4-Hydroxy-α-tetralone and its derivative as drug resistance reversal agents in multi drug resistant Escherichia coli

Article abstract of DOI:10.1111/cbdd.12263

The purpose of present investigation was to understand the drug resistance reversal mechanism of 4-hydroxy-α-tetralone (1) isolated from Ammannia spp. along with its semi-synthetic derivatives (1a-1e) using multidrug resistant Escherichia coli (MDREC). Th

Full text of DOI:10.1111/cbdd.12263

Chem Biol Drug Des 2014; 83: 482–492
Research Article
4-Hydroxy-a-Tetralone and its Derivative as Drug
Resistance Reversal Agents in Multi Drug Resistant
Escherichia coli
Gaurav R. Dwivedi1, Harish C. Upadhyay2,
Dharmendra K. Yadav3, Vigyasa Singh1,
Santosh K. Srivastava2, Feroz Khan3,
Received
4 July 2013, revised 15 October 2013 and
accepted for publication 15 November 2013
Nandan S. Darmwal4 and Mahendra P. Darokar1,*
The extensive use of antibiotics has raised serious health
concern throughout the world due to development of resis-
tance in bacteria against many classes of antibiotics (1).
Multidrug resistant (MDR) bacteria not only exert additional
burden of infection but also increase severity of infections
both in the hospital and in the community (2). MDR bacte-
ria over-express the efflux pumps, which expel a wide
group of unrelated compounds and antibiotics before they
reach the site of their action (3). In spite of outer mem-
brane barriers, the gram negative bacteria utilize efflux
pumps to achieve high degree of resistance (4). Hence,
these bacterial efflux pumps could be potential targets to
combat problematic infectious diseases caused by Escher-
ichia coli, Pseudomonas aeruginosa, Salmonella typhimuri-
um and Methicillin resistant Staphylococcus aureus (5).
1Molecular Bioprospection Department, CSIR-Central
Institute of Medicinal and Aromatic Plants, Kukrail Picnic
Spot Road, P.O. CIMAP, Lucknow, 226015, India
2Medicinal Chemistry Department, CSIR-Central Institute
of Medicinal and Aromatic Plants, Kukrail Picnic Spot
Road, P.O. CIMAP, Lucknow, 226015, India
3Metabolic & Structural Biology Department, CSIR-Centra
Institute of Medicinal and Aromatic Plants, Kukrail Picnic
Spot Road, P.O. CIMAP, Lucknow, 226015, India
4Department of Microbiology, Dr. R.M.L. Avadh University,
Hawai Patti, Allahabad Road, Faizabad, 224001, India
*Corresponding author: Mahendra P. Darokar,
mpdarokar@yahoo.com; mp.darokar@cimap.res.in
The purpose of present investigation was to understand
the drug resistance reversal mechanism of 4-hydroxy-a-
tetralone (1) isolated from Ammannia spp. along with its
semi-synthetic derivatives (1a–1e) using multidrug resis-
tant Escherichia coli (MDREC). The test compounds did
not show significant antibacterial activity of their own,
but in combination, they reduced the minimum inhibi-
tory concentration (MIC) of tetracycline (TET). In time kill
assay, compound 1 and its derivative 1e in combination
with TET reduced the cell viability in concentration
dependent manner. Compounds 1 and 1e were also
able to reduce the mutation prevention concentration of
TET. Both compounds showed inhibition of ATP depen-
dent efflux pumps. In real time polymerase chain reac-
tion (RT-PCR) study, compounds 1 and 1e alone and in
combination with TET showed significant down expres-
sion of efflux pump gene (yojI) encoding multidrug ATP
binding cassettes (ABC) transporter protein. Molecular
mechanism was also supported by the in silico docking
studies, which revealed significant binding affinity of
compounds 1 and 1e with YojI. This study confirms that
compound 1 and its derivative 1e are ABC efflux pump
inhibitors which may be the basis for development of
antibacterial combinations for the management of MDR
infections from inexpensive natural product.
Plants have always been a source of novel drugs and play
crucial role because their compounds may work as natural
blueprint or may be directly used as phytomedicine (6).
The plant secondary metabolites viz. terpenoids, alkaloids,
glycosteroids, flavonoids, polyphenols, tetralones etc. have
become alternate antimicrobial agents because of struc-
tural diversity. It has been observed that these plants
either contain antimicrobial compounds or possess com-
pounds that have no antibacterial activity of its own, but
possess the ability to enhance activity of antibiotics (7).
Synergism is known as the positive interaction of two
agents in combination, which exerts inhibitory effect
greater than the sum of their individual effects. This combi-
nation therapy has been useful (i) in the expansion of anti-
microbial spectrum (ii) prevention of the emergence of
resistant mutants and (iii) in minimization of toxicity. This
synergistic combination therapy may be used as an alter-
native to monotherapy for treating patients with invasive
and MDR infections (8).
