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Development of efflux pump inhibitors in antituberculosis therapy

Article  in  International journal of antimicrobial agents · May 2016

DOI: 10.1016/j.ijantimicag.2016.04.007

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 International Journal of Antimicrobial Agents 47 (2016) 421–429

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International Journal of Antimicrobial Agents

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j a n t i m i c a g

Review

Development of efflux pump inhibitors in antituberculosis therapy

Lele Song *, Xueqiong Wu *
Army Tuberculosis Prevention and Control Key Laboratory, Beijing Key Laboratory of New Techniques of Tuberculosis Diagnosis and Treatment,
Institute of Tuberculosis Research, The 309th Hospital of Chinese PLA, Beijing 100091, China

A R T I C L E I N F O A B S T R A C T

Article history: Resistance and tolerance to antituberculosis (anti-TB) drugs, especially the first-line drugs, has become
Received 26 January 2016 a serious problem in anti-TB therapy. Efflux of antimicrobial agents via bacterial efflux pumps is one of
Accepted 2 April 2016 the main reasons for drug resistance. Efflux pump inhibitors (EPIs) bind to efflux pumps to inhibit drug
efflux and thus enhance the drug effect and reduce drug resistance. Studies on EPIs targeting the efflux
Keywords: pumps of Mycobacterium tuberculosis (Mtb) help to understand Mtb resistance and to identify the po-
Tuberculosis
tential drug target and are of significance in guiding the development of new anti-TB drugs and optimal
Mycobacterium tuberculosis
combinations. Currently, there are many potential EPIs under study, but none of them has been used clin-
Efflux pump inhibitor
Verapamil ically for anti-TB therapy. In this article, we will provide an overview on the current development of EPIs
Reserpine targeting the efflux pumps of Mtb and discuss their potential clinical applications.
CCCP © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

1. Introduction tance (SMR) family, resistance–nodulation–division (RND) family,

Resistance of Mycobacterium tuberculosis (Mtb) to antitubercu-
losis (anti-TB) drugs is one of the intractable problems of anti-TB
therapy. The mechanisms of resistance have not yet been fully un-
derstood and several possible mechanisms are currently under
investigation, including (i) mutations in Mtb drug resistance-
associated genes encoding key enzymes or transcription factors; (ii)
overexpression of Mtb efflux pumps; (iii) changes in Mtb cell wall
permeability; and (iv) high expression of the Mtb two-component
system that regulates Mtb adaptation to intracellular and extracel-
lular environments [1–3]. This review will focus on the effects and
therapeutic potential of efflux pump inhibitors (EPIs) targeting
various Mtb efflux pumps.
Efflux is a self-protection phenomenon that exists widely in pro-
karyotic and eukaryotic cells. It is a physiological process extruding
endogenous metabolic waste and exogenous chemicals to main-
tain normal cell function [4–8]. Efflux pumps are key membrane
structures responsible for efflux and play crucial roles in maintain-
ing intracellular and extracellular material exchange and cellular
homeostasis [7,8]. They can recognise a variety of substances with
a wide range of physical and chemical properties, which is the cel-
lular basis of multidrug resistance [8]. Bacterial efflux pumps are
divided into six categories, including major facilitator superfamily
(MFS), ATP-binding cassette (ABC) family, small multidrug resis-
multidrug and toxic compound extrusion (MATE) family and drug
metabolite transporter (DMT) superfamily [7,9]. Efflux pumps of the
MFS, ABC, RND and SMR families have been found in Mtb.
Drug resistance can be divided into two types, comprising in-
trinsic resistance and acquired resistance. Intrinsic resistance refers
to efflux of antibiotics or toxic substances by induced expression
of intrinsic efflux pumps coded by bacterial genes or plasmids. In-
trinsic resistance is species-specific and allows bacterial cells to evade
adverse environments [10,11]. In contrast, acquired resistance can
develop by mutations that lead to high expression of efflux pump
genes or by the spread of plasmids carrying drug resistance genes.
As drug resistance is beneficial to bacterial survival, resistance will
spread in the population and will eventually become intrinsic re-
sistance. The expression of a variety of efflux pumps in a bacterial
population can lead to drug resistance to a broad spectrum of an-
timicrobial agents, leading to multidrug resistance [10,11].
EPIs are a type of molecule that binds to bacterial efflux pumps to
inhibit their efflux function. EPIs binding to Mtb efflux pumps were
shown to inhibit efflux of anti-TB drugs, to enhance Mtb killing, to
reverse Mtb drug resistance and to produce synergistic effects with first-
line anti-TB drugs [12,13]. Anti-Mtb EPIs can also reduce the Mtb load,
the dosage of chemotherapy drugs, and the time and relapse rate of
TB treatment in animal models [12,13]. In this article, the effects of po-
tential EPIs and their combined use with first-line anti-TB drugs are
reviewed, and the prospects of EPIs in anti-TB therapy are discussed.

