *En. cloacae* culture and PXD052024 for *P. aeruginosa* crude total lysate.

## Statistical analysis

All statistical analyses were performed using GraphPad Prism 8 (GraphPad Inc.) and are detailed in the figure legends. Differences were considered significant for calculated *p*-values ≤ 0.05.

## ACKNOWLEDGMENTS

This study was supported by the CNRS, INSERM, and Aix Marseille University. Proteomics analyses were carried out using the mass spectrometry facility of Marseille Proteomics (marseille-proteomique.univ-amu.fr) supported by IBISA, the Cancéropôle PACA, the Provence-Alpes-Côte d'Azur Region, the Institut Paoli-Calmettes, and Fonds Européen de Développement Regional (FEDER). Emma Forest PhD fellowship is supported by the Ministère de l'Enseignement Supérieur et de la Recherche. The authors would like to thank Pierre Santucci (LISM UMR7255 CNRS) for his help with Biorender.

## AUTHOR CONTRIBUTIONS

**Emma Forest:** Investigations; formal analysis; visualization; writing—original draft; writing—review and editing. **Jordan Lehoux:** Resources; writing—review and editing. **Alexandre Guy:** Resources; writing—review and editing. **Thierry Durand:** Resources; writing—review and editing. **Stéphane Audebert:** Data curation; investigation; writing—review and editing. **Luc Camoin:** Data curation; validation; writing—review and editing. **Christopher D. Spilling:** Resources; writing—review and editing. **Céline Crauste:** Resources; writing—review and editing. **Stéphane Canaan:** Formal analysis; writing—review and editing. **Jean Michel Branel:** Formal analysis; resources; writing—original draft; writing—review and editing. **Jean-Michel Bolla:** Conceptualization; methodology; project administration; supervision; validation; writing—original draft; writing—review and editing. **Jean-François Cavalier:** Conceptualization; formal analysis; methodology; project administration; supervision; validation; visualization; writing—original draft; writing—review and editing.

## ETHICS STATEMENT

This study did not involve human subjects and animals.

## CONFLICT OF INTERESTS

The authors declare no conflict of interests.

## DATA AVAILABILITY

All data generated or analyzed during this study are included in this article and its supplementary information files. The mass spectrometry proteomics data are available online through the ProteomeXchange Consortium (www.proteomexchange.org) with the dataset identifiers PXD053955 for *En. cloacae* culture and PXD052024 for *Pseudomonas aeruginosa* crude total lysate.

## SUPPORTING INFORMATION

Additional Supporting Information for this article can be found online at https://doi.org/10.1002/mlf2.70014.

## ORCID

*Emma Forest* https://orcid.org/0009-0000-9407-8703
*Jordan Lehoux* https://orcid.org/0000-0001-7369-0266
*Alexandre Guy* https://orcid.org/0000-0001-8206-8946
Thierry Durand ID https://orcid.org/0000-0001-6086-7296

Stéphane Audebert ID https://orcid.org/0000-0002-9409-2588

Luc Camoin ID https://orcid.org/0000-0002-1230-4787

Céline Crauste ID https://orcid.org/0000-0002-5714-8749

Stéphane Canaan ID https://orcid.org/0000-0001-7478-300X

Jean Michel Brunel ID https://orcid.org/0000-0002-9355-8980

Jean-Michel Bolla ID https://orcid.org/0000-0003-3892-4340

Jean-François Cavalier ID https://orcid.org/0000-0003-0864-8314

REFERENCES

1 Cook MA, Wright GD. The past, present, and future of antibiotics. *Sci Transl Med. 2022;14:eabo7793.

2 Aminov RI. A brief history of the antibiotic era: lessons learned and challenges for the future. *Front Microbiol. 2010;1:134.

3 O'Neill J. Tackling drug-resistant infections globally: final report and recommendations. The Review on Antimicrobial Resistance. 2016. Available from: https://amr-review.org/sites/default/files/160518_Final%20paper_with%20cover.pdf

4 Murray C, Ikuta KS, Sharara F, Swetschinski L, Robles Aguilar G, Gray A, et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. *Lancet. 2022;399:629-55.