The various species of genus Ammannia are frequently
used in traditional Chinese and Indian medicine (9–11). Am-
mannia baccifera Linn. is widely used in traditional Chinese
and Indian herbal formulations for the treatment of a number
of diseases including spinal disease, haemorrhoids, com-
mon cold, human female infertility and gastroenteropathy
Key words: 4-hydroxy-a-tetralone, ABC transporters, drug
resistance reversal, efflux pump, in silico docking, multidrug
resistant Escherichia coli
482
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12263

Synergistic Antibacterial Potential of Tetralones
Table 1: Antibiotic resistance/sensitivity pattern of different
cultures of Escherichia coli
(10). It has been reported to possess anticancer, antirheu-
matic, antidiuretic, antipyretic, antisteroidogenic, antimicro-
bial and rubefacient activities. Ammannia baccifera is also
used as an external remedy for ringworm and skin diseases
(11). In an earlier study, we reported bio-enhancing potential
of extracts and the compounds isolated from A. multiflora,
wherein the 4-hydroxy-a-tetralone and its various semi-
synthetic acyl and aryl derivatives showed bio-enhancing
potential against the nalidixic acid sensitive and nalidixic
acid resistant strains of E. coli (12). This prompted us to
carry out detailed biological investigations on 4-hydroxy-a-
tetralone and various semi-synthetic acyl/aryl derivatives
against the MDREC to investigate the possible mode of
action. This study is in continuation to our search for drug
resistance reversal agents from plants (12–14). The present
study reports drug resistance reversal potential of 4-
hydroxy-a-tetralone and its derivatives in combination with
TET using MDREC. The most active combinations were fur-
ther evaluated for time-kill kinetics, mutation prevention effi-
ciency, efflux pump inhibition, ATPase inhibition, real time
expression analysis and in silico docking studies.
MIC (lg/mL) of different antibiotics against sensitive,
mutants and clinical isolates of E. coli
Used
MDREC- MDREC- MDREC- MDREC-
antibiotics
KG4
KG1
EM5
EM7
MDSEC
Penicillin
400
400
50
200
200
50
100
25
50
12.5
6.25
25
50
3.125
6.25
6.25
0.39
6.25
12.5
6.25
6.25
50
Ampicillin
Polymyxin-B
12.5
25
Nalidixic acid 400
200
25
Ofloxacin
50
1.56
12.5
12.5
25
0.78
25
Rifampicin
100
25
Streptomycin 200
200
50
12.5
12.5
12.5
100
0.19
Kanamycin
Tetracycline
Erythromycin
Imipenem
100
800
200
0.19
400
200
0.19
25
100
0.19
0.19
1000–1.9 lg/mL and evaluated against test organisms. Min-
imum inhibitory concentration (MIC) values were determined
by 2-fold serial dilution broth assay with starting inoculums of
5 9 105 CFU/mL, incubated at 37 °C for 24 h and detected
from the observatory data as per CLSI guidelines (15). Exper-
imental observations were performed in triplicate to rule out
any error during the procedure. The antibiotics, tetracycline
(TET) and ofloxacin procured from Sigma Aldrich (USA) were
used as a positive control.
Methods and Materials
Bacterial strains and culture media
Multi drug resistant Escherichia coli clinical isolates
MDREC-KG4 and MDREC-KG1 were obtained from
Department of Microbiology, King George Medical Univer-
sity Lucknow, India whereas MDREC-EM5 and MDREC-
EM7 were the multi drug resistant mutants of E. coli iso-
lated from drug sensitive strain MDSEC trough ethyl
methyl sulfonate induced mutagenesis (13,14). Drug sensi-
tive E. coli (MDSEC, MTCC 739) was procured from
Microbial Type Culture Collection, Institute of Microbial
Technology Chandigarh, India. Antibiotic resistance/sensi-
tivity pattern of different E. coli strains is presented in
Table 1. Standard Mueller–Hinton agar and broth (MHA
and MHB, Hi-Media, Mumbai, India) were used as bacte-
rial culture media. Mueller Hinton Broth No. 2 (Control
Cations) was used for combination study. Colony counts
were determined using MHA plates.
Combination study
Combination studies were performed by broth checker-
board method (16). Cation-adjusted Mueller-Hinton broth
(150 lL) was added to each well of the 96-well plate. The
last four columns served as controls for E. coli growth and
plate sterility. The final concentrations ranged from 800 to
6. 25 lg/mL for TET and from 100 to 0.78 lg/mLfor test
compounds. Thus, each of the 64 wells had a unique com-
bination of antibiotics and test compounds. The final bacte-
rial inoculum in each well was 5 9 105 CFU/mL, except
the negative control. The plates were incubated at 37 °C
for 24 h. The MIC for each combination was recorded as
the last dilution without any turbidity as per CLSI guidelines.