* Corresponding authors. Army Tuberculosis Prevention and Control Key Laboratory,
Beijing Key Laboratory of New Techniques of Tuberculosis Diagnosis and Treatment,
Institute of Tuberculosis Research, The 309th Hospital of Chinese PLA, Beijing 100091,
China. Tel.: +86 10 6677 5109; +86 132 4014 9188; fax: +86 10 80115555.
E-mail addresses: xueqiongwu@139.com (X. Wu); songlele@sina.com (L. Song).
2. Major bacterial efflux pumps
Bacterial efflux pumps can be divided into two classes based on
their structure, energy source and substrate types. The first class

http://dx.doi.org/10.1016/j.ijantimicag.2016.04.007
0924-8579/© 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.
422 L. Song, X. Wu / International Journal of Antimicrobial Agents 47 (2016) 421–429

is the ABC transporters, which use the free energy of ATP hydro-
lysis to extrude drugs, whilst the second class is the secondary drug
transporters utilising the transmembrane electrochemical gradi-
ent of protons or sodium ions to extrude drugs. Based on size and
similarities in the primary and secondary structure, the second-
ary drug transporters can be subdivided into several distinct families,
including the MFS, SMR, RND and MATE families [9]. Efflux pumps
belonging to ABC, MFS, RND and SMR families have been found in
Mtb. Some of them have been confirmed to be functional mem-
brane proteins by multiple studies, whilst others appeared to be
putative Mtb efflux pumps suggested by comparative genomic
studies [8]. The major Mtb efflux pumps will be introduced in detail
in this section.
2.1. The ATP-binding cassette (ABC) family
The ABC efflux pump family is a group of transmembrane pro-
teins with a channel structure and cytosolic ATP-binding sites. ATP
hydrolysis provides energy for transport in the presence of mag-
nesium. P-glycoprotein (P-gp) is the most representative efflux pump
in the family. As shown in Fig. 1, P-gp is composed of two hydro-
phobic transmembrane domains (TMD1 and TMD2) and two
nucleotide-binding domains (NBD1 and NBD2) [14,15]. The TMD con-
tains six hydrophobic α-helices and forms the substrate recognition
sites and transport duct. The DrrABC complex (gene name Rv2936/
2937/2938) is one of the confirmed ABC family transporters in Mtb.
DrrA contains two NBDs, and DrrB contains six α-helices that are
structurally and functionally homological to P-gp [16]. The Rv2686c-
Rv2687c-Rv2688c and Rv1456c-Rv1457c-Rv1458c transport systems
are two ABC family efflux pumps identified recently in Mtb H37Rv.
High expression of the two pumps was observed in the presence
of first-line anti-TB drugs, leading to resistance of H37Rv strain to
at least one of the drugs from isoniazid (INH), rifampicin (RIF), strep-
tomycin (STM) and ethambutol (EMB) [17].
2.2. The resistance–nodulation–division (RND) family
The RND efflux pumps are proton-dependent efflux pumps com-
posed of an inner membrane protein, a membrane fusion protein
and an outer membrane factor, as shown in Fig. 1. The most rep-
resentative RND family efflux pump is the AcrAB–TolC pump in
Escherichia coli [18], formed by AcrB (inner membrane protein), AcrA
(membrane fusion protein) and TolC (outer membrane factor). AcrB
is a homotrimer and each subunit is composed of a prominent cap-
like structure and 12 α-helix transmembrane domains [19,20]. Drug
molecules bind to AcrB and induce the formation of a complete efflux
pump with AcrA and TolC [21,22]. Drug molecules are expelled by
TolC through the transmembrane domains of AcrB. This process re-
quires proton exchange to provide energy for AcrB to transfer drug
molecules to TolC [22–25]. The major RND family efflux pumps in

