Received: 28 November 2018 | Revised: 31 March 2019 | Accepted: 2 April 2019

DOI: 10.1002/med.21591

REVIEW ARTICLE

Efflux pump inhibitors of clinically relevant

multidrug resistant bacteria

Andraž Lamut | Lucija Peterlin Mašič | Danijel Kikelj |

Tihomir Tomašič

Chair of Pharmaceutical Chemistry, Faculty of

Pharmacy, University of Ljubljana, Ljubljana,
Slovenia
Correspondence
Tihomir Tomašič, Faculty of Pharmacy,
University of Ljubljana, Aškerčeva Cesta 7,
1000 Ljubljana, Slovenia.
Email: tihomir.tomasic@ffa.uni-lj.si
Funding information
Javna Agencija za Raziskovalno Dejavnost RS,
Grant/Award Numbers: Program Number:
0787‐P10208, 0787‐P10208
Abstract
Bacterial infections are an increasingly serious issue world-
wide. The inability of existing therapies to treat multidrug‐
resistant pathogens has been recognized as an important
challenge of the 21st century. Efflux pumps are important in
both intrinsic and acquired bacterial resistance and identi-
fication of small molecule efflux pump inhibitors (EPIs),
capable of restoring the effectiveness of available antibio-
tics, is an active research field. In the last two decades, much
effort has been made to identify novel EPIs. However, none
of them has so far been approved for therapeutic use.
In this article, we explore different structural families of
currently known EPIs for multidrug resistance efflux
systems in the most extensively studied pathogens (NorA
in Staphylococcus aureus, AcrAB‐TolC in Escherichia coli, and
MexAB‐OprM in Pseudomonas aeruginosa). Both synthetic
and natural compounds are described, with structure‐
activity relationship studies and optimization processes
presented systematically for each family individually. In
vitro activities against selected test strains are presented in
a unifying manner for all the EPIs described, together
with the most important toxicity, pharmacokinetic and in
vivo efficacy data. A critical evaluation of lead‐likeness

Abbreviations: AcrAB‐TolC, the major tripartite efflux pump system of E. coli; CFU, colony forming units; EPI, efflux pump inhibitor; EtBr, ethidium
bromide; FIC, fractional inhibitory concentration; HSA, human serum albumin; LBDD, ligand‐based drug design; MDR, multidrug resistance;
MES, multidrug efflux system; MexAB‐OprM, the major tripartite efflux pump system of P. aeruginosa; MRSA, methicillin‐resistant Staphylococcus aureus;
NorA, the major efflux pump of S. aureus; NorA ++, the S. aureus bacterial strain that overexpresses NorA; SAR, structure‐activity relationship.

Med Res Rev. 2019;1–45. wileyonlinelibrary.com/journal/med © 2019 Wiley Periodicals, Inc. | 1

2 | LAMUT ET AL.

characteristics and the potential for clinical development of

the most promising inhibitors of the three efflux systems is
described. This overview of EPIs is a good starting point for
the identification of novel effective antibacterial drugs.

KEYWORDS
AcrAB‐TolC, bacterial resistance, efflux pump inhibitor,
MexAB‐OprM, multidrug efflux systems, NorA, structure‐activity
relationship

1 | INTRODUCTION

“Any one of us could go into hospital in 20 years for minor surgery and die because of an ordinary infection that
can't be treated by antibiotics.” These are the words of the Chief Medical Officer in England, that reflects the
seriousness of the problem concerning bacterial resistance to antibiotics.1 Over the past few decades, large
numbers of articles have been published in the field of bacterial resistance development in various hospitals around
the world and this problem has come to the point where it has been recognized as an important challenge of the
21st century.2
In February 2017, the World Health Organization (WHO) revealed a list of antibiotic‐resistant “priority
pathogens”—the 12 bacterial families most threatening for humans that have acquired and developed various
resistance mechanisms and become multidrug resistant (MDR).3 Among them, Pseudomonas aeruginosa,
Acinetobacter baumannii, Enterobacteriaceae family (including Escherichia coli and Salmonellae), Enterococcus faecium,
Staphylococcus aureus, Helicobacter pylori, Campylobacter spp., and Neisseria gonorrhoeae constitute the leading cause
of life‐threatening nosocomial infections throughout the world and were, therefore, recognized as critical and high
priority pathogens.3–5
Only a few new chemical classes of antibiotics with new mechanisms of action have entered the clinic in the
past two decades, for example, oxazolidinones and lipopeptides that target only Gram‐positive bacteria.6 This is
due mainly to the decrease, for economic reasons, in discovery research in large pharmaceutical companies.
Namely, antibiotic regimens are typically of very limited duration, which makes them far less profitable than drugs
prescribed for chronic diseases, such as high blood pressure and hypercholesterolemia. In addition, newly approved
antibiotics are usually held in reserve and used only when currently available drugs are not sufficiently effective to
treat the infection, thus limiting initial investment return. Finally, strict regulatory requirements regarding adverse
side effects and many other hurdles in clinical trials have discouraged pharmaceutical companies from investing in
this area of research.6
Bacteria can be intrinsically resistant to certain antibiotics as a consequence of inherent structural or functional
characteristics, such as poor permeability of the bacterial cell wall and constitutive expression of efflux pumps that
are encoded by house‐keeping genes in the bacterial genome.7 On the contrary, bacteria can develop or acquire
resistance to antibiotics that is contributed to by several mechanisms, including plasmids coding multidrug
resistance genes, degradation or modification of the drug itself, alteration or protection of the drug target, and
minimization of drug concentration in intracellular compartments as a result of poor penetration into the bacterium
or of active efflux mechanisms.8 Among these, overexpression of efflux pumps is one of the most widely studied
resistance responses in bacteria, which itself can lead to clinically relevant resistance. In addition, it leads to
subinhibitory drug concentrations at the active site, thus contributing to the development of target‐based
resistance at a high‐level via mutations in drug target genes. The latter is especially difficult to handle as it results in
inefficiency of a whole class of existing antimicrobial agents that display their activity through this mode of action.9
LAMUT ET AL. | 3

Efflux‐mediated bacterial resistance involves efflux systems that expel antimicrobial agents from the cell
once they pass the membrane or enter the cytoplasm. They are capable of extruding a single class of
antibiotics (eg, the TetK determinants that convey resistance to tetracyclines and MsrA that exports only
certain macrolides) or multiple unrelated antibiotic classes.10,11 Efflux pumps can be expressed in different
forms in both Gram‐positive and Gram‐negative bacteria and various pumps can be present with different or
overlapping substrates.12,13
Multidrug efflux systems (MESs) are classified in six families, according to their structure (amino acid sequence
homology) and energy requirements. These are (i) the major facilitator superfamily (MFS), (ii) the small multidrug
resistance family (SMR), (iii) the multidrug and toxic compound extrusion family (MATE), (iv) the resistance‐
nodulation‐cell division superfamily (RND), (v) the adenosine‐triphosphate (ATP)‐binding cassette superfamily
(ABC), and (vi) the proteobacterial antimicrobial compound efflux family (PACE).14–20
Families (i) to (iv) are secondary transporters that use the proton gradient to drive the extrusion of
substrates by a proton/substrate antiport mechanism, except the MATE family that can also use Na+
membrane transport as a source of energy. On the contrary, ABC superfamily transporters are primary
transporters that use ATP for substrate extrusion. 21 The PACE family has only recently been identified in
some Gram‐negative bacteria. Its molecular mechanism of transport and mode of energization has, therefore,
not yet been established, but probably involves an electrochemical gradient.19 The first five families are
widely distributed in both Gram‐positive and Gram‐negative bacteria, except the RND family, which exists
only in Gram‐negatives. RND transporters are present in both prokaryotes and eukaryotes, but there is very
low homology between human and bacterial RND proteins, that share only 16% amino acid sequence identity,
so that overlap in substrate specificity is minimal.22 Since MFS, SMR, and RND pumps are all driven by the
proton motive force, they are also dependent on the pH gradient. For instance, it has been shown that E. coli
can expel ethidium bromide (EtBr) less efficiently at higher than at lower pH values. 23 The MES in bacteria can
consist of single gene products, such as NorA of S. aureus, or the multicomponent envelope translocases, such
as AcrAB‐TolC of E. coli and MexAB‐OprM of P. aeruginosa, that facilitate drug efflux across the outer
membrane of Gram‐negative bacteria.24,25 The intriguing efflux transport mechanism of the tripartite RND
efflux assemblies in Gram‐negative bacteria most probably operates via a peristaltic mode through individual
protomers of the trimeric RND component, as a consequence of consecutive functional cycling of these
protomers through three different states (loose, tight, and open), that correspond directly to substrate access,
binding and extrusion, respectively.26 Structural studies indicate that these tripartite efflux transporters can
capture their substrates directly from the periplasm or from the outer leaflet of the cytoplasmic membrane.27
Many efflux pumps have been characterized in hospital‐ and community‐acquired pathogens since their initial
discovery in the 1980s.28 A list of major efflux pumps of some critical and high priority pathogens is presented in
Table 1.12
Efflux pumps are crucial for bacterial survival in today's era of antibiotics. As a consequence, they are attractive
novel drug targets and identification of efflux pumps inhibitors (EPIs) is a promising and valid strategy for fighting
resistance to antibiotics.44,45 EPIs have been studied since the discovery, in 1976, of P‐glycoprotein, the first known
efflux transporter.46 There are many meaningful reasons for pursuing this area of research further. First, EPIs
restore susceptibility to antibacterials and coadministration of an EPI with an antibacterial compound could reduce
the degree of resistance to antibiotics in bacteria.22 Second, EPIs, in combination with antibiotics, increase the
latter's potency, could expand the spectrum of antibacterials and, further, reduce the frequency of emergence of
the target‐based resistance.47,48 Small molecule EPIs could prevent the emergence of new mutant strains and thus
weaken acquired and intrinsic resistance.49 Third, EPIs reduce the formation of biofilms and block the antibacterial
tolerance of biofilms.50 Fourth, plants produce both antibacterial compounds and EPIs that improve their
antibacterial activity, for example, antibiotic berberine and EPI 5′‐methoxyhydnocarpin (5′‐MHC) in Berberis
vulgaris, antibiotic α‐linolenic acid and isoflavone‐based EPIs in Lupinus argenteus, and antibiotic dalversinol A and
EPI methoxychalcone in Dalea versicolor, although EPI may not be a primary functional role of some plant natural
4 | LAMUT ET AL.

T A B L E 1 Efflux pumps in Gram‐positive and Gram‐negative bacteria

Pathogen MFS SMR MATE ABC RND PACE
Gram‐positive
Staphylococcus aureus11,12,29,30 NorA QacC MepA MsrA FarE
NorB QacD Sav1866
NorC Ebr AbcA
MdeA QacG VgaA
SdrM QacH VgaB
LmrS QacJ
QacA
QacB
Enterococcus faecium31–33 Tet(K) MsrC
Tet(L) EfrAB
EfmA
Gram‐negative
Acinetobacter baumanii34–38 Tet(A) AbeS AbeM AdeABC AceI
Tet(B) AdeDE
CmlA AdeFGH
MdfA AdeIJK
CraA AdeXYZ
AmvA
Helicobacter pylori13,39 MsbA HefABC
HefDEF
HefGHI
CznABC
Neisseria gonnorrhoeae13,40 Mef NorM MacAB MtrCDE
FarAB‐MtrE
Escherichia MdfA EmrE NorM MacAB‐TolC AcrAB‐TolC
coli13,41,42 EmrAB‐TolC SugE AcrAD‐TolC
EmrKY‐TolC TehAB YojI AcrEF‐TolC
EmrD MdtJI MdtABC‐TolC
Dep MdtEF‐TolC
Bcr OqxAB‐TolC
QepA YhiUV‐TolC
Mef(B)
Pseudomonas aeruginosa41,43 EmrE PmpM MexAB‐OprM
MexCD‐OprJ
MexEF‐OprN
MeXY‐OprM
MexJK‐OprM
MexGHI‐OpmD
CzrCBA
Abbreviations: ABC, adenosine‐triphosphate (ATP)‐binding cassette superfamily; MATE, multidrug and toxic
compound extrusion family; MFS, major facilitator superfamily; PACE, proteobacterial antimicrobial compound efflux
family; RND, resistancenodulation‐cell division superfamily; SMR, small multidrugresistance family.

products.51–53 In addition, EPIs may also have an impact on quorum sensing and virulence.54 Recent studies have
highlighted the importance of efflux pumps in the formation of bacterial persisters.55,56 A significantly higher
expression of multidrug efflux‐associated genes was found in persisters and their formation was largely reduced in
efflux knockout strains.55 Thus, EPIs could reduce the number of persisters and work effectively in combination
with antibiotics to treat chronic infectious diseases.55 Efflux pumps distribute asymmetrically in a growing
population of bacteria, which contributes to the heterogeneity in antibiotic resistance.57 Moreover, an increased
expression and activity of AcrAB‐TolC efflux pump is associated with a substantial reduction in expression of
bacterial DNA mismatch repair proteins, such as MutS, and higher spontaneous mutation frequency.58 Hence, EPIs
LAMUT ET AL. | 5

could also prevent the initial stages of permanent bacterial resistance development by preventing the acquisition of
drug‐resistance mutations.58
Many EPIs capable of potentiating the activity of antimicrobials have been identified in recent years.
Among the first were antihypertensive drugs reserpine and verapamil, although they were too weak EPIs to be
clinically relevant.59 Since then, a lot of effort has been made to identify novel EPIs, especially by
phytochemists, who have identified a series of structurally diverse natural compounds that have served as
starting points in the design of more potent and less toxic lead compounds. The most promising and studied
EPIs reported to date are presented in Figure 1. These are peptidomimetic PAβN (1), pyridopyrimidinone D13‐
9001 (2), pyranopyridine MBX2319 (3), representatives of the quinoline (4), and of the indole (5) derivatives
(Figure 1). However, only a few EPIs have reached clinical trials, where they have failed due mostly to toxic
effects at the concentrations essential for their activity, poor pharmacokinetic properties or low in vivo
efficacy.20,60–62 To the best of our knowledge, only for one EPI that has made its way into clinical
development, the structure is revealed. This is a bis(pyrimidine)sulfonamide MP‐601,205 (6; Figure 1) that was
tested in the form of aerosol formulation in combination with the approved antibiotic ciprofloxacin for the
treatment of respiratory infections in patients with cystic fibrosis and ventilator‐associated pneumonia,
caused by MDR Gram‐negative bacteria, such as P. aeruginosa.63,64 It was tested in phase I clinical trials, but
unfortunately its development was discontinued because of tolerability issues.65,66 A specific mode of action
for this compound has not yet been disclosed.
Nevertheless, extensive work in this field in the last few decades has resulted in the generation of a lot of
interesting information regarding the structure‐activity relationship (SAR) in different structural classes of EPIs
targeting NorA and RND efflux pumps. Moreover, the known crystal structures of individual subunits of the
tripartite efflux systems of E. coli AcrAB‐TolC and P. aeruginosa MexAB‐OprM, as well as the structures of their
whole tripartite assemblies, offer an opportunity for the further exploration and design of novel EPIs according to
available structural information.26
Basically, EPIs can bind to the same site as substrates and function as competitive inhibitors, impairing the
proper binding of substrates by a noncompetitive mechanism or act by hindering the functional movement of efflux
pumps.67–69 Moreover, EPIs can also act as cosubstrates for the efflux pump and are (preferably) coextruded from
the bacterial cell. Thus, when administered together with an antibiotic, they would prevent or at least lower the

