A novel molecular scaffold resensitizes multidrug-resistant S. aureus to fluoroquinolones

Article abstract of DOI:10.1039/c9cc03001h

Nosocomial infections arising from opportunistic pathogens, such as Staphylococcus aureus, are growing unabated, compounded by the rapid emergence of antimicrobial resistance. Herein, we demonstrate a new molecular design that exhibits excellent activity against multidrug-resistant S. aureus with no cytotoxicity and resensitizes fluoroquinolones (FQ) towards FQ-resistant methicillin-resistant S. aureus strains, with DNA gyrase B as the likely molecular target as determined by molecular dynamics (MD) simulations.

Full text of DOI:10.1039/c9cc03001h

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DOI: 10.1039/C9CC03001H
Received 00th January 20xx,
Accepted 00th January 20xx
COMMUNICATION
DOI: 10.1039/x0xx00000x
A novel molecular scaffold resensitizes multidrug-resistant S.
aureus to fluoroquinolones
Apurva Panjla,a Grace Kaul,b Manjulika Shukla,b Shubhandra Tripathi,a Nisanth N. Nair,a* Sidharth
Choprab* and Sandeep Vermaa*
Nosocomial infections arising from opportunistic pathogens, such degradation and scale-up issues have to be considered prior to
as Staphylococcus aureus, are growing unabated, compounded by full-scale clinical development. Needless to mention, there is
the rapid emergence of antimicrobial resistance. Herein, we considerable interest and urgent need to seek new scaffolds
demonstrate a new molecular design that exhibits excellent activity with selective antibacterial action to mirror success stories such
against multidrug-resistant S. aureus with no cytotoxicity and as teixobactin,9 and polymixin.10-12
resensitizes fluoroquinolones (FQ) towards FQ-resistant
The menace of multidrug resistant (MDR) bacterial strains
methicillin-resistant S. aureus strains, with DNA gyrase B as the poses serious health challenge necessitating prolonged
likely molecular target as determined by molecular dynamics (MD) chemotherapeutic intervention, which further adds to the pre-
simulations.
existing load of resistant strains. Given that the last line of
antibiotics have also reached the verge of MDR regime, it is
imperative that newer targets and active structures be unveiled
to support viable chemotherapeutic alternatives.13 In addition
to discovery of new antibiotic pharmacophores and active
structures, it is also recognized that adjuvants having potential
to enhance the efficacy of existing antibiotics or the ability to
resensitize antibiotic-resistant strains, also add to the molecular
repertoire against MDR strains.14,15 Thus, new entities
augmenting bactericidal activity of antimicrobials for
resensitization of resistant strains would be an added incentive
in antimicrobial drug development.
In this communication, we present design of a new
antibacterial based on tryptophan dipeptide platform,
conjugated to rhodamine B to impart cationic character and
membrane permeability,16 with a contribution from lipoic acid
fragment purported to provide protection toward oxidative
stress.17 It is notable that studies have documented favourable
effect of lipoic acid on membrane-bound renal enzymes against
gentamicin toxicity in bacteremic mice model, and recently, a
lipoic acid derivative was shown to suppress multidrug
resistance in clinical isolates of P. aeruginosa through anti-ROS
action.18,19 Based on this premise, new chemical entities 1 and 2
were designed to maintain a balance of charge, hydrophobicity
and lipophilicity, that could mimic AMPs followed by their
antibacterial assessment. Further, DL-lipoic acid in 1 was
substituted with R-lipoic acid for conjugation with L-tryptophan
and D-tryptophan containing dipeptides, to afford two
derivatives 1a and 1b, respectively (Figure 1). These analogs
were screened against the ESKAPE pathogen panel
(Enterococcus faecium, Staphylococcus aureus, Klebsiella
pneumoniae, Acinetobacter baumannii, Pseudomonas
aeruginosa, Enterobacter species) and the results are presented
in Table 1. Compounds 1, 1a and 1b exhibited antibacterial
activity against S. aureus (Table 1), while individual fragments
were devoid of activity.