Results were recorded in terms of fold reduction and frac-
tional inhibitory index. The interaction of each combination
was based on the fractional inhibitory concentration (FIC)
and fractional inhibitory concentration index (FICI). As clas-
sically defined, FICI of < 0.5 represented synergism, where
as FICI of 0.5–4.0 represented typical additive action, while
FIC index of > 4.0 represented antagonism (17). FICI was
calculated by adding the FICs (MIC of drug A in combina-
tion with drug B divided by MIC of drug A alone).
Test compounds and antibiotics
The compound 4-hydroxy-a-tetralone (1) a natural product
was earlier isolated from of Ammannia spp. The chemical
derivatization of compound 1 into its five acyl and aryl
derivatives; myristoyl (1a), palmitoyl (1b), cinnamoyl (1c),
m-anisoyl (1d) and 3, 4, 5-trimethoxy benzoyl (1e) was car-
ried out according to method available in our earlier publi-
cations (10,12). Chemical structures are given in Figure 1.
Determination of minimum inhibitory
concentration
The test compounds 4-hydroxy-a-tetralone (1) and its
derivatives were diluted into final concentrations of
Time kill assay
Time kill study of TET alone and in combination with
compound 1 and its derivative 1e against MDREC-KG4
was conducted using a previously reported method (18)
Chem Biol Drug Des 2014; 83: 482–492
483

Dwivedi et al.
Figure 1: Chemical structure of natural compound (1) and its semi-synthetic derivatives (1a–1e).
at different concentrations (TET 100, 200, 400, 800, 1600
and 3200 lg/mL). These concentrations were equivalent
to 1/8 MIC, 1/4 MIC, 1/2 MIC, MIC, 2MIC and 4MIC.
Each analysis was done in triplicate with a control. Time
kill curves were derived by plotting log10 CFU/mL against
time (h). Time kill kinetics was also studied in combina-
tions of antibiotic and the test compounds at the reduced
concentrations at which maximum synergy was observed.
25 lg/mL each. The EB-loaded bacterial suspension were
centrifuged at 16 060 9 g for 3 min, the supernatant
discarded and the pellet re-suspended in cold PBS (19).
The tubes then placed on ice. Aliquots of 0.095 mL of the
bacterial suspension were distributed to 0.3 mL 96 well
plates. Loss of fluorescence was recorded for 30 min at a
regular interval of 1 min at the excitation and emission
wavelength of 530 nm and 585 nm respectively using
spectrofluorometer (FLUO star omega; BMG Labtech,
Offenburg, Germany).
Selection of resistant mutants in vitro
The mutation prevention concentration (MPC) of TET
against E. coli MTCC 739 was determined as described
ATPase inhibitory assay
previously (19).
A
bacterial suspension of 1010 CFU
Bacterial membrane proteins were isolated by an earlier
reported method (21). ATPase assay was carried out in a
96 micro plate format using QuantichromTM ATPase Assay
kit (BioAssay Systems, Hayward, CA, USA). Initially optimal
enzyme concentration was determined by a series of dilu-
tions of enzyme (membrane protein containing ATPase) in
assay buffer. Enzyme and inhibitor was incubated first for a
certain period of time, before adding the substrate. Reac-
tions were set up to 40 lL containing 20 lL Assay Buffer,
5 lL enzyme(20 lg/lL), 5 lL Inhibitor, 10 lL (4 mM ATP)
and control with no inhibitor in separate wells. At the end of
reaction, 200 lL reagent was added and incubated for
30 min at room temperature. The improved malachite
green reagent forms a stable dark green color with liberated
inorganic phosphate(Pi), which was measured spectropho-
tometrically on a plate reader (620 nm) (22).
(0.1 mL) was plated onto MHA containing TET concentra-
tions equal to 29, 49, 89 and 169 MIC. The same con-
centrations of tetracycline were also tested in the
presence of compounds 1 and 1e at 25 mg/L. The muta-
tion frequency was calculated by counting the total num-
ber of colonies appearing after 48 h of incubation at 37 °C
on the drug-containing plate and then dividing the number
by the total number of CFU plated.