5 WHO. WHO publishes list of bacteria for which new antibiotics are urgently needed. 2017 [cited 2024 Aug 20]. Available from: https://www.who.int/news/item/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed

6 WHO. WHO bacterial priority pathogens list, 2024: Bacterial pathogens of public health importance to guide research, development and strategies to prevent and control antimicrobial resistance 2024 [cited 2024 Dec 19]. Available from: https://www.who.int/publications/i/item/9789240093461

7 De Oliveira DMP, Forde BM, Kidd TJ, Harris PNA, Schembri MA, Beatson SA, et al. Antimicrobial resistance in ESKAPE pathogens. *Clin Microbiol Rev. 2020;33:e00181-19.

8 Mancuso G, Midiri A, Gerace E, Biondo C. Bacterial antibiotic resistance: the most critical pathogens. *Pathogens. 2021;10:1310.

9 Beveridge TJ. Structures of Gram-negative cell walls and their derived membrane vesicles. *J Bacteriol. 1999;181:4725-33.

10 Delcour AH. Outer membrane permeability and antibiotic resistance. *Biochim Biophys Acta. 2009;1794:808-16.

11 Li XZ, Plésiat P, Nikaido H. The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria. *Clin Microbiol Rev. 2015;28:337-418.

12 Wright GD. Antibiotic adjuvants: rescuing antibiotics from resistance. *Trends Microbiol. 2016;24:862-71.

13 Douafer H, Andrieu V, Phanstiel O, Brunel JM. Antibiotic adjuvants: make antibiotics great again! *J Med Chem. 2019;62:8665-81.

14 Docquier JD, Mangani S. An update on β-lactamase inhibitor discovery and development. *Drug Resist Updat. 2018;36:13-29.

15 Dhanda G, Acharya Y, Haldar J. Antibiotic adjuvants: a versatile approach to combat antibiotic resistance. *ACS Omega. 2023;8:10757-83.

16 Pieren M, Tigges M. Adjuvant strategies for potentiation of antibiotics to overcome antimicrobial resistance. *Curr Opin Pharmacol. 2012;12:551-5.

17 Lieutaud A, Pieri C, Bolla JM, Brunel JM. New polyaminoisoprenyl antibiotics enhancers against two multidrug-resistant Gram-negative bacteria from enterobacter and salmonella species. *J Med Chem. 2020;63:10496-508.

18 Draveny M, Rose C, Pinet A, Ferrié L, Figadère B, Brunel J-M, et al. Scope and limitations of exploiting the ability of the chemosensitizer NV716 to enhance the activity of tetracycline derivatives against *Pseudomonas aeruginosa*. *Molecules. 2023;28:4262.

19 Delorme V, Diomandé SV, Dedieu L, Cavalier JF, Carrière F, Kremer L, et al. *MmPPOX* inhibits *Mycobacterium tuberculosis* lipolytic enzymes belonging to the hormone-sensitive lipase family and alters mycobacterial growth. *PLoS One*. 2012;7:e46493.

20 Nguyen PC, Delorme V, Bénarouche A, Guy A, Landry V, Audebert S, et al. Oxadiazolone derivatives, new promising multi-target inhibitors against *M. tuberculosis*. *Bioorg Chem.* 2018;81:414-24.

21 Point V, Malla RK, Diomande S, Martin BP, Delorme V, Carriere F, et al. Synthesis and kinetic evaluation of cyclophostin and cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases. *J Med Chem.* 2012;55:10204-19.

22 Madani A, Ridenour JN, Martin BP, Paudel RR, Abdul Basir A, Le Moigne V, et al. Cyclipostins and cyclophostin analogues as multitarget inhibitors that impair growth of mycobacterium abscessus. *ACS Infect Dis.* 2019;5:1597-608.

23 Spilling CD. The chemistry and biology of cyclophostin, the cyclipostins and related compounds. *Molecules*. 2019;24:2579.

