Oxadiazolone derivatives, new promising multi-target inhibitors against M. tuberculosis

Article abstract of DOI:10.1016/j.bioorg.2018.08.025

A set of 19 oxadiazolone (OX) derivatives have been investigated for their antimycobacterial activity against two pathogenic slow-growing mycobacteria, Mycobacterium marinum and Mycobacterium bovis BCG, and the avirulent Mycobacterium tuberculosis (M. tb) mc²6230. The encouraging minimal inhibitory concentrations (MIC) values obtained prompted us to test them against virulent M. tb H37Rv growth either in broth medium or inside macrophages. The OX compounds displayed a diversity of action and were found to act either on extracellular M. tb growth only with moderated MIC₅₀, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth. Of interest, all OX derivatives exhibited very low toxicity towards host macrophages. Among the six potential OXs identified, HPOX, a selective inhibitor of extracellular M. tb growth, was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP, in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 18 potential candidates, all being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA, TesA, KasA and MetA have been reported as essential for in vitro growth of M. tb and/or its survival and persistence inside macrophages. Overall, our findings support the assumption that OX derivatives may represent a novel class of multi-target inhibitors leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes involved in various important physiological processes.

Full text of DOI:10.1016/j.bioorg.2018.08.025

Accepted Manuscript
Oxadiazolone derivatives, new promising multi-target inhibitors against M. tu-
berculosis
Phuong Chi Nguyen, Vincent Delorme, Anaïs Bénarouche, Alexandre Guy,
Valérie Landry, Stéphane Audebert, Matthieu Pophillat, Luc Camoin, Céline
Crauste, Jean-Marie Galano, Thierry Durand, Priscille Brodin, Stéphane
Canaan, Jean-François Cavalier
PII:
S0045-2068(18)30722-3
DOI:
https://doi.org/10.1016/j.bioorg.2018.08.025
YBIOO 2482
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
16 July 2018
17 August 2018
20 August 2018
Please cite this article as: P. Chi Nguyen, V. Delorme, A. Bénarouche, A. Guy, V. Landry, S. Audebert, M. Pophillat,
L. Camoin, C. Crauste, J-M. Galano, T. Durand, P. Brodin, S. Canaan, J-F. Cavalier, Oxadiazolone derivatives, new
promising multi-target inhibitors against M. tuberculosis, Bioorganic Chemistry (2018), doi: https://doi.org/
10.1016/j.bioorg.2018.08.025
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Oxadiazolone derivatives, new promising multi-target inhibitors against M. tuberculosis
Phuong Chi Nguyen1§, Vincent Delorme2§ #, Anaïs Bénarouche1, Alexandre Guy3, Valérie Landry2,
Stéphane Audebert4, Matthieu Pophillat4, Luc Camoin4, Céline Crauste3, Jean-Marie Galano3,
Thierry Durand3, Priscille Brodin2, Stéphane Canaan1* and Jean-François Cavalier1*
1
Aix-Marseille Univ, CNRS, LISM, Institut de Microbiologie de la Méditerranée, Marseille,
France
2
Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL -
Center for Infection and Immunity of Lille, Lille, France
3
Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, Université de Montpellier,
CNRS, ENSCM, 15 Avenue Charles Flahault, 34093 Montpellier Cedex 5, France
4
Aix-Marseille Univ, Inserm, CNRS, Institut Paoli-Calmettes, CRCM, Marseille Protéomique,
Marseille, France
§ Authors have contributed equally to this work
Corresponding authors: jfcavalier@imm.cnrs.fr (J.-F. Cavalier); canaan@imm.cnrs.fr (S. Canaan)
#
Current Address: Tuberculosis Research Laboratory, Institut Pasteur Korea, Seongnam-si,
Gyeonggi-do, 13488 Republic of Korea.
1

ABSTRACT
A set of 19 oxadiazolone (OX) derivatives have been investigated for their antimycobacterial
activity against two pathogenic slow-growing mycobacteria, Mycobacterium marinum and
Mycobacterium bovis BCG, and the avirulent Mycobacterium tuberculosis (M. tb) mc26230. The
encouraging minimal inhibitory concentrations (MIC) values obtained prompted us to test them
against virulent M. tb H37Rv growth either in broth medium or inside macrophages. The OX
compounds displayed a diversity of action and were found to act either on extracellular M. tb
growth only with moderated MIC50, or both intracellularly on infected macrophages as well as
extracellularly on bacterial growth. Of interest, all OX derivatives exhibited very low toxicity
towards host macrophages. Among the six potential OXs identified, HPOX, a selective inhibitor of
extracellular M. tb growth, was selected and further used in a competitive labelling/enrichment
assay against the activity-based probe Desthiobiotin-FP, in order to identify its putative target(s).
This approach, combined with mass spectrometry, identified 18 potential candidates, all being
serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis.
Among them, Ag85A, CaeA, TesA, KasA and MetA have been reported as essential for in vitro
growth of M. tb and/or its survival and persistence inside macrophages. Overall, our findings
support the assumption that OX derivatives may represent a novel class of multi-target inhibitors
leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and
Cys-containing enzymes involved in various important physiological processes.
Keywords
Tuberculosis, Oxadiazolone, Lipolytic enzyme inhibitors, Activity-based probe (ABP)
2

Abbreviations
ABPP, activity-based protein profiling; CC50, compound concentration leading to 50% of cell
cytotoxicity; CyC, Cyclipostins & Cyclophostin analogs; ETO, ethionamide; FM, foamy
macrophages; HSL, hormone-sensitive lipase; ILI, intracytoplasmic lipid inclusions; INH,
isoniazid; MDR, multidrug-resistant strains; MIC50, minimal inhibitory concentration leading to
50% of growth inhibition; M. tb, Mycobacterium tuberculosis; pNP, para-nitrophenyl; REMA,
resazurin microtiter assay; RIF, rifampicin; TB, tuberculosis; TAG, triacylglycerols; TDR, totally
drug-resistant strains; XDR, extensively drug-resistant strains; xI, inhibitor molar excess related to 1
mol of enzyme; xI50, inhibitor molar excess leading to 50% enzyme inhibition.
3

