New approach to address antibiotic resistance: Miss loading of functional membrane microdomains (FMM) of methicillin-resistant Staphylococcus aureus (MRSA)

Article abstract of DOI:10.1016/j.micpath.2018.11.038

The synthesized potent piperazine analog ChDiPiCa was characterised by various spectroscopic techniques and for the first time evaluated functional membrane microdomain (FMM) disassembly in methicillin-resistant Staphylococcus aureus (MRSA). The ChDiPiCa showed excellent in vitro biocidal activity against MRSA at 26 μg/mL compared to the antibiotic streptomycin and bacitracin 14 μg/mL and 13 μg/mL at 10 μg concentration respectively. The membrane damaging property was confirmed by the SEM analysis. Further, we addressed the new approach for the first time to overcome antibiotic resistance of MRSA through membrane microdomain miss loading to lipids. By which, the ChDiPiCa confirms the significant activity in miss loading of FMM of MRSA which is validated by the fatty acid profile and lipid analysis. The result shows that, altered saturated (Lauric acid and Myristic acid), mono unsaturated (Oleic acid), and poly unsaturated (Linoleic acid and Linolenic acid) fatty acids and hypothesises, altered the membrane functional lipids. For the better understanding of miss loading of FMM by the ChDiPiCa, the in-silico molecular docking studies was analyzed and confirmed the predicted role. This suggests the way to develop ChDiPiCa in medicinal chemistry as anti-MRSA candidates and also this report opens up new window to treat microbial pathogens and infections.

Full text of DOI:10.1016/j.micpath.2018.11.038

Accepted Manuscript
New approach to address antibiotic resistance: Miss loading of functional membrane
microdomains (FMM) of methicillin-resistant Staphylococcus aureus (MRSA)
H.S. Nagendra Prasad, C.S. Karthik, H.M. Manukumar, L. Mallesha, P. Mallu
PII:
S0882-4010(18)31604-8
DOI:
https://doi.org/10.1016/j.micpath.2018.11.038
YMPAT 3275
Reference:
To appear in: Microbial Pathogenesis
Received Date: 17 September 2018
Revised Date: 22 November 2018
Accepted Date: 26 November 2018
Please cite this article as: Nagendra Prasad HS, Karthik CS, Manukumar HM, Mallesha L, Mallu P, New
approach to address antibiotic resistance: Miss loading of functional membrane microdomains (FMM)
of methicillin-resistant Staphylococcus aureus (MRSA), Microbial Pathogenesis (2018), doi: https://
doi.org/10.1016/j.micpath.2018.11.038.
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Graphical abstract

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New approach to address antibiotic resistance: Miss loading of functional
membrane microdomains (FMM) of methicillin-resistant
Staphylococcus aureus (MRSA)
H.S. Nagendra Prasada, C.S. Karthik a, H.M. Manukumara, L. Malleshab, P. Mallua*
aDepartment of Chemistry, Sri Jayachamarajendra College of Engineering, JSS Science and
Technology University, Mysuru-570 006, Karnataka, India
bPG Department of Chemistry, JSS College of Arts, Commerce and Science, Mysuru-
570025, Karnataka, India.
*Correspondence
Email: drmallu66@gmail.com

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Abstract
The synthesised potent piperazine analogue ChDiPiCa was characterised by various
spectroscopic techniques and for the first time evaluated functional membrane microdomain
(FMM) disassembly in methicillin-resistant Staphylococcus aureus (MRSA). The ChDiPiCa
showed excellent in vitro biocidal activity against MRSA at 26 µg/mL compared to the
antibiotic streptomycin and bacitracin 14 µg/mL and 13 µg/mL at 10 µg concentration
respectively. The membrane damaging property was confirmed by the SEM analysis. Further,
we addressed the new approach for the first time to overcome antibiotic resistance of MRSA
through membrane microdomain miss loading to lipids. By which, the ChDiPiCa confirms
the significant activity in miss loading of FMM of MRSA which is validated by the fatty acid
profile and lipid analysis. The result shows that, altered saturated (Lauric acid and Myristic
acid), mono unsaturated (Oleic acid), and poly unsaturated (Linoleic acid and Linolenic acid)
fatty acids and hypothesises, altered the membrane functional lipids. For the better
understanding of miss loading of FMM by the ChDiPiCa, thein-silico molecular docking
studies was analysed and confirmed the predicted role. This suggests the way to develop
ChDiPiCa in medicinal chemistry as anti-MRSA candidates and also this report opens up
new window to treat microbial pathogens and infections.
Key words: Piperazine; Pthalamide; MRSA; Antibiotic resistance; Membrane disassembly

