Identification of benzochromene derivatives as a highly specific NorA efflux pump inhibitor to mitigate the drug resistant strains of s. aureus?

Article abstract of DOI:10.1039/C6RA01981A

Increased expression of efflux transport proteins confers the Multi Drug Resistant (MDR) phenotype to drug resistant bacteria, which can be mitigated by efflux pump inhibitors (EPI). EPI's have the dual advantage of restoring efficacy of antibiotics and retarding the evolution of drug resistant mutants. In this study, 17 heterocyclic derivatives synthesized by a polyethylenimine (PEI) catalyzed one-pot protocol were evaluated for their EPI potential. Based on in silico studies and in vitro studies, 5 benzochromene (BC) based compounds (among 17 heterocyclic derivatives), were observed to significantly potentiate the effect of ciprofloxacin (CPX) and displayed a best modulation factor of 32, against the NorA overexpressed mutant SA-1199B. Toxicity analyses using the zebrafish model showed that, from all the benzochromene compounds evaluated, BC9 exhibited low toxicity. BC9 inhibited the NorA efflux pump at a minimum effective concentration (MEC) of 2 mg ml¹ (4.03 mM) and even at subinhibitory concentrations, and it was effective in reversing the CPX MIC of both the ATCC strain and a clinical isolate of MRSA by 128 and 4 fold respectively. Our results show that benzochromene derivative BC9 is a highly specific NorA inhibitor that can be employed to mitigate MDR strains of S. aureus that overexpress the NorA efflux pump.

Full text of DOI:10.1039/C6RA01981A

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Identification of benzochromene derivatives as a highly specific
NorA efflux pump inhibitor to mitigate drug resistant strains of S.
aureus.
Received 00th January 20xx,
Accepted 00th January 20xx
Asaithampi Ganesan,a‡ Lowrence Rene Christena,a‡ Himesh Makala Venkata Subbarao, a b
Ulaganathan Venkatasubramanian,a b Raman Thiagarajan,b Venkatabalasubramanian
Sivaramakrishnan a, Kabilan Kasilingam,a Nagarajan Saisubramanian,*ab and Subramaniapillai
Selva Ganesan*a
DOI: 10.1039/x0xx00000x
www.rsc.org/
Increased expression of efflux transport proteins confer Multi Drug Resistant (MDR) phenotype to drug resistant bacteria,
which can be mitigated by Efflux pump Inhibitors (EPI). EPI’s have a dual advantage of restoring efficacy of antibiotic and
retarding evolution of drug resistant mutants. In this study, 17 heterocyclic derivatives synthesized by Polyethylenimine
(PEI) catalyzed one-pot protocol were evaluated for its EPI potential. Based on in silico studies and in vitro studies, 5
benzochromene(BC) based compounds (among 17 heterocyclic derivatives), were observed to significantly potentiate the
effect of ciprofloxacin (CPX) and displayed a best modulation factor 32, against NorA overexpressed mutant SA-1199B.
Toxicity analyses using zebrafish model showed that, of the benzochromene compounds evaluated, BC9 exhibited low
toxicity. BC9 inhibited NorA efflux pump at a Minimum effective concentration (MEC) of 2µg/ml (4.03 µM) and even at
subinhibitory concentrations, it was effective in reversing CPX MIC of both ATCC strain and clinical isolate of MRSA by 128
and 4 fold respectively. Our results show that benzochromene derivative BC9 is a highly specific NorA inhibitor that can be
employed to mitigate MDR strains of S.aureus that overexpresses NorA efflux pump.
abrogate evolution of resistant mutants. In such a scenario, it
is imperative to urgently resort to alternate strategies to
curtail MDR bacteria. Hence of late, research focus has shifted
A Introduction
Multi drug resistant (MDR) bacterial strains render
antimicrobial agents ineffective and severely hamper effective
treatment which increases mortality and co morbidity. Hence,
World Health Organization (WHO) announced that MDR
strains are a great threat to public health. Among infections
caused by MDR strains, MRSA accounts for 80,461 invasive
infections annually in USA alone.1 Increased prevalence of
multi-drug resistance pathogens catalyzed by horizontal gene
transfer2 and fuelled by indiscriminate antibiotic use, cannot
be contained by the limited number of available antibiotics.
Above all due to evolutionary selection pressures, microbes
quickly develop resistance to even drugs like vancomycin3 and
lipopeptide daptomycin4 which were once considered to
from anti-bacterial agents to compounds that target drug
resistance mechanisms like beta lactamase inhibitors,5 efflux
pump inhibitors6 quorum sensing inhibitors, peptidomimetics,
phage therapies etc.7 Very recently both lytic and lysogenic
phages have been engineered with CRISPR Cas system to
selectively target antibiotic resistant bacteria.8
Drug resistance in microbial pathogens arises due to various
mechanisms like enzymatic inactivation, mutation of the target
site, decreased penetration or increased efflux,9 among these,
extrusion of diverse classes of structurally unrelated drugs
through the efflux pumps seems to be one of the favoured
mechanisms adopted by many pathogens which is evident
from the analysis of clinical isolates.6,10,11 A recent study has
shown that efflux serves as a primary mechanism to withstand
antimicrobial stress and confers resistance towards
fluoroquinolones and biocides in Staphylococcus aureus.12 The
ability of gram positive bacterium like S. aureus to thrive in gut
was recently attributed to the presence of MnhF efflux pump
that expels bile salts.13 Various reports have shown that efflux
pumps play an important role in virulence and lack of or defect
in efflux pump is closely associated with attenuated
virulence.14,15 By targeting efflux, EPI’s impart new lease of life
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to the existing array of antibiotic molecules that were initially
Benzochromene derivatives can be synthesized by low
rendered ineffective by MDR bacteria. By virtue of its ability to molecular weight triethylamine catalysed reaction under
increase intracellular drug concentration, EPI can resensitize sonication or reflux conditions.36 The previously reported
MDR bacteria to existing antibiotics and are likely to decrease amine catalysts have numerous disadvantages such as
the propensity of emergence of drug resistant mutants.16
difficulty in isolation of catalyst from the reaction medium,
toxic and volatile nature, difficulty in tuning the catalyst
Methicillin resistant Staphylococcus aureus (MRSA) in the form properties etc. Hence, we are also evaluating hyperbranched
of Health Care Associated Community Onset MRSA (HACO- polyethylenimine (PEI) as new catalyst to synthesise
MRSA), Heath care onset MRSA (HA-MRSA) and Community benzochromene derivatives. PEI shown in Figure 1 is non-toxic,
acquired MRSA (CA-MRSA) continues to be leading cause of hydrophilic, less-volatile highly branched polymeric amine
nosocomial infections in USA despite a substantial decrease in derivative with good catalytic potential. Moreover, the PEI
HACO-MRSA and HA-MRSA infections in 2011 relative to catalyst displayed good stability towards air and moisture and
2005.1 Estimates show that invasive MRSA infections account its properties can be altered favourably by functionalizing the
for 18,650 deaths annually.17 MDR phenotype due to efflux in amine groups.