1. Introduction
With 10.4 million new cases and 1.7 million deaths in 2016, tuberculosis (TB) caused by the
pathogenic species Mycobacterium tuberculosis (M. tb) remains the leading cause of death
worldwide from a single infectious agent [1]. Despite the quadritherapy treatment involving
isoniazid (INH), pyrazinamide, rifampicin (RIF) and ethambutol, the introduction of new molecules
on the market to strengthen or replace this first-line antibiotics regimen is a slow and tedious
process [2, 3]. Only a few drugs were able to pass the selection stages (e.g., bedaquiline [4],
delamanid [5] and PA-824 [6]). However, the appearance and spread of multidrug-resistant (MDR),
extensively drug-resistant (XDR) and totally drug-resistant (TDR) strains [7-10] highlights the
urgent need for finding new therapeutic options to fight against M. tb.
In this context, oxadiazolone-core (OX) compounds represent attractive tools. Compound S57
(Figure 1A), initially described in 1954 [11, 12] as being active against TB [13-15], was used as
template for the synthesis of 3,5-substituted 1,3,4-oxadiazole-2-one derivatives [16, 17]. These
molecules were found to exhibit interesting anti-mycobacterial activity against M. tb H37Rv, with
minimal inhibitory concentrations (MIC) of 1.25–8 µg/mL, comparable to those of INH
(0.5 µg/mL) and RIF (1.0 µg/mL) [16, 17]. Molecular modeling indicated that these compounds
possessed all necessary features to block the enzymatic activity of the mycobacterial cytochrome
P450-dependent 14-sterol demethylase (P45014DMs) [18], also a target for antifungal drug design
[19]. These findings thus suggest the potential use of such OX derivatives as alternative therapeutic
agents for TB [20].
Few years ago, we reported that the OX compound MmPPOX (Figure 1B), a reversible inhibitor
of the hormone-sensitive lipase (HSL) family of proteins [21, 22], was able to inhibit the growth of
M. tb with MIC values of around 15-25 µg/mL as determined on solid medium [23]. Keeping in
mind its strong affinity toward the HSL family member proteins (Lip-HSL), we further investigated
the in vitro inhibition of recombinant M. tb Lip-HSL. As expected, the nine purified Lip-HSL
enzymes tested were all strongly inhibited by MmPPOX, which reacted with the catalytic serine
4

residue by forming a covalent but (slowly) reversible bond (i.e., carbamate or thiocarbamate bond,
Figure 1B). Such an inhibitor could then be considered as a long-life substrate rather than a true
inhibitor as already observed with Orlistat (also named Tetrahydrolipstatine or THL), a
representative member of reversible serine hydrolase inhibitors [24-26].
Figure 1. Chemical structure of (A) 5-(pyridin-4-yl)-1,3,4-oxadiazol-2(3H)-one S57, and (B) 5-methoxy-3-
(3-phenoxyphenyl)-1,3,4-oxadiazol-2(3H)-one MmPPOX and its mode of action. (C) General procedure for
the one step preparation of 5-alkoxy-3-phenyl substituted-1,3,4-oxadiazol-2(3H)-one compounds from either
(3-phenoxyphenyl)hydrazine hydrochloride (1), (4-phenoxyphenyl)hydrazine hydrochloride (2) or
phenylhydrazine hydrochloride (3), giving OX derivatives 5a-k (i.e., RmPPOX), 6a,d,e,k (i.e., RpPPOX)
or 7a,d,e,k (i.e., RPOX), respectively. Reagents and conditions: i) Alkyl chloroformate 4a-k, Pyridine, 0 °C
to RT; ii) ClCO2CCl3, CHCl2, Pyridine, 0 °C to RT, 40-85%. Adapted from [27].
From these first results, a new series of 18 lipophilic OX derivatives based on MmPPOX core-
structure have been synthesized (Figure 1C) and tested for their anti-mycobacterial activities. More
precisely, each oxadiazolone molecule has been tested against M. tb H37Rv for i) its capacity to
inhibit in vitro growth; ii) its antitubercular activity on M. tb-infected macrophages, and iii) its
cytotoxicity towards macrophages. Interestingly, some analogs were found to inhibit M. tb growth
in vitro and/or inside macrophages without any significant toxicity to host cells. In addition, using
5

an activity-based protein profiling (ABPP) assay, the potential target enzymes of HPOX, acting
only on M. tb extracellular growth, were further identified.
6

2. Results and Discussion
2.1 Synthesis of oxadiazolone-core (OX) derivatives.
The set of new 18 lipophilic OX derivatives based on MmPPOX core-structure was designed by
varying the nature of the R chain and/or the positioning of the phenoxy group (in meta or para
position) when present (Figure 1C), and synthesized as previously reported [27]. The
(phenoxy)phenyl group, proposed to be responsible for strong hydrophobic interactions and
structural stiffening [27], was conserved in most of the new candidate inhibitors. In addition,
modifying the R chain born by the oxadiazolone ring allow an investigation of the influence of the
lipophilicity on the antibacterial activity exerted by these molecules. To remain consistent with
previous studies involving such compounds [23, 27], we have decided to use the specific
nomenclature already developed for these derivatives noted Rm(or p)PPOX; where m(or p)P
represents the meta (or para)-Phenoxy group when present; P the phenyl group; OX the
Oxadiazolone core; and R the alkyl chain (i.e., Be, benzyloxyethyl; M; methyl, E, ethyl; B, butyl;
iB, isobutyl; H, hexyl; O, octyl; Eh, 2-ethylhexyl; D, decyl; Do, dodecyl; Me, methoxyethyl).
2.2 Susceptibility testing on selected mycobacteria.
The antibacterial properties of the OX compounds were first evaluated towards three slow-growing
mycobacteria: M. marinum, M. bovis BCG and M. tb mc26230, a H37Rv strain with its RD1 region
and panCD genes deleted, resulting in an avirulent pan(−) phenotype [28]. The corresponding
MIC50 values, as determined by the REMA assay [29-32], are reported in Table 1. First, it is
noteworthy that the concentrations needed to inhibit 50% of the bacterial growth (MIC50) obtained
for rifampicin (RIF) and isoniazid (INH), used here as reference antibiotics, were in agreement with
literature data [33-35].
Nearly all 19 OXs were active against M. bovis BCG and M. marinum growth (Table 1). However,
with MIC50 values in the range 1.9-53 µM, M. marinum was nearly 2-times more sensitive to OX
compounds than M. bovis BCG (MIC50 from 3.5 to >120 µM). BePOX, HPOX and iBPOX, for
7

which the phenoxy substituent is absent, exhibited the most potent antibacterial activity towards M.
marinum, with mean MIC50 of 2.3 ±0.33 µM, comparable to that of RIF (1.4 µM). Interestingly,
these three OX compounds were also among the best inhibitors of M. bovis BCG growth; HPOX
being the best one (MIC50 = 3.5 µM).
From these encouraging data obtained using two slow-growing mycobacteria, it was tempting to
extrapolate that these OXs would behave in a similar way against M. tb growth and conclude that
the same three compounds, i.e. BePOX, HPOX and iBPOX, would then be promising anti-TB
molecules.
Drug susceptibility testing of the 19 OXs was thus further assessed using the non-virulent M. tb
mc26230 strain. Among all tested compounds, 14 OXs were active against M. tb mc26230. The two
best growth inhibitors obtained were iBpPPOX and BePOX, which displayed similar MIC50 value
(mean 32.1 ±1.0 µM). In all other cases, MIC50 values were indicative either of a weak (mean
MIC50 = 84.1 ±9.1 µM for MmPPOX, MPOX and MemPPOX), or a moderate (mean MIC50 =
46.9 ±5.1 µM for BmPPOX, iBmPPOX, HmPPOX, HpPPOX, HPOX, OmPPOX, EhmPPOX,
BemPPOX and BepPPOX) antibacterial activity (Table 1). From these data, M. tb mc26230 was
found nearly 13- and 5-times less sensitive to the OX compounds than M. marinum and M. bovis
BCG, respectively.
Surprisingly, iBPOX, which differs from BePOX and HPOX by the length of its R substituent,
was not active against this mycobacteria. Moreover, no clear trends or rules in terms of structure-
activity relationships (SAR) have emerged regarding the potency of these oxadiazolone-core
compounds against M. marinum and M. bovis BCG. Indeed, increasing the lipophilicity by varying
the nature of the R chain on the oxadiazolone ring and/or the positioning of the phenoxy group
when present had no real impact on the anti-mycobacterial activity.
Interestingly, with M. tb mc26230, some SAR tendencies can however be set up. First, and as
mentioned above, the positioning of the phenoxy group in meta or para position has no real impact
on the antibacterial activity of the corresponding compounds (i.e., MmPPOX vs. MpPPOX;
8