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Introduction
Staphylococcus aureus is a Gram-positive and one of the most notorious ESKAPE
(Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter
baumanii, Pseudomonas aeruginosa, and Enterobacter species) clinical bacterial
pathogenscolonizes 30% of healthy human individuals, causing community-acquired and
nosocomial infections worldwide. [1-4] Since 1948, the first discovery of antibiotic
methicillin and bacteria S. aureus acquired a resistance determinant called ‘mecA’ in its
genome to become methicillin-resistant S. aureus (MRSA) and quickly become predominant
master infectious pathogen with increased global prevalence. [1]Because of its higher drug-
resistance rate, now MRSA becoming global problem to treat,which leads to higher morbidity
and mortality than the methicillin-susceptible S. aureus (MSSA). [5-7]Notorious MRSA
evolved with a wide range of strategies to colonize and invade human system to cause death,
despite the presence of multiple host defence mechanisms. The death rate was increased from
700,000 per year, and it become10 million by 2050; hence, MRSA was renamed as “super
bug”. [8, 9]
Diverse fields approaching new improvements in discovery and development of new
antibacterial compounds but, due to cost and microbial resistance it has been slow and arsenal
effective antibiotics were diminishing. [10]Many classes of antibacterial drugs were approved
by the Food Drug Administration (FDA) from 1939 to 2017 such as Sulfonamides,
Aminoglycosides, Penicillins, Tetracycline, Polypeptides, Lincosamides, Oxazolidinones,
Quinolones, Macrolides, Cephalosporins, Rifamycins, Tuberactinomycins, Carbapenems,
Glycopeptides, Monobactams, Streptogramins, Lipoglycopeptides, Lipopetides, and
Macrocyclins are the leaders in the antibiotics. [11] These types of antibiotics common
mechanism by act on bacterial target sites such as cell wall (β-lactams- Ceftaroline), cell
membrane (Daptomycin), cell wall and cell membrane (Semisynthetic lipoglycopeptides-

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Telavancin), DNA and/or RNA synthesis (Fluroquinolones- Delafloxacin), protein synthesis
(licosamides- Tedizolid targeting 50S ribosome; tetracyclines- Eravacycline), and folate
synthesis (sulphonamides- Sulfamethoxazole; folate inhibitors- Trimethoprim). [12] Due to
unintended consequences of antibiotic misuse and overuse, cited as major driving force to
develop resistance in one of human pathogen MRSA. The lack of effective therapies into
jeopardizes of modern medical practices, which attract to understanding mechanism of new
and innovative approaches in antibiotic development for effective and tomorrow better
treatments. [13-15] In this regard, there is an urgent need for the new class of alternative
candidates to target new bacterial biomolecules are the leading therapeutics to treat perilous
pathogens.
Since 1940s, bacterial cell wall biosynthesis is the alternative target pathways for the
synthesis of novel target antibiotic candidates and they are proved their importance in clinical
use because; there are no homologous structures and pathways for anologs in humans. The
unique protective structure of bacteria and its integrity is crucial for its viability. This was
maintained by the organized multi-enzyme complex machineries spanning extra- and
intracellular compartments. The inhibitors of bacterial cell wall show high specific target
affect and low side effects, which results in the effective treatment and good safety profile are
most widely used antibiotic.At present drug discovery research, besides inhibiting the
enzyme required for the cell wall biosynthesis, specially looking at possible ways to block
membrane-coupled biosynthetic intermediates has a tremendous importance and left armor in
resistant bacteria like MRSA. After immense research, the precursor of peptidoglycans
(PGN) lipid II paused as weak spot in Gram-positive bacteria (MRSA) and considered as
“Achilles heel” in cell wall biosynthesis in bacteria. [16-18]
The pioneering fluid mosaic model of cell suggest, the various lipid species separated
into microdomains important in formation of lipid raft which is crucial for cell membrane