gram positive bacterial pathogens is predominantly conferred
by Major Facilitator Superfamily (MFS) of efflux pumps Herein, we report hyperbranched PEI catalyzed novel
comprised of either 12 or 14 transmembrane segments. NorA, benzochromene derivatives by facile one-pot synthesis and its
NorB, MdeA and Tet38 are examples of MFS pumps in S. subsequent evaluation as a NorA efflux pump inhibitor in S.
aureus of which; NorA has been widely studied.18-20 Sampling aureus relative to reserpine using in silico & in vitro methods.
of MRSA from different geographical locations revealed that Mutation prevention concentration and mutation frequency
these strains typically upregulate NorA efflux pumps and were used to assess ability of putative EPI to resist generation
exhibit spat002 genotype.21 Many instances of treatment of drug resistant mutants. Membrane permeability and
failure in MRSA infections was attributed to the poor membrane integrity studies were used to discern mechanism
bactericidal activity of vancomycin,22 hence enhancing of action of non-toxic putative EPI. Finally as a proof of
bactericidal activity of antimicrobial agents like ciprofloxacin concept, the selected non-toxic EPI were evaluated to
(CPX) by combination therapy with putative EPI’s, will help in potentiate the effect of CPX against ATCC culture of MRSA and
better therapeutic management of MRSA infections.
a clinical isolate of MRSA. We have employed both cell culture
based and zebrafish based toxicity testing to evaluate the
NorA functions as a proton mediated antiporter and uses toxicity of the selected molecules.
diverse
structurally
unrelated
molecules
including
fluoroquinolones,23 biocides and dyes as its substrates.
B Results and Discussion
Diverse classes of molecules including plant metabolites
reserpine,24
piperine,25,26
capsaicin,26
organic
flavones,27
a
Chemistry
flavonolignans,28
heterocyclic
compounds
The PEI with number average molecular weight (Mn) 1200 was
used for our synthesis. The number of primary, secondary and
tertiary amine groups (NH2, NH and N) present in the PEI was
calculated from the inverted gate 13C-NMR spectroscopy. The
PEI was converted to the corresponding secondary PEI
derivatives (PEI-Bz) and tertiary PEI derivatives (PEI-Me) by
closely following the method previously developed in our
laboratory.37 The structure of the catalyst was given in Figure
1. Representative synthesis of benzochromene derivative was
given in Figure 1. The detailed information on characterization
of PEI and synthesis of PEI derivatives were given in the
supplementary information.
phenothiazines and thioxanthines29 piperazinyl linked
fluoroquinolones30 and even biogenic metal nanoparticles31
were earlier identified as NorA/efflux inhibitors in S. aureus.
The structural diversity of molecules that serve as NorA
substrates/inhibitors reveal its broad specificity and implies
that NorA should bind their substrates through hydrophobic
and electrostatic interactions rather than a precise network of
non-covalent interactions as suggested earlier.32,33 Broad
substrate specificity in turn affords credibility for screening
novel classes of inhibitors. In addition, many of the molecules
reported earlier were not taken up further due to their toxicity
and/or less potency to employ them clinically as observed with
reserpine.34 Thus there is need for potent NorA inhibitors with
lower inherent toxicity and better minimum effective
concentration (MEC) values.
Benzochromene derivatives have been identified to have
pronounced impact on multi-drug resistant tumor cells.35 In
general, multi-drug resistance mechanism in cancer cells were
circumvented by shutting-off ATP dependant efflux pumps.
Hence, it is of interest to investigate the efflux pump inhibitory
activity of benzochromene derivatives in
a highly drug
resistant S. aureus model strains.
2 | J. Name., 2012, 00, 1-3
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R1 R2
N
R1 R2
N
R3
N
(R1 = R2 = R3 = H)
PEI
Evaluation of EPI effect
N
N
N
R1
R1
PEI-Bz (R1 = CH2Ph, R2 = R3 = H)
PEI-Me (R1 = R2 = R3 = Me)
N
R3
N
n
N
R2
N
R2
R3
N
All the 17 derivatives synthesized belonging to 4 different
series viz., pyrazole (PZ), pyranopyrazole (PP), benzochromene
(BC), and cyclic ketone (CK) were screened for inhibitory
activity against efflux pump NorA by virtual screening. Earlier
report showed that 49 % (151/309) of clinical S. aureus strains
including MRSA typically overexpress NorA efflux pump38
hence, targeting NorA efflux pump could effectively target
MRSA strain of S. aureus. Since crystal structure of NorA, an
efflux pump belonging to MFS family has not yet been
determined, in silico screening was performed using model
generated by homology modelling. The structure was
modelled using its closest resembling homologue MFS (PDB ID:
R2
N
R1
R1
N
R2
N
R3
R2
CN
O
X
R1
CHO
R2
PEI-Me
Methanol
OH
R1
O
N
X
80° C:1.5 h
N
H
HN
H2
O
R1 = H; X = CH; R2
R1 = Br; X = CH; R2
R1 = Br; X = N; R2 = H (BC9)
=
=
); 3-OMe (
)
BC1
4-Cl (
BC2
); 4-Br ( ); 3-NO2
BC3
(
BC4
); 2,4-dichloro (
BC5
4-(2-naphthyl) (BC6); 4-Cl (BC7); 2,4-dichloro (BC8); 3-N (BC9)
Figure 1. PEI catalysed synthesis of benzochromene derivatives.