iBmPPOX vs. iBpPPOX; HmPPOX vs. HpPPOX; BemPPOX vs. BepPPOX). Remarkably,
iBPOX bearing the short chain isobutyl has no activity as compared to the phenoxyphenyl
derivatives iBpPPOX and iBmPPOX. This is however not the case with the medium chains hexyl
and benzyloxyethyl OXs, for which the respective activity of HPOX and BePOX is retained and
even slightly better than HmPPOX & HpPPOX on the one hand, and BemPPOX & BepPPOX on
the other hand. Finally, in absence of the bulky phenoxy group, the best MIC50s against M. tb
mc26230 were also obtained with HPOX and BePOX vs. MPOX and iBPOX. In brief, the R chain
length thus seems to affect the potency of the tested compounds. More globally, the best growth
inhibitors were found to carry a middle chain length of around 6 to 9 carbon atoms (i.e., hexyl, 2-
ethylhexyl, octyl or benzyloxyethyl chains). Longer or shorter chain’s OXs (i.e., methyl,
methoxyethyl, ethyl, decyl or dodecyl) exhibited no or only very poor activity.
In summary, iBpPPOX displayed the best, while still moderate, antibacterial activity against M. tb
mc26230. The reason why such differences exist between the activity of the OX compounds against
M. marinum and M. bovis BCG, compared to M. tb, is not clear and will need further studies to be
elucidated; but differences in membrane composition and permeability are likely playing a role in
this phenotype.
2.3 High-content screening assay on virulent M. tb H37Rv.
As recently observed for another class of potent M. tb growth inhibitors, the Cyclipostins &
Cyclophostin analogs (CyC) [31], MIC50 values determined against M. tb mc26230 do not
necessarily correlate with the activity against M. tb H37Rv, particularly the activity against
intracellular bacteria.
Consequently, each of the 19 OX derivatives have been further evaluated for their specific
antitubercular activity against extracellularly- and intracellularly-growing virulent M. tb H37Rv-
GFP strain, using a high-content screening assay based on the fluorescence measurement of GFP-
expressing bacteria. In vitro growth (i.e., extracellular assay) of M. tb H37Rv-GFP was first
9

monitored after 5 days at 37°C in presence of increasing concentrations of candidate inhibitors [31,
36-38]. Intracellular growth of M. tb H37Rv-GFP was also assessed following a 5-day exposure of
infected Raw264.7 murine macrophage cell line to the various OX compounds. In this case, the
percentage of infected cells, the total number of bacteria, the number of bacteria per infected cells,
as well as the number of living host cells allowed us to determine the MIC50 of the compound [36,
39, 40]. In subsequent experiment, the concentration leading to 50% of host cell cytotoxicity, i.e.
CC50, were also determined in absence of infection.
Among all tested molecules, 6 potential OX candidates exhibited interesting antitubercular
properties (Table 2 and Figure 2). BePOX and HPOX impaired exclusively M. tb growth in
culture broth medium with the same moderate MIC50 (30.8 and 44.6 µM, respectively) than
obtained previously on M. tb mc26230. In contrast, iBPOX, HpPPOX and BepPPOX showed a
clear preference against intracellularly-replicating mycobacteria with similar MIC50 values (3.5-
17.1 µM) than the first line antibiotics. Of interest, only iBpPPOX exhibited moderate (32.0 µM) to
quite good (8.5 µM) activity against both extracellular and intramacrophagic M. tb, respectively
(Figure 2).
Beside antibacterial activity, significantly, all these 6 OX inhibitors exhibited very low toxicity
towards host macrophages with CC50 > 100 μM, similarly to INH (CC50 > 150 µM) and
ethionamide (CC50 ≥ 120 µM), two potent anti-TB drugs (Table 2). Their respective selectivity
index (SI = CC50/MIC50) on intramacrophagic M. tb vs. Raw264.7 cells was thus found to be in a
range from 5.8 and up to 28. While these are preliminary results that would need to be confirmed in
other cell types including hepatocytes, they are encouraging for further improvement of the OX
compounds. This absence of cytotoxicity was actually not obvious, given the number of (Ser/Cys)-
enzymes present in host cells and the predicted potency of the OX compounds to target these
classes of enzymes.
Interestingly, the observed differences in the behavior of OX compounds; in particular the higher
activity against intracellular bacteria than against extracellular ones, have also been reported in the
10

case of the CyC compounds family [31]. As previously discussed above, inhibition of M. tb H37Rv
may result from several (and probably different) mechanisms of action or penetration of the OX
derivatives. With the hexyl and benzyloxyethyl chain, the absence/presence of the phenoxy group in
para position clearly shifted the activity of the corresponding OX from extracellular- (i.e., HPOX
& BePOX) to intracellular-replicating bacilli (i.e., HpPPOX & BepPPOX). On the other hand,
with the short isobutyl chain, both iBpPPOX and iBPOX are found most active against
intramacrophagic-replicating M. tb.
From these findings, it is tempting to assume that these OX compounds thus lead to the inhibition
of specific but most likely distinct mycobacterial target enzymes between intramacrophagic- vs.
extracellularly-replicating bacilli.
11

Figure 2. In vitro and ex vivo dose-response activity of the OX derivatives against M. tb H37Rv. (A)
Activity of BePOX, iBpPPOX and HPOX against GFP-expressing M. tb replicating in broth medium,
expressed as normalized relative fluorescence units (RFU%). The dashed line represents the level of
inhibition (~70%) reached with 1 µg/mL (=7.3 µM) INH as control. The MIC50 of BePOX, iBpPPOX and
HPOX replicating in culture broth medium were 30.8 μM, 32.0 µM and 44.6 μM, respectively. (B) Activity
of iBpPPOX, BepPPOX, HpPPOX and iBPOX against M. tb replicating inside Raw264.7 macrophages.
Results are expressed as the percentage of infected macrophages after 5 days post-infection. The dashed line
represents the level of inhibition (~87%) reached with 10 µg/mL (=73 µM) INH as control. The MIC50 of
BepPPOX, iBpPPOX, HpPPOX and iBPOX replicating inside macrophages were 3.5 μM, 8.5 µM, 9.5 µM
and 17.1 μM, respectively. Each value is the mean ± SD for a triplicated dose-response. Experiments were
conducted at least twice with consistent results.
12