24 Wang G, Brunel JM, Rodriguez-Villalobos H, Bolla JM, Van Bambeke F. The polyamino-isoprenyl potentiator NV716 revives disused antibiotics against Gram-negative bacteria in broth, infected monocytes, or biofilms, by disturbing the barrier effect of their outer membrane. *Eur J Med Chem.* 2022;238:114496.

25 Troudi A, Fethi M, Selim El Asli M, Bolla JM, Klibi N, Brunel JM. Efficiency of a tetracycline-adjuvant combination against multidrug resistant *Pseudomonas aeruginosa* Tunisian clinical isolates. *Antibiotics*. 2020;9:919.

26 Wang G, Brunel JM, Bolla JM, Van Bambeke F. The polyaminoisoprenyl potentiator NV716 revives old disused antibiotics against intracellular forms of infection by *Pseudomonas aeruginosa*. *Antimicrob Agents Chemother.* 2021;65:e02028-20.

27 Gray DA, Wenzel M. Multitarget approaches against multiresistant superbugs. *ACS Infect Dis.* 2020;6:1346-65.

28 Huemer M, Mairpady Shambat S, Brugger SD, Zinkernagel AS. Antibiotic resistance and persistence-implications for human health and treatment perspectives. *EMBO Rep.* 2020;21:e51034.

29 Uddin TM, Chakraborty AJ, Khusro A, Zidan BRM, Mitra S, Emran TB, et al. Antibiotic resistance in microbes: history, mechanisms, therapeutic strategies and future prospects. *J Infect Public Health*. 2021;14:1750-66.

30 Cavalier JF, Spilling CD, Durand T, Camoin L, Canaan S. Lipolytic enzymes inhibitors: a new way for antibacterial drugs discovery. *Eur J Med Chem.* 2021;209:112908.

31 Madani A, Mallick I, Guy A, Crauste C, Durand T, Fourquet P, et al. Dissecting the antibacterial activity of oxadiazolone-core derivatives against *Mycobacterium abscessus*. *PLoS One*. 2020;15:e0238178.

32 Nguyen PC, Delorme V, Bénarouche A, Martin BP, Paudel R, Gnawali GR, et al. Cyclipostins and cyclophostin analogs as promising compounds in the fight against tuberculosis. *Sci Rep.* 2017;7:11751.

33 Barelier S, Avellan R, Gnawali GR, Fourquet P, Roig-Zamboni V, Poncin I, et al. Direct capture, inhibition and crystal structure of HsaD (Rv3569c) from *M. tuberculosis*. *FEBS J.* 2023;290:1563-82.

34 Nguyen PC, Madani A, Santucci P, Martin BP, Paudel RR, Delattre S, et al. Cyclophostin and Cyclipostins analogues, new promising molecules to treat mycobacterial-related diseases. *Int J Antimicro Agents.* 2018;51:651-4.

35 Sarrazin M, Martin BP, Avellan R, Gnawali GR, Poncin I, Le Guenno H, et al. Synthesis and biological characterization of fluorescent cyclipostins and cyclophostin analogues: new insights for the diagnosis of mycobacterial-related diseases. *ACS Infect Dis.* 2022;8:2564-78.

36 Weseler A, Geiss HK, Saller R, Reichling J. A novel colorimetric broth microdilution method to determine the minimum inhibitory concentration (MIC) of antibiotics and essential oils against *Helicobacter pylori*. *Pharmazie*. 2005;60:498-502.

37 Tunney MM, Ramage G, Field TR, Moriarty TF, Storey DG. Rapid colorimetric assay for antimicrobial susceptibility testing of
*Pseudomonas aeruginosa*. *Antimicrob Agents Chemother*. 2004; 48:1879-81.

38 Borselli D, Brunel JM, Gorgé O, Bolla JM. Polyamino-isoprenyl derivatives as antibiotic adjuvants and motility inhibitors for *Bordetella bronchiseptica* porcine pulmonary infection treatment. *Front Microbiol*. 2019;10:1771.