Among the three molecules, PEI-Bz was identified as suitable
catalyst for the synthesis of pyrazole (PZ), pyranopyrazole (PP),
and cyclic ketone (CK) derivatives and PEI-Me for the synthesis
of benzochromene (BC) derivatives. The catalytic efficiency of
PEI-Me in BC1 synthesis was found to be superior to the
conventional organic and inorganic bases (Ex: Piperidine, DBU,
pyridine, K2CO3 etc.). The detailed analyses of the results were
given in of the supporting information (Table S1). PEI
derivatives readily sequester acidic protons from active
methylene group substrates and thereby facilitate the facile
formation of PZ and CK in good yields. Similarly, three-
component reaction of active methylene group substrates,
aldehydes with malononitrile or 2-naphthol yielded PP and BC
respectively. After completion of reaction, the catalyst was
easily isolated from the reaction medium by filtration and was
reused up to five times without significant loss in catalytic
activity. The detailed information on screening of the reaction
conditions, title compounds synthesis, recyclability studies on
representative compounds were given in the supplementary
information. The synthesized target molecules are given in
figure 2.
3WDO). Based on in silico screening results, top 6 compounds
(binding energy higher -9.5 kcal/mol) and 3 compounds with
low binding energy (for validating the docking results) were
chosen for further in vitro studies (Table S2). Among the
selected compounds, two compounds belonged to cyclic
ketone (CK) series, one compound pertained to pyrazole (PZ)
series and rest of the five belonged to benzochromene (BC)
series. Compounds CK1, BC1 and BC3 binds to one site while
compounds BC2 & BC4 bind to a different site about 15 Å away
from this. One compound BC9 binds to a third site that is in
between to these two sites with a slight overlap to those sites
(Figure S1). As proposed earlier,27 hydrophobic interactions
contribute mostly to ligand binding with aromatic π-π
interactions as preferred interactions. Compound BC9 forms a
hydrogen bond with Ser215 side chain (Figure 3).
Anti-Staphylococcal Effect
While screening the chosen compounds for their EPI effect, it
becomes essential that they need to have lower intrinsic anti-
bacterial effect since it could interfere with the EPI
measurements. Hence, the selected 9 compounds were
evaluated for its anti-staphylococcal activity. All compounds
displayed an MIC of 64 µg/ml or > 64 µg/ml (Table S3) implying
that the all the selected compounds displayed minimal anti-
staphylococcal activity.
Pyranopyrazoles
Pyrazole derivative
Cyclic ketones
R
R
Cl
O
O
H3C
CH3
N
CH3
CN
NH2
N
N
N
N
N
OH OH
O
OH OH
Ph
Ph
Ph
R = Cl (CK1); H
(CK2); OCH3 (CK3)
R = Cl (PP1); NO2 (PP2);
CH3 (PP3); H (PP4)
PZ1
Benzochromens
R2
R1 = H; R2
=
4-Cl (
); 4-Br (
BC1
BC2
);
R1
3-NO2 (BC3); 2,4-dichloro (BC4); 3-OMe (BC5)
R1 = Br; R2 = 4-(2-naphthyl) (BC6); 4-Cl (BC7);
O
BC8
BC9
)
2,4-dichloro ( ); 3-N (
HN
H2N
O
Figure 2. Synthesized target molecules by polyamine catalysis
Figure 3. Benzochromene derivative BC9 interacts favorably with NorA homolog.
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Ciprofloxacin MIC reversal
(Figure S2) showed that BC3 was least toxic followed by BC2
and BC6 for the entire concentration range tested. Both BC1
and BC9 were toxic beyond 8 µg/ml (Figure S2). Since testing
toxicity in a whole animal model would be a holistic approach
relative to cell culture based testing,40 toxicity of all five
chosen compounds were evaluated in zebrafish model, which
we had recently adopted for evaluating toxicity and efficiency
of putative EPI’s. Fishes in groups of 5 were exposed to six
heterocyclic ring derivatives at a concentration of 16 µg/ml [8
times its minimum effective concentration (MEC)] the
compounds were dissolved in methanol and later dispersed in
water and toxicity due to compound was assessed by
mortality/ survival of the fishes within the group, 48 h post
exposure. Based on mortality based scoring BC9 was observed
to be non-toxic whereas rest of the compounds either
displayed reduced solubility and or exhibited varying level of
toxicity (Table S4). Interestingly BC2 and BC3 that was non-
toxic in cell culture testing were observed to be toxic in
zebrafish model. Whereas BC1, BC6 and BC9 that displayed
varying levels of toxicity were observed to be non-toxic in
zebrafish model (Table S4). This could be partly attributed to
the metabolism of the compounds by liver enzymes and partly
by excretion, which highlights the previous observation that
many compounds identified as non-toxic in cell culture fail in
clinical trials due to their toxicity in animal models.40 As BC9
was found to exhibit toxicity beyond 8µg/ml in cell culture but
was observed to be non-toxic in zebrafish model, we
performed detailed toxicity analysis of liver carboxylesterase
and brain acetylcholinesterase activities for BC9 in zebrafish as
reported in methods section. As shown in (Figure S3), BC9 did
not have any adverse effect on liver α- and β-carboxylesterase
activities (Figure S3.1) and brain acetylcholinesterase activities
(Figure S3.2) relative to their respective untreated control,
implying lack of any significant toxicity to zebrafish exposed to
BC9. Despite the fact that BC9 was toxic beyond 8 µg/ml in cell
culture, BC9 was chosen further because it was non-toxic in
both mortality based scoring and by liver and brain enzyme
assays in zebrafish model, which can be further accounted by
the fact that cell culture is a static system where the
compound does not get eliminated whereas whole animal
models are dynamic where the half-life of the compound is
determined by its metabolism/excretion from the system. In
addition minimum effective concentration (MEC) determined
by checkerboard method42 showed that BC9 had an MEC of
2µg/ml (Corresponding to ~4.03 µM), which is 4 times lower
than the concentration at which BC9 might pose toxicity to
cultured cells but not in a zebrafish model.