F I G U R E 1 Structures of representative promising efflux pump inhibitors (EPIs) (1‐5) belonging to different
structural families and a clinical candidate (6)
6 | LAMUT ET AL.

rate of antibiotic extrusion, which would, consequentially, lead to a sufficiently high intracellular concentration of
the drug, which is the basis of effective treatment of bacterial infections (Figure 2).
It has been established that an ideal EPI active against Gram‐negative bacteria should meet the following
criteria. It should (i) overcome the outer membrane barrier, (ii) enhance the activity of antibiotics that are efflux
pump substrates, (iii) show no activity in mutants lacking the respective efflux pumps, (iv) decrease extrusion and
increase accumulation of efflux pump substrates, and (v) not affect the proton gradient across the cytoplasmic
membrane.71
Increasing numbers of review articles during the past few years speak clearly about the increasing interest
in this field of research.1,20,72–75 The present article is focused on the currently known EPIs and explores,
where possible, the SAR of each individual family of EPIs in the most typical and extensively studied WHO
priority pathogens (S. aureus, E. coli, and P. aeruginosa), together with their corresponding MDR efflux systems NorA,
AcrAB‐TolC, and MexAB‐OprM. The most significant achievements are here summarized and knowledge
concerning recent advances in the research on EPIs upgraded. The structural features and activities of the most
active analogs are presented for each family, with emphasis on the structures interesting for drug development.
Small synthetic molecules, as well as natural compounds, are described, together with critical evaluation of their
lead‐likeness characteristics and capability to be progressed into clinical development.

2 | P H YS I O L O G I C A L R O L E S O F E F F L U X P U M P S

There has been some speculation about the physiological role of efflux pumps in bacteria. The in vivo role of efflux
pumps is believed to be very complex, since they play an important role in bacterial physiology, metabolism, and
pathogenicity.76 Bacteria may use efflux pumps to eliminate toxic endogenous metabolites, for the secretion of
virulence determinants, to participate in the cell stress response and in biofilm formation. Moreover, they can be
responsible for removing toxins that are encountered in the environment (heavy metals, biocides) and can serve as
protective tools that enable bacteria to survive the colonization process during infection in a host, when it is being
attacked by noxious agents.27,77 It has been reported that RND type MES play a role in adherence, invasion, and

F I G U R E 2 A schematic presentation of efflux pump inhibitory activity of EPIs. MFS transporters in

Gram‐positive bacteria (A) and RND transporters in Gram‐negative bacteria (B) use the proton gradient to drive
the extrusion of an antibiotic by a proton/drug antiport mechanism. Blocking of antibiotic efflux by the action of an
EPI increases antibiotic concentration in the cytosol of resistant bacteria, thus increasing its binding to the
target and restoring its effectiveness.22,68,70 EPI, efflux pump inhibitor; MFS, major facilitator superfamily;
RND, resistance‐nodulation‐cell division superfamily [Color figure can be viewed at wileyonlinelibrary.com]
LAMUT ET AL. | 7

colonization of the host cell.78,79 Hence, EPIs may also reduce bacterial virulence in vivo, as was proven lately in an
insect model of infection,54 and act as antivirulence determinants that lower the pathogenic potential of
microorganisms.75,80 An interesting example of how efflux pumps serve to extrude physiological compounds made
by the cells themselves is that of the N‐acylhomoserine lactones, which have been shown, in P. aeruginosa, to diffuse
through media to other cells of the population and activate many processes, thereby serving as quorum‐sensing
signals.81,82

3 | METHODS FOR BIOLOGICAL EVALUATION OF EFFLUX PUMP

I N H IB I T O R S

Different methods, listed below, can be used for evaluation of EPI. Among them the most widely used method for
determining the specific mode of action (ie, EPI), as well as for target validation, is measurement of the fluorescence
of different fluorescent molecules.83 An example of the latter is EtBr, which is often used as a model substance,
since active efflux is the only known mechanism of bacterial resistance to this compound. Usually, cells are loaded
with EtBr in the presence of an inhibitor and are placed in a fluorimeter cuvette with fresh medium. EtBr emits
fluorescence only when inside the cell, where it intercalates into DNA. A substantial decrease in fluorescence is
detected when no inhibitor is present, since the EtBr is effluxed from the cells. An increase in fluorescence after
addition of EPI is an indicator of EPI.84,85 Other examples of fluorescent molecules used for determination of EPI
are the hydrophobic dye Hoechst H33342, which emits intense fluorescence inside the cell upon binding to DNA,86
1,2′‐dinaphthylamine, which becomes fluorescent when partitioned into a phospholipid bilayer, and others, such as
1‐anilinonaphthalene‐8‐sulfonate and acriflavine.83 Efflux inhibition can also be studied at the level of a single cell
by direct evaluation of bacterial efflux activity.87–90 In this case, individual bacterial cells are isolated in a femtoliter
droplet and analyzed by the fluorescence measurement. Fluorescein‐di‐β‐D‐galactopyranoside is used for this
purpose, being hydrolyzed by β‐galactosidase in the cytoplasm of a bacterial cell to produce fluorescein.88
The general method for testing the efficacy of EPIs is measurement of the growth inhibition in different
bacterial strains by selected antibiotics in the presence and absence of EPI. Potential inhibitors are first inspected
for their intrinsic antibacterial activity to prove that reduction in MIC of an antibiotic is not a consequence of other
mechanisms of antibacterial activity. In these assays, wild‐type, efflux‐knockout, and efflux‐proficient strains are
often used. A greater synergistic effect in the efflux‐proficient strain compared to the wild‐type strain and lack of
growth inhibition in the efflux‐knockout strain is an indicator of EPI. In addition, different efflux pump
overexpresser strains are used to evaluate the role of the specific efflux pump in the inhibitory activity of EPIs.91
A typically used Gram‐positive bacterial strain in efflux inhibition tests is the well characterized S. aureus
SA1199B strain that overexpresses NorA. The IC50 values of NorA inhibition, measured using the EtBr efflux assay
in SA1199B, are reported as 10 µM for positive control compounds like reserpine, thioridazine, and
prochlorperazine, and 25 µM for paroxetine.92–94 On the contrary, the most widely used Gram‐negative strains
are AcrAB‐TolC overexpressers Enterobacter aerogenes EA‐27 and CM‐64, as well as MexAB‐OprM overexpressers
P. aeruginosa PAM1032 and PAM1723. A broad spectrum EPI phenylalanine‐arginine β‐naphthylamide (PAβN, 1;
Figure 1) is routinely used as a positive control in investigating various P. aeruginosa and E. coli efflux systems. It has
been used as a chemical probe to study antimicrobial resistance and in the evaluation of novel identified EPIs from
both synthetic and natural origins.1
The most frequently used antibiotics in Gram‐positive bacterial assays are ciprofloxacin and, in many cases,
berberine. Berberine itself is a plant secondary metabolite and a phytoalexin (antimicrobial and/or antioxidative
substance synthesized de novo by plants) produced in response to stressors, such as the invasion of microbes.95,96
Berberine alkaloids are amphiphilic cations and well‐known substrates for MDR transporters.97 Antibiotic
compounds used in Gram‐negative assays are chloramphenicol, which is a substrate for the AcrAB‐TolC efflux
system, and levofloxacin, which is expelled by MexAB‐OprM, MexEF‐OprN, MexCD‐OprJ, and MexXY‐OprM.83 It
8 | LAMUT ET AL.

has been reported that chloramphenicol also acts as an inducer of the MDR in E. coli and E. aerogenes by inducing
the overexpression of AcrAB‐TolC.98
The fractional inhibitory concentration (FIC) index is often used to describe the degree of synergy between two
antibacterial compounds, reflecting the efficacy of the EPIs used. For two interacting compounds A and B, the FIC
index is defined as99,100

MIC of A in combination with B MIC of B in combination with A

FIC index = +
MIC of A alone MIC of B alone

FIC index values between 0.5 and 1 indicate additivity between the compounds, while synergy is defined as an
FIC index below 0.5; the lower the value the higher is the degree of synergy.100,101 However, results are usually
expressed either as n‐fold potentiation of antibiotic activity or as minimum inhibitory concentration (MIC)
reduction at specific concentrations of both the antibiotic and the EPI. Minimum potentiation concentration (MPC)
is conveniently used for this purpose and is defined as the lowest concentration of an EPI that causes n‐fold
reduction in MIC of an antibiotic (expressed as MPCn for n‐fold reduction).102,103

4 | SE LEC TED C LINICA LL Y REL EVA NT PATH OGENS A ND T HEIR EFF LU X

PU MPS

4.1 | Staphylococcus aureus

S. aureus is a strain of Gram‐positive cocci and one of the major pathogenic bacteria causing many life‐threatening
infections. It exhibits a diversity of resistance mechanisms against antimicrobial agents. Methicillin‐resistant
S. aureus (MRSA) strains in particular may cause severe and, often, fatal infections,104–106 making them among the
most frequent nosocomial pathogens in developed countries.107 To date, 14 multidrug efflux pumps have been
described for S. aureus, encoded either in the chromosomes or in plasmids.11 The most extensively studied efflux
pumps in S. aureus are those belonging to the MFS family, with NorA as a representative example (Table 1).29,30
MRSA isolates frequently overexpress NorA and NorB. In addition, MepA is often overexpressed in S. aureus clinical
isolates and NorC, NorD, FarE, and AbcA are recognized as being important in S. aureus pathogenicity.11,12
NorA is a single polypeptide chain, 388 amino acid protein that comprises 12 transmembrane (TM) segments
(α‐helices) with C‐ and N‐terminal domains arranged in a pseudo‐twofold symmetry. Both C‐ and N‐terminal
domains consist of 6 TM α‐helices connected by a long hydrophilic cytoplasmic loop. According to the homology
model built by Bhaskar et al,108 a large hydrophobic binding cleft is accompanied by eight TM helices and composed
of nonpolar amino acid residues evolutionarily conserved among MFS transporters. NorA exhibits 44% amino acid
sequence identity with Bmr MDR efflux pump from Bacillus subtilis and 24% sequence identity with the tetracycline
efflux pump Tet(A) from E. coli. It is a chromosomally encoded efflux system and increased resistance to
fluoroquinolones has been associated with increased expression of the norA gene.109 NorA can extrude a diverse
range of structurally and chemically dissimilar compounds, such as fluoroquinolones (ciprofloxacin and norfloxacin),
puromycin, pentamidine, quaternary ammonium compounds, and antiseptics (berberine, tetraphenylphosphonium,
benzalkonium chloride, and cetrimide), phenothiazines, thioxanthenes, verapamil, omeprazole, and terpenoids
(totarol, ferruginol, salvin, and carnosic acid), reserpine and dyes (such as EtBr, rhodamine, acridines, and
biocides).29,92,110–117 NorA is embedded in the membranes of prokaryotes. To date, its crystal structure has not
been resolved and its substrate binding pocket not defined.108 It is suggested, however, that the NorA substrate
binding site is large and hydrophobic, which could explain the broad substrate specificity. Such characteristics of a
binding region do not favor the establishment of a precise network of hydrogen bonds but, rather, allow substrates
to associate with NorA through a combination of electrostatic attraction and hydrophobicity.118 The NorA efflux
pump has been recognized as a potential target for the development of EPIs.108 However, the mechanism of its
LAMUT ET AL. | 9

ability to recognize a wide range of structurally dissimilar compounds remains unknown. As a consequence, there
can be no structure‐based approach to the design of inhibitors of this transporter.84 Several EPIs have been
reported to target NorA.119–123 However, these compounds have not entered the clinic because of the unwanted
side effects observed, especially following long‐term administration. The large number of known inhibitors and
their broad structural diversity reflect the low substrate specificity of NorA, similar to that shown for the
mammalian multidrug transporter P‐glycoprotein.124