Fig. 1. Schematic structures of ATP-binding cassette (ABC), resistance–nodulation–division (RND), major facilitator superfamily (MFS) and small multidrug resistance (SMR)
efflux pump systems in Mycobacterium tuberculosis. The structure of the P-glycoprotein (P-gp) efflux pump is used to illustrate the ABC family, and the structure of the AcrAB–
TolC efflux pump is used to illustrate the RND family. A detailed description of each individual family can be found in Section 2. The binding sites for each individual drug
reviewed in this article are listed under the figure. The solid circles represent binding to a certain type of efflux pump supported by the literature, whilst N/A represents
that the binding site information is not available for a drug. CCCP, carbonyl cyanide m-chlorophenyl hydrazone; DNP, 2,4-dinitrophenol; CPZ, chlorpromazine; TZ, thior-
idazine; GEQ, Genz-10850.
 L. Song, X. Wu / International Journal of Antimicrobial Agents 47 (2016) 421–429
Mtb include mycobacterial membrane proteins (MmpL) and my-
cobacterial small membrane protein (MmpS), and homology was
found between them. It was shown that MmpL7 has strong resis-
tance against INH, which can be antagonised by the EPIs reserpine
(RES) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP)
[26,27], whilst MmpS4 is required for the production and efflux of
cell surface glycolipids [28].
2.3. The major facilitator superfamily (MFS) family
The MFS efflux pumps are dimers containing 12 transmem-
brane domains. Regions determining the transporter specificity are
located at the end of the transmembrane domains as helices and
extracellular loops, as shown in Fig. 1 [29–32]. MFS efflux pumps
expel drug molecules through the substrate/H+(Na+) antiporter
system [33]. Tet and Mef efflux pumps are the most representa-
tive efflux systems in the MFS family, shown to be associated with
macrolide resistance [34,35]. LfrA is the first MFS efflux pump found
in Mycobacterium, which has a high degree of homology with the
Staphylococcus aureus QacA (Rv2846) efflux pump [36,37]. The Tap
efflux pump (Rv1258c) is also a member of the MFS family, exhib-
iting resistance against aminoglycoside and tetracycline antibiotics
[38,39]. The p55 efflux pump was shown to be responsible for a re-
duction in bacterial susceptibility to the first-line anti-TB drugs RIF
and clofazimine in the M. bovis Bacillus Calmette–Guérin (BCG)
model, whilst CCCP and valinomycin (VLM) were shown to be EPIs
targeting the p55 efflux pump [40].
2.4. The small multidrug resistance (SMR) family
The SMR family efflux pumps are small proteins (100–200 amino
acids) composed of four transmembrane helical structures con-
taining conserved amino acids, which directly interact with
hydrophobic regions of aminoglycoside and macrolide antibiotics
[41]. Mmr (Rv3065) is the first identified SMR family efflux pump,
exhibiting high homology with the QacEu (Staphylococcus) and EmrE
(E. coli) efflux pumps, and mediates efflux of erythromycin and eth-
idium bromide (EtBr) [42]. The Rv3789 gene was also suggested to
encode a potential SMR efflux pump [43].
3. Development of potential efflux pump inhibitors targeting
M. tuberculosis efflux pumps
Potential anti-TB drugs can be divided into two groups, namely
synthetic drugs and drugs from plant extracts. Each individual drug
may target one or more efflux pumps. The EPI classification, EPI
names, targeted efflux pumps and the effects of EPIs are summarised
and compared in Table 1. The potential effects of each individual
EPI in anti-TB therapy are introduced in detail in this section.
3.1. Synthesised drugs
3.1.1. Verapamil (VER)
VER is by far the most studied anti-TB EPI. The main effects of
VER were suggested to be related to its inhibition of Mtb efflux
pumps. Studies with INH- or RIF-resistant clinical samples or Mtb
strains showed that combined use of VER with INH or RIF reduced
the minimum inhibitory concentration (MIC) of both drugs and re-
versed the Mtb drug resistance against both drugs [40,44–47].
Similarly, combination of VER with levofloxacin decreased the MIC
of levofloxacin 2–8-fold in clinical samples resistant to levofloxacin,
whilst no effect was observed in levofloxacin-sensitive clinical
samples [48]. VER can also inhibit efflux pumps to increase the re-
tention of drugs in Mtb cells, and its effect was shown to be stronger
than CCCP, RES and chlorpromazine [49]. Bedaquiline is the first anti-
multidrug-resistant (MDR) Mtb drug approved by the US Food and
423
Drug Administration (FDA); however, drug resistance to bedaquiline
has already been reported [50,51]. It was found that VER can reduce
the MIC of bedaquiline 8–16-fold and also reduce the MICs of
moxifloxacin and clofazimine [50,52,53]. An isomer of VER, namely
norverapamil, exhibited less inhibition of calcium channels but
similar inhibition of macrophage-induced resistance. Therefore,
norverapamil and its derivatives represent a promising option in
reducing drug side effects and improving drug tolerance [54]. In
general, VER exhibited synergistic effects with a series of anti-TB
drugs, decreased drug MICs, reduced drug resistance and in-
creased drug retention in Mtb. Inhibition of Mtb efflux pumps by
VER could be the common mechanism underlying the above effects.
In addition, studies with animal models found that VER enhanced
Mtb removal in Mtb-infected macrophages, inhibited the growth
and tolerance of intracellular Mtb, reduced the drug dosage, short-
ened the duration of therapy [45,53,54] and reduced the recurrence
rate [45] in combination chemotherapy. These animal studies provide
evidence and experience for the application of VER in future clin-
ical anti-TB therapy [55].
3.1.2. Protonophores
Protonophores are a type of ionophore that moves protons across
the cell membrane. Ionophores are lipid-soluble complexes that
transport ions across the cell membrane [56]. There are two main
types of ionophores, including carrier ionophores and channel
formers. The carrier ionophores bind to a particular ion, carry the
ion through the hydrophobic interior of the lipid membrane and
release the ion to the other side of the membrane, whilst the channel
formers are usually large proteins introducing a hydrophilic pore
into the membrane and allowing ions to move across the hydro-
phobic membrane through the pore [57]. Since transmembrane ion
concentration gradients are required for maintaining the mem-
brane potential, ionophores have important physiological roles for
living organisms [58]. The properties of ionophores have been used
in developing antibiotics, such as macrocyclics [59]. Protonophores
belong to the carrier ionophores [60]. The protonophores that have
potential anti-TB effect include CCCP, 2,4-dinitrophenol (DNP) and
VLM.
3.1.2.1. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Studies
on CCCP mainly focus on its synergistic effect with other anti-TB
drugs, specifically on the inhibitory or killing effects of the com-
bined therapy on Mtb. Silva et al first reported that CCCP can reduce
the MIC of STM and tetracycline and strengthen the anti-Mtb effect in
PAZ22 strain by inhibiting the MFS type efflux pump P55 [61]. Sim-
ilarly, Ramón-García et al also found that CCCP inhibited P55-
related resistance to RIF and clofazimine in M. bovis BCG, suggesting
that transmembrane proton motive force and electrochemical gra-
dient are the energy source of drug efflux [62]. CCCP also decreased
the MIC of tetracycline in M. smegmatis by inhibiting the Tap efflux pump
of the MFS family [63]. In addition, studies with clinical samples
proved that CCCP enhanced the anti-TB effect by inhibiting MFS efflux
pumps. Singh et al showed that CCCP decreased the MIC of ofloxacin
in ofloxacin-resistant Mtb in clinical samples whereas it exhibited
no effect in ofloxacin-sensitive Mtb, suggesting that CCCP can inhibit
Mtb resistance to quinolones [48]. Gupta et al demonstrated that
CCCP reduced the MIC of INH and EMB by inhibition of the JefA efflux
pump of the MFS family in a recombinant Mtb strain [64]. Apart
from the MFS efflux pump, CCCP was also shown to act on the ABC
family efflux pump. Pasca et al found that CCCP reduced resis-
tance to ciprofloxacin by depleting the intracellular ATP of
M. smegmatis and inhibiting the ABC family efflux pump Rv2686c-
Rv2687c-Rv2688c [65]. Recent studies showed that CCCP significantly
reduced the MIC of INH and RIF in MDR Mtb, and even completely
restored the sensitivity of MDR Mtb to RIF, whilst the MIC change
showed a significant difference compared with the drug-sensitive
424 L. Song, X. Wu / International Journal of Antimicrobial Agents 47 (2016) 421–429

Table 1
Names, classifications, targets, main effects and clinical impact of Mtb efflux pump inhibitors.