All nine chosen compounds were assessed for its ability to
potentiate the antibacterial effect of ciprofloxacin (CPX),
fluoroquinolone antibiotic and a known substrate for NorA efflux
pump.23 S. aureus strain SA 1199B (NorA ++, A116E Grl A) that
overexpressed NorA efflux pump was chosen for this study, apart
from overexpression of NorA efflux pump, this strain also carries a
mutation in its gyrase gene making it more resistant to CPX. We
surmised that if the tested compound(s) could potentiate effect of
CPX in pump overexpressed mutant (SA-1199B), it can effectively
mitigate other S. aureus strains. The selected compounds were
evaluated at 16 µg/ml, which is 1/4 of MIC of putative EPIs. Among
the nine compounds evaluated, five benzochromene compounds
(BC1, BC2, BC3, BC6, BC9) displayed a very high modulation factor
(MF) of 32, BC5 displayed a MF of 8. Cyclic ketone (CK1) showed a
very low MF of 2 whereas CK2 and pyrazole derivative (PZ1) did not
exhibit any change in CPX MIC and hence the MF was 1 (Table 1).
Table 1. Putative EPI’s identified by virtual screening potentiated
the effect of CPX against NorA overexpressed mutant (SA1199B).
Fold reduction
Ciprofloxacin
Ciprofloxacin
MIC
(µg/ml)
in Ciprofloxacin
MIC
Compound
designation
MIC in
combination with
BC 9 (µg/ml)
(Modulation
Factor)
CK1
CK2
BC1
BC2
BC3
BC5
BC6
BC9
PZ1
8
8
8
8
8
8
8
8
8
4
2
1
8
0.25
0.25
0.25
1
32
32
32
8
0.25
0.25
8
32
32
1
Since five out of six BC compounds were highly effective in
potentiating antibacterial effect of CPX by 32 fold; it is likely
that benzochromene backbone itself contributes to highly
specific NorA inhibitory effect. This corroborates with docking
results as these compounds have the highest binding energy
and, presence of benzochromene group could help in forming
the π-π interactions (Figure 3).
Previous report showed that 2- phenylquinone derivatives
caused a 16 fold CPX MIC reversal in SA-1199B.28 Similarly, an
earlier study had showed that celecoxib derivative
Pyrazolo[4,3c][1,2]benzothiazines 5,5-dioxide also caused a 16
39
fold MIC reversal for ciprofloxacin in SA-1199B On the other
hand with another S. aureus strain SA-K2378 that only
EtBr MIC reversal
overexpresses NorA efflux pump, without possessing
a
As efflux is the only known mechanism of resistance to EtBr41
reversal of EtBr MIC indicates role of tested compound as an
efflux pump inhibitor. Efflux inhibitory potential of the non-
toxic benzochromene derivative BC9 was evaluated by EtBr
MIC reversal studies in following S. aureus strains viz., NorA
overexpressed strain (SA-1199B), NorA knock out strain (K-
1758) and wild type strain of the same genotype (SA-1199).
EtBr displayed an MIC of 64 µg/ml with NorA overexpressed
mutation in gyrase gene like SA-1199B, a drastic 32 fold
reversal was observed.33
Toxicity Evaluation
We evaluated toxicity of the highly effective compounds (BC1,
BC2, BC3, BC6 and BC9) at a concentration range of 2-64 µg/ml
by MTT assay in human pancreatic cell lines. Our results
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strain, 32 µg/ml with wild type and 4 µg/ml with NorA knock
out strain. Based on the previous report that EtBr MIC of >
12.5 µg/ml implies increased probability for the
overexpression of efflux pump,21 it is likely that even wild type
strain might be upregulating efflux pumps. As expected, BC9
caused increased intracellular accumulation of EtBr and caused
a maximal MF of >32 in NorA overexpressed strain followed by
MF of 32 in wild type strain and relatively lower MF of >4 in
NorA knock out strain (Table 2). By virtue of being more
effective with NorA overexpressed strain and least effective
with NorA knock out strain, it is evident that BC9 is highly
specific inhibitor of NorA efflux pump.
b)
Table 2. Benzochromene derivative BC9 causes maximal reversal of
EtBr MIC in NorA overexpressed strain
EtBr +
EtBr MIC
(µg/mL)
BC9
MIC
Modulatio
n Factor
Strains
Figure 4: Benzochromene derivative BC9 inhibits EtBr efflux in wild type and
NorA overexpressed strain of S. aureus. Residual fluorescence of EtBr in a)
NorA overexpressed strain of S.aureus (SA1199B) and b) wild type S. aureus
(SA1199) over the time course of 0-10 min after 1h treatment with
BC9,BC5,CK2,PZ1 and reserpine. Data presented is the average from three
independent experiments. Error bars represent standard error of the mean. (BC
– Benzochromene, CK- cyclic ketone, PZ- Pyrazole and Res – reserpine.)