Staphylococcus aureus, a Gram-positive bacteria, is a
versatile opportunistic pathogen possessing a remarkable
tenacity to overcome antibiotic action, leading to emergence of
drug-resistant phenotypes which are responsible for causing
infections under communities as well as under hospital settings
worldwide.1-3 S. aureus biofilms are known to escape host
immune response to cause endocarditis, osteomyelitis, skin and
soft tissue infections to sepsis, all of which are becoming
increasingly difficult to treat due to rapid spread of
antimicrobial resistance (AMR).4 Given the current trends in
discovery and approval of new antibiotics, it is estimated that
drug-resistant superbugs could cause up to 10 million deaths
worldwide by 2050. Such an alarming situation necessitates
that in addition to continued discovery of broad-spectrum
antibiotics, emphasis should also laid on new molecules that
could synergize with known antibiotics and could potentiate
them through favourable action on a variety of mechanistic
targets.5 Thus, continuous discovery of innovative antibacterial
molecules, acting via novel mechanisms is an urgent unmet
need. In this context, antimicrobial peptides and peptide-based
scaffolds represent an effective strategy for the discovery of
new antimicrobials with broad-spectrum action and
decelerated emergence of drug-resistant strains.6 Such peptide-
containing structures exhibit significant therapeutic potential
through an interplay of hydrophobicity and cationic charges,7
and as an added advantage, they are potentially less prone to
resistance, can counter virulence factors and modulate host
immune response.8 However, limitations such as toxicity,
a. Department of Chemistry, Indian Institute of Technology, Kanpur
E-mail: sverma@iitk.ac.in, nnair@iitk.ac.in
b. Department of Microbiology, Central Drug Research Institute, Lucknow
Email: skchopra007@gmail.com
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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Journal Name
S
S
S
View Article Online
S
DOI: 10.1039/C9CC03001H
HN
HN
O
NH
O
NH
H
N
H
N
O
O
O
N
O
N
N
NH
N
O
NH
O
N
O
1
N
N
O
N
1a
1a: R-lipoic acid, L-tryptophan
1:DL-lipoic acid, L-tryptophan
S
S
S
S
Figure 2: Time kill kinetics of 1b against S. aureus (ATCC 29213). Each experiment was
OH
done in replicates and the values represent SD
HN
O
NH
O
NH
H
N
H
N
O
O
O
N
O
N
N
O
NH
N
O
OH
N
O
N
N
O
2
N
1b
1b:R-lipoic acid, D-tryptophan
2:DL-lipoic acid, L-tyrosine
Figure 1: Molecular structures of 1, 1a, 1b and 2
Figure 3: MIC values of levofloxacin and 1b are shown against S. aureus ATCC 29213
along with the number of passages
Cytotoxicity of active compounds toward Vero cells was
determined and the selected compounds were found to be non-
toxic with high CC50 values, offering Selectivity Index >100
(Table 1). 3, a control compound without dipeptide residue,
with higher MIC value, was also toxic (Scheme S1, ESI; Table 1).
Given the favourable antimicrobial activity and selectivity index
of these three compounds, we continued with 1b as presence
of D-amino acid offers more stability. Consequently,
antimicrobial action of 1b was determined against several
clinically drug-resistant strains of S. aureus. 1b showed
equipotent activity against methicillin-resistant S. aureus
(MRSA) and vancomycin resistant S. aureus (VRSA), with MIC
ranging from 2-4 μg/mL (Table 2). These results clearly
suggested that observed antibacterial actions are possibly
operating via other novel molecular target(s).
resistance.20,21 Upon examination, 1b was found to exhibit conc.
dependent bactericidal activity with ~6 log reduction in colony
forming unit (CFU) in 24h, which was comparable to
levofloxacin.
We further assessed the potential emergence of bacterial
resistance against 1b. The propensity of bacteria to generate
resistance can be evaluated using serial exposure of organisms
to antimicrobial agents.22 We exposed S. aureus ATCC 29213 to
serial passages of 1b and levofloxacin and monitored the
changes in MIC values over a period of 25 days. Despite
repeated culturing in the presence of sub-inhibitory
concentrations of 1b, a stable resistant mutant was not isolated
for 1b and the MIC increased by only 2-fold (Figure 3).
Comparatively, S. aureus expressed high level resistance to
levofloxacin with a MIC of 32 and 64 µg/mL, within 17 and 20
days of culture, respectively. It must be noted that 1b exhibits
lower propensity to generate resistance as compared to
levofloxacin.