39 Troudi A, Bolla JM, Klibi N, Brunel JM. An original and efficient antibiotic adjuvant strategy to enhance the activity of macrolide antibiotics against Gram-negative resistant strains. *Int J Mol Sci*. 2022;23:12457.

40 Wang G, Brunel JM, Preusse M, Mozaheb N, Willger SD, Larrouy-Maumus G, et al. The membrane-active polyaminoisoprenyl compound NV716 re-sensitizes *Pseudomonas aeruginosa* to antibiotics and reduces bacterial virulence. *Commun Biol*. 2022;5:871.

41 Ferrand A, Vergalli J, Bosi C, Pantel A, Pagès JM, Davin-Regli A. Contribution of efflux and mutations in fluoroquinolone susceptibility in MDR enterobacterial isolates: a quantitative and molecular study. *J Antimicrob Chemother*. 2023;78:1532-42.

42 Schindler PRG, Teuber M. Action of polymyxin B on bacterial membranes: morphological changes in the cytoplasm and n the outer membrane of *Salmonella typhimurium* and *Escherichia coli* B. *Antimicrob Agents Chemother*. 1975;8:95-104.

43 Sautrey G, El Khoury M, Dos Santos AG, Zimmermann L, Deleu M, Lins L, et al. Negatively charged lipids as a potential target for new amphiphilic aminoglycoside antibiotics. *J Biol Chem*. 2016;291: 13864-74.

44 Cabrini G, Verkman AS. Potential-sensitive response mechanism of diS-C3-(5) in biological membranes. *J Membr Biol*. 1986; 92:171-82.

45 Borselli D, Lieutaud A, Thefenne H, Garnotel E, Pagès JM, Brunel JM, et al. Polyamino-isoprenic derivatives block intrinsic resistance of *P. aeruginosa* to doxycycline and chloramphenicol in vitro. *PLoS One*. 2016;11:e0154490.

46 Bohnert JA, Schuster S, Szymaniak-Vits M, Kern WV. Determination of real-time efflux phenotypes n *Escherichia coli* AcrB binding pocket phenylalanine mutants using a 1,2'-dinaphthylamine efflux assay. *PLoS One*. 2011;6:e21196.

47 Blair JMA, Piddock LJV. How to measure export via bacterial multidrug resistance efflux pumps. *mBio*. 2016;7:e00840-16.

48 Piddock LJV. Multidrug-resistance efflux pumps—not just for resistance. *Nat Rev Microbiol*. 2006;4:629-36.

49 Larson G. Some aspects of the chemistry of alkynylsilanes. *Synthesis*. 2018;50:2433-62.

50 Panichakul D, Lim YW, Mathey F. Reactivity of 2-silyl- and 2-stannyl-substituted phosphirenes. *Organometallics*. 2010;29: 1985-7.

51 Ogata H, Goto S, Sato K, Fujibuchi W, Bono H, Kanehisa M. KEGG: kyoto encyclopedia of genes and genomes. *Nucleic Acids Res*. 1999;27:29-34.

52 Kanehisa M, Sato Y, Kawashima M, Furumichi M, Tanabe M. KEGG as a reference resource for gene and protein annotation. *Nucleic Acids Res*. 2016;44:D457-62.

53 Díez-González S. Chapter Three—Copper(I)—acetylides: access, structure, and relevance in catalysis. In: Pérez PJ editor, Advances in organometallic chemistry. 66, London: Academic Press; 2016. p. 93-141.

54 Whiteside MD, Winsor GL, Laird MR, Brinkman FSL. OrthologeDB: a bacterial and archaeal orthology resource for improved comparative genomic analysis. *Nucleic Acids Res*. 2013; 41:D366-76.

55 Luo H, Lin Y, Gao F, Zhang CT, Zhang R. DEG 10, an update of the database of essential genes that includes both protein-coding genes and noncoding genomic elements. *Nucleic Acids Res*. 2014;42:D574-80.