(µg/mL)
1
SA1199 (Wild type)
K1758 (NorA knock
out)
32
4
32
< 1
< 2
> 4
SA1199 B (NorA
Overexpressed )
64
> 32
residual fluorescence both in wild type and NorA
overexpressed strain, which was higher than that affected by
reserpine (Figure 4). On the other hand, among derivatives
with lower binding energy BC5 caused discernible efflux
inhibition in both wild type and NorA overexpressed strain but
the efflux inhibitory potential of BC5 was much lower than that
caused by either reserpine or BC9. Whereas PZ1 and CK2 failed
to inhibit efflux in NorA overexpressed strain but in wild type
strain a low but perceptible efflux inhibition was noted,
implying that both PZ1 and CK2 might inhibit efflux pump
other than NorA in S.aureus. Thus our RTE studies reveal that
BC9 is a better and non- toxic inhibitor of NorA relative to
reserpine.
EtBr MIC reversal for Nor A overexpressed strain by CK2, PZ1
and BC5 showed that CK2 and PZ1 caused a 2 fold reversal
whereas BC5 caused a 16 fold reversal in EtBr MIC which is in
line with in silico studies wherein compounds with lower
binding energies were on an average less effective in their
efflux inhibitory effect relative to those with higher binding
energy.
Real Time Efflux
In order to confirm and quantitate the EPI effect of BC9, Real
time efflux (RTE) studies were performed with both wild type
S. aureus (SA-1199) and NorA overexpressed strain of S. aureus
(SA-1199B). Reserpine a known a NorA inhibitor24 was used as
a positive control. Inhibition of EtBr efflux by the screened
compound results in higher residual fluorescence relative to
untreated cells. RTE studies show that BC9 causes enhanced
Dose response curve for EtBr accumulation showed that BC9
relative to reserpine caused 2-3 fold increase in EtBr
accumulation in the entire concentration range tested (2-
16µg/ml), which is 1/32 to 1/4 of its MIC (Figure 5a and 5b).
Lower minimum effective concentration (MEC) of 2 µg/ml
(4.03µM) displayed by BC9, its enhanced efflux inhibitory
effect relative to reserpine and its less toxic nature even at
higher concentrations tested (16 µg/ml) in zebrafish model
imply therapeutic potential of BC9.
a)
a)
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Figure 6: Benzochromene BC9 potentiates the bactericidal effect of ciprofloxacin in
NorA overexpressed mutant S. aureus (SA-1199B). NorA overexpressed S.aureus strain
was incubated along with Ciprofloxacin (4µg/ml) and Cipro in combination with various
derivatives. Cells were plated at various time intervals on TSA plates and colony counts
were estimated after 24h of incubation. (BC- Benzochromene; PZ- Pyrazole; CK- Cyclic
ketone)
b)
evaluated even though BC9 was used at 3 fold lower concentration
relative to reserpine (Figure 6). As expected, among
derivatives with lower binding energy, BC5 was less effective
than reserpine but was better than treatment with CPX alone,
whereas derivatives CK2 and PZ1 were unable to potentiate
the effect of CPX for the entire duration evaluated and in fact
by 24h, they displayed CFU equivalent to that of untreated
control. Thus based on time-kill studies, increased efficacy
displayed by BC9 at a 3 fold lower concentration and its low
toxicity in zebrafish model imply that BC9 is a more potent and
safe EPI than reserpine.
Synergy testing
Synergistic interactions of BC9 with CPX was determined by
checkerboard assay for all three S. aureus strains (NorA
overexpressed, knock out and wild type), and the results show that
BC9 did not exhibit any synergy with both NorA knock out (FIC index
=8.00) and wild type strain (FIC Index = 25.03), whereas it displayed
synergy with CPX against NorA overexpressed strain (FIC Index =
0.14). BC9 exhibiting synergy only with NorA overexpressed strain
but not with NorA knock out and wild type strain reveals that BC9 is
highly specific EPI of NorA efflux pump. During synergy testing by
checkerboard method we observed that BC9 at its MEC of 2µg/ml
(4.03µM) was observed to reduce MIC of CPX in NorA
overexpressed strain from 8µg/ml to 1µg/ml which underscores the
NorA efflux inhibitory potential of BC9
Figure 5: Benzochromene derivative BC9 causes dose dependent accumulation of EtBr
in Nor A overexpressed mutant strain of S.aureus (SA1199B). a) Dose-dependent
uptake of EtBr in NorA overexpressed strain of S.aureus (SA-1199B) in the presence of
a) BC9 and b) Reserpine (RES). The test compounds have been tested at the following
concentrations (1, 2, 4, 8 and 16 µg/ml). The experiments were carried out over 30min
of time and the residual fluorescence quantified by spectrofluorimetry with an Ex
530nm and Em 580nm. The error bars represents standard error of the mean from
three independent experiments.
Time-Kill studies
Time kill curve studies with NorA overexpressed strain showed
that CPX at 4 µg/ml concentration (Half the MIC) did not cause
significant bactericidal effect although it prevented further
growth for 24 h. However, when CPX (4µg/ml) was used in
conjunction with BC9 (8 µg/ml), more than 3 log fold reduction
in Colony forming units (CFU) was observed proving that BC9
has synergistic interaction with CPX and potentiates
bactericidal effect of CPX. The positive control reserpine
(25µg/ml) also potentiated the bactericidal effect of CPX
(4µg/ml), however, the potentiation of CPX caused by BC9 was
consistently higher than reserpine for the entire time range
EPI effect of BC9 against MDR strain
Since BC9 functioned as a highly specific inhibitor of NorA and
did not display synergy with NorA knock out strain, we were
interested in evaluating if it could indeed curtail MRSA strains.
Towards this end, ability of BC9 to cause CPX MIC reversal in
an ATCC culture of MRSA and a clinical isolate of MRSA was
evaluated. Our results showed that as expected, BC9 at 2X
MEC (4 µg/ml) re-sensitized MRSA to CPX and reduced MIC of
CPX in ATCC culture by 128 fold (from 32 to 0.25µg/ml) and in
clinical isolate by 4 fold (from 128 to 32 µg/ml) proving its
utility in a clinical setting to tackle MDR MRSA strains.