Drug type Drug name Putative drug targets Main drug effects Rating of clinical relevance References

Rating Evidence Evidence Other evidence

in vitro in vivo

Synthesized Verapamil and ABC:DrrAB,PstB,Rv2686c • Decrease the MIC of anti-TB drugs ☆☆☆☆☆ Strong Strong FDA-approved [40,44–55]
drugs norverapamil -2687c-2688c, • Reduce drug dosage EPI, but further
MFS:lfrA,Rv1634,Rv1258c, • Exhibit synergistic effect with anti-TB studies are
Rv1877,Rv2846c drugs needed for TB
RND:Rv1145,Rv1146, • Increase the retention of anti-TB drugs in therapy in
Rv0678 Mtb animals and
SMR:Rv3065(mmr) • Enhance Mtb removal in macrophages human
• Inhibit the growth and tolerance of Mtb
• Shorten the therapy duration
Protonophore: ABC:Rv2936-Rv2937(DrrAB), • Decrease the MIC of anti-TB drugs ☆☆☆ Strong Limited N/A [27,47–49,
CCCP Rv0933(PstB),Rv2686c- • Reduce Mtb resistance to anti-TB drugs 61–66]
2687c-2688c • Exhibit synergistic effect with anti-TB
MFS:lfrA, Rv2459(jefA), drugs
Rv1410c(P55),Rv1634,Rv1258c,
Rv1410c,Rv1877,Rv2846c
RND:Rv1145,Rv1146,
Rv0676c-Rv0677c
(MmpS5-MmpL5)
SMR:Rv3065(mmr)
Protonophore: ABC:Rv2936-Rv2937(DrrAB), • Decrease the MIC of anti-TB drugs ☆☆ Some Lack N/A [48,61,62]
DNP Rv0933(PstB),Rv2686c-
2687c-2688c
MFS:lfrA,Rv1634,Rv1258c
Protonophore: MFS:Rv1410c(P55) • Increase the retention of anti-TB drugs in ☆☆ Strong Lack N/A [62,67–72]
valinomycin Mtb
• Enhance the Mtb sensitivity to anti-TB
drugs
Phenothiazine: RND:Rv1145,Rv1146 • Exhibit killing activity against drug- ☆☆☆ Strong Limited Used clinically, [12,44,
chlorpromazine MFS:Rv1877,Rv2846c sensitive and resistant Mtb but antipsychotic 73–80]
SMR:Rv3065(mmr) • Exhibit synergistic effect with anti-TB side effects need
drugs to be considered
• Enhance the Mtb sensitivity to anti-TB seriously
drugs
Phenothiazine: RND:Rv3160c-Rv3161c • Exhibit killing activity against drug- ☆☆☆ Strong Limited Used clinically, [12,44,47,
thioridazine sensitive and resistant Mtb but antipsychotic 73–80]
side effects need
to be considered
seriously
Capuramycin and Phosphor-N-acetylmuramyl- • Killing MDR and non-MDR Mtb ☆☆ Strong Limited N/A [81–87]
analogues pentapeptide-translocase • Reduced the bacterial load in mouse lungs
(translocase I) • Exhibit synergistic effect with anti-TB
drugs
GEQ compound RND:Rv2942(MmpL7) • Inhibit Mtb growth ☆ Lack Lack N/A [88]
Phe-Arg-β- CmlA(cmlR1),FloR(cmlR2), • Inhibit Mtb resistance to anti-TB drugs ☆ Some Lack N/A [89,90]
naphthylamide levofloxacin-specific RND • Decrease the MIC of anti-TB drugs
(MC-207110, efflux pumps
MC-02595,MC-
04124,BU-005)
Spectinamides MFS:Rv1258c • Exhibit synergistic effect with anti-TB ☆☆ Some Limited N/A [91,92]
drugs
• Decrease of Mtb loads in mouse lung
• Mtb killing in acute tuberculosis infection
SILA 421 mdr-1 • Exhibit synergistic effect with anti-TB ☆☆ Some Limited N/A [93–95]
drugs
• Enhance macrophage ability of Mtb killing
• Inhibit Mtb resistance to anti-TB drugs
Timcodar ABC:DrrAB,PstB,Rv2686c • Exhibit synergistic effect with anti-TB ☆☆ Some Limited N/A [48,96]
-2687c-2688c drugs
MFS:lfrA,Rv1634,Rv1258c • Inhibit Mtb growth
Pgp and/or MDR-associated • Reduce Mtb loads in lung
protein MRP
Plant extracts or Reserpine ABC:Rv2936-Rv2937- • Decrease the MIC of anti-TB drugs ☆☆☆ Strong Limited Used [13,26,
derivatives Rv2938(DrrABC),Rv0933(PstB), • Reduce or reverse the Mtb resistance to clinically, but 49,50,
Rv2686c-Rv2687c-Rv2688c anti-TB drugs antihypertension 92–103]
RND:Rv0678,Rv1145,Rv1146, • Elevate the concentrations of anti-TB drugs side effects need
Rv2942(mmpL7) in Mtb to be considered
MFS:Rv1410c(P55),Rv1877,
Rv2846c
SMR:Rv3065(mmr)
Piperine MFS:Rv1258c • Decrease the MIC of anti-TB drugs ☆☆☆ Strong Some The intake of [109–113]
• Reduce the CFU in mice piperine in daily
• Upregulation of cellular immunity diet favours the
use of the natural
compound
Berberine NorA,RamR • Inhibit the drug resistance of MDR bacteria ☆☆ Some Limited N/A [114–117]
Quercetin SMR:Rv3065(mmr),Isocitrate • Inhibit the Mtb growth ☆ Some Lack N/A [118–120]
lyase
Tetrandrine MFS:Rv2459(jefA),Rv3728 • Decrease the MIC of anti-TB drugs ☆ Some Lack N/A [121,122]
SMR:Rv3065(mmr) • Exhibit synergistic effect with anti-TB
drugs
Farnesol Not determined • Exhibit synergistic effect with anti-TB ☆ Lack Lack N/A [123]
drugs
• Increase the accumulation of anti-TB drugs
in Mtb
Phenylpropanoids RND:Rv1145,Rv1146 • Decrease the MIC of anti-TB drugs ☆☆ Strong Lack N/A [49,124,
MFS:Rv1877,Rv2846c • Increase the accumulation of anti-TB drugs 125]
SMR:Rv3065(mmr) in Mtb
Compound 1 MurE ligase,NorA • Inhibit the growth of Mtb ☆ Lack Lack N/A [126]
 L. Song, X. Wu / International Journal of Antimicrobial Agents 47 (2016) 421–429
Mtb. Furthermore, a series of ABC and MFS family efflux pumps were
shown to play roles in Mtb resistance to INH and RIF, and
overexpression of these efflux pumps was observed in the pres-
ence of CCCP in INH- or RIF-resistant Mtb, indicating the induction