Ethidium bromide efflux studies
The fluorometric determination of ethidium bromide efflux
was performed as per reported method (20). To get meta-
bolically active cells the culture of MDREC-KG4 was grown
in 10 mL MHB (pH 7.3 Æ 0.2) with optical density (OD) of
0.6 at 600 nm. The cells were collected by centrifugation
at 16 060 9 g for 3 min and washed with phosphate buf-
fer saline. Ethidium bromide (25 lg/mL) wad added in the
bacterial suspension and incubated for 60 min at 25 °C in
the absence/presence of test compounds (1 and 1e) at
qRT– PCR analysis of efflux pump gene
The transcriptional profile of efflux pump gene (yojI) encod-
ing multidrug ATP binding cassettes (ABC) transporter
484
Chem Biol Drug Des 2014; 83: 482–492

Synergistic Antibacterial Potential of Tetralones
protein was analyzed in treated and non treated cells of
MDREC-KG-4. Cells were grown to mid-log phase in the
presence of sub-inhibitory concentration (1/4 MIC) of tetra-
cycline, compound 1 and its derivative 1e alone and in
combination. The real-time quantification of the RNA tem-
plates was analyzed by SYBR GreenER qPCR super mix
(Invitrogen, Grand Island, NY, USA) using 7900HT fast real
time PCR system (Applied Biosystems, Grand Island, NY,
USA). Observations were recorded in terms of LogRQ after
normalization of indigenous gene (GAPDH) expression.
Results
Two clinical isolates and 2 mutants were found to be
resistant to all the clinically used antibiotics except imipe-
nem. The compound 4-hydroxy-a-tetralone (1) and its
semi-synthetic derivatives (1a-1e) exhibited MIC in the
range of 250–1000 lg/mL against multi drug resistant
clinical isolates MDREC-KG4 and MDREC-KG1, multi
drug resistant mutants MDREC-EM5, MDREC-EM7 and
one multi drug sensitive MDSEC strain of E. coli and the
results are presented in Table 2. When these compounds
were tested in combination with TET, the MIC of TET
was reduced up to eight fold. Further, these observations
were validated by using another antibiotic ofloxacin
against all multi drug resistant strains of E. coli wherein
reduction in the MIC of ofloxacin up to 8 folds was
recorded (Table S1). On the basis of FICI, compounds 1,
1c and 1e showed synergistic interaction with TET
(Table 2). Compound 1e exhibited best interaction with
TET in terms of reduction in the MIC against MDREC-
KG4 and MDREC-KG1 that showed high level of resis-
tance towards TET.
In silico docking study
Protein structure prediction of YojI using
homology modeling
Homology model was developed for YojI protein of E. coli
(23), since no crystallographic data is available at present
in Protein Data Bank (PDB) (www.rcsb.org) for molecular
docking studies by employing GENO3D, an automatic
web server for protein structure modeling (www.geno3d-
pbil.ibcp.fr). Template protein maltose/maltodextrin transport
ATP-binding protein (acc.No. NP_142200.1) showed 32.9%
sequence identity with YojI a multidrug ATP binding cassettes
(ABC) transporter protein (acc. No. YP_490449.1). Best
model based on highest number of residues present in the
allowed region of Ramachandran plot was selected.
The treatment of MDREC-KG4 with TET at various con-
centration (1/8MIC, 1/4MIC, 1/2 MIC, MIC, 2MIC and
4MIC) reduced the viability of E. coli significantly at MIC,
2MIC and 4MIC (Figure 2A). However, the reduction in via-
bility of MDREC-KG4 could be achieved at lower concen-
trations (1/4MIC and 1/8MIC) of TET when tested in
combination with compound 1 and 1e (Figure 2B).
In silico molecular modeling and docking
parameters
TET at 50 lg/mL (8 9 MIC) did not allow the growth of
E. coli MTCC 739 indicating its mutation prevention con-
centration. However, MPC was reduced to 25 lg/mL and
12.5 lg/mL when tested in combination with compound 1
and 1e respectively (Table 3).
Molecular docking, construction, geometry optimization
and energy minimization of reserpine a well established
efflux pump inhibitor was used as control (24–27) and the
under study compounds 1 and its derivatives 1a–1e was
performed with YojI receptor by using the Sybyl-X v1.3
molecular modeling and drug discovery software (Tripos
International, USA). All the molecules were initially designed
in Sybyl, while the molecular construction, geometry optimi-
zation, and energy minimization process was performed by
using HP XW4600 workstation, running the Red Hatꢀ
Enterprise Linux 4.0 (32-bit) operating system (Silicon
Graphics Inc., Fremont, CA, USA). The Tripos force field
with a distance-dependent dielectric and Powell gradient
algorithm with a convergence criterion of 0.001 kcal/mol
was used for optimization. Partial atomic charges were
To evaluate the efflux pump inhibitory effect, compounds 1
and 1e were subjected to fluorescence based ethidium
bromide efflux assay using MDREC-KG4. As evident from
Figure 3, significant decrease in fluorescence was
observed in non treated control cells. While in presence of
compounds 1 and 1e, the loss of fluorescence was signifi-
cantly reduced (Figure 3).
Further, both compounds 1 and 1e were found to inhibit
ATPase at 25 lg/mL concentration at which optimal
reduction in the MIC of TET was observed (Figure 4). The
known drug reserpine also inhibited ATPase at 25 lg/mL
indicating involvement of these compounds in the inhibition
of ATP dependent efflux pumps.