Although based on our data we can surmise that BC9 would be
effective as an EPI with NorA overexpressing strains of MRSA,
its efficacy against MRSA in vivo and also against strains that
overexpresses efflux pumps other than NorA remains to be
explored.
MPC and Mutation frequency
Mutation Prevention Concentration (MPC) is the lowest
concentration of the compound that fails to give rise to
mutants.43 MPC was tested for CPX alone and CPX in
combination with BC9 against wild type S. aureus and NorA
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uncorrected.
Subsequently NMR analyses (H1 & C13) were
determined by a Bruker Av-300MHz spectrometer. All the reactions
were conducted to the 10 ML round bottom flask with a magnetic
stirrer. Both PEI-Bz and PEI-Me are prepared by previously reported
procedure.37 Hyper branched polyamine with number average
molecular weight (Mn) 1200 was chosen for studies. The number of
primary (1o), secondary (2o), tertiary (3o) amine group present in the
hyperbranched polyamine is calculate based on inverted-gate 13C-
NMR spectroscopy.45
overexpressed mutant (SA-1199B) and we noted that MPC for
CPX was at 4 x MIC of CPX, whereas when used in combination
with BC9, MPC of CPX was reduced to 2x MIC for both wild
type and mutant strains. Similarly, BC9 in combination with 2x
MIC of CPX exhibited a significantly lower mutation frequency
-6
(1x 10-9) relative to treatment with CPX alone (8 x 10 for the
-6
wild type and 2.2 x 10 for NorA resistant mutant). By virtue
of exhibiting a lower MPC and mutation frequency BC9 as an
EPI can restrict the selection of CPX resistant mutants.
General procedure for the synthesis of cyclic ketons (CK 1-3) or
pyrazole (PZ1): A mixture of substituted aldehyde (1 mmol), 1,3
cyclohexadienone (or) 3-Methyl-1-phenyl-2-pyrazoline-5-one (348.4
mg; 2 mmol) and PEI-Bz polyamine catalyst (100 mg) were taken in
the 5 ml of ethanol and it was stirred at RT. The progress of the
reaction was monitored through thin layer chromatography (TLC)
using ethyl acetate and petroleum ether (3: 7) as eluent mixture.
After completion, the reaction mixture was poured in to crushed ice
and filtered through the Whatman filter paper. The solid mass was
collected and further stirred in hexane (1 x 10 mL) for 10 minutes
and filtered through Whatman filter paper. The product thus
obtained was essentially pure.
Membrane Permeability & Membrane Integrity
Membrane permeability assay was performed to assess
whether BC9 affects the membrane permeability/ membrane
integrity with propidium iodide (PI) in wild type S.aureus (SA-
1199) strain as reported earlier.44 Our results showed that
relative to untreated cells, BC9 treatment caused 36% increase
in membrane permeability and enhancement in permeability
was independent of dose dependent effect of BC9 (Table 3).
Thus it is likely that BC9 accesses its target efflux pump NorA
by altering bacterial cell membrane permeability.
Procedure for recyclability of PEI-Bz on CK1 synthesis
Table 3: Treatment with BC9 perturbs membrane
permeability in S.aureus
Synthesis of CK1 was carried out as mentioned in the above
section. After completion of reaction, the reaction mixture was
poured in to crushed ice and filtered through the Whatman filter
paper. The combined filtrate was concentrated under reduced
Average
Treatments
Fluorescence
Intensity (AU)
Permeability (%)
pressure and extracted with chloroform (2
× 15 mL). The combined
Untreated
organic extracts were dried over anhydrous sodium sulfate, filtered
and concentrated under reduced pressure. The recycled catalyst
was without further purification.
2.66 ± 0.13
5.59 ± 0.23
-
-
(Cells +PI)
CTAB Treated
Normalized
Fluorescence
(CTAB
General procedure for the synthesis of pyranopyrazoles (PP 1-4). A
mixture of aromatic substituted aldehyde (1 mmol), 3-Methyl-1-
2.93
100
phenyl-2-pyrazoline-5-one
(174.20
mg,
1mmol),
treatment)
BC9 (2µg/ml)
BC9 (4 µg/ml)
BC9 (8 µg/ml)
malononitrile(66mg, 1 mmol) and secondary polyamine (50mg)
were taken in the 3 ml of ethanol and was stirred at 80°C for 45 min
to 1 h. The progress of the reaction was monitored through thin
layer chromatography (TLC) using chloroform and methanol (9:1) as
eluent mixture. After completion, the reaction mixture was cooled
to room temperature and the reaction was quenched with distilled
water (5 mL) followed by extracted with ethyl acetate (2×5 mL),
washed with anhydrous brine solution and dried over sodium
sulphate. The organic layer was filtered and concentrated under
reduced pressure. The crude reaction mass further purified through
column chromatography (Chloroform: Methanol (9:1)).further
concentrated under reduced pressure. The crude reaction mass
further purified through column chromatography (Chloroform:
Methanol (9:1)).
3.77 ± 0.39
3.69 ± 0.21
3.72 ± 0.38
37.8
35.1
36.2
Membrane integrity studies revealed that BC9 did not cause
significant change in extracellular concentration of DNA and
protein relative to untreated bacterial cells, whereas
treatment with non-ionic detergent (Triton X 100) caused a 6
fold increase in concentration of extracellular protein and
nucleic acid relative to treatment with BC9, revealing that
membrane integrity is not compromised by BC9.