of efflux pump expression by CCCP [47,66]. In addition to the MFS
and ABC families, CCCP also exhibited inhibitory effects on efflux
pumps of the RND family. A study by Milano et al showed that CCCP
decreased resistance to azoles in all BCG strains by inhibiting the
RND family MmpS5–MmpL5 efflux pump [27]. It was also shown
that CCCP inhibited the efflux of acetoxyeugenol acetate and EtBr
in M. smegmatis. Its effect was weaker than VER but stronger than
RES and chlorpromazine [49].
3.1.2.2. 2,4-Dinitrophenol (DNP). DNP acts as an EPI similar to CCCP.
It was shown that DNP reduced the MIC of ofloxacin in ofloxacin-
resistant Mtb clinical isolates, making Mtb more sensitive to
ofloxacin, whilst no effect was observed with ofloxacin-sensitive Mtb.
DNP may produce an effect by inhibiting the MFS and ABC family
efflux pump, and this effect of DNP may be applied to all quinolones
drugs [48].
3.1.2.3. Valinomycin (VLM). VLM is a depsipeptide ion transporter
with high selectivity for potassium [67–70]. Earlier studies showed
that VLM significantly inhibited the efflux of pyrazinoic acid and
led to its retention in pyrazinamide-resistant M. smegmatis or Mtb,
suggesting complete or partial inhibition of proton-motivated com-
ponents in VLM inhibition [71]. Choudhuri et al also found that VLM
caused the retention of INH in M. smegmatis by dissipation of the
proton motive force, suggesting the same mechanism as that re-
ported by Zhang et al [71,72]. Further studies revealed that VLM
enhanced Mtb sensitivity to RIF and novobiocin by inhibiting the
energy source of the P55 efflux pump involving transmembrane
proton and electrochemical gradients. Furthermore, P55 expres-
sion was significantly reduced in the presence of VLM [62,68].
3.1.3. Phenothiazines
Phenothiazine drugs are currently used in clinical treatment of
psychiatric diseases; chlorpromazine and thioridazine are the two
drugs most studied in anti-TB research. Crowle et al first found that
chlorpromazine exhibited an inhibitory effect on Mtb, in which the
intracellular effect was stronger than the extracellular effect [73].
This observation was confirmed by Amaral et al, who further found
that thioridazine exhibited a similar effect as chlorpromazine in
killing drug-resistant Mtb [74]. Bettencourt et al investigated the
killing activity of several phenothiazine drugs against Mtb and ranked
the activity as chlorpromazine = thioridazine > promethazine > pro-
mazine = desipramine, whilst their effective concentrations were all
>1 mg/L, which cannot be reached in clinical practice. However, as
phenothiazine drugs could be concentrated by macrophages, they
may still have the potential to become anti-TB drugs in MDR Mtb
therapy [75]. Studies by Ordway et al confirmed the speculation.
It was found that chlorpromazine and thioridazine can kill both drug-
sensitive and -resistant Mtb in vitro at concentrations below those
present in the plasma of patients treated with these drugs, and they
were not toxic to macrophages nor did they affect in vitro cellular
immune processes [76]. Further studies showed that chlorproma-
zine exhibited a synergistic effect with many anti-TB drugs, including
INH, RIF, STM, pyrazinamide and rifabutin, and enhanced the killing
effect of these drugs on intracellular Mtb [44,74]. This was also con-
firmed in studies by Viveiros and Amaral in which chlorpromazine,
thioridazine and promethazine enhanced the anti-Mtb effect against
pandrug-resistant (PDR) Mtb when combined with RIF or STM at
concentrations that are minimally effective when employed sepa-
rately [77]. Studies by Martins et al also supported that thioridazine
and its derivatives enhanced the killing of Mtb [78]. In addition, chlor-
promazine was also found to enhance the sensitivity to RIF in RIF-
425
resistant Mtb [47]. In summary, phenothiazines (chlorpromazine and
thioridazine) are potential drugs against drug-resistant Mtb, whilst
their antipsychotic side effects need more study if they are to be
used in combination therapy [12,79,80].
3.1.4. Capuramycin and analogues
Capuramycin is an inhibitor of bacterial phospho-N-acetyl-
muramyl-pentapeptide-translocase (translocase I). Its antimicrobial
spectrum is narrow and it only inhibits Streptococcus pneumoniae
and M. smegmatis. Therefore, it has not been studied as an anti-TB
drug but has been used as a prototype molecule in the develop-
ment of new anti-MDR Mtb drugs [81]. A series of capuramycin
derivatives, including RS-118641 [82], SQ997, SQ922, SQ641 [83–85]
and UT-01320 [86], exhibited anti-TB activity against MDR Mtb. RS-
118641 was shown to inhibit MDR and non-MDR Mtb and to reduce
the bacterial load in mouse lungs. It was shown to be effective against
intracellular Mtb and M. avium infection [82]. SQ997, SQ922 and
SQ641 were shown to have potential to kill Mtb much faster than
the current first-line anti-TB drugs. The recovery time after a single
dose of SQ641 treatment was significantly longer than that of the