2.4 Targets identification by Activity-based protein profiling.
Based on the aforementioned results, and taking into account their strong affinity for Serine and/or
Cysteine (Ser/Cys)-based enzymes (Figure 1B), one can hypothesize that OX inhibitors might
target and impair the activity of various enzymes involved in several processes of M. tb pathogenic
life cycle, thus resulting in bacterial death without any cytotoxicity towards host cells. Accordingly,
target(s) identification experiments were conducted by applying an activity-based protein profiling
(ABPP) approach [41-44]. In order to take into account the penetration/diffusion of the inhibitor
through the mycobacterial cell wall, all experiments have been performed on living bacterial cells
and not with a lysate, as previously described [31].
Here, HPOX, that selectively inhibits M. tb growth only in culture broth medium, was selected for
such experiments. M. tb mc26230 cells were grown to log phase and then incubated with HPOX
compound or DMSO as a control. After cell lysis, part of the lysate was used for competitive probe
labelling/enrichment assay using the Desthiobiotin-FP probe, targeting (Ser/Cys)-based enzymes
[43] (Figure 3). In parallel, the remaining lysate was incubated with TAMRA-FP, also targeting
(Ser/Cys)-based enzymes [43], to reveal the candidates presumably reacting with HPOX on SDS-
PAGE gel, using fluorescence scanning [31]. Around 9 distinct bands labelled by TAMRA-FP were
visible in the fluorescence readout (Figure 3B –lane E) and could also be detected by silver
staining after release of the enzymes captured by Desthiobiotin-FP (Figure 3B – lane B). In
contrast, pre-treatment with HPOX (Figure 3A) resulted in a decrease in fluorescence intensity of
all visible bands, as exemplified by the black arrows in Figure 3B – lane D. Indeed, the enzymes
previously inactivated by HPOX inhibitor will thus be unable to further react with the probes. The
respective enriched mixtures (Figure 3B – lanes A-B) were digested with trypsin and the resulting
peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS)
followed by subsequent label free quantification analysis. The proteins that were also found in the
13

control experiment (i.e., Figure 3B - lane A: DMSO alone for unspecific binding to streptavidin-
magnetic beads) were not taken into account.
Figure 3. Activity based protein profiling (ABPP) workflow for the identification of the proteins
covalently bound to OX inhibitors. (A) Cell culture of M. tb mc26230 was pre-treated with selected HPOX
inhibitor prior to cell lysis and further incubation with TAMRA-FP or Desthiobiotin-FP probe. In this latter
case, samples were then treated with streptavidin-magnetic beads for the capture and enrichment of labelled
proteins. (B-C) Equal amounts of proteins obtained in A were separated by SDS-PAGE and visualized by in-
14

gel fluorescence (right panel - lanes D-E: TAMRA detection) or silver staining (left panel – lanes A-C).
Enzymes whose TAMRA-FP labelling is impeded because of the presence of HPOX in their active-site are
shown by arrowheads. (D) Tryptic digestion followed by tandem mass spectrometry analyses and subsequent
differential proteomics analysis allowed identifying the target proteins.
15

The resulting mycobacterial targets of HPOX were displayed as volcano plot (Figure 4). Only
proteins identified with a permutation false discovery rate (pFDR) of 5% and a score threshold
value ≥ 60 were selected, therefore leading to a panel of 18 distinct proteins (Table 3). These
identified enzyme candidates ranged in their functional category from intermediary
metabolism/respiration (6 proteins), lipid metabolism (5 proteins), cell wall/cell processes (6
proteins), and virulence / detoxification / adaptation (1 protein) (Table 3).
Figure 4. Volcano plot of proteomic analysis of HPOX in M. tb mc26230 culture. Volcano plot showing
the significance two-sample t-test (-Log p-value) versus fold-change (Log2 (LFQ normalized intensity in
HPOX versus Desthiobiotin-FP condition) on the y and x axes, respectively. Here the plot is zoomed on the
positive difference between the two conditions. The full line is indicative of protein hits obtained at a
permutation false discovery rate of 1% (pFDR). Only protein hits obtained at a pFDR of 5% (dashed line)
and with a score threshold value ≥ 60 were selected, and are highlighted in black (non-essential) or in red
(essential). Data results from two different experiments processed three times.
16

As expected, the identified proteins targeted by HPOX were all (Ser/Cys)-based enzymes. Among
them, a variety of Ser/Cys hydrolases were detected. These included the putative β-lactamase
Rv1367c possibly involved in cell wall biosynthesis; two amidases AmiC (Rv2888c) and AmiD
(Rv3375); BpoC (Rv0554) a putative serine hydrolase [43]; two members of the lipase family Lip
(LipH [45] and LipV [46]); three Cutinase-like proteins (Cfp21, Cut2 and Cut3) [47]; and the
monoacylglycerol lipase Rv0183 [48].
More interestingly, 5 out of 18 identified proteins have been annotated as essential enzymes [49]
(Table 3). These include the antigen 85 complex, Ag85A (Rv3804c), Ag85B (Rv1886c) and
Ag85C (Rv0129c) [50]; the thioesterase TesA (Rv2928) [51]; the carboxylesterase CaeA
(Rv2224c) [52]; the beta-ketoacyl synthase KasA (Rv2245) [53]; and the sole putative α/β-
hydrolase MetA (Rv3341) belonging to the homoserine O-acetyltransferase family proteins in M. tb
[54].
2.5 TesA, Rv0183 and Cfp21 are inhibited by HPOX.
In order to validate some targets of HPOX inhibitor, we further investigated its ability to efficiently
inhibit the activity of three identified proteins; i.e., TesA, Cfp21 and Rv0183. Coding sequences
were amplified from M. tb genome, cloned in E. coli and the enzymes produced in recombinant
form and purified as previously reported [48, 55, 56]. Purified proteins were then individually
incubated for 30 min at room temperature with HPOX at various inhibitor molar excess (xI). The
residual enzyme activity was then measured using para-nitrophenyl caprylate (pNPC-8) as substrate
for TesA [56] and Cfp21 [55], and monoolein as substrate in the case of Rv0183 [48]. The variation
in the residual enzyme activity allowed determination of the inhibitor molar excess leading to 50%
enzyme inhibition, i.e., xI50 value [27, 57]. Thereby, a xI50 value of 0.5 is synonymous with a 1:1
stoichiometric ratio between the inhibitor and the lipolytic enzyme, and is therefore the highest
level of inhibitory activity that can be achieved.
17