General procedure for the synthesis of benzochromenes (BC1-9):
A
mixture of substituted aldehyde (1 mmol), Substituted 3-
Cyanoacetyl indole (1 mmol) and 2-naphthol (1 mmol) containing
PEI-Me catalyst (50 mg) were taken in the 5 ml of methanol and it
was stirred at 80°C in appropriate time. The progress of the reaction
was monitored through thin layer chromatography (TLC) using
methanol and Chloroform (1:9) as eluent mixture. After completion,
Experimental
General
All the chemicals were purchased from Sigma Aldrich, Loba
chemicals, Merck, Avra synthesis, and SD Fine chemicals and all the reaction mixture was cooled to room temperature, the solid
obtained was filtered through the Whatman filter paper and
used without any other further purification. Melting points were
taken in the microscopic melting point meter and were
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Journal Name
washed with cold ethanol. The product thus obtained was PI-CTAB assay: Propidium iodide accumulates only in membrane
essentially pure.
compromised cells; hence accumulation of PI indicates enhanced
membrane permeability. og-phase cells of S.aureus suspended in
L
Antimicrobial studies: Antibacterial effect of the selected benzo
chromen derivatives identified by virtual screening were discerned
by evaluating its MIC (minimum inhibitory concentration) and MBC
(minimum bactericidal concentration) by two fold broth
microdilution method as reported earlier.46 Briefly the compounds
were dissolved in DMSO, serially diluted from 0.25µg/ml to 64µg/ml
and inoculated with S.aureus strains in Mueller-Hinton broth and
incubated at 37°C for 18-24h. The lowest concentration of
compound which prevented growth (visible turbidity) was assumed
to be MIC. Samples (10 µl) from the tubes that lacked visible
turbidity were plated on MH agar plates and the concentration that
gave rise to 3 or fewer colonies were deemed as MBC.
PBS were treated with BC9 and propidium iodide (PI) for 1-2h.
Fluoresence due to uptake and binding of PI to DNA can be
quantified (Ex 500nm Em 600nm) using
a Jasco FP-8200
spectrofluorimeter (Jasco, Tokyo, Japan). Cetyltrimethylammonium
bromide (CTAB) was used as a positive control and the change in
permeability is expressed as ratio of PI accumulation in CTAB -
treated to CTAB-untreated cells.
Membrane Integrity
In order to discern membrane damage, cell membrane integrity was
studied in the presence or absence of BC9 as reported earlier.48
Synergy testing Checkerboard Analysis: The interactions between
non-toxic putative EPI BC9 (benzochromen derivative) and
fluoroquinolone antibiotic Ciprofloxacin (CPX) was discerned using
checkerboard analysis on a 96-well plate by the standard method
reported earlier.47 One of the components BC9 was diluted along
the y-axis and the other CPX was diluted along x-axis. The FIC was
calculated according to the equation: FIC index = FICA + FICB = (MIC
of drug A in combination/MIC of drug A alone) + (MIC of drug B in
combination/ MIC of drug B alone). An FIC index ≤ 0.5, indicated
synergy, an FIC index between 0.5-2.0 indicated additive effect and
an FIC ≥ 2.0 indicated antagonism.
Briefly, cells after treatment were collected at various time intervals
(0-4h), pelleted at 13,250 rcf for 5 min. The release of DNA and
proteins due to cell damage caused by BC9 was quantified at
260nm and 280nm respectively. 0.5 % triton X 100 treated cells
was used as control.
Homology modeling and Protein Preparation
The Protein sequence NorA of Staphylococcus aureus (Accession
No: P0A0J7) was retrieved from the UniProtKB database
(http://www.uniprot.org/). The 3D protein structure was predicted
using MUSTER (Multi-source Protein structure Threader).49 The
predicted structure had best similarity with Crystal Structure of
Major facilitator superfamily MFS (PDB ID: 3WDO) having a
resolution of 3.15 Å. The Predicted 3D Protein structure was
validated using Ramachandran plot and Prosa. The template and
target protein structures were studied for their similarity by super
imposing using SuperPose Version 1.0. The validated protein
structure was subjected to non-Polar hydrogens and Gasteiger
charges using Autodock 4.0 (ScrippsResearch Institute, USA).
MIC reversal: The ability of BC9 to potentiate the effect of CPX was
evaluated with the following strains SA1199 (wild-type S.aureus),
SA1199B (S.aureus overexpressing NorA), K1758 (NorA knock out
strain of S.aureus). BC9 was used at 0.5X MIC along with
ciprofloxacin in varying concentrations in cation adjusted Mueller-
Hinton broth and incubated at 37°C for 18-24 hrs. The fold-change
in MIC of ciprofloxacin due to BC9 in wild type and Nor A mutant
Real-time efflux assay was used to monitor time dependent efflux
of EtBr from S.aureus. Overnight cultured cells of SA1199, SA1199B
and K1758 were re-inoculated into nutrient broth and allowed to
reach log phase (0.2-0.3 OD). Cells were harvested by centrifugation
and suspended in sterile PBS (phosphate buffer saline pH 7.0)
Aliquots were withdrawn and subjected to treatment with 1µg/ml
of EtBr and 0.4% glucose along with BC9 at 0.5X MIC for 1 h. Post
treatment, cells were harvested, washed twice and re-suspended in
PBS. EtBr efflux was assessed by quantitating fluorescence for
10min using JASCO- FP-8200 spectrofluorometer (Ex 530nm and Em
585nm). Reserpine was employed as a positive control.
Ligand Preparation
The energy minimization for the 31 ligands drawn using chemdraw
ultra 8.0 was done by Avagadro software and subsequently the
ligands were subjected to addition of Gastegier charges through
Autodock 4.0.
Molecular Docking Studies:
The grid box was generated by keeping the grid points 101x101x101
in X, Y, Z directions where the target protein (NorA) was covered to
let the ligand find its binding pocket in its free state. Docking
studies were performed between the target protein and prepared
ligands by applying Lamarckian genetic algorithm50 in Autodock 4.0.
The docking protocol was then implemented on the ligands in the
dataset. The study was carried for 20 GA runs, which was found to
be optimum to reproduce the pose in its crystal. The other GA
parameters like the population size and the genetic operators were
kept at their default values. The results were interpreted by
observing the least binding energy (kcal/mol) and hydrogen bonds;
stacking interactions involving between the active site residues of
the target and ligand molecules were analyzed using Pymol
molecular visualization tool. The RMSD was calculated to ensure
that the selected ligands have conformational similarity.