Time killing kinetics activity of 1b with S. aureus ATCC 29213
was investigated, compared to ceftazidime and levofloxacin to
determine whether observed antimicrobial activity was
bacteriostatic or bactericidal in nature (Figure 2). Bactericidal
antibiotics are desired over bacteriostatic since they eliminate
infection faster as well as suppress development of drug
DNA gyrase and topoisomerase IV are required for the
processing of topological changes in bacterial DNA structure.
Table 1: Minimum Inhibitory Concentration (MIC) in μg/mL and Selectivity Index (SI) values against ESKAPE pathogen panel of compounds 1, 2, 3, 1a, and 1b and their components
Compound
E. coli
ATCC
S. aureus
ATCC 29213
K. pneumoniae
BAA-1705
A. baumannii
BAA 1605
P. aeruginosa
ATCC 27853
Cell cytotoxicity
(CC50) against
Selectivity
Index
25922
Vero cells (μg/mL)
(CC50/MIC)
1
2
1a
1b
3
>64
>64
>64
>64
>64
>64
>64
0.015
2
16
4
4
8
>64
>64
0.125
>64
>64
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
>64
>64
>64
8
>64
>64
>64
>64
>64
>64
>64
0.5
> 1000
<100
>400
>1000
<80
ND
ND
ND
>500
<6.25
>100
>250
<10
ND
ND
ND
Rhodamine B
DL-lipoic acid
Levofloxacin
ND-Not determined
2 | J. Name., 2012, 00, 1-3
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Table 2: MIC (μg/mL ) of 1b against clinical multidrug resistant S. aureus strains including MRSA and VRSA along with control antibiotics
View Article Online
DOI: 10.1039/C9CC03001H
Bacteria
Strain
1b
Levofloxacin
Meropenem
Methicillin
Vancomycin
S. aureus
Vancomycin
resistant
S. aureus (VRSA)
Methicillin resistant
S. aureus (MRSA)
ATCC 29213
VRS 1
VRS 4
VRS 12
4
4
4
4
4
<0.5
32
>64
32
<0.5
>64
>64
32
2
1
>64
>64
>64
>64
>64
>64
>64
1
NRS100
<0.5
>64
NRS119
NRS 129
NRS 186
NRS 191
NRS 192
NRS 193
NRS 194
NRS 198
4
4
4
4
4
4
2
4
4
<0.5
4
16
8
32
<0.5
32
>64
32
32
>64
64
>64
8
>64
>64
>64
>64
>64
>64
32
1
1
1
1
1
1
1
1
>64
>64
DNA gyrase are heterotetrameric proteins composed of two
On the other hand, intercalation of 1b in DNA gyrase A (in
subunits, referred as A and B, and they serve as a validated complex with DNA) was found to be unfavourable (Figure S1,
antibacterial target for existing antibiotics as fluoroquinolones ESI). Further, the binding affinities of 1b and novobiocin against
(FQ) that target DNA gyrase A.23,24 In comparison, there is no ParE were also found to be similar (-9 kcal/mol, Table S5 and
clinically utilized antibiotic targeting gyrase B. In order to Figure S3, ESI). To further validate the binding mechanism, an
understand the possible mechanism of action of 1b, all-atom molecular dynamics (MD) simulation was performed at
computational studies were carried out to explore the binding 300 K for the docked conformation of 1b in gyrase B, which was
of 1b with DNA gyrase A (PDBID 5CDQ), DNA gyrase B (PDBID fully solvated with explicit water molecules. During the 50 ns
4P8O), and ParE (Topoisomerase IV; PDBID 4URN). Molecular trajectory, 1b was found to reside at the active site of gyrase B
docking studies were first conducted with 1b against DNA without intermittent detachments (Figure 4; Figure S4, Movie
gyrase A and gyrase B, which revealed that it favourably binds S1, ESI), which indicates a strong affinity between 1b and gyrase
to gyrase B ATP binding site with a binding energy of -11 B, Asp53:OD1--N6:1b, Glu50:OE1/OE2--N4:1b, and Asn54:ND2-
kcal/mol (Table S4, ESI). Interestingly, the affinity of 1b was - O1:1b (Figure 4; Figure S2, Table S6, ESI). Cumulatively, the
higher compared to known gyrase B inhibitors such as aforementioned hydrophobic and hydrophilic interactions of 1b
novobiocin and aminobenzimidazole by ~2-3 kcal/mol (Table with DNA gyrase B active site residues, as deciphered from
S4, ESI).
molecular dynamics simulation, explain strong binding affinity
observed in our docking computations.