€
assessed by using Gasteiger-Huckel method. 2D structures
were converted to 3D structures using the program Con-
cord v4.0 and maximum number of iterations performed in
the minimization was set to 2000. Further geometry optimi-
zation was done through MOPAC-6 package using the
semi-empirical PM3 Hamiltonian method. To find the possi-
ble bioactive conformations of control, compound 1 and its
derivative 1e, the Sybyl-X v1.3 interfaced with Surflex-Dock
program was used to dock the ligands into the active site
of E. coli YojI protein. During docking procedure all param-
eters were assigned to there default values (28).
ATP dependent efflux pump inhibition was further validated
by studying the expression of multidrug ATP binding cas-
settes (ABC) transporter protein gene (yojI). As evident from
Figure 5, treatment of TET (1/4 MIC) increased the expres-
sion of yojI. On the other hand, treatment of 1e significantly
Chem Biol Drug Des 2014; 83: 482–492
485

Dwivedi et al.
Table 2: In vitro interactions of plant compounds and TET against different cultures of Escherichia coli
MIC (lg/mL)
MIC (mM)
Alone
Reduction (n-
folds) in the
MIC of TET
Organisms
Agents
Alone
Combination
Combination
FICI
Outcome
MDREC-KG4
TET
1
800
500
1000
1000
500
1000
250
800
–
1.801
3.086
2.688
2.500
1.712
3.378
0.702
1.314
0.900
3.086
2.688
2.500
1.712
3.378
0.702
1.314
0.056
3.086
2.688
2.500
1.712
3.378
0.702
1.314
0.028
3.086
2.688
2.500
1.712
3.378
0.702
1.314
0.014
3.086
2.688
2.500
1.712
3.378
0.702
1.314
–
–
<0.5
–
–
4
2
2
4
2
8
2
–
4
2
2
4
2
8
2
–
4
2
2
4
2
4
2
–
4
2
2
4
2
4
2
–
2
2
2
2
2
4
2
25/200
50/400
50/400
25/200
50/400
25/100
25/400
–
50/100
50/200
50/200
50/100
50/200
25/50
0.154/0.450
0.134/0.900
0.125/0.900
0.085/0.450
0.169/0.900
0.070/0.225
0.041/0.900
–
0.308/0.225
0.134/0.450
0.125/0.450
0.171/0.225
0.169/0.450
0.070/0.112
0.082/0.450
–
0.154/0.014
0.134/0.028
0.125/0.028
0.085/0.014
0.169/0.028
0.070/0.014
0.082/0.028
–
0.154/0.007
0.134/0.014
0.125/0.014
0.085/0.007
0.169/0.014
0.070/0.007
0.082/0.014
–
Synergy
Addition
Addition
Synergy
Addition
Synergy
Addition
–
Synergy
Addition
Addition
Synergy
Addition
Synergy
Addition
–
Synergy
Addition
Addition
Synergy
Addition
Synergy
Addition
–
Synergy
Addition
Addition
Synergy
Addition
Synergy
Addition
–
1a
0.5–4.0
0.5–4.0
<0.5
0.5–4.0
<0.5
0.5–4.0
–
<0.5
0.5–4.0
0.5–4.0
<0.5
0.5–4.0
<0.5
0.5–4.0
–
<0.5
0.5–4.0
0.5–4.0
<0.5
0.5–4.0
<0.5
0.5–4.0
–
<0.5
0.5–4.0
0.5–4.0
<0.5
0.5–4.0
<0.5
0.5–4.0
–
0.5–4.0
0.5–4.0
0.5–4.0
0.5–4.0
0.5–4.0
1b
1c
1d
1e
RES
TET
1
MDREC-KG1
MDREC-EM5
MDREC-EM7
MDSEC
400
500
1a
1000
1000
500
1000
250
800
25
500
1000
1000
500
1000
250
800
12.5
500
1000
1000
500
1000
250
800
6.25
500
1b
1c
1d
1e
RES
TET
1
50/200
–
25/6.25
50/12.5
25/12.5
25/6.25
50/12.5
25/6.25
50/12.5
–
25/3.12
50/6.25
50/6.25
25/3.12
50/6.25
25/3.12
50/6.25
–
25/3.12
50/3.12
50/3.12
25/3.12
50/3.12
25/1.56
50/3.12
1a
1b
1c
1d
1e
RES
TET
1
1a
1b
1c
1d
1e
RES
TET
1
0.154/0.007
0.134/0.007
0.125/0.007
0.085/0.007
0.169/0.007
0.070/0.003
0.082/0.007
Addition
Addition
Addition
Addition
Addition
Synergy
Addition
1a
1000
1000
500
1000
250
1b
1c
1d
1e
<0.5
0.5–4.0
RES
800
lowered the expression level of yojI in MDREC-KG4 alone
and in combination with TET.
with the target protein YojI. Docking results for control
compound reserpine against YojI showed significant bind-
ing affinity as indicated by total score of 6.5866 and
formed two hydrogen bonds with ARG-122 and THR-48
of length 2.2 and 1.8 respectively (Table 4).