INH [83]. However, the water solubility for SQ997, SQ922 and SQ641
appeared to be poor and their antibacterial activity was not satis-
factory by oral administration. Nikonenko et al therefore attempted
to dissolve SQ641 in the water-soluble vitamin E analogue tocopheryl
polyethylene glycol 1000 succinate (TPGS) or conjugated SQ641 with
TPGS particles, which greatly improved the efficacy of SQ641 in a
TB mouse model [84]. Bogatcheva et al improved the killing of H37Rv
strain in mouse macrophages by conjugating SQ997, SQ922 and
SQ641 with aminoundecanoic acid or decanoic acid [85]. More re-
cently, another derivative of capuramycin (UT-01320) exhibited a
synergistic anti-TB effect with SQ641. Apart from inhibition of trans-
ferase I, capuramycin was also found to inhibit bacterial RNA
polymerase [86]. In addition, Wang et al modified the capuramycin
molecule using protecting groups from chiral methanol and im-
proved its anti-TB effect [87].
3.1.5. Genz-10850 (GEQ)
GEQ is an inhibitor of the InhA enzyme and its inhibitory effect
on Mtb growth was weak [88]. The N-benzoyl-piperazine central
core is the key structure to ensure good inhibition of InhA enzy-
matic activity. Chollet et al synthesised and evaluated 25 GEQ
analogues by chemical modification of the piperazine ring, the amide
group, the aryl moiety and the fluorene moiety [88]. Compound 33b
with an additional hexyloxy chain on the fluorene moiety dis-
played improved activity both against InhA enzyme and Mtb growth.
These data suggest that GEQ derivatives have the potential to inhibit
Mtb growth after the above chemical modifications [88].
3.1.6. Phe-Arg β-naphthylamide (PAβN)
PAβN (MC-207,110) was discovered to be an EPI for the
levofloxacin-specific RND family. More recent studies revealed that
it was also an inhibitor for MFS family CmlA and FloR efflux pumps.
It inhibited the resistance of Streptomyces coelicolor against chlor-
amphenicol, florfenicol and thiamphenicol [89,90]. BU-005, MC-
02595 and MC-04124 are synthetic derivatives of MC-207,110 with
similar effects [89]. MC-207,110 decreased the MIC of chloram-
phenicol against S. coelicolor 8-fold [90], whilst BU-005 was three
times more potent than MC-207,110 in suppressing S. coelicolor re-
sistance to chloramphenicol [89]. These studies suggest that MC-
207,110 may act as an EPI for the MFS and RND families and is a
potential inhibitor of Mtb.
3.1.7. Spectinamides
An in vitro study showed that spectinamide analogues with a
modified structure exhibited killing activity on MDR and PDR Mtb,
evasion of efflux by Rv1258c, and no cross-resistance with current
426 L. Song, X. Wu / International Journal of Antimicrobial Agents 47 (2016) 421–429
anti-TB drugs. They also showed a significant decrease of Mtb loads
in the lung in a mouse model [91]. The synthesised compound
spectinamide1599 exhibited synergistic effects with clarithromycin,
doxycycline and clindamycin. An in vivo study with a mouse model
proved that the combination of spectinamide1599 with
clarithromycin enhanced Mtb killing in acute TB infection but not
in chronic Mtb infection [92]. The study also found that distinct drug
combinations induced a synergistic effect and drug resistance: in-
appropriate drug exposure may lead to drug resistance, whilst
appropriate combination produced a synergistic effect [92].
3.1.8. SILA-421
In macrophages infected with MDR Mtb H37Rv, SILA-421 alone
can transform macrophages without MDR killing activity into mac-
rophages with MDR killing activity and is free of cell toxicity [93].
In vitro experiments demonstrated that it had a concentration- and
time-dependent antimicrobial activity [94] and its effect was similar
to that of thioridazine [95]. Further studies showed that SILA-421
enhanced the ability of macrophages for Mtb killing by inhibiting
the efflux pump Mdr-1 and was effective for several MDR strains
including H37Rv [95]. SILA-421 can also work synergistically with
INH or RIF to strengthen their activity, inhibit Mtb resistance to INH,
and completely eradicate RIF-resistant Mtb [94].
3.1.9. Timcodar
A study from Grossman et al showed that timcodar exhibited
weak inhibition of Mtb growth when it was used alone in broth
medium, whilst it exhibited a synergistic effect and a ten-fold in-
crease in Mtb inhibition when used in combination with RIF,
moxifloxacin or bedaquiline in macrophage culture [96]. Timcodar
was shown in a mouse model to enhance the effects of RIF and INH,
to reduce Mtb loads in the lung, and to decrease the probability of
chronic infection recurrence when it was used in combination with
RIF or INH [96].
3.2. Plant extracts and derivatives
3.2.1. Reserpine (RES)
RES is an indole alkaloid present in a variety of Rauwolfia
serpentina plants. It is widely used for the treatment of mild and
moderate hypertension with sedation effects [13,97–99]. Earlier
studies showed that RES is an antagonist of the P-gp efflux pump