As depicted in Figure 5, a clear dose-dependent inhibition was observed with the three enzymes.
TesA, Rv0183 and Cfp21 were indeed strongly inactivated, with 97.6% and 88.5% inhibition at xI =
20, respectively (Figure 5, inset). Interestingly, HPOX was found to react almost stoichiometrically
with pure TesA and Rv0183 as confirmed by their respective xI50 values of around 0.60. These
results clearly demonstrate that these three lipolytic enzymes are effective targets of HPOX.
Figure 5. HPOX efficiently impairs TesA, Cfp21 and Rv0183 activities in vitro. Variation of the residual
activities of TesA, Cfp21 and Rv0183 as a function of increasing molar excess (xI) of HPOX. Each enzyme
was pre-incubated at various inhibitor molar excess (xI) for 30 min at 25 °C. The inset displays the
determined values of xI50 and the level of inhibition at xI = 20. Kinetic assays were performed as described in
Experimental Section. Results are expressed as mean values ± SD of at least two independent assays.
The fact that these OXs derivatives behave against M. tb extracellular growth similarly to two other
well-known non-specific (Ser/Cys)-enzyme inhibitors, namely Orlistat (MIC ~ 25 µM) [41, 58] and
the human lysosomal acid lipase inhibitor lalistat (MIC ~ 25–50 μM) [42], support the assumption
that HPOX, and certainly all the other active OX compounds, may act as multi-target inhibitors by
impairing the activities of multiple non-essential lipolytic enzymes as well as essential proteins
involved in various important physiological pathways of M. tb life cycle.
18

Overall, it is now acknowledged that the lipolytic enzymes containing a catalytic Ser or Cys residue
in their active site are not only involved in the host-pathogen cross-talk [59], but also play several
roles in the physiopathology of the disease, in particular by recycling fatty acids from host lipids, a
key element favoring M. tb reactivation [48, 60] and survival in dormancy [61]. M. tb indeed
induces the formation of lipid bodies (LB) inside infected macrophages, giving the cells a foamy
appearance. In foamy macrophages (FM), bacilli accumulate lipids within intracytoplasmic lipid
inclusions (ILI) [62-65], which allow the bacteria to persist in a non-replicating state. In FM,
several mycobacterial lipolytic enzymes of M. tb hydrolyze host triacylglycerols (TAG) from LB
and the resulting fatty acids are stored within ILI as newly synthesized TAGs. Consequently,
although they exhibited moderate MIC50 values against M. tb H37Rv as compared to classical
antibiotics or the more recent CyC analogs [31]; OX compounds may however represent attractive
chemical tools for identifying such (Ser/Cys)-containing enzymes in living mycobacteria, studying
the regulation of ILI formation in infected FM [66], and thus provide a better understanding of how
bacilli can persist inside lipid-rich FM.
3. Conclusion
In conclusion, we have developed a new series of promising oxadiazolone-core compounds, active
against three pathogenic mycobacteria. By blocking extracellular and/or intracellular M. tb growth,
we anticipate that these OX probes could thus provide interesting insights in the mechanisms
operating during mycobacterial replication, persistence and/or reactivation, which are major issues
for deciphering the susceptibility and the general development of TB. Moreover, as these
compounds target bacterial pathway yet unexploited by anti-TB compounds, the identification of
the mycobacterial enzymes inhibited by these OX compounds in vivo may contribute to background
information for the development of new therapeutic strategies for elimination of either actively
replicating or latent bacilli from infected individuals. Accordingly, given the importance of such
19

(Ser/Cys)-enzymes for M. tb viability during infection, they should represent new attractive drug
targets. Such experiments are currently underway, and will be reported in due course.
4. Experimental Section
4.1. Chemistry.
The first 13 oxadiazolone derivatives 5a-k, 6k and 7k were synthesized as described previously [27,
67]. The new six derivatives 6a,d,e and 7a,d,e were prepared from commercial (4-
phenoxyphenyl)hydrazine hydrochloride (2) and phenylhydrazine hydrochloride (3), respectively,
by performing both the coupling reaction with alkyl chloroformate 2a-k (step i) and the cyclization
reaction with diphosgene (step ii) in a one-pot two-steps reaction [27]. All compound were at least
98% pure as determined by HPLC analysis [27]. Stock solutions (4 mg/mL) in which the
oxadiazolone compounds were found to be completely soluble in dimethyl sulfoxide (DMSO), were
prepared prior to drug susceptibility testing. See Supplementary Material for NMR, HPLC analysis
and HRMS spectra of the new six OX derivatives.
4.1.1 General procedure for the one step preparation of 5-alkoxy-3-aryl-1,3,4-oxadiazol-2(3H)-one
compounds.
4.1.1.1. 5-(2-(benzyloxy)ethoxy)-3-(3-phenoxyphenyl)-1,3,4-oxadiazol-2(3H)-one (6a = BepPPOX).
(4-phenoxyphenyl)hydrazine hydrochloride [67] 2 (8.2 g, 34.6 mmol, 1 equiv.) and 1-methyl
pyrrolidone (2.41 ml, 31.1 mmol, 0.9 equiv.) were dissolved in dry pyridine (700 mL). The solution
was cooled in an ice bath to 0 °C. Then, 2-benzyloxyethyl chloroformate 4a (6.87 mL, 38.6 mmol,
1.1 equiv.) was added dropwise over a period of 30 min at 0-5 °C and allowed to stir for 1 h at 0 °C
and 1 h at room temperature. The reaction mixture was diluted by addition of methylene chloride
(300 mL) and dry pyridine (70 mL) and the mixture was cooled at -10 °C using an ice-salt bath. A
solution of diphosgene (6.26 mL, 51.8 mmol, 1.5 equiv.) in methylene chloride (30 mL) was added
dropwise using a syringe pump over a period of 1 h while maintaining -10 °C with an ice-salt bath.
20