Molecular docking study of compound 1 with YojI receptor
showed better binding affinity as indicated by Sybyl Sur-
flex-Dock scores (total score) of 5.2635 which is similar to
reserpine (control) and formed H-bonds (hydrogen-bonds)
ꢀ
of length 2.0, 2.5 and 2.2 A with the hydrophobic residues
Discussion
namely, ALA-140, ALA-142 and ARG-122. Similarly,
molecular docking results for compound 1e against target
protein YojI, also showed good binding affinity as indicated
by total score of 6.6932 which is similar to reserpine and
MDR organisms are known to exhibit high degree of resis-
tance to an array of drugs through the involvement of
ATP-dependent and ATP-independent efflux pumps (2,29).
In order to reduce the selection pressure of antibiotics on
microbes and for the better efficacy of existing antibiotics,
the need of the hour is to search for agents that are able
ꢀ
form H-bond of length 2.0 A with the hydrophobic residue
ARG-146. The other derivatives showed varying degree of
binding affinity (docking scores from 3.6546 to 4.5132)
486
Chem Biol Drug Des 2014; 83: 482–492

Synergistic Antibacterial Potential of Tetralones
Figure 2: Time–kill curves of
MDREC-KG4 showing the dose
dependent bactericidal effect of (A)
Tetracycline (B) Tetracycline in
combination with compounds 1
and 1e.
of hydroxyl group with cinnamoyl (1c) did not affect the
drug resistance reversal potential of parent compound (1).
But careful analysis of the combination study results
revealed that further substitutions of anisoyl (1d) benzene
ring by two more methoxy groups (3, 4, 5-trimethoxy ben-
zoyl derivative, 1e) exhibited two times increased drug
resistance reversal potential. Hence it may be concluded
that esterification of 4-hydroxy-a-tetralone (1) with 3, 4, 5-
trimethoxy benzoyl group significantly enhanced the drug
resistance reversal potential. On the basis of FICI, com-
pound 1 and its derivatives 1c and 1e synergistically
reduced MIC of TET, while other derivatives showed addi-
tive interactions. Several natural products and derivatives
have been reported that they did not possessed antibacte-
rial activity but in combinations they were able to reduce
the dose of partner drugs many folds (34,35). Compound
1 and its derivative 1e were further explored for their pos-
sible mode of action because they were found to be the
best in terms of reducing the MIC of TET.
Table 3: Mutation frequency of Escherichia coli (MTCC 739)
Mutation frequency of E. coli with TET alone and in
combination with compounds
2
4
8
16
Agents
MIC
MIC
MIC
MIC
MIC
TET
3.7 9 1010 1.2 9 1010 0.3 9 1010 <1010 <1010
(Alone)
TET + 1
1.4 9 1010 0.2 9 1010 <1010
<1010
2.3 9 1010 0.5 9 1010 <1010
<1010 <1010
<1010 <1010
<1010 <1010
TET + 1e 0.8 9 1010 <1010
TET
+ RES
to reverse the drug resistance by inhibiting these efflux
pumps. Such compounds, which do not possess antimi-
crobial activity of their own, but when given in combination
with an antibiotic, enhances the activity and availability
many folds (12,13,30,31). These compounds do not exert
any selection pressure and on the other hand, reduce
dosage of antibiotics many folds so that their ill effects are
minimized. Thus, these compounds substantially delay the
resistance development process and enhance the life-span
of novel and existing antibiotics (13,32).
These compounds not only increased the intrinsic suscep-
tibility of E. coli to TET, but also significantly reduced the
emergence of tetracycline resistant mutants of E. coli. The
combinations of TET with compounds 1 and 1e had bac-
tericidal effect and reduced the CFU, whereas TET alone
exhibited the same effect at higher concentration. Other
plant compounds such as piperine and capsaicin are
known to possess these properties (35,36).
On the basis of MIC data, the compound 1 and the semi-
synthetic derivatives 1a–1e were found inactive against all
tested organisms as their MIC was higher than that of
stringent activity criteria (33). In a combination study, the
compound 1 reduced the MIC of TET by four fold. This
prompted us to carry out chemical transformation of 1 and
study the structure activity relationship (SAR). Compound
1 was derivatized into five lipophilic acyl and aryl deriva-
tives, of which, myristoyl (1a), palmitoyl (1b) and m-anisoyl
(1d) derivatives reduced MIC of the TET by two fold i.e.
the drug resistance reversal potential was reduced to half
to that of the parent compound (1). However, substitution
Accumulation and efflux of ethidium bromide are the indi-
cators of efflux pumps involvement in multidrug resistance
development, particularly in gram negative bacteria such
as E. coli (37). In a fluorescence based ethidium bromide
efflux assay, compound 1e showed significant (even better
than reserpine) inhibition of the efflux pump. The efflux
pump inhibitors/modulators might be working through any
one or the combinations of; (i) altering regulatory steps that
govern the expression of efflux pumps, (ii) inhibiting the
Chem Biol Drug Des 2014; 83: 482–492
487

Dwivedi et al.