and can completely reverse the drug efflux in MDR 1R4 bacteria
[100]. RES can also inhibit the Tet(K) efflux pump of meticillin-
resistant S. aureus (MRSA) and decrease the MIC of tetracycline in MRSA
from 128 μg/mL to 32 μg/mL [101]. A study by Viveiros et al showed
that RES reduced Mtb resistance to INH 100-fold, suggesting the pres-
ence of a RES-sensitive efflux pump system(s) [102]. Subsequently,
studies of other authors pointed out that RES can reverse Mtb re-
sistance to INH and EtBr, possibly by inhibiting the MmpL7, IniA and
P27-P55 efflux pumps [26,103,104]. Roy et al further compared the
inhibitory effects of RES, VER, CCCP and chlorpromazine on
M. smegmatis and showed that VER had the strongest inhibitory
effect, followed by CCCP, RES and chlorpromazine [49]. The MIC was
consistent with that from previous reports [105,106]. Similarly, Sun
et al compared the inhibitory and reversal effects of RES, VER and
CCCP on ofloxacin resistance of eight extensively drug resistant (XDR)
Mtb, 40 MDR Mtb and 38 PDR Mtb [107]. Results showed that the
three drugs can decrease the MIC of ofloxacin 2–32-fold, in which
CCCP exhibited the strongest effect by reducing the MIC of 83% of
the samples >2-fold, whilst RES and VER reduced the MIC of 55%
and 74% samples >2-fold, respectively [107]. More recently, Huang
et al showed that RES can elevate the ciprofloxacin concentration
in Mtb and reduce its MIC, whilst it had no effect on some
ciprofloxacin-resistant Mtb containing gyrA gene mutations [108].
Andries et al showed that RES and VER reduce the MIC of bedaquiline
and clofazimine in vitro, whilst VER cannot enhance the anti-Mtb
effect of bedaquiline or reverse Mtb resistance in a mouse model
in vivo [50].
3.2.2. Piperine
Piperine is an alkaloid widely present in the fruits of many pepper
plants. A study found that a dietary dose of piperine exhibited an
anti-TB effect by inhibiting the drug metabolising enzyme and in-
creasing the concentration of plasma P-gp substrates phenytoin and
RIF [109]. Piperine can also decrease the MIC of ciprofloxacin in MRSA
[110]. Studies with RIF-resistant Mtb H37Rv strain and clinical iso-
lates found that piperine can reduce the MIC of RIF by inhibiting
the overexpression of efflux pump Rv1258c [111]. In M. smegmatis,
piperine at 32 μg/mL reduced the MIC of EtBr 2-fold and in-
creased its accumulation in cells, suggesting the involvement of efflux
pump inhibition [112]. More interestingly, recent studies found that
piperine can promote the growth of T-cells and B-cells, increase Th-1
cell factor and enhance macrophage activity. Piperine also induced
the differentiation of Th-1 subtype CD4 + /CD8 + T-cells and in-
creased the secretion of interferon-gamma (INFγ) and interleukin-2
(IL-2) in mice infected with Mtb. Compared with RIF alone, the com-
bination of RIF and piperine further reduced the lung number of
CFU in mice. In addition, upregulation of Th1 cell immunity exhib-
ited a synergistic effect with RIF, which improved the therapeutic
effect of TB in immunosuppressed patients [113].
3.2.3. Berberine
Berberine is a quaternary ammonium salt from the
protoberberine group of isoquinoline alkaloids and exists widely in
Berberis plants. Early studies on ciprofloxacin-resistant S. aureus
showed that berberine inhibited the multidrug resistance of S. aureus
by inhibiting the NorA efflux pump [114]. Subsequent studies found
that the Berberis compound 5’-methoxyhydnocarpin also inhib-
ited the drug resistance of S. aureus by inhibiting the NorA efflux
pump. It exhibited no antimicrobial activity when used alone, but
enhanced the effect of berberine when the two compounds were
combined [115]. A recent crystallographic study also confirmed that
berberine can inhibit the drug resistance of MDR bacteria by binding
to NorA and RamR efflux pumps [116,117]. Although there is no
direct evidence that berberine has an inhibitory effect on Mtb, it
can be speculated that berberine may also inhibit the growth of MDR
Mtb, as the abovementioned efflux pumps are also expressed in MDR
Mtb.
3.2.4. Quercetin
Quercetin belongs to the flavonoids and is present in many veg-
etables, fruits, leaves and grains. Latest research indicated that
quercetin exhibited an anti-TB effect. The molecular model of protein
binding sites from Suriyanarayanan et al suggested that quercetin
exhibited stable binding with Mtb Mmr and E. coli EmrE efflux
pumps, and the molecular interaction between quercetin and the
pumps appeared to be more stable than that with VER, RES and
chlorpromazine, suggesting that quercetin may reduce efflux and
become a non-antibiotic drug in TB adjuvant treatment [118]. Dey
et al further demonstrated that quercetin has antimicrobial effects
on a series of Mtb and Klebsiella pneumoniae producing β-lactamase
[119], and Shukla et al also showed that quercetin inhibited the utili-
sation of acetate by inhibiting the Mtb H37Rv isocitrate lyase, and