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organization which serves as platform for controlling protein-protein interactions and cellular
signal transduction. [19, 20] However, the prokaryotic lipid-rafts organized region is called
‘functional membrane microdomains’ (FMM), similar to eukaryotic counterparts. Still it is
unclear, how FMM formed by unknown aggregation of the biosynthesized isoprenoid
membrane lipids. [21, 22] The presence of mecA gene in MRSA develops a resistance and
does not allow methicillin, penicillin, and ring like penicillin-like antibiotics to bind
transpeptidase enzyme involved in the cell wall of MRSA and allow it to replicate
normally.This gene encodespenicillin binding protein 2A(PBP2A) has low affinity to β-
lactam antibiotics and active site of this protein is located in deep pocket and inaccessible to
β-lactam antibiotics. [23, 24]These β-lactams are structure analogues of peptidoglycan
precursor’s cell wall and intern act as suicide substrates of the transpeptidases involved in the
two step peptidoglycan cross-links reaction. [25, 26]
Till now four different peptide antibiotics have been targeted the Lipid II of cell wall
synthesis such as 1. glycopeptides (vancomycin and teicoplanin), 2. lantibiotics (nisin and
mersacidin), 3. depsipeptide antibiotics (ramoplanin and enduracidins) and 4. cyclic peptides
(mannopeptimycins, plusbacin and katanosin B). [27-32] Also, recent research addressed the
sulfonyl piperazine and pyrazole compounds to inhibit the lipid A biosyntehsis and inhibit the
transport of lipoprotein to outer membrane in Escherichia coli. Hydrophilic candidates such
as rifamycins, macrolides, and aminoglycosides can enter the cell through outer membrane
bilayer; whereas hydrophilic candidates like β-lactams and fluoroquinolones impaired by the
lipopolysaccharide (LPS) and porins are the only way to get in to the cell. [34, 35] The efflux
pump (resistance-nodulation-division (RND)) inhibitors such as 1-(1-naphthylmethyl)-
piperazine (NMP) and phenyl-arginine-β-naphthylamide (PAβN) have been addressed in
Vibrio cholerae. [36] With this background, we conducted to address the piperazine based
phthalimide derivative (ChDiPiCa) for the first time to investigate the membrane lipid-raft

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miss assembly nature against MRSA. This evidence was established through assessing the
disassembled lipid profile of MRSA and validated by molecular docking approaches. The
results suggest that, the disassembled microdomains of FMM organization may cause a
susceptible to overcome the antibiotic resistance in MRSA and was hypothesized for the first-
time clinical treatment in future.
Materials and methods
Chemistry
All chemicals and reagents were obtained from Sigma Aldrich and Merck Chemical
Pvt-Ltd and utilized without further purification.NMR spectra were recorded in CDCl3 using
400MHz, all chemicals shift are reported in ppm relative to TMS,Mass spectroscopic analysis
was performed on a Water micro TOF QII mass spectrometer.
Procedure for the synthesis of piperazine compound (5)
2-(2-(piperazin-1-yl)ethyl)isoindoline-1,3-dione
Equimolar concentrations of isobenzofuran-1,3-dione (phthalic anhydride) (1) and 2-
(piperazine-1-yl)ethamine(2) were refluxed for 6 hrs in methanol (25 ml) and 2-3 drops of
glacial Acetic acid was added to the mixture. The reaction completion was confirmed by
TLC. The solvent was concentrated and washed with water. The solid product was dried and
recrystallized from methanol. The greenish yellowish product (3) was obtained with good
yield (Scheme.1).
N-(4-chlorophenyl)-4-(2-(1,3-dioxoisoindolin-2-yl)ethyl)piperazine-1-carbothioamide
(ChDiPiCa) (5)
A solution of 2-(2-(piperazin-1-yl)ethyl)isoindoline-1,3-dione(3) (1.0 eq) in DCM
was taken and cooled to 0-5 °C in an ice bath. Triethylamine (3.0 eq) was added to the cold

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reaction mixture and stirred for 10 min and then 4-Chloro phneolisothiocyantes (4) (2.0 eq)
were added. The reaction mixture was refluxed for 6hrs (Scheme.1). The progress of the
reaction was monitored by TLC. Upon completion, the solvent was removed under reduced
pressure and residue was taken in water and extracted with ethyl acetate. The organic layer
was washed with 1 % hydrochloric chloride and finally water wash was given to organic
layer and dried with anhydrous sodium sulphate.
Petroleum ether / ethyl acetate = 2: 3 (v / v) as eluent for column chromatography. Off
white solid, 85% yield.1H NMR: (400 MHz, CDCl3) 7.82 (d, 2H,Ar-H) 7.67 (d, 2H, Ar-H)
7.39 (d, 2H, Ar-H) 6.93 (d, 2H,Ar-H) 4.21 (s, 1H,N-H) 3.25 (d, 4H,pip-H) 3.24 (t, 2H,C-H)
2.90 (d, 4H,Pip-H) 2.62 (t, 2H,C-H).13C NMR: (400Hz, DMSO) 184.19, 168.25, 155.61,
135.13, 132.14, 129.45, 134.28, 110.21, 109.45, 102.52, 58.78, 58.52, 46.27, 40.84. LCMS
m/z Calculated for C21H21ClN4O2S: (M-H) 428.94: found 428.87, IR Vmax(cm-1) 3360(N-H),
1740(C=O), 1678 (C=C),1345(C-N), 1250(C-N),1210(C=S), 754(C-Cl) Elemental Analysis:
Calculated : C, 64.57; H, 6.66; N, 19.85; Experimental: C, 64.56; H, 6.56; N, 19.81.
(Supporting information)
Scheme 1: Synthetic route for compound ChDiPiCa (5).