EtBr efflux assays: SA1199 cells were grown in nutrient broth till
they reached an OD of 0.2-0.3. Cells were harvested at 6000rpm for
15min and suspended in PBS. These cells were distributed into
replica tubes. BC9 was added in increasing concentrations
(10µg/ml- 25µg/ml) along with EtBr at 1µg/ml and 0.4% glucose.
Tubes were incubated for 1 h for maximum uptake of EtBr. Post
incubation, cells were again harvested and washed twice with PBS
to remove any traces of EtBr and resuspended in EtBr free PBS. The
rate of EtBr efflux was immediately quantified for over 30min at
5min intervals using Jasco FP-8200 spectrofluorimeter (Jasco,
Tokyo, Japan) with Ex 530nm and Em 580nm. Reserpine was used
as positive control.
Membrane Permeability studies:
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MTT Assay
resistant mutants. Its hydrophobic nature might explain
increased permeability and enhanced binding to NorA
Human pancreatic cells (MIA-Pa-Ca-2) were cultured in 96 well
plates in the presence of varying concentrations (1-64 µg/ml) of
benzochromene compounds (BC1, BC2, BC3, BC6 and BC9). After 24
h of growth MTT was added and after 4h of incubation, MTT was
solubilized using DMSO and the absorbance was measured at 590
nm
exhibited by BC9. As
a proof of concept BC9 caused
remarkable reversal in CPX MIC in two MRSA strains. Added
with its ease of facile one pot synthesis and relatively low
toxicity in zebrafish model, BC9 has potential to be considered
as lead molecule that specifically targets NorA mediated drug
efflux in S. aureus and is suitable for evaluating EPI effect in
vivo in appropriate animal models.
Fish toxicity studies
Adult zebrafish (Danio rerio) irrespective of sex, measuring 4 to 5
cm in length, weighing approx. 300 mg, were purchased from a
local aquarium. Animal acclimatization was performed following
established protocols.51 Since the putative EPIs exhibited low
antimicrobial activity, the compounds were evaluated at 8X MEC
(16 µg/ml). For toxicity evaluation, a total of 5 fish were exposed to
various concentrations of the compound for 48 h and mortality of
the fish was monitored. The toxicity experiment was repeated
twice. The compounds were first prepared as stocks in dimethyl
sulfoxide (DMSO) and later mixed directly in water for fish
exposure. A separate DMSO alone control was maintained for a
similar duration but there were no mortality or significant
behavioural changes (data not shown). Water and compounds were
renewed every day. Based on the mortality during the course of
exposure and the number of fish alive at the end of 48 hours in
both the exposures, selected derivatives were chosen for further
analyses. The study consisted of three groups: control, DMSO alone
and compounds (1 L/tank).
Authors contributions
AG- synthesized compounds; LRC carried out the biological
studies; HMV- carried out the docking studies; UV- designed in
silico studies; TR designed zebra fish studies; VS- designed cell
culture studies; KK- contributed for synthesis and
characterization; NS and SSG designed biology and chemistry
part of the work respectively and NS,SSG, UV and TR wrote the
manuscript.
Acknowledgements
The Authors sincerely thank TRR funding provided by
management, SASTRA University to NS & UV to carry out this
work. S.S.G. thanks DST for DST-Fast Track Grant (No:
SR/FT/CS-09/2011). TR is thankful the Science and Engineering
Research
Board
(SERB),
Government
of
India,
Tissue preparation
(SERB/F/1266/2012-13, dated 31st May 2012) for financial
support. DST- FIST funding (Grant No SR/FST/ETI-331/2013)
provided by DST, Govt of India to SCBT, SASTRA University is
gratefully acknowledged. Infrastructure facility (CRF)
established through R&M funds (Grant No R&M/0021/SCBT-
007/2012-13) of SASTRA to carry out this work is gratefully
acknowledged. R&M grant provided to Microbiology
Department by Management SASTRA University is gratefully
acknowledged. Research fellowship provided by UGC to L.R.C.
Maulana Azad National Fellowship, UGC, New Delhi, India
(MANF-2012-13-CHR-WES-13462) is gratefully acknowledged.
we thank Dr. Vairaprakash for useful discussion on
characterization of hyperbranched polyamines, Dr.S.Banudevi
for providing cell lines and Mrs M. Elamathi for technical help
with cell culture.
To check the effect of selected compounds on select biochemical
parameters of zebrafish, a total of 10 fish were exposed to 8mg/L of
the respective compounds for 48 h. At the end of exposure (48 h),
fish were sacrificed (anesthetized by 150mM MS-222 and
euthanized by decapitation), skin removed and the liver from two
fish from the same group were pooled and homogenized in ice-cold
buffer (Tris-HCl, 0.1M, pH 7.4). The homogenate was centrifuged
º
(10,000 x g, 10 min, 4 C) and supernatant used for all analyses in
duplicates. Brain was homogenized for acetylcholinesterase (AChE)
assay. From the homogenate prepared from liver or brain pooled
from two fishes, duplicates were derived for each assay. Protein
was estimated by the method of Lowry et al.52 Estimation of
carboxyl esterase was essentially as described by Argentine and
53
James and acetylcholinesterase (AChE) activity was measured by
Edmann’s degradation.54
Conclusions
Notes and references
In conclusion we have adopted a combination of in silico and in
vitro studies and identified that benzochromene BC9 displayed
low intrinsic anti staphylococcal activity and highly specific
efflux inhibitory effect against NorA pump of S. aureus. MIC
reversal studies show that even at concentrations as low as ¼
of its MIC, BC9 caused a drastic 32 fold CPX MIC reversal in
NorA overexpressed strain SA-1199B. BC9 displayed a low MEC
of 2µg/ml corresponding to 4.03 µM. Real time efflux and time
kill studies proved that BC9 potentiated effect of CPX better
than reserpine and exhibited synergy with CPX in NorA
overexpressed strain. Most importantly MPC and mutation
frequency showed that BC9 restricted selection of drug
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