Figure 3: Inhibition of ethidium
bromide efflux by compounds 1
and 1e.
ABC family efflux pumps while others are nominal such as
P type ATPase, F type ATPases and H + ÀATPase (38).
The presence of higher number of ABC family efflux
pumps in E. coli encouraged us to search ATPase inhibi-
tors having similar properties like ouabain, reserpine and
phenothiazine (39–41).
Compounds 1 and 1e not only enhanced the intrinsic sus-
ceptibility of MDREC-KG4 towards TET, but also signifi-
cantly reduced the expression of ATP dependent efflux
pump system. Tetracycline is known to induce higher
expression of different efflux pump genes (42,43), but in
presence of 1 and 1e the expression level of yojI was sig-
nificantly down regulated when used alone or in combina-
tion with TET. YojI was studied because it is one of the
important ATP dependent multidrug ATP binding cassettes
(ABC) transporter proteins in E. coli (38). In our study,
compound 1 and 1e have affected the transcriptional reg-
ulation of yojI. It might be possible that the compounds
have additional effect either on functional assembly of the
multi-component pump, outer membrane channels, or col-
lapsing the energy of efflux pumps. RND efflux pumps (tri-
partite complex) have been reported as key players for
multidrug resistance in gram negative bacteria but other
efflux pumps are also crucial for the discovery of efflux
pump inhibitors/modulators (38,42–44).
Figure 4: ATPase inhibitory activity of compounds 1 and 1e.
functional assembly of the multi-component pump, (iii)
blocking the outer membrane channel, (iv) collapsing the
energy of efflux, (v) creating competitive or non-competitive
inhibition with a non antibiotic molecule to the affinity sites
of the efflux pump, and (vi) changing the chemical design
of previous antibiotics (3). Many plant compounds have
been reported to reduce the MIC of antibiotics through
involvement of either one or the combinations of the above
mechanisms (3,34–37).
In order to further support the above observation, molecu-
lar docking studies of compound 1 and its derivatives 1a–
1e were carried out by exploring the binding site interact-
ing residues. In docking site of compound 1 with YojI
receptor, the chemical nature of binding site residues
Further, to understand whether these compounds interfere
with ATP dependent efflux pump, compound 1 and its
derivative 1e were evaluated for ATPase inhibitory activity
in clinical isolate MDREC-KG4 because it showed resis-
tance to all the tested antibiotics. Compounds 1 and 1e
were found to inhibit ATPase, which indirectly measures
the activity of efflux transporters. Among 74 ATP depen-
dent transporters known so far in E. coli, 69 belong to
ꢀ
within a radius of 4 A from bound compound were: acidic
(polar, negative charged) e.g. GLU-94, GLU-144 (Glutamic
acid); basic (polar, hydrophobic and positive charged) e.g.
ARG-122, ARG-146 (Arginine), LYS-96, (Lysine); and
488
Chem Biol Drug Des 2014; 83: 482–492

Synergistic Antibacterial Potential of Tetralones
Figure 5: Expression pattern of
yojI transcript of MDREC-KG4 in
presence of TET, compounds 1
and 1e alone and in combinations.
Table 4: Comparison of docking score and binding site residues of studied compounds against the antibacterial target YojI receptor
Length of Amino acid residue No. of
S.
Total
score
hydrogen involved in Docking Hydrogen
ꢀ
ꢀ
No. Compound
Biding pocket residue in (4 A)
bond (A)
interaction
Bond (H)
1.
Compound-1 5.2635 LEU-86, GLU-94, LYS-96, ARG-122, ILE-123, ALA-140,
2.0
2.5
2.2
–
ALA-140
ALA-142
ARG-122
–
3
ALA-141, ALA-142, ALA-143, GLU-144, ARG-146
2.
3.
1a
1b
4.5132 THR-48, LEU-50, ARG-74, PHE-77, SER-78, ALA-79,
PHE-81, TRP-85, LEU-86, ALA-141, GLU-144, ARG-146
3.7412 THR-48, LEU-50, ARG-74, LYS-75, PHE-77, SER-78,
ALA-79,VAL-80, PHE-81, TRP-85, LEU-86, ALA-141,
GLU-144, ARG-146
–
–
–
–
4.
5.
6.