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Biology
Bacterial strains and growth conditions, assay protocols and biological data
General information
The lyophilized reference bacterial strain (Staphylococcus aureus-MTCC96) from
Microbial Typing Culture Collection (MTCC), Chandigarh, India was used, which is gift of
Mr. Anand, A. P. Ganesh Analytical Services and Consultancy, Mysuru. The strain was
cultured in recommended broth according to the protocol provided by MTCC. The bacteria
wascultured into Tryptic Soy Broth (TSB) (Sigma Aldrich, 22092), Agar plate (Sigma-
Aldrich, A1296), and the TSB agar plate was incubated for 20 h, to form single bacterial
coloniesat and 37 °C. The overnight bacterial suspension was adjusted by OD600
measurements inside a spectrophotometer under static conditions and diluted to possess a
final bacterial density of 106 CFU/mL for furtherstudy.
Antimicrobial activity
An overnight grown MRSA culture wasused for disc diffusion method to deduce the
ChDiPiCa minimum inhibitory concentration (MIC) according to the earlier report [37]. The
bacterial suspension was prepared from the overnight culture and 1x106 CFU/mL cells and
were inoculated on to Mueller-Hinton agar, then sterile 5mm disc were placed, to which 5 µL
of different serial concentrations of ChDiPiCa was added. Control was performed without
o
any test sample and incubated at 37 C for 24 h to examine zone of inhibition. Assay
performed in triplicates and repeated thrice.
Quantitative and qualitative evaluation of antibacterial efficiency
The antibacterial activity of MRSA was evaluated by incubating 500 µL of bacterial
suspension to each well overnight (for 18 h) at 37 °C. At the end of experiment, the viable
planktonic bacteriaremains in the medium was examined through the SPM (spread plate

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method) namely, bacterial suspensions were serially diluted with fresh PBS, evenly spread on
agar plates, and incubated overnight at 37 °C). The number of CFU on each agar plate was
calculated and typical experimental results were photographically recorded for qualitative
evaluation of the planktonic bacteria as per the previous results [38, 39]. Each experiment
was repeated three times.
Bacterial cell microscopy
Membrane damage study
To understand the effect of sample on MRSA membrane, scanning electron
microscopy (SEM) wascarried by treating MIC of ChDiPiCa for 2 h, then pelleted cells were
o
centrifuged (10,000 rpm for 5 min) at 4 C. Further, cells were fixed by using 2.5%
glutaraldehyde in PBS, pelleted and smeared on glass slide, dried followed by stepwise
treatment of 30% to 100% ethanol. After drying under room temperature,the sample analyzed
for SEM as per the protocol of Manukumar et al. [37, 40].
Total lipid extraction
The membrane fatty acid of MRSA control and treated was analyzed by centrifuging
the broth for 10 min at 500 rpm, harvested pellets immediately used for Born-Trifluoride
(BF3) extraction described by Manukumar et al. [37]. Briefly, cell pellet was washed in sterile
2 mL distilled water in 50 mL tube and add (3.75 × 2) mL of methanol/chloroform (2:1, v/v)
then mix for 2 h. After a incubation, supernatant (S1) was collected by centrifuge at 2500 rpm
for 15 min. Remaining pellet suspended in (4.75 × 2) mL of methanol/chloroform/water
(2:1:0.8, v/v), vortexes, then leave for 2 h to collect supernatant (S2) by centrifugation. Pool a
S1 and S2 and added ((S1 + S2)/3.8) mL of chloroform followed by equal volume of water.
Finally, after vortex and settle of 2 phase system, the lower lipid contains chloroform fraction
was collected,warmed and evaporate under nitrogen gas. The final small volume of
chloroform was added, sealed and stored at 4 °C for short term, −20 °C for long-term storage.