4.2 | Escherichia coli

E. coli is Gram‐negative bacterium and a well‐recognized human pathogen. Though only some serotypes are
pathogenic, it is the most common causative pathogen of urinary tract infections worldwide. A serious emergence
of food‐ and water‐borne infections (such as that with O157:H7) and enterohaemorrhagic E. coli (O104:H4), as well
as the worldwide pandemic clone O25:H4ST131 with multidrug‐resistance phenotype virulence genes, constitute a
serious threat to public health.125–127
E. coli possesses a large number of efflux pumps belonging to various families,13,41,42 some of which are
presented in Table 1. Seven MDR efflux systems have been identified in biological studies, and many others,
identified by comparative amino acid sequence analysis.24,25 A well‐studied example and the major efflux pump of
E. coli is the tripartite AcrAB‐TolC complex, which belongs to the RND superfamily. It is the predominant MES for
antibiotic extrusion and presumably the best characterized E. coli efflux pump.13 This pump assembly comprises the
trimeric outer‐membrane channel (TolC), the trimeric secondary transporter located in the inner membrane (AcrB),
and the hexameric periplasmic protein adaptor (AcrA), that bridges these two integral membrane proteins. As
proposed by Du et al,128 there is no direct interaction between AcrB and TolC, but AcrA interacts with both of
them independently.
AcrAB‐TolC presents a broad substrate specificity (polyselectivity) and is able to extrude numerous compounds
including antibiotics (eg, chloramphenicol and fluoroquinolones), dyes (acriflavin and dinaphthylamine), detergents
(sodium dodecyl sulfate and Triton), biocides and a wide variety of natural compounds that exhibit major
differences in their structure and activity.129–131 Antibiotics enter the periplasm by diffusion through the lipid
bilayer or through a porin channel, and interact with the substrate‐binding pocket of AcrB. The AcrB transporter
then uses the proton gradient to extrude the compound into the TolC channel and then out of the cell.132
The available crystal structures for the individual subunits of the efflux pump have provided an insight into
AcrAB‐TolC pumping mechanism.133–136 However, details of their interactions and stoichiometry, as well as how
they are organized within the pump remained a matter of debate until the first pseudoatomic structure of tripartite
efflux complex was published128 and the overall shape of the pump, as well as the relative arrangements of its
components, revealed by electron microscopy studies.26,70,137 Just recently, the near‐atomic resolution cryoEM
structures of the full AcrAB‐TolC assembly in both resting and drug transport states, with associated tertiary and
quaternary structure changes, were reported in detail, providing a better understanding of the function of this
efflux pump machinery.138 It has been suggested that antibiotics enter the AcrB substrate binding site either at the
level of the outer layer of the inner membrane or of the periplasmic space.68,139 Inner membrane entry is assumed
for hydrophobic drugs with relatively low molecular weights, whereas periplasmic entry could be preferred for
taking up hydrophilic drugs with relatively high molecular weights from the periplasm (ie, acting as a periport).
These channels then merge at the proximal pocket, which is separated from the subsequent distal binding pocket
by a flexible G‐loop. Reduction of the volume of the proximal pocket results in the moving of compounds toward
the distal pocket, resembling a peristaltic motion. Finally, compounds are squeezed out of the cell through the TolC
channel as a consequence of conformational changes of the pump.68
In 2013, the first X‐ray structure of AcrB in complex with the inhibitor D13‐9001 (2; Figure 1) was published.140
This makes AcrB a good model for studying compounds with EPI properties in silico.69,141–143 The AcrB substrate
binding pocket is flexible, large and rich in phenylalanine residues. These residues define the binding pocket and
10 | LAMUT ET AL.

enable the substrate molecules to interact via ring‐stacking (eg, π‐π, cation‐π) and hydrophobic interactions.136,144
However, molecular docking and molecular dynamics simulation studies have shown that there is no common
binding motif of efflux pump substrates.145,146 A hypothesis suggesting that the protonation/deprotonation of
three charged amino acid residues (Asp407, Asp408, and Lys940) is essential for substrate binding or extrusion has
been proposed, since their substitution led to complete loss of resistance to drugs.147 It was also shown that
proteins such as AcrB can accommodate various ligands by using different subdomains within the binding pocket. In
some cases, the simultaneous binding of two drugs can also occur.145
On the contrary, TolC plays a major role in the export of compounds and the key to the function of TolC is the
periplasmic tunnel entrance opening.148 Crystal structures of TolC have been determined, enabling a profound
insight into its structure and function. The TolC tunnel helices are constrained at the entrance by a circular network
of hydrogen bonds and electrostatic bridges. Tyr and Phe aromatic residues cluster and form a ring around the base
of the TolC β‐barrel, which is important in the closed states of TolC because of hydrogen bonds. The TolC channel is
forced to close when the strength of hydrogen bonds between the essential sites is increased, which creates an
opportunity for designing EPIs.149 However, there are two main drawbacks of TolC as a target for the development
of EPIs: (i) AcrAB‐TolC assembly is able to tolerate significant depletion of TolC protein conditioned by mutational
modifications or changes in its expression, and (ii) despite different whole cell screening assays, not a single
compound that could block TolC channel activity in vivo has so far been identified.150
It has been shown that overexpression of the AcrAB‐TolC pump contributes to the selection of
fluoroquinolone‐resistant mutants that exhibit mutated targets (DNA gyrase and/or topoisomerase IV) in a
variety of Gram‐negative bacteria involved in severe human diseases.151,152 Moreover, AcrAB‐TolC contributes to
the MDR mechanism in E. aerogenes and many other Enterobacteriaceae. Gene sequence analysis of E. coli and
E. aerogenes AcrAB‐TolC efflux systems have revealed a high degree of similarity between these two bacteria, since
both AcrA proteins possess 85% sequence identity in a 399 amino acid overlap. AcrB proteins show 87% sequence
identity in a 1049 amino acid overlap and TolC proteins show 82% sequence identity in a 495 amino acid overlap.
However, E. aerogenes TolC protein appears to be slightly smaller than that of E. coli.153

4.3 | Pseudomonas aeruginosa

P. aeruginosa is a Gram‐negative opportunistic pathogen and a leading cause of nosocomial infections as well as of
chronic lung infections in patients with cystic fibrosis.154,155 The outer membrane of P. aeruginosa differs from that
of other Gram‐negative bacteria and is much less permeable. Efflux pumps in P. aeruginosa expel antibiotics more
effectively than those of E. coli, since its main outer membrane porin OprF has about two orders of magnitude
lower permeability than OmpF of E. coli; this is the basis of the much higher P. aeruginosa intrinsic resistance
levels.34 Eradication of this pathogen is, therefore, even more challenging.156
RND type efflux pumps play a very important role in extruding antibiotics from the cell in P. aeruginosa. To date,
at least 10 RND type MESs are known to exist in this microorganism and its genome sequences show the presence
of several additional RND‐type systems (Table 1).41,43 Among them, MexAB‐OprM, MexCD‐OprJ, MexEF‐OprN,
and MexXY‐OprM contribute most significantly to the antibiotic resistance.157,158 Substrates for these efflux
pumps overlap, but they are not completely identical. For example, fluoroquinolones, having a positive charge, are
transported most efficiently by the MexCD‐OprJ, whereas quinolones without the fluorine atom are preferably
extruded by MexEF‐OprN.83 MexAB‐OprM and MexXY‐OprM are the main efflux systems responsible for bacterial
survival during infection and intrinsic antibiotic resistance.159 MexAB‐OprM is expressed constitutionally in
P. aeruginosa, thus accounting for its pathogenicity and intrinsic resistance to fluoroquinolones.160 This system was
the first MES to be described in this pathogen, having been identified originally during research on siderophore‐
mediated iron transport systems.64 It is widely overexpressed in clinical isolates and contributes to the resistance
of wild‐type strains to antibiotic drugs.63 MexAB‐OprM exhibits the broadest substrate specificity of all known
MESs of P. aeruginosa. The list of substrates includes various antimicrobial agents (eg, β‐lactams, chloramphenicol,
LAMUT ET AL. | 11

trimethoprim, macrolides, older and newer fluoroquinolones), dyes (acriflavine, acridine orange, crystal violet, and
EtBr), detergents, tea tree oil components (α‐terpineol), organic solvents, and triclosan.41,161
MexAB‐OprM is a system homologous to AcrAB‐TolC from E. coli. It is composed of three parts an inner
membrane protein MexB, an outer membrane protein OprM, and the periplasmic membrane fusion protein MexA.
The outer membrane pore ensures that the extruded compound does not remain in the periplasm, thus preventing
its return to the cytosol.162 AcrB and MexB share 70% amino acid sequence identity, AcrA and MexA share 57%
identity, and TolC and OprM 19% identity.163 Moreover, AcrB can substitute for MexB and is also able to form an
active drug complex with natural partners of MexB, namely MexA and OprM.164 Like its homologue TolC, OprM is
able to function in multiple efflux systems and can also work independently of MexAB.165 Despite their high
functional and structural similarity, AcrB and MexB appear to possess discrete variants responsible for several
unique features and for substrate selectivity.164 Crystal structures have been reported for all three parts of the
MexAB‐OprM efflux system,166–168 although the structure of an assembled tripartite complex has not yet been
determined. However, the reconstitution of native MexAB‐OprM in a lipid nanodisc system has recently been
reported, demonstrating the intrinsic ability of the native components to self‐assemble into a stable tripartite
complex.26

4.4 | Efflux pumps of other WHO priority pathogens

Most of the vancomycin‐resistant Enterococci are E. faecium, for which MsrC, EfrAB and EfmA efflux pumps have
been reported to be the most important in conferring MDR (Table 1).31–33 Overproduction of AmvA and RND
efflux pumps, such as AdeABC, AdeFGH, and AdeIJK, is associated with resistance to antibiotics in Acinetobacter
baumanii (Table 1).34–36 In addition, AbeM is present in many clinical isolates of A. baumannii and has been shown to
increase the MICs of antibiotics.37,38 Of the several RND efflux systems found in H. pylori, HefABC contributes to
MDR,13,39 while the clinical importance of three MESs was demonstrated for N. gonorrhoeae, namely MtrCDE,
MacAB, and NorM, which recognize antibiotics relevant for the treatment of gonococcal infections (Table 1).13,40

5 | EFFL UX P UMP INH IBITORS OF SELE CTED CLIN IC ALL Y RE LEV ANT
PA THOGEN S

Basically, there are two broad categories of EPIs, depending on the discovery approach used. The most commonly
used approach is via cell‐based screening in which one or more specific antibiotics are added to the growth medium
at subinhibitory concentrations. Potential EPIs with the ability to resensitize the test strain to the corresponding
antibiotic have been identified. The second approach to searching for novel EPIs is based on the simple fact, which
is actually the main problem for the development of resistance, that antibiotics are specific substrates for their
cognate efflux pump systems. In this case, the antibiotic scaffold is derivatized to yield compounds that are devoid
of antimicrobial activity but are readily effluxed through the same efflux systems. Such compounds could serve as
competitive substrates which, in turn, decrease the expulsion of an antibiotic from bacteria and maintain its
intracellular concentration above the threshold needed for inhibition of bacterial growth.169 A good example of this
is the series of tetracycline derivatives, including 13‐cyclopentylthio‐5‐hydroxytetracycline (13‐CPTC, 7; Figure 3),
as the specific tetracycline efflux protein Tet(B) inhibitor described by Nelson et al170 Some successful substrate
competitors were also found for E. aerogenes.171 However, no candidates for clinical development have been
identified from this category of EPIs to date.
12 | LAMUT ET AL.

F I G U R E 3 13‐CPTC (7) showing modification of the initial tetracycline scaffold (marked green) [Color figure
can be viewed at wileyonlinelibrary.com]

5.1 | Staphylococcus aureus

5.1.1 | Aryl alkenyl amides
One of the early studies on NorA inhibitors was performed by Markham et al,84 who screened a large, structurally
diverse chemical library and identified NorA inhibitors belonging to different structural classes. In this study,
INF240 (8; Figure 4) was identified as a promising compound that displayed enhanced antibacterial activity against
NorA overproducing S. aureus strain (SA1199B), with an FIC index of 0.28, and a more than 50‐fold decreased
frequency of emergence of resistance to ciprofloxacin. In addition, the results from toxicity assessment assays in
HeLa cell line showed moderate toxicity of INF240 with an EC50 value of 5.7 µM.100
Piperine (9; Figure 4) is a plant alkaloid, isolated from black pepper (Piper nigrum). It inhibits human
P‐glycoprotein172 as well as NorA.173 It inhibits the efflux of EtBr in the same manner as reserpine and has the
ability to lower the ciprofloxacin MIC in ciprofloxacin‐resistant S. aureus mutants.173 Some SAR studies were
performed with synthesized piperine analogs, revealing that (i) antibiotic potentiating activity is higher with
introduction of a short alkyl group (ethyl, n‐propyl) at the C4 position of the pentadienoic side chain, (ii)
replacement of the piperidine group with aromatic amines possessing electron‐withdrawing groups (eg, CN)
enhanced antibiotic potentiating activity, while that with electron‐donating groups was deleterious to this activity,
and (iii) amide groups are important in potentiating antibacterial activity.174,175
Taken as a representative structure, SK‐20 (10; Figure 4) was found to be more potent than piperine with an
MPC8 value of 19.9 µM for ciprofloxacin against SA1199B. Toxicity assays in mice revealed that SK‐20 was
tolerated well when administered orally (LD50 > 1 g/kg body mass).174 The same MIC reduction of ciprofloxacin was
observed for compound 11 (Figure 4) at 3.1 µM, while compound 12 (Figure 4) had an MPC4 of 0.14 µM.119,176
Cinnamamide derivatives were also reported to potentiate the activity of ciprofloxacin against SA1199B, with an
MPC4 of 19.1 µM for the most active compound.177