1c
1d
1e
4.2403 THR-48, LEU-50, SER-78, ALA-79, PHE-81, TRP-85,
LEU-86
3.6546 MET-45, THR-48, LEU-50, SER-78, ALA-79, PHE-81,
TRP-85, LEU-86
6.6932 SER-78, ALA-79, PHE-81, TRP-85, LEU-86, GLU-94,
LYS-96, ARG-122, ILE-123, ALA-137, ALA-140, ALA-141,
ALA-143, GLU-144, ARG-146.
–
–
–
–
1
–
–
2.0
ARG-146
7.
Reserpine
6.5866 MET-45, THR-48, LEU-50, ARG-74, SER-78, ALA-79,
VAL-80, PHE-81, TRP-85, LEU-86, GLU-94, LYS-96,
LEU-107, ARG-122, ILE-123, ALA-137, ALA-140,
ALA-141, ALA-143, GLU-144, ARG-146
2.2 1.8
ARG-122 THR-48
2
Surflex-Dock scores (total scores) were expressed in –log10(Kd) units to represent binding affinities.
hydrophobic e.g. LEU-86 (Leucine), ILE-123 (Isoleucine);
ALA-140, ALA-141, ALA-142, ALA-143 (Alanine), thus
compound 1 showed good binding affinity and strong
hydrophobic interactions which may lead to more stability
and activity (Figure 6A, Table 4).
hydrophobic and positive charged) e.g. ARG-122 (Argi-
nine); aromatic (hydrophobic) e.g. PHE-81, (Phenylalanine);
acidic (polar, negative charged) e.g. GLU-144 (Glutamic
acid); basic (polar, hydrophobic and positive charged) e.g.
ARG-146 (Arginine), LYS-96, (Lysine); and hydrophobic
amino acid TRP-85 (Tryptophan), therefore compound 1e
showed good binding affinity and strong hydrophobic
interaction with YojI receptor, indicating enhanced stability
and activity of this compound (Figure 6B).
In docking sites of compound 1e with YojI receptor, the
conserved binding site pocket amino acid residues within
ꢀ
a selection radius of 4 A from bound ligands were: nucleo-
philic (polar, hydrophobic) e.g. SER-78 (Serine); hydropho-
bic e.g. LEU-86 (Leucine), ILE-123 (Isoleucine); ALA-137,
ALA-140, ALA-141, ALA-143(Alanine); acidic (polar, nega-
tive charged) e.g. GLU-144 (Glutamic acid); basic (polar,
However, in case of docking pose of reserpine (positive
control) with target protein YojI, the conserved binding site
pocket amino acid residues within a selection radius of
Chem Biol Drug Des 2014; 83: 482–492
489

Dwivedi et al.
(A)
(B)
(C)
ꢀ
Figure 6: In silico molecular docking showing the binding site residues of YojI protein within a selection radius of 4 A from bound ligands
revealing the hydrophobic pocket of active conformation. Binding sites of: (A) compound 1, (B) compound 1e and (C) reserpine.
ꢀ
4 A from bound ligands were nucleophilic (polar, hydro-
Acknowledgments
phobic) e.g. SER-78 (Serine); hydrophobic e.g. LEU-50,
LEU-86, LEU-107 (Leucine), ILE-123 (Isoleucine), MET-45
(Methionine), THR-48 (Threonine), VAL-80 (Valine); ALA-
79, ALA-137, ALA-140, ALA-141, ALA-143 (Alanine);
acidic (polar, negative charged) e.g. GLU-94, GLU-144
(Glutamic acid); basic (polar, hydrophobic and positive
charged) e.g. ARG-74, ARG-122, ARG-146 (Arginine),
LYS-96, (Lysine); and aromatic (hydrophobic) e.g. PHE-81
(Phenylalanine), TRP-85 (Tryptophan). When docking
results of reserpine were compared with that of com-
pounds 1 and 1e, it was revealed that the basic and
hydrophobic residue arginine was common in all while
interacting with target protein (Figure 6C).
The authors are thankful to Director, CSIR-CIMAP for pro-
viding the facilities. Fellowship to GRD from CSIR, New
Delhi is gratefully acknowledged. Financial support from
CSIR Network project BSC 0121 is gratefully acknowl-
edged. The help of Dr. Mastan Singh and Dr M.K. Gupta,
King George Medical University, Lucknow in terms of pro-
viding multi drug resistant clinical isolates of E. coli is
gratefully acknowledged.
Conflict of Interest
The authors declare that they have no conflict of interest.
The observations recorded in this study justify the hypoth-
esis according to which, the molecules that target the cell
membrane or cell walls are most likely to synergize with
conventional antibiotics or antiseptics by inhibiting the
efflux pumps, weakening the cell envelope and increasing
cellular permeability (45). As already mentioned by Higgins
(2007), probably, the time has come not to fight on differ-
ent types of drug resistance but to find alternatives to
avoid and avert it (46). In this regard, these plants based
compounds will be a promising candidate as ABC trans-
porter blockers to manage the multidrug resistance.
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492
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