Drug resistance in bacteria can occur through
mutations in the antibiotic’s target; the acquisition of enzymes that modify or degrade the drug,
such as aminoglycoside-modifying enzymes or
b-lactamases; their active expulsion from the
bacteria; or alterations of the cell permeability (6).
Sometimes, antibiotic resistance can be reversed—
for example, through restoration of the antimicrobial activity of b-lactams by clavulanic acid,
which inhibits enzymes responsible for their
degradation (7). Unfortunately, there are so far
no other examples of this 40-year-old paradigm.
Some of the most effective anti-TB antibotics
require bioactivation by Mycobacterium tuberculosis
enzymes to acquire their antibacterial effect. These
pro-antibiotics not only include the 40-year-old
compounds isoniazid (INH), pyrazinamide (PZA),
p-aminosalicylic acid (PAS) and ethionamide (ETH),
but also the recently approved drug delamanid
(OPC-67683) and the under-clinical-evaluation compound pretomanid (PA824). However, bioactivation of pro-antibiotics is vulnerable to mutational
inactivation or attenuation of the corresponding
bioactivating enzymes, as observed for INH-, PZA-,
and ETH-resistant clinical isolates with mutations
in katG (8), pncA (9), and ethA (10, 11), respectively. Similarly, experimentally generated and
clinical resistance to delamanid and to pretomanid
pointed to enzymes and coenzymes involved
in their bioactivation (12–14). Resistance to PAS
also involves mutations in enzymes, such as di-hydrofolate synthase, which is implicated in its
We have discovered a spiroisoxazoline family
of Small Molecules Aborting Resistance (SMARt)
that induces expression of an alternative bioactivation pathway of ETH, reverting acquired resistance of M. tuberculosis to this antibiotic.
The bioactivation of ETH in M. tuberculosis
is normally catalyzed by the Baeyer-Villiger mono-
oxygenase EthA (10, 11, 16). Transformation of
ETH by EthA into highly reactive intermedi-
ates leads to the formation of a stable covalent
adduct of ETH and nicotinamide adenine di-
nucleotide (NAD) (10, 17). This adduct binds to
and inhibits the enoyl reductase InhA involved
in mycolic acid biosynthesis, one of the essential
components in the mycobacterial cell wall (18, 19).
The production of EthA is regulated by the TetR-
type transcriptional repressor EthR (20). Previous-
ly, we have shown that small-molecule inhibitors
of EthR stimulate the transcription of the ethA
gene (21–24), which improves the bioactivation of
ETH and consequently boosts its antibiotic ac-
tivity, both in vitro and in vivo (25). These booster
molecules, such as BDM41906, reduce or reset
the innate resistance of M. tuberculosis to ETH;
however, as expected, they were unable to boost
the bioactivation of ETH in strains harboring mu-
tations in ethA (Table 1, panel B).
During optimization of first-generation EthR
inhibitors, most derivatives revealed a good cor-
relation between binding to EthR and ETH-
boosting activity against the bacteria (21–24).
However, unexpectedly, the replacement of the
1Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille,
U1019-UMR8204-CIIL–Center for Infection and Immunity of
Lille, F-59000 Lille, France. 2Université Lille, Inserm, Institut
Pasteur de Lille, U1177–Drugs and Molecules for Living
Systems, F-59000 Lille, France. 3Division of Translational
Medicine and Chemical Biology, Science for Life Laboratory,
Department of Medical Biochemistry and Biophysics,
Karolinska Institutet, Stockholm, Sweden. 4National
Reference Center for Tuberculosis and Mycobacteria,
Bacterial Diseases Service, Operational Direction
Communicable and Infectious Diseases, Scientific Institute of
Public Health (WIV-ISP), Brussels, Belgium. 5Tuberculosis
Research Laboratory, Institut Pasteur Korea, South Korea.
6Bioversys AG, Hochbergerstrasse 60C, 4057 Basel,
Switzerland. 7Biozentrum, University of Basel, Basel,
Switzerland. 8Laboratoire des Biopolymères et des
Nanomatériaux Supramoléculaires, Université Libre de
Bruxelles, Brussels, Belgium. 9VIB Center for Structural
Biology, VIB, Pleinlaan 2, 1050 Brussels, Belgium.
10Structural Biology Brussels, Vrije Universiteit Brussel
(VUB), Pleinlaan 2, 1050 Brussels, Belgium. 11Swiss Tropical
and Public Health Institute, Basel, Switzerland. 12University
of Basel, Basel, Switzerland.
*These authors contributed equally to this work. †Corresponding
author. Email: email@example.com (B.D.); nicolas.willand@
univ-lille2.fr (N. W.); firstname.lastname@example.org (A.R.B.)
Table 1. Impact of BDM41906 and SMARt-420 on the ethionamide susceptibility of a selection of clinical strains. (Panel A) Antibiotic profile. Threshold concentrations above which bacteria
are considered clinically resistant are indicated. The drug-sensitivity status of each strain is reported;
green indicates “under the threshold concentration,” and red indicates “above the threshold concentration.” Specifically, for ETH, MICs have been defined by MGIT960 and are reported (values in
micrograms per milliliter). All selected strains except the reference pan-susceptible laboratory strain
H37Rv (group 1) are multidrug-resistant (INH- and RIF-resistant). Group 2 includes ETH-sensitive strains.
Group 3 contains ETH-resistant strains without mutation in ethA. Group 4 contains ETH-resistant
strains mutated in ethA. (Panel B) MIC of ETH in the presence of 10 mM first-generation booster
BDM41906. (Panel C) MIC of ETH in the presence of 10 mM SMARt-420.