After the addition is complete the reaction mixture stirred 1 h at -10 °C and 2 h at room
temperature. The reaction mixture was diluted with water (1 L) and extracted with diethyl ether
(3× 250 mL). The combined organic layers were washed with water (2× 250 mL) and brine (3× 100
mL), dried over MgSO4, and filtered. Purification by column chromatography using
cyclohexane/ethyl acetate (98/2 to 95/5, v/v) as eluent gave the title compound 6a (BepPPOX) as a
yellow oil (9.94 g, 71%). Analytical data for BepPPOX: Rf (AcOEt/Cyclohexane 1:3, v/v) 0.36.
1
HRMS (ESI) m/z [M+H]+ calcd. for C23H21N2O5: 405.1445 Da ; found : 405.1446 Da. H NMR δ
7.71 (dd, J = 9.1 Hz, J = 2.2 Hz, 2H), 7.31 (m, 7H), 6.98-7.13 (m, 5H), 4.60 (s, 2H), 4.54 (m, 2H),
13
3.83 (m, 2H). C NMR δ 157.14 (s), 155.23 (s), 154.77 (s), 148.29 (s), 137.43 (s), 131.56 (s),
129.85 (2 × d), 128.55 (2 × d), 127.99 (d), 127.79 (2 × d), 123.47 (d), 119.83 (2 × d), 119.47
(2 × d), 118.74 (2 × d), 73.42 (t), 70.56 (t), 66.99 (t).
4.1.1.2. 5-isoButyloxy-3-(4-phenoxyphenyl)-1,3,4-oxadiazol-2(3H)-one (6d = iBpPPOX). Prepared
using Isobutyl chloroformate 4d applying similar method as described above for 6a. Analytical data
for iBpPPOX: pale yellow oil (87%). Rf (AcOEt/Cyclohexane 1:3, v/v) 0.55. HRMS (ESI) m/z
1
[M+H]+ calcd. for C18H19N2O4: 327.1339 Da ; found : 327.1342 Da. H NMR δ 7.72 (dd, J = 9.2
Hz, J = 2.3 Hz, 2H), 7.30 (m, 2H), 6.97-7.12 (m, 5H), 4.14 (t, J = 6.6 Hz, 2H), 2.15 (m, 1H), 1.0 (d,
13
6H). C NMR δ 157.17 (s), 155.42 (s), 154.67 (s), 148.36 (s), 131.66 (s), 129.83 (2 × d), 123.42
(d), 119.79 (2 × d), 119.49 (2 × d), 118.69 (2 × d), 71.39 (t), 22.75 (t), 18.70 (2 × q).
4.1.1.3. 5-Hexyloxy-3-(4-phenoxyphenyl)-1,3,4-oxadiazol-2(3H)-one (6e = HpPPOX). Prepared
using Hexyl chloroformate 4e applying similar method as described above for 6a. Analytical data
for HpPPOX: yellow oil (85%). Rf (AcOEt/Cyclohexane 1:3, v/v) 0.95. HRMS (ESI) m/z [M+H]+
calcd. for C20H23N2O4: 355.1652 Da ; found : 355.1652 Da. 1H NMR δ 7.72 (dd, J = 9.0 Hz, J = 2.0
Hz, 2H), 7.30 (m, 2H), 6.97-7.12 (m, 5H), 4.37 (t, J = 6.6 Hz, 2H), 1.80 (m, 2H), 1.30-1.58 (m, 6H),
13
0.90 (t, J = 6.8 Hz, 3H). C NMR δ 157.16 (s), 155.33 (s), 154.67 (s), 148.38 (s), 131.65 (s),
21

129.83 (2 × d), 123.42 (d), 119.78 (2 × d), 119.49 (2 × d), 118.68 (2 × d), 71.79 (t), 31.36 (t), 28.35
(t), 25.17 (t), 22.50 (t), 13.99 (q).
4.1.1.4. 5-Benzyloxyethoxy-3-phenyl-1,3,4-oxadiazol-2(3H)-one (7a = BePOX). Prepared using
2-benzyloxyethyl chloroformate 4a and phenylhydrazine hydrochloride 3 applying similar method
as described above for 6a. Analytical data for BePOX: pale yellow oil (79%). Rf
(AcOEt/Cyclohexane 1:3, v/v) 0.36. HRMS (ESI) m/z [M+H]+ calcd. for C7H17N2O4: 313.1183 Da ;
found : 313.1183 Da. 1H NMR δ 7.75 (m, 2H), 7.18-7.73 (m, 8H), 4.60 (s, 2H), 4.54 (m, 2H), 3.83
13
(m, 2H). C NMR δ 155.23 (s), 148.23 (s), 137.44 (s), 136.17 (s), 129.12 (2 × d), 128.55 (2 × d),
127.98 (d), 127.79 (2 × d), 125.56 (d), 117.95 (2 × d), 73.41 (t), 70.55 (t), 66.98 (t).
4.1.1.5. 5-isoButyloxy-3-phenyl-1,3,4-oxadiazol-2(3H)-one (7d = iBPOX). Prepared using Isobutyl
chloroformate 4d and phenylhydrazine hydrochloride 3 applying similar method as described above
for 6a. Analytical data for iBPOX: white crystals (77%). Mp: 76-77°C. Rf (AcOEt/Cyclohexane
1:3, v/v) 0.55. HRMS (ESI) m/z [M+Na]+ calcd. for C12H14N2O3Na: 257.0897 Da ; found :
1
257.0895 Da. H NMR δ 7.80 (m, 2H), 7.42 (m, 2H), 7.25 (m, 1H), 4.18 (d, J = 6.6 Hz, 2H), 1.85
13
(m, 1H), 1.05 (d, 6H). C NMR δ 155.63 (s), 148.51 (s), 136.44 (s), 129.29 (2 × d), 125.67 (d),
118.14 (2 × d), 77.57 (t), 27.94 (d), 18.89 (2 × q).
4.1.1.6. 5-Hexyloxy-3-phenyl-1,3,4-oxadiazol-2(3H)-one (7e = HPOX). Prepared using Hexyl
chloroformate 4e and phenylhydrazine hydrochloride 3 applying similar method as described above
for 6a. Analytical data for HPOX: white crystals (71%). Mp: 34-35°C. Rf (AcOEt/Cyclohexane 1:3,
v/v) 0.59. HRMS (ESI) m/z [M+H]+ calcd. for C14H19N2O3: 263.1390 Da ; found : 263.1390 Da. 1H
NMR δ 7.80 (ddd, J = 8.4 Hz, J = 2.3 Hz, J = 0.75 Hz, 2H), 7.42 (dd, J = 7.6 Hz, J = 1.7 Hz, 2H),
13
7.22 (t, J = 7.4 Hz, 1H), 4.40 (t, J = 6.6 Hz, 2H), 1.85 (m, 2H), 1.33-1.49 (m, 6H), 0.93 (t, 3H). C
NMR δ 155.54 (s), 148.53 (s), 136.45 (s), 129.30 (2 × d), 125.67 (d), 118.13 (2 × d), 71.97 (t),
31.45 (t), 28.55 (t), 25.36 (t), 22.68 (t), 14.17 (q).
22