56 Brogden RN, Carmine A, Heel RC, Morley PA, Speight TM, Avery GS. Amoxycillin/clavulanic acid: a review of its antibacterial activity, pharmacokinetics and therapeutic use. *Drugs*. 1981; 22:337-62.

57 Stokes JM, MacNair CR, Ilyas B, French S, Côté JP, Bouwman C, et al. Pentamidine sensitizes Gram-negative pathogens to antibiotics and overcomes acquired colistin resistance. *Nat Microbiol*. 2017;2:17028.

58 MacNair CR, Farha MA, Serrano-Wu MH, Lee KK, Hubbard B, Côté JP, et al. Preclinical development of pentamidine analogs identifies a potent and nontoxic antibiotic adjuvant. *ACS Infect Dis*. 2022;8:768-77.

59 Zurawski DV, Reinhart AA, Alamneh YA, Pucci MJ, Si Y, Abu-Taleb R, et al. SPR741, an antibiotic adjuvant, potentiates the in vitro and in vivo activity of rifampin against clinically relevant extensively drug-resistant *Acinetobacter baumannii*. *Antimicrob Agents Chemother*. 2017;61:e01239-17.

60 Vaara M. Polymyxin derivatives that sensitize Gram-negative bacteria to other antibiotics. *Molecules*. 2019;24:249.

61 Eckburg PB, Lister T, Walpole S, Keutzer T, Utley L, Tomayko J, et al. Safety, tolerability, pharmacokinetics, and drug interaction potential of SPR741, an intravenous potentiator, after single and multiple ascending doses and when combined with β-lactam antibiotics in healthy subjects. *Antimicrob Agents Chemother*. 2019; 63:e00892-19.

62 Krishnamoorthy G, Leus IV, Weeks JW, Wolloscheck D, Rybenkov VV, Zgurskaya HI. Synergy between active efflux and outer membrane diffusion defines rules of antibiotic permeation into Gram-negative bacteria. *mBio*. 2017;8: e01172-17.

63 Wesseling CMJ, Martin NI. Synergy by perturbing the Gram-negative outer membrane: opening the door for Gram-positive specific antibiotics. *ACS Infect Dis*. 2022;8:1731-57.

64 Hansen LH, Jensen LB, Sørensen HI, Sørensen SJ. Substrate specificity of the OqxBmultidrug resistance pump in *Escherichia coli* and selected enteric bacteria. *J Antimicrob Chemother*. 2007;60:145-7.

65 Chiaradia L, Lefebvre C, Parra J, Marcoux J, Burlet-Schiltz O, Etienne G, et al. Dissecting the mycobacterial cell envelope and defining the composition of the native mycomembrane. *Sci Rep*. 2017;7:12807.

66 Daffé M, Marrakchi H. Unraveling the structure of the mycobacterial envelope. *Microbiol Spectr*. 2019;7:e0027-2018.

67 Gutsmann T, Hagge SO, David A, Roes S, Böhling A, Hammer MU, et al. Lipid-mediated resistance of Gram-negative bacteria against various pore-forming antimicrobial peptides. *J Endotoxin Res*. 2005; 11:167-73.

68 Deutsch EW, Bandeira N, Sharma V, Perez-Riverol Y, Carver JJ, Kundu DJ, et al. The ProteomeXchange consortium in 2020: enabling 'big data' approaches in proteomics. *Nucleic Acids Res*. 2020;48:1145.

69 Perez-Riverol Y, Bai J, Bandla C, García-Seisdedos D, Hewapathirana S, Kamatchinathan S, et al. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. *Nucleic Acids Res*. 2022;50:D543-52.

**How to cite this article:** Forest E, Lehoux J, Guy A, Durand T, Audebert S, Camoin L, et al. The polyamino-isoprenyl enhancer NV716 enables the antibacterial activity of two families of multi-target inhibitors against the ESKAPEE bacterium *Enterobacter cloacae*. *mLife*. 2025; https://doi.org/10.1002/mlf2.70014