Finally, we performed synergy experiments with FQ and 1b
against FQ-susceptible and FQ-resistant (FQ-R) MRSA NR10193
and NR10198 strains (Table S1-S3, ESI; Table 3), to give insight
whether FQ and 1b could "resensitize" FQ-resistant strain to FQ
in active screens. 1b and FQ synergized to "resensitize"
NR10193 and NR10198 to various FQ drugs, when used in
combination, to suggest putative medicinal applications of 1b
and FQ combination in treating FQ-resistant MRSA infections.
To further validate the observed synergy, time kill analysis
was performed using 1X MIC of ciprofloxacin and 1b against
NRS10193 and NRS10198. The results of drug combination time
kill analysis are depicted in Figure 5. As can be seen in Figure
5(a), the combination of 1X MIC 1b (4 µg/mL) and ciprofloxacin
(128 µg/mL) reduces ~1.5 log10 more CFU than either drug alone
against NRS10193 at 24 h. A similar trend is also visible in
NRS10198 where the combination reduces ~0.85 log10 more
CFU than either compound alone at 24 h (Figure 5b). Taken
together, it is clear that the combination of 1b and FQ is
synergistic and “resensitizes” FQ against FQ-resistant strains of
S. aureus. Thus, 1b and derivatives could serve as adjunct
therapeutics for potentiating established antibiotics, since
former targets a novel antibacterial target (gyrase B), thereby
offering a double-pronged strategy for resistant strains.
Figure 4: Snapshot from MD simulation of solvated 1b: gyrase B complex. Crucial
interactions of the ligand with the protein are identified. Hydrogen bond with protein are
indicated in red dotted lines, while water mediated hydrogen bonds are drawn in blue
dotted lines
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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Table 3: Synergism of 1b against fluoroquinolones against FQ-resistant NRS10193 and NRS10198 (FIC: Fractional Inhibitory Concentration)
Drug in
FQ-resistant NRS10193
FQ-resistant NRS10198
View Article Online
DOI: 10.1039/C9CC03001H
combination of 1b
FIC A
FIC B
FICI----(FIC-A +FIC-B)
Inference
FIC A
FIC B
FICI----(FIC-A +FIC-B)
Inference
Levofloxacin
Ciprofloxacin
Moxifloxacin
0.5
0.5
0.25
0.003
0.001
0.001
0.50
0.50
0.25
Synergic
Synergic
Synergic
0.25
0.25
0.25
0.007813
0.5
0.25
0.25
0.75
0.5
Synergic
No interaction
Synergic
FIC A = MIC of 1b in the combination with drug/MIC of 1b alone, FIC B = MIC of drug in the combination with 1b/MIC of drug alone, FICI-FIC Index
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In conclusion, 1b demonstrated potent in vitro MIC against
multiple multidrug-resistant S. aureus strains, concentration-
dependent rapid S. aureus bactericidal effect and found
nontoxic toward mammalian cells up to 1000 μg/mL, as well as
exhibited low propensity of generating resistance. In addition,
we also demonstrated that 1b potentiates FQ against FQ-
resistant S. aureus with a possibility of developing combination
therapy regime.25 Taken together with molecular dynamics
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SV would like to thank J C Bose Fellowship for financial support.
This work is also partially supported by DST Nano Mission. AP
thanks CSIR, India, for Senior Research Fellowship. MS thanks
UGC and GK thanks DST-INSPIRE for Research fellowship.
Authors thank HPC IIT Kanpur and Department of Chemistry for
computational facilities. ST acknowledges financial support
from Institute Postdoctoral Fellowship of IIT Kanpur
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Conflicts of interest
There are no conflicts of interest.
Notes and references
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Microbiol. 2007, 10, 428-435.
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