FIGURE 4 Aryl alkenyl amides 8 to 12 displaying NorA inhibitory activity

LAMUT ET AL. | 13

5.1.2 | Indole derivatives

Reserpine is a plant‐derived indole alkaloid from the roots of Rauwolfia spp. and was discovered serendipitously to
be an effective inhibitor of the tetracycline efflux caused by the Bmr efflux pump of B. subtilis. It has been reported
that reserpine can also eradicate the resistance conferred by the NorA of S. aureus and demonstrate a fourfold
decrease in tetracycline MIC values for two MRSA isolates.110 Moreover, an MPC4 of 32.9 µM was observed in the
presence of reserpine for ciprofloxacin, moxifloxacin, and sparfloxacin against various S. aureus clinical isolates.178
Unfortunately, application of reserpine clinically, in combination with antibiotics, cannot be achieved since the
concentrations needed for effective NorA inhibition are neurotoxic.179 In general, reserpine is considered to be an
EPI of MFS and RND efflux pumps; it also inhibits P‐glycoprotein.180,181
Among the number of other active indole derivatives, 5‐nitro‐2‐phenylindole INF55 (13; Figure 5) has been
recognized as a promising lead, possessing an MPC4 of 6.3 µM for ciprofloxacin against SA1199B.84 On the basis of
these observations, Samosorn et al synthesized a series of 5‐nitro‐2‐aryl‐1H‐indoles. The most potent was
compound 14 (Figure 5), which effectively blocked EtBr efflux in S. aureus K2361 (NorA++) and exhibited an MPC16
of 13.4 µM for norfloxacin. It lowered the MIC of berberine 250‐fold, being more potent than INF55.121
Other INF55 analogs were investigated by Ambrus et al182 First, they explored the importance of the 5‐NO2
group in an attempt to identify inhibitors that would avoid the known toxicity problems of aromatic nitro
compounds. Second, they were interested in analogs bearing hydroxymethyl groups, since they could be suitable
moieties for the cleavable ester linkages of NorA inhibitor‐antibacterial drug hybrids. Unfortunately, all compounds
lacking 5‐NO2 were significantly less potent than INF55 in potentiating berberine antibacterial activity. Compound
15 (Figure 5), bearing a 3′‐COOMe group, proved to be the most potent compound identified in this study,
displaying a slightly higher potency than INF55 against all three S. aureus strains (wild type, ΔNorA, and NorA++).
Nevertheless, the important finding was that compound 16 (Figure 5), bearing a 4′‐CH2OH group, was almost
equipotent to INF55 and therefore suitable for the preparation of hybrid molecules.182
The SAR for the described indole derivatives was established as follows: (i) the electron withdrawing group at
C5 of the indole was essential for activity (eg, NO2, CN, and halogen), but carbonyl‐based electron‐withdrawing
groups and sulfonic acids, esters and amides at C5 abolished the activity; (ii) the halogen group at C5 and the
CH2NHBoc side chain at C3 position potentiate the activity; (iii) the indole NH is not essential for activity and could

FIGURE 5 Indole‐based NorA inhibitors

14 | LAMUT ET AL.

be replaced by a sulfur atom; (iv) 3′‐COOMe or 4′‐CH2OH group substitution on the 2‐aryl ring retains the activity,
while other substitutions are less favorable and lead to loss of antibacterial activity potentiation.122,182
Furthermore, another research group was investigating indole nucleus as a promising scaffold for the synthesis
of novel antimicrobial agents.183 Among the prepared indole derivatives, compound 17 (Figure 5) displayed the
most promising NorA inhibitory activity with an MPC4 of 1 µM for ciprofloxacin against SA1199B.184 In addition,
even more potent bis‐indole compounds were disclosed in the patent application with compound 5 (Figure 1) as a
representative example, which showed an MPC4 below 0.4 µM for ciprofloxacin against SA1199B. Cytotoxicity
assays against three different human cell lines were performed and 5 was considered as noncytotoxic at a
concentration of 1 µM.185 A series of further bis‐indole derivatives was prepared with the best compounds
displaying NorA inhibitory activity equipotent to 5.186 However, there have been no data for in vivo efficacy or
progress of these potent NorA inhibitors toward clinical evaluation.
Recently, two potent indole‐based NorA EPIs (18 and 19; Figure 5), with some unexpected features of shape
and size, were reported.187 They were shown to be equipotent, with IC50 values around 2 µM and were able to
restore ciprofloxacin activity against SA1199B with FIC index values of 0.27 and 0.25, respectively. On the basis of
these observations, it was concluded that NorA is also able to bind quite bulky molecules.187
On the basis of the promising inhibition of NorA by INF55 (13; Figure 5), some hybrid molecules were also
synthesized. The first was SS14 (20; Figure 6), a conjugate of INF55 and berberine connected via a methylene
group. It was shown to be a superior antibacterial compound with over 200‐fold greater activity against S. aureus
(NorA++) than berberine itself. SS14 showed good efficacy in a Caenorhabditis elegans in vivo model of infection,
curing worms of the enterococci.188 Meta (21; Figure 6) and para (22; Figure 6) regioisomers of SS14 displayed
very similar antibacterial and worm‐curing effects.189 However, both isomers, unlike SS14, also possessed
MDR‐inhibitory activity (proved by observing the effect on EtBr uptake and efflux), suggesting that they are true
dual‐targeting antibacterials. Each hybrid accumulated in S. aureus cells more than either berberine alone or
berberine in the presence of INF55.189 Elongation of the methylene linker by the introduction of an oxygen atom
increased the antibacterial activity and MDR pump inhibitory potency. The MIC of compound 23 (Figure 6) was
1.7 µM, nearly twofold better than that of SS14 against S. aureus (NorA++) strain K2378, and the intracellular
concentration of EtBr was nearly twofold higher than that of SS14 in all tested strains. In addition, structural
simplification of the indole moiety turned out to be unfavorable for the antibacterial activity of hybrids, although
it was shown that the indole nucleus is not necessary for NorA EPI.190 One may quickly conclude that berberine‐
INF55 hybrids elicit their antibacterial effects through the combination of activities of two functionally distinct
components (antibacterially active berberine and NorA inhibitor INF55), but a study suggests that the
exact mechanism of action, in this case, differs from that when berberine is co‐administered with INF55‐based
EPIs.191
To date, several potent indole‐based NorA inhibitors were discovered and optimized, but their efficacy still
needs to be evaluated in clinical settings.

FIGURE 6 Berberine‐NorA inhibitor hybrids

LAMUT ET AL. | 15

5.1.3 | Quinolines and quinolones

On the basis of structural similarities with 3‐phenyl‐1,4‐benzothiazines, which were reported to be NorA EPIs,192
different subclasses of aryl quinolones were screened and 3‐aryl‐4‐methyl‐2‐quinolones were selected for further
optimization.193 Analogs were synthesized, taking into account the substitution pattern of flavonoids, well‐known
NorA inhibitors. The most promising compound was 24 (Figure 7) which showed an MPC2 of 3.9 µM for
ciprofloxacin against SA1199B. Although 24 possessed some intrinsic antibacterial activity (MIC between 62.5 and
125 µM) against various bacterial strains, it was proven to be a NorA inhibitor, because no synergism against
S. aureus K1712 (NorA knock‐out strain) and no potentiation of the bactericidal activity of several non‐NorA
substrates (oxacillin, erythromycin, tetracycline, and vancomycin) by 24 was observed. Moreover, it inhibited EtBr
efflux with an IC50 value of 18 µM, comparable to that of reserpine.193 Toxicity assays revealed that 24 was not
toxic up to 100 µM concentration in HME‐1 (immortalized human breast epithelial) cells in vitro and no weight loss
in vivo or death was observed with BALB/cByJ mice treated with 24. In addition, a very simple structure with low
MW and an easy chemical synthesis (two steps with high yield) makes it a promising lead compound for further
investigation.73
Inspired by the flavone nucleus of previously described EPIs, Sabatini et al194 synthesized a series of quinoline
derivatives To identify more potent NorA inhibitors. This concept was based on the hypothesis that the
2‐phenylquinolone nucleus possesses all the known features responsible for EPI activity, including the ability to
form electrostatic interactions and a suitably large hydrophobic area.195 Moreover, it is a mimic of the well‐known

F I G U R E 7 Quinolone (24), quinolines (25, 26), and representative quinolone‐based hybrids (28‐31) possessing
NorA inhibitory activity
16 | LAMUT ET AL.

quinolone antibacterials and is suitable for simple chemical modification. The most active compound was 25
(Figure 7), which, with an MPC16 of 32 µM against SA1199B, reduced the MIC of ciprofloxacin.194
A pharmacophore model was built for NorA EPIs on the basis of published data and used to design novel
inhibitors by means of ligand‐based drug design (LBDD).196 Of these, an analog of 25 bearing a piperazine ring
instead of piperidine was one of the most potent EPIs with an MPC16 of 8 µM for ciprofloxacin against SA1199B.
Further, when tested against SA K2378 (another S. aureus strain that overexpresses NorA), this compound showed
an MPC16 of 4 µM for ciprofloxacin. Preliminary SAR studies, performed for this class, showed that (i) introduction
of 2‐ethylamino alkyl chains, linked to the C4 hydroxy group, enhances the activity; (ii) inclusion of a side‐chain
N‐atom in an aliphatic ring maintained the activity, which tends to decrease when a second heteroatom (O, S) is
introduced into the ring; (iii) activity was completely lost when a side chain N‐atom was included in an aromatic
ring, phthalimide moiety or disubstituted with benzyl groups (mainly because of reduction of the nitrogen
nucleophilicity and/or steric hindrance); (iv) mixed substitution of the side chain N‐atom with benzyl and ethyl was
well tolerated, and (v) a compound with a propoxy group at the C4′ position of the C2 phenyl ring displayed the
most potent inhibitory activity.196 Another study from the same research group confirmed the 2‐phenylquinoline
nucleus as being a suitable scaffold on which to design novel NorA inhibitors.197
The recently reported optimization of this series led to significantly improved potency against SA1199B strain.
A benzyloxy group was attached to 25 at the C6 position of the core quinoline moiety and derivative 26 (Figure 7)
displayed an MPC4 of 1.6 µM for ciprofloxacin.198 Furthermore, replacement of the benzyloxy with a methoxy
group gave an even more potent analog 4 (Figure 1) with an MPC8 value of 1.9 µM.199 The latter was also devoid of
any significant intrinsic antibacterial effect and was demonstrated to be nontoxic at concentrations required for
effective NorA inhibition, with CC50 value of 100 µM when tested on human liver epithelial (HepG2) cells and more
than 238 µM against human monocytic cell line (THP‐1). Cytotoxicity studies of other analogs suggested that
the position of methoxy group on the core quinolone moiety is not significant in terms of toxicity, but piperidine
ring plays an important role, making compounds safer.199 Moreover, preliminary pharmacokinetic studies were
performed and 4 was shown to be metabolically stable in in vitro experiments with mouse liver microsomes. In
addition, it did not inhibit the cytochrome CYP3A4 at 10 µM concentration. Furthermore, 4 displayed also
inhibition of MepA efflux pump of S. aureus. This is an advantage in terms of designing broad spectrum EPIs
targeting different S. aureus efflux pumps at once, thus preventing overexpression of other efflux pumps and
reversal of resistance as a consequence of selective NorA inhibition.199 Further characterization of these potent
2‐phenylquinoline derivatives in in vivo studies is needed, which will reveal their true therapeutic potential and
possibility to progress into clinical development.
Further, German et al114 synthesized hybrid molecules incorporating structural features of the previously
described bisaryl urea‐based NorA inhibitors, like INF271 (27; Figure 7), which showed an MPC4 of 5.1 µM for
ciprofloxacin against SA1199B,84 and fluoroquinolones (ciprofloxacin or ofloxacin) conjugated via the C7 position
of the fluoroquinolone core structure. The idea behind this was to convert broad‐spectrum efflux substrates into
expanded spectrum EPIs. Compound 28 (Figure 7) at 0.5 µM inhibited EtBr efflux by 74% in SAK2361 (a NorA
overexpressing strain). It showed no intrinsic antibacterial activity when tested against the same strain, which can
be explained by the lack of the necessary piperazine ring with a basic terminal nitrogen, which is a common
requisite feature for most fluoroquinolones. Nevertheless, it is one of the most potent NorA EPIs reported to date,
although it has not been reported to be tested for potentiation of antibacterial activity of the known antibiotic.
Modifications of SAR revealed that the potent inhibition of NorA is not due just to the presence of the bisaryl urea
motif attached to ofloxacin, but is a consequence of the fluoroquinolone‐EPI conjugate structure as a whole. On the
contrary, compound 29 (Figure 7) resulted in threefold lower MIC than ciprofloxacin when tested against SAK2361
but, since it lacked the EtBr efflux inhibition, it was concluded that this is a consequence of evading the efflux
pumps rather than actively inhibiting them.114
As a continuation of the fluoroquinolone‐EPI molecule hybrids series, some bicyclic bulky fragments
were attached to the C7 position in an attempt to obtain new quinolone derivatives with potent activity against
LAMUT ET AL. | 17

Gram‐positive bacteria.200 First, synthesized compounds were tested against a variety of bacterial strains, when
6‐amino‐8‐methylquinolone was identified as the most promising scaffold. In particular, 30 (Figure 7) exhibited an
8‐fold lower MIC against SA1199B than did ciprofloxacin. To avoid intrinsic antibacterial activity, which is
conferred by a carboxylic acid group at C3 position, esters of the best analogs were tested in an EtBr efflux
inhibition assay. The best compounds resulted in an EtBr inhibition comparable to that induced by reserpine. Good
synergistic activity in reducing ciprofloxacin MIC was demonstrated for compound 31 (Figure 7), since it was able
to reduce ciprofloxacin MIC against SA1199B with an MPC16 of 104 µM. It was concluded, that the
thiopyranopyridine moiety is a key structural feature that leads to NorA inhibition and restores the activity of
ciprofloxacin.200
To sum up, quinoline derivatives are among the most potent NorA inhibitors reported to date. However, in vivo
validation is required to give the proof‐of‐concept and required information about clinical usefulness of these
promising EPIs.