4.2. Biological evaluation
4.2.1. Bacterial strains and growth condition.
M. marinum ATCC BAA-535/M, M. bovis BCG Pasteur 1173P2 and M. tb mc26230 (H37Rv RD1
panCD [28]) strains were routinely grown in Middlebrook 7H9 broth (BD Difco) supplemented
with 0.2% glycerol, 0.05% Tween 80 (Sigma-Aldrich), 10% oleic acid, albumin, dextrose, catalase
(OADC enrichment; BD Difco) (7H9-S) and 24 µg/mL D-panthothenate (M. tb mc26230). For
further intra and extracellular assays, M. tb H37Rv expressing GFP [36] was grown for 14 days in
7H9-S supplemented with 50 µg/mL hygromycin B (Euromedex). All cultures were kept at 37 °C
without shaking, except M. marinum which was grown at 32 °C.
4.2.2. Susceptibility testing on M. marinum, M. bovis BCG and M. tb mc26230.
The concentrations of compound leading to 50% of bacterial growth (MIC50) were first determined
using the resazurin microtiter assay (REMA) [29, 30]. Briefly, log-phase bacteria were diluted to a
cell density of 5 × 106 cells/mL and 100 µL of this inoculum was grown in a 96-well plate in the
presence of serial dilutions of compounds. After 7-14 days incubation, 20 µL of a 0.025% (w/v)
resazurin solution was added to each well (200 µL) and incubation was continued until the
appearance of a color change (from blue to pink) in the control well (bacteria without antibiotics).
Fluorescence of the resazurin metabolite resorufin (excitation, 530 nm; emission, 590 nm) was then
measured [30] and the concentration leading to 50% growth inhibition was defined as the MIC50.
See Supplementary Material for detailed protocol.
4.2.3. High-content screening assay - Extracellular assay.
A 14 days old culture of M. tb H37Rv-GFP was washed twice with PBS and resuspended in 7H9
medium containing 10% OADC, 0.5% glycerol, 0.05% Tween 80 and 50 µg/mL hygromycin B.
Bacteria were seeded in 384 well plates (7 × 105 bacteria/mL) containing 2-fold dilutions of the
compounds in DMSO. The final volume of DMSO was kept under 0.3%. Plates were incubated at
23

37 °C, 5% CO2 for 5 days. Bacterial fluorescence levels (RFU) were recorded using a fluorescent
microplate reader (Victor X3, Perkin-Elmer). The MIC50 of all tested compounds were determined
using ten-point dose-response curves. In each plate, negative control with 1% DMSO; and positive
controls containing 1 µg/mL INH and RIF were also included.
4.2.4. High-content screening assay in infected macrophages - Intracellular assay.
The growth of M. tb H37Rv-GFP strain in macrophages was monitored by automated fluorescence
confocal microscope (Opera, Perkin-Elmer) as already described [36, 68]. Briefly, bacteria were
washed twice with PBS and resuspended in RPMI 1640 medium (Invitrogen) supplemented with
10% heat-inactivated fetal bovine serum (FBS, Invitrogen). Murine (Raw264.7) macrophages
(American Type Culture Collection TIB-71) were infected at a multiplicity of infection (MOI) of
2:1 and incubated 2 hours at 37 °C in RPMI 1640 medium containing 10% FBS. Cells were then
washed, treated with 50 µg/mL amikacin (Euromedex) for 1 hour at 37 °C to kill all extra-cellular
bacteria, washed again and finally seeded in 384-well plates (5 × 105 cells/mL), containing 2-fold
dilutions of compounds in DMSO. The final volume of DMSO was kept under 0.3%. Plates were
incubated for 5 days at 37 °C, 5% CO2. Infected cells were stained for 30 min using Syto60 dye
(Invitrogen) at a final concentration of 5 µM before reading using fluorescence confocal microscope
(20X water objective; GFP: ex 488 nm, em 520 nm; Syto60: ex 640 nm, em 690 nm). Dose-
responses were fitted using Prism software (sigmoidal dose-response, variable slope model). The
MIC50 was determined using ten-point dose-response curves as an average of the MIC50 of 4
parameters, the ratio of infected cells, the total area of bacteria, the cells number and the bacterial
area per infected cell. In each plate, negative control with 1% DMSO (i.e., infected macrophages
only); as well as positive controls containing 10 µg/mL INH, ETO and RIF were also included.
4.3. HPOX target enzymes identification
4.3.1. Activity-based protein profiling (ABPP). Homogeneous bacterial suspension of M. tb
mc26230 in 7H9-S was adjusted at an OD600 of 60 and then incubated with the selected HPOX
24

inhibitor (400 µM final concentration) or DMSO (control) at 37 °C for 2-3 h. under gentle shaking
at 75 rpm. Bacteria were then washed 3 times with PBS containing 0.05% Tween 80, resuspended
in PBS buffer at a 1:1 (w/v) ratio and then lysed by mechanical disruption on a BioSpec Beadbeater.
Both HPOX-treated M. tb mc26230 and DMSO-control lysate samples (750 µL – 0.75 mg total
proteins) were labeled with 2 µM Desthiobiotin-FP probe for 90 min at room temperature. Samples
were enriched for biotinylated proteins using Nanolink streptavidin magnetic beads 0.8 µm
(Solulink), according to the manufacturer’s instructions. The resulting captured biotinylated
proteins solution was mixed with 5X Laemmli reducing sample buffer, and heated at 95 °C for 5
min. The released denatured proteins were subjected to tryptic digestion, peptide extraction, and
LC-MS/MS analysis as described below.
Alternatively, the HPOX-treated M. tb mc26230 and DMSO-control lysate samples (100 µL –
100 µg total proteins) were incubated with 2 μM ActivX TAMRA-FP probe (Thermo Fisher
Scientific) for 90 min at room temperature and in absence of light. The reaction was stopped by
adding 5X Laemmli reducing sample buffer and boiling at 95 °C for 5 min. The labeled proteins
were further separated by SDS-PAGE electrophoresis. TAMRA fluorescence (TAMRA: λex 557
nm, λem 583 nm) was detected using a ChemiDoc MP Imager (Bio-Rad). Detailed Materials and
Methods is given in Supplementary Material.
4.3.2. Protein identification and quantification. Protein extract were loaded and stacked on a
NuPAGE gel (Life Technologies). Stained bands were submitted to an in-gel trypsin digestion [69].
Peptides extracts were reconstituted with 0.1% trifluoroacetic acid in 4% acetonitrile and analyzed
by liquid chromatography (LC)-tandem mass spectrometry (MS/MS) using an Orbitrap Fusion
Lumos Tribrid Mass Spectrometer (Thermo Electron, Bremen, Germany) online with a an Ultimate
3000RSLC nano chromatography system (Thermo Fisher Scientific, Sunnyvale, CA). Protein
identification and quantification were processed using the MaxQuant computational proteomics
platform, version 1.5.3.8 [70] using a UniProt M. tuberculosis ATCC 25618 database (date
2018.01; 2164 entries). The statistical analysis was done with Perseus program (version 1.5.6.0).
25