in turn, inhibited the glyoxylate shunt [120].
3.2.5. Tetrandrine
Tetrandrine is a type of natural substance purified from the roots
of plants Fourstamen stephania. It showed a similar effect to the
calcium channel blocker VER [121]. Tetrandrine decreased the MIC
both of INH and EMB in INH and EMB dual-resistant Mtb with an
effective rate of 82%. Therefore, combined use of tetrandrine with
 L. Song, X. Wu / International Journal of Antimicrobial Agents 47 (2016) 421–429
INH or EMB may improve the anti-TB effect and help to reduce the
drug dose and side effects [122].
3.2.6. Farnesol
Farnesol is a widely used spice that exists naturally in essential
oils such as lemon grass oil and citronella oil. Studies have shown
that farnesol significantly increased the accumulation of EtBr in
M. smegmatis, decreased the MIC of EtBr up to 8-fold and exhib-
ited a synergistic effect with RIF. These results suggest that farnesol
achieved its anti-TB effect through inhibition of Mtb efflux pumps
[123].
3.2.7. Phenylpropanoids
Phenylpropanoids have been suggested to be strong EPIs. Study
found that namely 1′-S-1′-acetoxychavicol acetate, 1′-S-1′-
acetoxyeugenol acetate and trans-p-coumaryl diacetate can reduce
the MIC of EtBr and increase the accumulation of EtBr in
M. smegmatis [49]. Chen et al also found that 2,3-diacetoxy-1-
methoxy-5-allylbenzene and 3-acetoxy-4-hydroxy-1-allylbenzene
exhibited an anti-TB effect through the inhibition of H37Rv efflux
pumps [124,127]. Phenylpropanoids may become anti-TB drugs for
MDR Mtb [125].
3.2.8. Compound 1
Compound 1 is a compound extracted from Hypericum olympicum
L. cf. uniflorum. A study found that it can inhibit the growth of
M. bovis BCG by inhibiting the ATP-dependent MurE ligase. It can
also act on the S. aureus NorA efflux pump to suppress drug efflux.
These findings support the potential use of Compound 1 as an EPI
in anti-TB therapy [126].
3.3. Clinical impact of potential antituberculosis drugs
We systematically evaluated the clinical impact of each drug re-
viewed in this article and provided a rating for them, as shown in
Table 1. The rating is based on currently available evidence includ-
ing in vitro and in vivo studies. A five-star system was developed
to perform the assessment, with more stars representing a stron-
ger clinical impact.
VER exhibited a very strong clinical impact, as both in vitro and
in vivo experiments showed positive anti-TB effects, and the FDA
also approved it as an EPI for antimicrobial therapy. Its in vivo anti-
TB effects have been confirmed in animal models, including studies
in larval zebrafish [54] and mouse models [52,53]. VER also reduced
macrophage-induced tolerance to numerous anti-TB compounds,
including the newly FDA-approved bedaquiline and moxifloxacin
[53]. Combined use of VER may shorten the therapy periods for
bedaquiline and clofazimine and may lower the bedaquiline doses
and reduce the toxicities in treatment [52]. Therefore, VER appears
to be an effective anti-TB drug with acceptable safety; however, its
side effects in future TB therapy are still a critical aspect requiring
investigation.
Apart from VER, some other drugs in Table 1 also exhibited po-
tential in anti-TB therapy. However, in vivo evidence from animal
studies are limited for most of them, although some of them showed
very promising in vitro effects, such as CCCP, phenothiazines, RES
and piperine (those with three stars). Further investigations are
needed for these drugs on their in vivo effects and side effects.
4. Conclusions
In this article, current development of anti-TB EPIs has been sys-
tematically reviewed. Most drugs exhibited enhancement of the first-
line anti-TB drugs and inhibitory or reversal effects on drug
resistance; however, most of them are still under investigation at
basic research stage, whilst only a few have demonstrated prom-
427
ising animal experiment data. Although bedaquiline and VER are
approved by the FDA as EPIs, their application as anti-TB drugs still
needs further investigation. The dosage in combined therapy with
the existing first-line anti-TB drugs needs to be determined and the
exact combination and drug side effects remain to be clarified. Drugs
currently used in clinical therapy for other diseases, such as VER,
phenothiazines and RES, have a promising future in anti-TB therapy
owing to their definite properties regarding safety. However, their
side effects could be an issue in future combination therapy. Natural
substances of plant origin, such as piperine and berberine, also have
great potential as they are part of the diet. Future research should
focus on new drugs and regimens of anti-TB therapy with com-
bined use of multiple drugs.
Funding: This study was supported by the Serious Infectious Dis-
eases Special Foundation of China [grant 2012ZX10003008002].
Competing interests: None declared.
Ethical approval: Not required.
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