5.1.4 | Sulfur heterocycles and other sulfur‐containing compounds

Phenothiazines (such as chlorpromazine) have an intrinsic antibacterial effect92 as well as the ability to potentiate
the activity of various antibiotics, including levofloxacin, erythromycin, and azithromycin, by interfering with the
bacterial inner membrane proton gradient,201 and NorA inhibition.92 To identify novel and more potent S. aureus
NorA inhibitors, a series of 1,4‐benzothiazine derivatives were designed on the basis of minimization of the
phenothiazine MDR EPIs structural template.192 In this series, compound 32 (Figure 8) displayed the best activity,
with an MPC8 of 18.5 µM for ciprofloxacin against SA K2378. Although it possessed some intrinsic antibacterial
activity against S. aureus ATCC 25923 (wild type strain) with an MIC of 95 µM, the absence of ciprofloxacin MIC
reduction in the NorA deleted strain SA K1902 and 70% EtBr efflux inhibition in SA1199B strongly
suggest its inhibition of NorA. SAR studies revealed that (i) the benzothiazine moiety is a key feature for activity;
(ii) substituents on the C3 substituted phenyl ring play an important role and that potent compounds contain a
methoxy, propoxy, thiomethyl, or nitro group at the para position, and (iii) N4 methylation gave a compound with
weaker activity.192
Some correlation between physicochemical properties calculated in silico for seven selected compounds and
NorA inhibitory activity has been found, demonstrating that cLogP is correlated directly with efflux inhibition and,

FIGURE 8 Sulfur‐containing compounds as NorA inhibitors

18 | LAMUT ET AL.

on the contrary, that intrinsic water solubility and the percentage of uncharged molecules at pH 4 are strongly
inversely correlated. Other molecular descriptors, such as polar surface area, molecular weight, molecular volume,
globularity, and hydrophilic accessible volume are less well correlated with NorA inhibition.192
A benzothiophene derivative, 33 (Figure 8), was also found to inhibit NorA.122 It was designed by Fournier
et al, based on the known very potent NorA inhibitor INF55 (13; Figure 5), bearing in mind that the activity of
INF55 was less dependent on the nitro functionality than on the 2‐arylindole structural part. They, therefore,
synthesized sulfur analogs of INF55. Compound 33 restored the activity of ciprofloxacin against SA1199B at
52.5 µM and was able to inhibit EtBr efflux as strongly as reserpine. Moreover, cytotoxicity assays in three different
cell lines (KB, MCF7, and MCF7R) revealed a moderate level of toxicity for this compound at 10 µM. However, 33
contain a reactive and chemically unstable aldehyde functional group, which could contribute to the problems of
stability and/or toxicity and hinder its use in further optimization and development.122
Some other representative sulfur‐containing compounds acting as NorA inhibitors are presented in Figure 8.
Compound 34 (Figure 8) was found by in silico screening of an array of natural compounds.202 It exhibited an MPC8
of 68.2 µM for ciprofloxacin against SA1199B and 93% inhibition of EtBr efflux at concentrations above 30 µM in
the same strain. However, unexpected activities against two S. aureus wild type strains (ATCC25923 and SA1199)
led the authors to the conclusion that 34 is also able to block efflux pumps other than NorA. SAR studies suggested
that (i) limited modifications of the sulfone or amide group do not affect inhibitory activity significantly,
(ii) modifications of the imidazole ring substituents reduce the activity drastically, and that (iii) changing the
1‐methyl‐pyrrole ring to a furan ring leads to a slight decrease in inhibitory activity.202
Celecoxib has been reported to increase the sensitivity of S. aureus strains to different antibacterials203 and
another study confirmed its inhibitory activity against the NorA efflux pump, displaying an IC50 value slightly higher
than 40 µM in an EtBr efflux inhibition assay against SA1199B.204 On the basis of the NorA in silico model
described above,202 NorA inhibitory activity of an analog of celecoxib (35; Figure 8) was also discovered. It
inhibited NorA mediated efflux of EtBr with an IC50 similar to that of reserpine and showed strong synergism with
ciprofloxacin, displaying an MPC8 of 13.8 µM against SA1199B, which is superior to the reference compounds
reserpine and paroxetine. Nevertheless, it also showed modest intrinsic antibacterial activity (MIC of 110 µM
against S. aureus SA1199). Moreover, it was assayed for cytotoxicity in HepG2 cells and found to be nontoxic, with
CC50 > 221 μM, showing results comparable to those previously reported for ciprofloxacin.204,205

5.1.5 | Boronic acids

Fontaine et al evaluated boronic acid derivatives for their synergism with ciprofloxacin against SA1199B. Of all the
compounds, the most promising NorA inhibitory activity was shown by a series of pyridine‐3‐boronic acid and
benzene‐3‐boronic acid derivatives, such as 36 and 37 (Figure 9). They showed MPC4s of 4.4 and 2.2 µM,
respectively. Both compounds were noncytotoxic in a KB cell (epidermal carcinoma cells) cytotoxicity assay and
were thus confirmed as inhibitors of the NorA efflux pump.120 Detailed SAR studies for pyridine‐3‐boronic acid and
benzene‐3‐boronic acid derivatives showed that (i) a boron atom is essential for EPI activity; (ii) shifting
substituents on pyridine‐3‐boronic acid derivatives from para to meta position with respect to the boronic acid
moiety decreased the activity slightly, but was still well tolerated, while ortho substituted analogs showed a
significant loss of activity; (iii) cyclization at positions C5, C6 of pyridine and C3, C4 of benzene analogs or
introduction of a methyl group at C5 and C3 was well tolerated.120
SAR also revealed the importance of an (aryl)alkoxy side chain at the C6 position of pyridine‐3‐boronic acids.
Further optimization of this series included an extension of the alkyl chain at C6, and compounds 38 and 39 (Figure
9) were found to reduce the MIC value of ciprofloxacin against SA1199B with MPC4s of 15.6 and 14.8 µM,
respectively. These compounds possessed no significant intrinsic antibacterial activity and were able to promote
accumulation of EtBr inside the cells. Moreover, they did not inhibit the mammalian P‐gp efflux pump and 39
(Figure 9) showed a low level of toxicity in KB cells at 10 µM. However, they were still less effective than the
LAMUT ET AL. | 19

FIGURE 9 Boronic acids as NorA inhibitors

positive control, reserpine.206 In addition, boronic acids are also considered as potent β‐lactamase inhibitors and
possess a variety of other biological effects.120,207 Thus, selectivity for efflux pumps should be addressed in the
future development of this class of EPIs. Alternatively, dual inhibition of efflux pumps and β‐lactamase can be used
as an advantage by targeting both resistance mechanisms at the same time.

5.1.6 | Flavones and chalcones

A number of flavonoids were reported to potentiate the activity of norfloxacin against SA1199B,208 suggesting that
a flavone nucleus could be suitable when designing novel NorA inhibitors. Interestingly, the Berberis plant species
that makes berberine also produce an MDR inhibitor, flavolignan 5′‐MHC (40; Figure 10) that strongly potentiates
the action of berberine.51 It has been found as a potent NorA efflux inhibitor, reducing the MIC of berberine by
16‐fold when coadministered. Another weak, plant‐derived, inhibitor is baicalein (41; Figure 10), which is isolated
from Scutellaria spp. and is widely used in Chinese medicine for treating bacterial infections.73 It was shown
to decrease the MIC value of oxacillin against MRSA (strain SAST239) with an MPC128 of 118 µM and to
inhibit SA1199B strain with prolongation of postantibiotic effects from 4 to 24 hours. Kaempferol rhamnoside
(42; Figure 10), isolated from Persea lingue, potentiated the MIC of ciprofloxacin against SA1199B with an MPC16 of
2.15 µM and an IC50 value of 2 µM, as determined in the EtBr efflux‐inhibition assay, which is almost fivefold lower
than that for reserpine (IC50 of 9 µM). As proposed by Holler et al,209 based on the toxicity profile of analogs, it is

FIGURE 10 Flavones acting as NorA inhibitors

20 | LAMUT ET AL.

not likely to exert acute toxicity.210 Coumarin 43 (Figure 10), isolated from Mesua ferrea, was shown to be a potent
NorA inhibitor with a stronger NorA efflux pump inhibitory activity than reserpine and verapamil against SA1199B
and MRSA831. It showed an MPC32 of 62 µM and MPC8 of 31 µM for norfloxacin against these two strains,
respectively. On the basis of synthesized derivatives, the presence of phenyl, 2‐methylbutanoyl and prenyl groups
at C4, C6, and C8, respectively, on the coumarin scaffold, is a structural requirement for EtBr efflux inhibition.211
Moreover, isoflavone biochanin A (44; Figure 10) showed an MPC16 of 35.2 µM for berberine and MPC4 of 35.2 µM
for norfloxacin when administered together with each individual antibiotic against wild‐type S. aureus (8325‐4).52
Inspired by the structural similarities with the coumaroyl substituents, a series of chalcones were tested as
NorA inhibitors. Among them, compounds 45 and 46 (Figure 11) decreased the MIC of ciprofloxacin against
SA1199B with MPC4s of 8.1 and 51.8 µM, respectively.212,213 SARs revealed that (i) on ring A of the chalcone
scaffold (Figure 11), a favorable substituent for synergistic activity is methoxy at C2, while an additional methoxy at
either C3, C4, or C5 does not affect the activity; (ii) on ring B of the chalcone scaffold (Figure 11), a favorable
substituent for synergistic activity is dimethylaminoethoxy at C4, while OH at this position leads to a loss of
inhibitory activity. The latter supports the hypothesis of Guz et al,214 who have investigated the SAR of several
flavolignans and flavones with NorA affinity. They discovered that a methoxy group is more favorable than a
hydroxyl substituent at the C4′ position of flavones.

5.1.7 | Polyphenols and terpenoids

Polyphenol inhibitors, isolated from green tea extracts, were proved to reverse MRSA resistance.215 For example,
epicatechin gallate (47; Figure 12) decreased the MIC of norfloxacin against SA1199B with an MPC4 of 45.2 µM.216
The phenolic diterpene totarol (48; Figure 12) potentiates the activity of norfloxacin against the same strain
with an MPC4 of 3.5 µM and inhibits EtBr efflux with an estimated IC50 of 15 µM.217 Another terpenoid, ferruginol
(49; Figure 12), was shown to be a weaker EPI, suggesting that the position of isopropyl and hydroxyl groups is
important for activity. However, ferruginol showed an excellent, 80‐fold, potentiation of oxacillin activity against
the epidemic MRSA strain, EMRSA‐15, comparable with the activity of epicatechin gallate against the same strain
and, therefore, restoring oxacillin efficacy.218

5.1.8 | Other structural classes

The NorA inhibitors pheophorbide A (50; Figure 13), ginsenoside Rh2 (51; Figure 13), and the heterocyclic
oligosaccharide orizabins all show how structurally complex inhibitors can bind to NorA, showing the broad
substrate diversity of this efflux transporter.123,219,220 Orizabin IX (52; Figure 13) completely reversed the
resistance to norfloxacin against SA1199B at a concentration of 1.2 µM. NorA‐mediated efflux of EtBr and
norfloxacin could also be altered with protonophores such as carbonyl cyanide m‐chlorophenyl hydrazone, which
dissipates the membrane proton gradient of the NorA efflux pump.109,110,113

FIGURE 11 Chalcone scaffold and chalcone‐based NorA inhibitors

LAMUT ET AL. | 21

FIGURE 12 Examples of a polyphenol (47) and terpenoid‐like (48, 49) NorA inhibitors

FIGURE 13 Structures of complex molecules acting as NorA inhibitors

5.2 | Escherichia coli

5.2.1 | Pyranopyridines
Pyranopyridines provide a good example of how to move from low toward high potency scaffolds. MBX2319 (3;
Figure 1) is structurally not similar to any of the EPIs previously described and was identified through a cell‐based
high‐throughput screening campaign to be a potent inhibitor of AcrAB‐TolC with activity against a panel of
Enterobacteriaceae strains, namely E. coli, K. pneumoniae, E. aerogenes, Enterobacter cloacae, Salmonella enterica, and
Shigella flexneri.221 It decreased the MICs of ciprofloxacin, levofloxacin, and piperacillin in E. coli AB1157 (wild type
E. coli strain) with an MPC2 of 1.56 µM for all three antibiotics. Moreover, it did not possess intrinsic antibacterial
activity and was not active against AcrAB‐TolC deficient strains, which proves that this efflux pump is a target of
MBX2319. In E. coli strains 285 and 287 (efflux overexpressers) it decreased the MIC of ciprofloxacin with MPC8
and MPC4 values of 3.13 µM, respectively. At the same concentration, MBX2319 in combination with a minimal
bactericidal concentration of ciprofloxacin decreased the viability (colony forming units [CFU]/mL) of E. coli
AB1157 by 10 000‐fold after 4 hours of exposure when compared to ciprofloxacin alone. Toxicity assays showed
that CC50 against HeLa cells was more than 100 µM, thus defining MBX2319 as being nontoxic. MBX2319 also
exhibited activity against P. aeruginosa ATCC 27853 (clinical isolate), with an MPC6 of around 25 µM for
cefotaxime.72 This is not surprising, since AcrB and MexB are very similar in primary structure (69.8% of amino acid
identity and 83.2% amino acid similarity) as well as in three‐dimensional protein structure (root‐mean‐square
deviation of 1.4 Å).166
Opperman et al studied this scaffold further and synthesized 60 new analogs of MBX2319 (3; Figure 1) to gain
information about the SAR and to locate sites on the pyranopyridine scaffold that could be varied to improve
solubility, metabolic stability, and potency. Compounds 53 to 56 (Figure 14) were shown to be the most promising
in this series, reducing the MICs of levofloxacin and piperacilin against E. coli AB1157 with MPC4 values in the
22 | LAMUT ET AL.

range of 0.05 to 0.8 µM, which is about an order of magnitude lower than that of previously described EPIs acting
on the AcrAB‐TolC efflux pump system. All of them possessed improved aqueous solubility compared to that of
MBX2319 and were also more stable in a human liver microsome stability assay.222 The SAR was established by the
following: (i) nitrile, dimethylenesulfide, and geminal dimethyl groups are important for maintaining potency;
(ii) nonacidic substituents in the phenyl moiety improve potency and in vitro pharmacokinetic properties; (iii) an
additional dimethyl group on the morpholine ring improves microsomal stability and replacement of the
morpholinyl group with the (2‐methoxyethyl)piperazinyl moiety yields moderately stable compounds with
significantly improved aqueous solubility. To summarize, modification of the phenyl ring had greater effects on
potency and CYP450 inhibition, whereas modification of the morpholinyl group had greater effects on solubility
and microsomal stability.222
MBX2319 (3; Figure 1) selectively inhibits AcrB, the most plausible mechanism being by competitive inhibition
and/or blocking of access to the substrate binding site of AcrB.221 A structural comparison of MBX2319 and other
potent EPIs, for example, the peptidomimetic PAβN (1; Figure 1) and pyridopyrimidine D13‐9001 (2; Figure 1),
shows that they all consist of at least two hydrophobic ring systems that can interact with hydrophobic amino acid
residues in the substrate binding site. Detailed computational modeling confirmed this hypothesis and, further,
suggested that the pyridine ring forms π‐stacking interactions with the phenylalanine residues.141 In addition, high‐
resolution crystal structures of inhibitors 3, 54, 55, and 56 (Figures 1, 14, and 15) bound to the AcrB periplasmic
domain have been published,69 providing a deeper insight into the mechanism of action as well as interactions of
pyranopyridine compounds in the substrate binding site of AcrB. Crystal structures have shown that
pyranopyridines sterically block the access of substrates into the deep binding pocket of AcrB. They bind within
a phenylalanine‐rich cage (Figure 15), which extends from the deep binding pocket, forming an extensive network
of hydrophobic interactions and thus effectively preventing the functional rotation of the AcrB trimer. The
increased potency of inhibitors correlates with the formation of a delicate protein‐ and water‐mediated hydrogen
bond network, as in the case of MBX3132 (55; Figure 14) and MBX3135 (56; Figures 14 and 15).69
The data described above have, together, led to the conclusion that pyranopyridines are potent EPIs and
potential adjunctive therapeutic agents for the treatment of infections caused by Gram‐negative pathogens.221 The