Differential proteins were detected using a two-sample t-test at 0.01 and 0.05 permutation based
FDR. The mass spectrometry proteomics data, including search results, have been deposited to the
ProteomeXchange Consortium (www.proteomexchange.org) [71] via the PRIDE partner repository
with
the
dataset
identifier
PXD010255
(Reviewer
account
details.
username: reviewer87937@ebi.ac.uk / password: Hhpb9H0Y). Detailed Materials and Methods is
given in Supplementary Material.
4.4. Inhibition assays on pure recombinant proteins.
The three lipolytic enzymes from M. tb, the thioesterase TesA, the monoacylglycerol lipase
Rv0183, and the Cutinase-like protein Cfp21 were produced and purified as previously reported
[48, 55, 56].
The lipase−inhibitor pre-incubation method was used to test, in aqueous medium and in the absence
of substrate, the possible direct reactions between lipases and inhibitors as previously described [27,
57, 72]. Briefly, an aliquot of each enzyme was pre-incubated at 25 °C with HPOX at various
inhibitor molar excess (xI) ranging from 1 to 40 related to 1 mol of enzyme. A sample of the
incubation medium was collected after 30 min incubation period and the residual enzyme activity
was measured. The variation in the residual lipase activity allowed determination of the inhibitor
molar excess which reduced the enzyme activity to 50% of its initial value (xI50) [27, 57, 72]. In
each case, control experiments were performed in the absence of inhibitor. The respective
enzymatic activity of TesA and Cfp21 were assessed using para-nitrophenyl (pNP) ester assay with
pNP caprylate (pNP-C8) as substrate, as described previously [23]. Rv0183 residual activity was
determined using monoolein as substrate, as reported in [48, 57]. Dose-response curves were fitted
in Kaleidagraph 4.2 Software (Synergy Software). Results are expressed as mean values ± SD of at
least two independent assays.
26

Figure captions
Figure 1. Chemical structure of (A) 5-(pyridin-4-yl)-1,3,4-oxadiazol-2(3H)-one S57, and (B) 5-
methoxy-3-(3-phenoxyphenyl)-1,3,4-oxadiazol-2(3H)-one MmPPOX and its mode of action. (C)
General procedure for the one step preparation of 5-alkoxy-3-phenyl substituted-1,3,4-oxadiazol-
2(3H)-one compounds from either (3-phenoxyphenyl)hydrazine hydrochloride (1), (4-
phenoxyphenyl)hydrazine hydrochloride (2) or phenylhydrazine hydrochloride (3), giving OX
derivatives 5a-k (i.e., RmPPOX), 6a,d,e,k (i.e., RpPPOX) or 7a,d,e,k (i.e., RPOX), respectively.
Reagents and conditions: i) Alkyl chloroformate 4a-k, Pyridine, 0 °C to RT; ii) ClCO2CCl3, CHCl2,
Pyridine, 0 °C to RT, 40-85%. Adapted from [27].
Figure 2. In vitro and ex vivo dose-response activity of the OX derivatives against M. tb
H37Rv. (A) Activity of BePOX, iBpPPOX and HPOX against GFP-expressing M. tb replicating
in broth medium, expressed as normalized relative fluorescence units (RFU%). The dashed line
represents the level of inhibition (~70%) reached with 1 µg/mL (=7.3 µM) INH as control. The
MIC50 of BePOX, iBpPPOX and HPOX replicating in culture broth medium were 30.8 μM, 32.0
µM and 44.6 μM, respectively. (B) Activity of iBpPPOX, BepPPOX, HpPPOX and iBPOX
against M. tb replicating inside Raw264.7 macrophages. Results are expressed as the percentage of
infected macrophages after 5 days post-infection. The dashed line represents the level of inhibition
(~87%) reached with 10 µg/mL (=73 µM) INH as control. The MIC50 of BepPPOX, iBpPPOX,
HpPPOX and iBPOX replicating inside macrophages were 3.5 μM, 8.5 µM, 9.5 µM and 17.1 μM,
respectively. Each value is the mean ± SD for a triplicated dose-response. Experiments were
conducted at least twice with consistent results.
Figure 3. Activity based protein profiling (ABPP) workflow for the identification of the
proteins covalently bound to OX inhibitors. (A) Cell culture of M. tb mc26230 was pre-treated
with selected HPOX inhibitor prior to cell lysis and further incubation with TAMRA-FP or
27

Desthiobiotin-FP probe. In this latter case, samples were then treated with streptavidin-magnetic
beads for the capture and enrichment of labelled proteins. (B-C) Equal amounts of proteins obtained
in A were separated by SDS-PAGE and visualized by in-gel fluorescence (right panel - lanes D-E:
TAMRA detection) or silver staining (left panel – lanes A-C). Enzymes whose TAMRA-FP
labelling is impeded because of the presence of HPOX in their active-site are shown by
arrowheads. (D) Tryptic digestion followed by tandem mass spectrometry analyses and subsequent
differential proteomics analysis allowed identifying the target proteins.
Figure 4. Volcano plot of proteomic analysis of HPOX in M. tb mc26230 culture. Volcano plot
showing the significance two-sample t-test (-Log p-value) versus fold-change (Log2 (LFQ
normalized intensity in HPOX versus Desthiobiotin-FP condition) on the y and x axes, respectively.
Here the plot is zoomed on the positive difference between the two conditions. The full line is
indicative of protein hits obtained at a permutation false discovery rate of 1% (pFDR). Only protein
hits obtained at a pFDR of 5% (dashed line) and with a score threshold value ≥ 60 were selected,
and are highlighted in black (non-essential) or in red (essential). Data results from two different
experiments processed three times.
Figure 5. HPOX efficiently impairs TesA, Cfp21 and Rv0183 activities in vitro. Variation of the
residual activities of TesA, Cfp21 and Rv0183 as a function of increasing molar excess (xI) of
HPOX. Each enzyme was pre-incubated at various inhibitor molar excess (xI) for 30 min at 25 °C.
The inset displays the determined values of xI50 and the level of inhibition at xI = 20. Kinetic assays
were performed as described in Experimental Section. Results are expressed as mean values ± SD
of at least two independent assays.
28

Tables
Table 1. Antibacterial activities of the oxadiazolone derivatives against M. marinum, M. Bovis
BCG and M. tb mc26230 using the REMA method a
MIC50 (µM)
M. marinum M. bovis BCG M. tb mc26230
Compounds
INH
RIF
20.5
1.4
0.71
0.027
1.5
0.015
7.0
11.5
52.9
15.0
39.9
>120
71.3
>120
89.8
MmPPOX
MpPPOX
MPOX
3.4
2.7
4.1
7.1
9.4
6.6
>120
91.2
52.2
EmPPOX
MemPPOX
BmPPOX
13.6
6.9
2.5
4.4
8.5
5.7
57.2
31.1
>120
iBmPPOX
iBpPPOX
iBPOX
11.1
3.4
2.6
5.8
14.0
3.5
48.8
41.3
40.5
HmPPOX
HpPPOX
HPOX
5.6
10.1
1.9
10.5
18.5
6.6
49.5
44.5
33.2
BemPPOX
BepPPOX
BePOX
8.0
11.0
44.7
OmPPOX
8.3
8.1
43.3
EhmPPOX
10.5
19.5
80.8
22.6
>120
>120
DmPPOX
DomPPOX
a
Experiments were performed as described in Experimental Section. MIC50: compound minimal
concentration leading to 50% of growth inhibition, as determined by the REMA assay. The best MIC50
obtained for each strain are highlighted in bold. Values are mean of at least two independent assays
performed in triplicate. INH, isoniazid; RIF, rifampicin.
29