FIGURE 14 Pyranopyridines—the most potent AcrAB‐TolC efflux pump inhibitors known to date
LAMUT ET AL. | 23

F I G U R E 1 5 Binding mode of MBX3135 (56, in green sticks) in the AcrB efflux pump (in gray) ligand binding pocket
(PDB entry: 5ENR)69 determined by X‐ray crystallography. For clarity, only amino acid residues forming hydrophobic
interactions and hydrogen bonds with inhibitor are shown as gray sticks. Water molecules are presented as red spheres.
Hydrogen bonds are indicated by black dotted lines [Color figure can be viewed at wileyonlinelibrary.com]

pyranopyridine series is in the process of lead optimization and, to date, there have been no reports of in vivo
activity of these compounds.72

5.2.2 | Indoles
3‐Aminoindole‐6‐carboxylic acid (57; Figure 16) and 3‐nitro‐6‐aminoindole (58; Figure 16), both similar to INF55
(13; Figure 5), have been shown to potentiate the activity of four different antibiotics against E. coli FJ307 (a highly
drug efflux clinical strain, overexpressing TolC). They displayed an MPC32 of 0.5 mM and MPC64 of 0.5 mM,
respectively, for chloramphenicol and, both of them, MPC8 of 0.5 mM for ciprofloxacin. Indole derivatives 57 and
58 were designed by quantum chemistry in keeping with the known TolC structure and were established to be
AcrAB‐TolC inhibitors acting on the TolC subunit. The aim was to incorporate in one molecule a pair of proton‐
donor and proton‐acceptor functional groups, so that they could link Asp and Tyr together by hydrogen bonds. The
molecules reduced drug export by constraining the tunnel at the distal end of TolC.148,223

5.2.3 | Quinolines and quinazolines

A number of quinoline derivatives have been studied for their ability to restore the activity of antibiotics expelled
by an AcrAB‐TolC efflux system. Their selection as potential EPIs was based on the screening test with clinical
isolates of E. aerogenes and on structural similarity to fluoroquinolones, the main efflux substrates. It is proposed
that this family of molecules acts as competitive inhibitors of antibiotic transport inside the AcrB subunit.224 The
compounds were assayed for their ability to decrease the level of resistance to chloramphenicol in E. aerogenes

FIGURE 16 Indole derivatives as AcrAB‐TolC inhibitors

24 | LAMUT ET AL.

resistant isolates. Among various quinoline‐based compounds tested, 59 (Figure 17) and its close structural analogs
were particularly effective in potentiating the chloramphenicol activity in vitro, with an eightfold and 16‐fold
decrease in the chloramphenicol MIC against resistant strains, which is comparable to the activity of PAβN
(1; Figure 1). This effect was observed at concentrations of inhibitors at which no intrinsic antibacterial activity was
present.224,225 In details, compound 59 proved to be the most effective inhibitor of the series, with an MPC16 of
200 µM for chloramphenicol against EA27 (an E. aerogenes clinical isolate overexpressing AcrAB‐TolC). The specific
value of the side chain volume was found to be associated with the ability of the molecule to reduce the MIC of
chloramphenicol, supporting the hypothesis concerning the comparability of this volume to the diameter of the
internal pocket located in the AcrB channel.134,226 The SAR of these molecules was determined, leading to
the observation that (i) the alkyl side chain plays an important role in achieving inhibitory activity (true in particular
of alkoxy‐ and thioalkoxy‐quinolines possessing piperidinoethyl chains), and (ii) the connecting heteroatom (O, S, or
N) and the location of substituents on the heterocyclic moiety appear to be important for the EPI activity.27,224
In addition, an antimalarial drug mefloquine was identified as an AcrAB‐TolC inhibitor, showing an increase
in accumulation of EtBr in a dose‐ and time‐dependent manner.227
Mahamoud et al went further with optimization to synthesize a series of structurally similar quinazoline
derivatives. Compound 60 (Figure 17) was found to be the most effective enhancer of chloramphenicol activity,
exhibiting an MPC16 of 2.5 mM against CM‐64 (the E. aerogenes chloramphenicol‐resistant strain that
overexpresses AcrAB‐TolC) and exhibits an MPC8 of 2.5 mM against EA27. It also showed a reduction of MIC
for different fluoroquinolones (especially nalidixic acid and ciprofloxacin) against CM‐64 comparable to that caused
by PAβN (1; Figure 1), as well as increased chloramphenicol activity against a panel of resistant Klebsiella
pneumoniae and P. aeruginosa isolates.228

5.2.4 | Arylpiperazines
On the basis of the results of high‐throughput screening, arylpiperazines were identified as potential EPIs, since
they were able to reverse the multidrug resistance in E. coli overexpressing RND‐type efflux pumps.229 Of these,
the most potent was 1‐(1‐naphthylmethyl)‐piperazine (NMP, 61; Figure 17) that reduced the MIC of levofloxacin
with an MPC8 of 442 µM in the E. coli strain 3‐AG100MKX (that overexpresses AcrAB) and was also active against
E. aerogenes, K. pneumoniae, and A. baumannii.230,231 However, the mechanism of action of NMP and related
compounds has not yet been elucidated, although it has been suggested that NMP interferes with the functional
assembly and movement of the G‐loop of the AcrB subunit that plays an important role in the extrusion of
substrates.146 Arylpiperazines had limited success as EPIs due to their low potency. Moreover, because these
compounds are structurally similar to the phenylpiperidine‐based SSRI class of antidepressants, they are also likely
to be too toxic for use in mammals.27,94

FIGURE 17 Quinoline (59), quinazoline (60), and arylpiperazine (61) displaying AcrAB‐TolC inhibitory activity
LAMUT ET AL. | 25

5.2.5 | Hydantoins
Handzlik et al suggested that a hydantoin scaffold could be suitable for the development of novel EPIs, since the
hydantoin core could be viewed as a cyclic peptide analog of the peptide sequence located in the central part of a
well‐known EPI, PAβN (1; Figure 1). Of the synthesized hydantoin derivatives, compounds 62 and 63 (Figure 18)
have been shown to be the most active. They displayed MPC4s of 63 µM for nalidixic acid, sparfloxacin, and
chloramphenicol against E. aerogenes CM‐64.232 However, some of the compounds tested, including 63, decreased
the MIC of sparfloxacin significantly more in the reference strain ATCC13084 (E. aerogenes wild type) than in the
CM‐64 strain, suggesting that additional anti‐MDR mechanisms distinct from AcrAB‐TolC pump inhibition are
involved in their mode of action. The extensive SAR performed in the study showed amphiphilic character of the
molecules to be crucial for the expected activity on AcrAB‐TolC (hydrophilic basic amine fragment and hydrophobic
aromatic moiety in the case of the most active compounds, such as 63) and for the strong electron‐donating
properties of substituents on the benzylidene moiety responsible for the similar properties observed in the assays
in the CM‐64 strain.232 Nevertheless, since this type of compound possesses an α,β‐unsaturated carbonyl
functionality, which is considered to be a good Michael acceptor, it could be toxic for human cells, so further studies
on safety are needed.

5.2.6 | Geraniol derivatives

Lorenzi et al233 screened various plant extracts for their ability to decrease resistance in MDR Gram‐negative
bacteria. They identified a monoterpene, geraniol (64; Figure 19), that could inhibit efflux pump activity in E. coli,
P. aeruginosa, E. aerogenes, and A. baumannii. To understand the relationship between the structure of geraniol and
its inhibitory activity they examined compounds with similar structures. In the presence of geraniol and
geranylamine (65; Figure 19), the MIC values of chloramphenichoweol against E. aerogenes EA289 MDR strain were
significantly lower. However, geraniol, but not geranylamine, was identified as a substrate of the AcrAB‐TolC efflux
pump and was considered as a competitive substrate inhibitor. Furthermore, the protein‐compound interaction
studies performed by Lieutaud et al demonstrated that geranylamine binds directly to purified AcrB. It might,
therefore, act on AcrB but at a different site, far from the chloramphenicol‐binding site. This means that its
inhibitory properties involve a noncompetitive mechanism so that, mechanistically, geranylamine is a different type
of EPI. In this study, no structural motif was clearly identified as a specific ligand‐binding structure for AcrB.234
According to the structure of geraniol, the terpene core was essential for biological activity, whereas the
hydroxyl group could be replaced by an amine, which also improved solubility in aqueous media. Bearing this in
mind, compounds 66 and 67 (Figure 19) were identified as effective AcrAB‐TolC inhibitors, displaying an MPC16 of
31 µM and MPC8 of 62.5 µM, respectively, for chloramphenicol against EA289.235 However, Yasuda et al236
demonstrated that polyamine compounds increase the permeability of the outer membrane of E. coli and potentiate
antimicrobial activities of novobiocin and erythromycin, presumably by displacing divalent cations (Ca2+ and Mg2+)
found in the membrane, thus altering its integrity. Although it was shown that geranylamine does not permeabilize
the outer membrane,234 further studies of polyamino‐isoprene derivatives, which are structurally related to 66 and
67, showed that these compounds tend to destabilize the P. aeruginosa outer membrane.237 Thus, the antibiotic

FIGURE 18 Structures of hydantoins

26 | LAMUT ET AL.

FIGURE 19 Structures of geraniol derivatives as AcrAB‐TolC inhibitors

potentiating activity of this class of EPIs may be due to both EPI and increased permeability of the outer membrane
of E. coli, which could be connected to their cationic character at physiological pH.

5.2.7 | Catechols
In keeping with the systematic development of EPIs in Gram‐negative bacteria, some polyphenolic compounds,
that were identified as inhibitors of AcrB on in silico screening, should also be noted.238 A database of 50
phytochemicals was evaluated and binding to AcrB was studied with docking experiments. The docking of the
known inhibitor PAβN (1; Figure 1) was used for comparison. Nordihydroguaretic acid (NDGA, 68; Figure 20), a
compound with antioxidant properties derived from creosote bush (Larrea tridentata), was the most effective EPI
described. It decreased the MIC values of erythromycin, chloramphenicol, and tetracycline with an MPC4 of
847 µM against E. coli BW25113 (a wild type, drug‐resistant strain). Although this compound is still of low potency
it is a good example of how useful the in silico approach can be in identifying novel, structurally diverse inhibitors
that could serve as lead compounds in the design of more potent EPIs.238 However, NDGA also possesses activity
against several other enzymes with different biological functions at even lower doses than those required for EPI,
which hinders its further development as a selective EPI.239,240

5.2.8 | 3,4‐Dibromo‐1H‐pyrrole‐2,5‐diones
The interesting small compound 69 (Figure 20), isolated from the marine bacterium Pseudoalteromonas piscicida,
was discovered on screening a variety of marine microbial extracts for efflux inhibition in MDR bacterial strains.241
It was found to decrease, between twofold and 16‐fold, the MICs of various antibiotics in E. coli strains, including
ciprofloxacin, levofloxacin, chloramphenicol, kanamycin, erythromycin, oxacillin, and tetracycline, overexpressing
three archetype RND transporters (MexXY‐OprM, MexAB‐OprM, and AcrAB‐TolC). In particular, it showed an
MPC4 of 7.85 µM for kanamycin and MPC4 of 251 µM for chloramphenicol against E. coli strain AG102

FIGURE 20 Structures of other AcrAB‐TolC inhibitors: catechol (68) and dibromopyrrole (69)
LAMUT ET AL. | 27

(overexpressing AcrAB‐TolC). Moreover, it displayed an MPC4 of 502 µM for chloramphenicol and MPC16 of
251 µM for erythromycin against E. coli strain MG1655 ΔBC/pABM (overexpressing MexAB‐OprM). It was also
able to cause increased accumulation of the fluorescent efflux pump substrate Hoechst 33342 in a dose‐dependent
manner in wild‐type E. coli AG100. It exhibited an IC50 value of 3 µM in an efflux inhibition assay, confirming its EPI
activity. Moreover, it did not possess intrinsic antibacterial activity on tested strains. It was postulated that 69
(Figure 20), as a hydrophobic ring system, interacts with hydrophobic residues located near or in the substrate‐
binding site. This molecule therefore deserves further attention as a promising scaffold to develop effective EPIs
utilizing a fragment‐based drug design approach.241

5.3 | Pseudomonas aeruginosa

5.3.1 | Peptidomimetics
One of the first known EPIs in Gram‐negative bacteria was PAβN (MC‐207,110) (1; Figure 1), a low molecular
weight dipeptide amide, which was identified by assaying an array of natural product extracts and synthetic
compounds using P. aeruginosa MES over‐expressing strains in the presence of levofloxacin.102 It decreased the MIC
of levofloxacin with an MPC8 of 22.4 µM against P. aeruginosa PAM1032 strain that overexpresses the MexAB‐
OprM efflux pump. It was validated as a broad‐spectrum EPI, since it was also able to effectively inhibit other efflux
systems in different P. aeruginosa strains overexpressing either MexCD‐OprJ, MexEF‐OprN, or MexXY‐OprM.83
Moreover, it has been shown that PAβN also possesses activity against the AcrAB‐TolC pump of E. coli and several
other Enterobacteriaceae species, although it is not very potent.242 PAβN was shown to increase the permeability
of the outer membrane of the P. aeruginosa PAM2035 strain, that lacks the functional MexAB‐OprM pump, in a
dose‐dependent manner due to its dicationic character, which has raised concerns concerning toxicity.72 In vivo
cytotoxicity data showed that PAβN was toxic to mice (LD50 < 25 mg/kg)243 and its further developed analogs were
proven to be nephrotoxic as a result of their accumulation in lysosomes.244,245
To determine the initial SAR, more than 500 analogs were synthesized and tested. Analog 70 (Figure 21)
showed an MPC8 of 11.9 µM for levofloxacin against PAM1032 and nearly twofold increase in activity against all
the strains tested when compared to the parent compound PAβN (1; Figure 1), while retaining the broad‐spectrum
inhibitory characteristics.102 In addition, compound 71 (Figure 21), with improved stability to serum proteases due
to the N‐methyl functionality, was assessed for its activity in an in vivo murine neutropenic thigh model against
P. aeruginosa PAM1032. When administered in combination with levofloxacin, a 3‐log reduction in CFU was
observed for approximately 4 hours followed by regrowth. In contrast, when no EPI was present, bacterial growth
was similar to that of the untreated control animal.102 The initial SAR suggested that (i) a basic amino acid with a
primary amino group is a prerequisite for biological activity; (ii) ornithine could replace Arg; (iii) methylation of the
NH group that links both amino acids increased the serum stability of the compound without affecting its potency;
(iv) replacement of an L‐ by a D‐amino acid is acceptable and also increases serum stability; (v) an appropriately
substituted aromatic amino acid and the appropriate length of its side chain is important for the activity (replacing
Phe with homoPhe resulted in improved potency); (vi) the best aromatic ring on the right hand side of the molecule
was shown to be 3‐aminoquinoline, like in 70 (Figure 21) which also showed less cytotoxic effects to mammalian
cells in vitro.102
PAβN (1; Figure 1) is able to restore the activity of many unrelated antibiotics, such as levofloxacin,
erythromycin, and chloramphenicol.22 The mechanism of action of PAβN and its analogs were at first proposed to
operate through competitive EPI. In this case, efflux pumps could recognize PAβN and its analogs as substrates
instead of the target antibiotics (eg, levofloxacin or ciprofloxacin) and, as long as they would expel these EPIs from
the cell, there will be no possibility of the antibiotic escaping outside the bacteria. Thus its intracellular
concentration would remain high, enabling an effective therapeutic outcome.22,246 However, as shown recently by
molecular dynamics simulation experiments and docking studies, PAβN most probably induces conformational
changes in the substrate extrusion channel, and thereby preventing the binding of other substrates to this
28 | LAMUT ET AL.

FIGURE 21 Structures of peptidomimetics with potent MexAB‐OprM inhibitory activity

site.141,146 In addition, PAβN could also straddle and prevent the movement of the G‐loop, an important gateway
that facilitates substrate extrusion.72,247
To overcome the drawbacks of the previously described analogs, compound 72 (Figure 21) was synthesized by
the same research group,248 incorporating all the known SAR findings and maintaining all the favorable biological
features of the original series, including potentiation of levofloxacin activity against PAM1032. On the basis of the
activity of some more derivatives, an important conclusion from this study was that a peptide backbone is not
essential for activity. It was suggested that a dicationic compound with an appropriate range of lipophilicity,
coupled with the topological features of compound 72, could be favorable for inhibiting P. aeruginosa MDR efflux
pumps.248
However, when administered by intravenous bolus injection, many of the key peptidomimetic compounds,
including PAβN and 72 (Figure 21), displayed appreciable toxic properties in mice and rats, with LD50 < 25 mg/kg. In
general, the less lipophilic the compounds were, the less toxic they were, but also less potent. The basic residue of
the ornithine group was recognized as the major contributor to the observed toxicity. Since this functional group is
also responsible for the activity, Renau et al successfully uncoupled the two effects by modifying the ornithine
group to give proline derivatives. The most promising example was compound 73 (Figure 21), which was more than
fourfold less toxic in rodents than 72, together with a retained broad‐spectrum and potency against all the
P. aeruginosa strains tested, namely MexAB‐OprM, MexCD‐OprJ, and MexEF‐OprN. In addition, 73 displayed
improved pharmacokinetics and similar efficacy in combination with levofloxacin in an in vivo model of infection.243
Furthermore, inhibitor 74 (Figure 21) was identified through a screening of various peptidomimetic analogs.
It showed an MPC8 of 1.19 µM for levofloxacin against P. aeruginosa PAM1723 (a strain that overexpresses
MexAB‐OprM). Although it was not the most potent analog, it displayed, in particular, a favorable rat serum
pharmacokinetic profile (free drug clearance 0.81 L/h/kg with Cmax 4.0 µg/mL at 2 mg/kg dose).73,249
Currently, PAβN derived EPIs are the most studied and developed family of inhibitors against P. aeruginosa and
other efflux systems. SAR studies are quite complete and AcrB has been successfully cocrystallized with a variety of
substrates.128,136,144,250–252 The main advantage is that it is difficult for bacteria to develop resistance against this
type of compound, since any mutation of the efflux pump will also render the pump unable to expel the substrate
(antibiotic) effectively from the bacterial cell. The main drawback, however, still remains the issue of tissue
LAMUT ET AL. | 29

accumulation and subsequent toxicity, along with the relatively high concentrations needed for effective inhibition
of RND efflux pumps (eg, 50 µM), which hinders their clinical application and further development as drugs.27,69,72

5.3.2 | Pyridopyrimidinones
From the original family of diamine EPIs, such as the peptidomimetics described above, several other structural
classes have been explored in extensive design, synthesis and characterization studies.103,253–258 Nakayama et al
reported compound 75 (Figure 22) as the first example of a MexAB‐OprM specific EPI.103 It was discovered in a
high‐throughput screening campaign for levofloxacin potentiation, using the PAM1032 strain. The activity is
impressive, with an MPC8 of 1.55 µM for 70% bacterial growth inhibition. Moreover, 75 was not active against
MexCD‐OprJ, MexEF‐OprN, and MexXY‐OprM overexpressing strains, which indicates its MexAB‐OprM specific
inhibition. However, the physicochemical properties of this molecule are problematic (poor water solubility, high
lipophilicity, high affinity for serum albumin) and it lacked in vivo activity in a murine sepsis model. Using
pharmacophore modeling it was shown that a thiazole moiety and an acidic group in appropriate positions were
essential for activity. Therefore, while maintaining all these features, the central molecular scaffold was redesigned
and the pyridopyrimidinone derivatives were discovered. The rationale was to introduce a scaffold that is
commonly observed in the therapeutic area of anti‐infectives and will, as a consequence, most probably display
favorable physicochemical properties and pharmacokinetic profile. In particular, the best compound in this series
was 76 (Figure 22), with an MPC8 of 27.1 µM for levofloxacin against PAM1032. It also showed reduced binding to
serum proteins. Although 76 was not the most potent analog, it displayed the best in vivo efficacy with 100%
survival rate after 7 days at a 50 mg/kg dose, in combination with a 15 mg/kg dose of levofloxacin in a murine

FIGURE 22 Pyridopyrimidinones as effective MexAB‐OprM inhibitors

30 | LAMUT ET AL.

neutropenic sepsis model with PAM1723.253 Extensive SAR studies were performed, leading to the discovery that
incorporation of a hydrophobic moiety at the 2‐position of the pyridopyrimidinone scaffold significantly enhances
activity due to its occupying an additional hydrophobic binding pocket. In this context, 6‐membered cyclic amines
such as morpholine and piperidine derivatives (eg, 77; Figure 22) generated the most active compounds. In addition,
hydrophilic substitution of a piperidine moiety at the 2‐position was feasible without compromising activity and the
latter was therefore recognized as a potential site for the enhancement of aqueous solubility.254
Several compounds were then prepared with improved biological properties, including activity, stability, and
reduced toxicity.255–257 For example, vinyl tetrazoles with 2‐piperidinyl substituents, such as in 78 (Figure 22), and
acrylic acid derivatives bearing aryl residues in place of piperidine, such as in 79 (Figure 22), were prepared.
Compound 78 showed high potency in vitro, but the solubility was very low. In contrast, 79 had improved solubility
and showed a reasonable safety profile in an acute toxicity assay, but displayed only moderate activity in
vivo.255,257 Compounds 76 to 79 were all considered as MexAB‐OprM specific EPIs.257
Incorporating SAR knowledge accumulated from both series, a quaternary ammonium moiety was introduced
into the molecule 78 (Figure 22) to give compound D13‐9001 (2; Figure 1), which exhibited good efficacy in vivo, a
good safety profile in an acute toxicity assay and high solubility.258 Moreover, human serum albumin (HSA) did not
significantly reduce the in vitro activity of D13‐9001. The latter potentiated MIC of levofloxacin with MPC8 values
of 2.94 µM (without HSA) and 5.88 µM (in the presence of 0.125% HSA) against PAM1723 and the MIC of
aztreonam with MPC8 of 2.94 µM against the same strain. In vivo experiments showed a significantly improved
survival rate at the end of day seven, when used at 1.25 mg/kg with 1000 mg/kg of aztreonam, compared with
treatment with 1000 mg/kg aztreonam alone. This experiment was conducted in a lethal pneumonia model in rats
infected with wild‐type P. aeruginosa strain PAM1020 with a basal expression of MexAB‐OprM. Pharmacokinetic
parameters in rats were fairly linear, not affecting the serum levels of aztreonam. Plasma concentration of
D13‐9001 after 2 hours was 0.262 µg/mL at 1.25 mg/kg dose. The calculated half‐life was 0.23 hours. In addition,
fairly linear pharmacokinetic parameters were also reported in monkeys, with serum levels exceeding those
observed in rats at the same administered dose of 5 mg/kg. The plasma concentration after 1 hour was 1.22 µg/mL
at 1 mg/kg dose and the calculated half‐life was 0.28 hours. According to pharmacokinetic studies, in vivo efficacy in
mammals is expected to be obtainable at even lower doses when compared to that in rats. D13‐9001 was not lethal
to mice at 100 mg/kg.258
The mechanism of action of D13‐9001 (2; Figure 1) was deduced in one of the first crystal structure studies of
inhibitor bound to an efflux transporter.140 Crystallographic data of D13‐9001 bound to MexB trimer have shown
that this inhibitor is inserted deeply and binds tightly to a narrow pit composed of a phenylalanine cluster located in
the distal pocket. In this context, pyridopyrimidine ring forms π–π interactions with the phenylalanine residues
without large conformational changes. As a consequence, D13‐9001 sterically hinders the functional rotation of
MexB. Moreover, it was also demonstrated that this molecule is only exported a little by efflux transporters (unlike
PAβN, 1; Figure 1), at least at the concentrations used for its inhibitory activity.140 In addition, crystal structure of
D13‐9001 bound to AcrB was solved, showing that binding sites for this compound in AcrB and MexB are
similar.140 D13‐9001 and other pyridopyrimidine derivatives are specific AcrAB‐TolC and MexAB‐OprM inhibitors
that do not inhibit MexXY‐OprM efflux system. The specificity is determined by fitting into the hydrophobic pit of
the distal binding pocket, which is in the case of MexY occupied by bulky indolyl moiety of tryptophan that
sterically hinders binding of these inhibitors.68,259
In conclusion, the advantage of specificity of D13‐9001 and other pyridopyrimidine analogs turned out to be
their main drawback and limitation for clinical usefulness to treat MDR P. aeruginosa infections. Inhibition of
MexXY‐OprM would, in this case, actually be beneficial, since intrinsic antibiotic tolerance and survival of
P. aeruginosa during infection is strongly correlated with the proper activity of MexAB‐OprM and MexXY‐OprM
efflux systems.68,159
LAMUT ET AL. | 31

5.3.3 | Natural products

Microorganisms have evolved to produce some interesting compounds that target MDR efflux pumps as a part of
their chemical arsenal for combating other competing species. Two of these are EA‐371α (80; Figure 23) and EA‐
371δ (81; Figure 23), which were identified through screening of a library of 78,000 microbial fermentation
extracts.260 They are produced by a strain of the Streptomyces family closely related to Streptomyces velosus , and
were demonstrated to be potent and specific P. aeruginosa MexAB‐OprM efflux system inhibitors, with MPC8
values of 4.29 µM (EA‐371α) and 2.15 µM (EA‐371δ) for levofloxacin against PAM1032. They did not show intrinsic
antibacterial activity in any of the strains tested. In an efflux pump substrate accumulation assay with the substrate
that is enzymatically hydrolyzed inside the cells to a fluorescent product, 80 was capable of increasing the
accumulation of the substrate in a dose‐dependent manner in PAM1032, but was devoid of activity in PAM1626
(triple‐pump‐deleted P. aeruginosa strain). In addition, both compounds were inactive against MexCD‐OprJ and
MexEF‐OprN analogoverproducing P. aeruginos strains. However, 80 is not suitable as a lead compound and was
shown to be moderately cytotoxic.260

6 | F UT U R E P E RS P E CTI VE S

EPIs have emerged as a research area largely in the past two decades. Interactions between antibacterial
compounds and efflux pumps are complex and the generation of effective EPIs is, therefore, a challenge.83 Due to
broad substrate specificities, MDR efflux pumps usually possess large binding sites, as was shown in the case of the
RND family of efflux transporters.68 There is no single property, according to molecular weight, logP, net charge or
polar surface area (PSA) that defines whether a compound will be an effective MES substrate or an inhibitor. In fact,
adequate inhibitory activity could be achieved by a compound that is actually an MES substrate and is
preferentially effluxed from the bacteria. Such “competitive substrate inhibitors” offer an opportunity to reverse
the classical approach of developing inhibitors and to design good efflux pump substrates that will saturate MES in
bacteria and thus restore the efficacy of available antibiotics when coadministered. At present, NorA inhibitors can
be generated only by the LBDD approach, since the crystal structure for NorA has not yet been resolved. Some
structural predictions have been made using homology models to define the substrate binding site,108 but the
mechanism underlying the ability of NorA to recognize and extrude an array of structurally and chemically
dissimilar compounds remains unknown. The lack of information regarding specific interactions between substrates
and EPIs with NorA also makes it difficult to rationally design potent inhibitors with computational methods.
However, the LBDD approach has been applied successfully in the case of 2‐phenylquinoline derivatives as NorA
inhibitors by Sabatini et al196,261 They used a library of the most potent NorA EPIs described in the literature to
build a pharmacophore model in an attempt to find their common characteristics. The generated pharmacophore
model revealed four chemical features of potent NorA EPIs: one H‐bond acceptor, one positive charge, one
aromatic ring and one other aromatic ring or hydrophobic region.261

FIGURE 23 Representative natural products displaying MexAB‐OprM inhibitory activity

32 | LAMUT ET AL.

To develop high potency inhibitors, it is most appropriate to start from small molecules that possess
physicochemical properties according to the “rule of 3” (molecular weight ≤ 300, clogP ≤ 3, rotatable bonds ≤ 3,
H‐bond donors ≤ 3, H‐bond acceptors ≤ 3, PSA ≤ 3), since they are suitable for the process of optimization. As an
example, we can point to some compounds, described in this article, like the INF series (INF240; Figure 3 and
INF55; Figure 4), that have served as starting points for the design of more promising NorA inhibitors, such as
piperine analogs and indole derivatives. The most potent NorA inhibitors described to date are compounds 5
(Figure 1) and 12 (Figure 4). Moreover, aryl quinolines (eg, 4; Figure 1) also constitute promising lead‐like scaffolds.
Comparison of the structural features of the most promising NorA inhibitors described in this article, underlines
the importance of a methoxy group, electron withdrawing groups (such as halogens, CN, or NO2) and aromatic ring
systems in achieving high potency. In particular, an indole nucleus, with electron withdrawing groups at C5 position
and a 2‐phenylquinoline nucleus are suitable scaffolds on which to design novel EPIs. However, optimization efforts
should not rely only on increasing potency toward a corresponding target, but should also take into account the
ADMET properties of synthesized inhibitors to obtain good clinical candidates. Therefore, structure‐property
relationships with problems of permeability and solubility also need to be addressed as being equally important in
the process of EPIs discovery.262
Alternatively, structure‐based drug design can be applied to identify AcrAB‐TolC and MexAB‐OprM inhibitors,
bearing in mind the structures of individual subunits of both tripartite efflux systems. Nevertheless, their structural
similarity could be used as advantageous in the design of broad spectrum EPIs, which could target both efflux
systems at the same time, thereby restoring the activity of a larger panel of antibacterials. Among the AcrAB‐TolC
inhibitors, a group of pyranopyridine derivatives has been clearly shown to be the most promising, with optimized
compounds 53 to 56 (Figure 14) as the most advanced examples. Undisputedly the best MexAB‐OprM inhibitors to
date are found in the series of pyridopyrimidinones, where extensive efforts were made to optimize an initially
discovered molecular scaffold. Above all, the most promising analogue, D13‐9001 (2; Figure 1), is a well‐optimized
molecule, which had good clinical candidate potential, but could not progress into clinics because it was able to
inhibit only P. aeruginosa efflux system MexAB‐OprM, but not MexXY‐OprM. Peptidomimetics however have been
studied in detail, with optimization of ADMET properties, starting from the basic PAβN (1; Figure 1) scaffold. PAβN
derived EPIs are currently the most studied and developed families of inhibitors against P. aeruginosa.
Many promising compounds have been patented, for example, pyranopyridines, peptidomimetics, and indole
derivatives.185,249,263 However, no EPI has been clinically approved to date, mainly because of low in vivo efficacy,
poor pharmacokinetic properties and/or toxicity problems.20,61,62 There have been some attempts to separate
toxicity issues from EPI activity at stages of lead optimization. For example, peptidomimetic analogs, in which the
basic ornithine group was recognized as the major contributor to the observed in vivo toxicity but was also crucial
for potentiation activity. After conformational restriction of the amino group to yield proline derivatives, these
compounds were less toxic but retained their potency (eg, compare 72 and 73; Figure 21).243 Bis‐indole derivatives
constitute another example in which an iodine atom was considered as a potentially labile functionality which could
manifest toxicity after its release. Newly synthesized potent analogs thus possessed a chlorine or bromine atom
instead (compare 17; Figure 5 and 5; Figure 1).184–186 In addition, the position of the methoxy group on the
2‐phenylquinoline nucleus was recognized as being unimportant in terms of toxicity, but piperidine in the side chain
was favored over a N,N‐diethyl group, thus creating safer compounds (4; Figure 1).199 Many natural products have
been investigated so far in the attempt to obtain less toxic inhibitors, but the main drawback of natural compounds
is their structural complexity, often associated with difficulties in establishing a good SAR, thus hindering their
further development. However, some of them, such as kaempferol rhamnoside (42; Figure 10), orizabin IX
(52; Figure 13) and ginsenoside Rh2 (51; Figure 13), possess quite good antibiotic potentiating activity. Other
natural molecules of lower complexity showed somewhat moderate activity, with the exception of MexAB‐OprM
inhibitors EA‐371α (80; Figure 23) and EA‐371δ (81; Figure 23). Nevertheless, many natural compounds have
served as model structural motifs for the development of more potent synthetically derived EPIs. As an example,
reserpine, piperine (9; Figure 4) and geraniol derivatives (Figure 19) can be quoted.
LAMUT ET AL. | 33

Besides the classical EPIs described above, antibacterially acting hybrid molecules bearing structural features
of both a representative antibacterial agent (eg, berberine, ciprofloxacin, and ofloxacin) and the corresponding EPI
(eg, INF55, INF271, the thiopyranopyridine moiety) showed some encouraging results such as compounds 21
(Figure 6) and 30 (Figure 7). Rapid consideration leads to the conclusion that these molecules act as dual inhibitors,
but their exact mechanism of antibacterial activity still needs to be elucidated in detail.
The structural similarities of EPIs described here, show some common features. For example, the indole moiety
appears to work well for both NorA and AcrAB‐TolC efflux pumps, as shown for the INF55 derivatives (Figure 5)
and other independently designed indole analogs derived by the quantum chemistry approach (57 and 58; Figure
16). In the same manner, quinolines are constituents of the most potent NorA inhibitors (4; Figure 1), but they were
also proven to be AcrAB‐TolC inhibitors. Interestingly, the quinoline moiety is also a part of the most active and
patented peptidomimetic analogs (72‐74; Figure 21). Furthermore, the p‐coumaric acid structural fragment is
present in the potent plant‐derived NorA inhibitor kaempferol rhamnoside (42; Figure 10). The coumarin
compound 43 (Figure 10) is also structurally similar to the p‐coumaric acid, as are also the chalcone derivatives
(45 and 46; Figure 11) that are NorA inhibitors. Comparison of the structures of the potent Gram‐negative EPIs,
such as MBX2319 (3; Figure 1), PAβN (1; Figure 1) and D13‐9001 (2; Figure 1), reveals that they all contain at least
two hydrophobic ring systems, which interact with hydrophobic amino acid residues in the substrate binding
site.221
Exploring EPI scaffolds further, pyranopyridines (Figure 14) possess a nitrile group attached to an aromatic
pyridine moiety, basic side chain nitrogens (eg, 53; Figure 14) and methoxy groups (eg, 54; Figure 14). In the case of
peptidomimetics (Figure 21), basic side chain nitrogen is again present, with a pyridine ring as part of the quinoline
moiety. The latter is similar in the case of pyridopyrimidinones (Figure 22), but these molecules possess instead
an acidic carboxyl or tetrazolyl side chain group. Interestingly, these properties are also present among promising
NorA inhibitors. The only exception here is the acidic groups that are found only in NorA molecular hybrids
(Figure 7). Some of these general characteristics are present in the structure of MP‐601,205 (6; Figure 1), a
compound that progressed into phase I of clinical development. In detail, it contains aromatic moieties including
two pyrimidine rings and possesses a methoxy group. At first sight, a sulfonamide functionality appears to be rather
unusual, but other sulfonamide EPIs have also been reported, as described in chapter 5.1.4 (eg, 34 and 35; Figure 8).
In summary, potent EPIs possess some general structural characteristics, which can be described as follows:
(i) they contain at least two hydrophobic ring systems, of which at least one contains a nitrogen atom, (ii) the ring
system can be quinoline, quinolone, benzene, pyridine, pyranopyridine, pyrimidine, pyridopyrimidinone, or indole,
(iii) electron withdrawing groups such as halogens, CN, and NO2 are attached to the ring systems and contribute to
the EPI activity, (iv) a methoxy group is often present, most frequently attached to the benzene or quinoline ring,
(v) a side chain contains either a basic amine group (secondary, tertiary, or primary), such as guanidine, piperazine
or piperidine, or an acid functionality, such as carboxylic acid or tetrazole (Figure 24).
In Gram‐negative bacteria, the design of novel EPIs still presents a great challenge, because penetration of the
outer membrane is controlled by porins, which favor zwitterionic and smaller hydrophilic molecules. In contrast,
the major RND efflux pumps prefer larger hydrophobic molecules. In this case, a suitable position on EPIs can be
identified by a rational design in which charged groups are attached where they would improve the penetration
into bacteria. However, compounds could also penetrate through the outer membrane of Gram‐negative bacteria
by routes other than those taken by porins, such as diffusion through a lipid bilayer.264,265
It is of great concern that many newly discovered antibacterial agents that carry the necessary structural features
for antibacterial activity are already substrates for efflux pumps, as shown recently by Omosa et al.266 The
compounds were more efficient in bacterial tests than standard drugs, but they should be combined with EPIs to
achieve sufficient effectiveness against MDR bacterial infections. In a recent study researchers determined molecular
properties that are important for activities of antibiotics in E. coli and P. aeruginosa. Molecular properties selected by
active efflux and the outer membrane barriers have been found to be different for the two pathogens. Moreover, in
several cases descriptor values corresponding to active antibiotics and significant barrier effects overlapped, which
34 | LAMUT ET AL.

F I G U R E 2 4 General structural characteristics of potent EPIs, identified by comparing different structural

classes of known NorA, AcrAB‐TolC and MexAB‐OprM inhibitors. EPI, efflux pump inhibitor [Color figure can be
viewed at wileyonlinelibrary.com]

highlights the synergy between the two barriers. Further, it suggests that molecular properties of antibiotics should
be optimized in parallel to achieve favorable outer membrane permeability and to evade efflux.267
It seems paradoxical that a single, low molecular weight and drug‐like compound could exist that would counter
the activity of drug efflux, when the MDR efflux pumps are well crafted to handle such structurally diverse
compounds.128 Resolved crystal structures and homology models enable a deeper insight into the structure and
function of the major efflux pumps present in WHO priority pathogens, which will hopefully yield a clinically useful
EPI. This review is a contribution to the search for novel antibacterial agents that provides an overview of the
current state‐of‐the art and some future directions for the design of EPIs. Although the therapeutic utility of EPIs
still needs clinical validation, this approach holds promise for prospective treatment of bacterial infections.

A C K N O W L E D GM E N T S

This study was supported by Slovenian Research Agency (Program Number: 0787‐P10208). The authors thank
Prof. Dr. Roger Pain for proofreading the manuscript.

OR CID

Andraž Lamut http://orcid.org/0000-0003-3147-4180

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A U T H O R’ S B IOG RA P HIE S

Andraž Lamut, MPharm, received his Master's degree in Pharmacy from the Faculty of Pharmacy, University of
Ljubljana. He is a PhD student at the Chair of Pharmaceutical Chemistry at the Faculty of Pharmacy, University of
Ljubljana. He is involved in the studies on how to overcome antimicrobial drug resistance. His research is focused
mainly on the design and synthesis of novel DNA gyrase B inhibitors with antibacterial activity and Hsp90
inhibitors as antiviral agents.

Prof Dr Lucija Peterlin Mašič is a full professor of Medicinal Chemistry and assistant professor of Toxicological
Chemistry at the University of Ljubljana, Faculty of Pharmacy. She obtained her PhD in 2003 in Medicinal
Chemistry under the supervision of Prof. Dr. Danijel Kikelj. She did a post‐doctoral education in 2009 in
AstraZeneca, Mölndal, Sweden, in the department for Safety Assessment under the supervision of dr. Scott
Boyer. After her PhD degree, she continued her research career in two research areas medicinal chemistry and
toxicology. In the field of medicinal chemistry, she is involved in studies how to overcome the resistance of
bacterial and cancer cells. In the last years, she has been focused on the design and synthesis of new DNA
gyrase B and Mur ligase inhibitors as new potential antibacterial compounds. In the field of toxicological
chemistry, she is involved in the introduction of different toxicological assays in the early stages of the drug
discovery programs. She has also studied different mechanisms of toxic action of endocrine disrupting
chemicals with different in vitro methods.

Prof Dr Danijel Kikelj obtained his PhD in pharmaceutical chemistry at the University of Heidelberg, Germany
in 1988. Since 2000 he has been working at the Faculty of Pharmacy, University of Ljubljana as full professor of
medicinal chemistry. Since 1999 he has been the head of a research group and the head of the research
program Medicinal chemistry financed by the Slovenian Research Agency. He has participated in two EU
projects from the 7th Framework Program and from Horizon 2020 and in ongoing Marie Sklodowska Curie
ETN INTEGRATE. He was one of the founders of Paul Ehrlich MedChem Euro‐PhD Network and acted as a
coordinator of the Network from 2012‐2015. The main areas of his research are medicinal chemistry,
LAMUT ET AL. | 45

peptidomimetics, structure‐based design of dual antithrombotic compounds, pharmaceutical leads based on

marine bioactive compounds and most recently discovery of inhibitors of bacterial topoisomerases with
activity against Gram negative bacteria.

Assoc Prof Dr Tihomir Tomašič received his degree in pharmacy in 2006 and his PhD in pharmaceutical
sciences in 2011 from the University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia. He did his
postdoctoral research at Inte:Ligand, Vienna, Austria, under the supervision of Prof Dr Sharon D. Bryant. At
present he is an associate professor of medicinal chemistry at the Faculty of Pharmacy, University of Ljubljana.
His main research interests concern computer‐aided design, synthesis and biological evaluation of biologically
active compounds, particularly with antibacterial and antiviral activity. His research focus at present is on the
development of DNA gyrase inhibitors targeting resistant ESKAPE pathogens and the identification of new
efflux pump inhibitors.

How to cite this article: Lamut A, Peterlin Mašič L, Kikelj D, Tomašič T. Efflux pump inhibitors of clinically
relevant multidrug resistant bacteria. Med Res Rev. 2019;1‐45. https://doi.org/10.1002/med.21591