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Analysis of fatty acid
FAME: The transmethylation was carried out for extracted samples to analyse their acyl
groups as fatty acids methyl esters (FAME) by gas–liquid chromatography (GLC). To the
extracted total lipid fraction add 2 mL of H2SO4(2.5%, v/v) in dry methanol (stored over
anhydrous sodium sulphate). Heated 2 h at 70 °C, then the reaction was stopped by addition
of 3 mL of NaCl (9%, v/v) in water. The FAME was extracted with 3 × 2 mL of
light petroleum spirit, then extracts were evaporated under a steam of nitrogen gas and
residue was re-suspended in a small volume of light petroleum for GLC analysis.
Gas–liquid chromatography
The prepared FAME samples were analyzed using gas chromatography having glass
column (1 m × 4 mm) packed with 10% SP2330 on 100–120 supersport. The oven
temperature of 160 °C was adjusted and the injector/detector was adjusted to 200 °C. The
carrier nitrogen gas was adjusted with pressure of 2 kPa. Separated FAME was calculated
using following formula:
FAME=Retention time on column/Peak height
Peaks were identified by their retention time relative to those of authentic standards.
Purification of staphyloxanthin lipids
The overnight cultured MRSA pellet was recovered by centrifuging10000 rpm for 10
min at 4 °C and pellet was resuspended in 10 mL methanol for 30 min at 60 °C agitating
continuously according to previous report [41]. Cell debris were removed by centrifugation
and supernatant was concentrated. Then caratinoid was extracted using 2 mL ethyl
acetate/1.7MNaCl (1:1 v/v)and ethyl acetate phase was recovered two times. The collected
extract was dried and stored at -20 oC for further study. To purify staphyloxanthin, extracted
lipids were resuspended in chloroform and separated by TLC.The staphyloxanthin band

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scratched off the TLC and re-extracted with 4 mL ethyl acetate/1.7 M NaCl (1:1 v/v) then
purity was confirmed its mass by confirmed by its mass by LC-MS/MS.To compare the
FMM-enriched and -depleted fractions of caratenoid, (DRM) and (DSM) fractions
resuspended in 1 volume H2O, 1 volume chloroform and methanoland centrifuged to separate
organic and aqueous phases. Finally, the chloroform caratenoid phase was dried in vacuo and
stored at -20 oC.
Cell fractionation and purification of DRM
The overnight cultured MRSA pellet was harvested by centrifuging speed of 10000
rpm for 10 min at 4 °C and pellet and resuspended in PBS buffer with 1 mM
phenylmethylsulfonylfluoride (PMSF) and 5 mL DNaseI (2000 U/mL). The 15 mL
lysostaphin (1 mg/ml) was used to lysis the cells by incubating for 15 min at 37 °Cand
disturbed using French press.The unbroken cells and debris were removed by centrifugation
o
(10 min, 10,000 rpm, 4 C) supernatant ultracentrifuged (1 h, 15,000 rpm) to separate the
membrane fraction. Then the pellet was dissolved in lysis buffer and FMM isolation protein
was extracted. Finally, the DRM and DSM fractions were used for further study [42].
Lipid analysis
The dried DRM and DSM samples in analyzed using ultra-performance liquid
chromatogram coupled to a quadrupole/time-of-flight hybrid mass spectrometer equipped
with electrospray ionization source (LC-MS/MS). Processing of chromatograms, peak
detection, and integration were performed using MassLynx software (version 4.1, Waters).
Molecular docking validation
Molecular docking protocol was followed as per the above report with slight
modifications [43]. Briefly, The structural drawing and geometry cleaning of novel potential
leads of ChDiPiCa were performed in Maestro 9.3 of Schrodinger suite 2012 platform and

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then subjected to other parameters viz energy minimization by using OPLS 2005 force field,
addition of hydrogen atoms, neutralization of charged groups, generation of ionization states
and set pH 7.5 using Epik. Generation of tautomers and stereoisomers of 32 per ligand and
low-energy ring conformations and optimize the geometries followed by generating low
energy ring conformation per ligand were computed, optimized by LigPrep and used for
molecular docking.
The co-ordinates of membrane-bound transglycosylase in complex with a lipid II
analogand monofunctional glycosyltransferase in complex with moenomycin from S. aureus
were obtained from the RCSB Protein Data Bank, whose PDB IDS are 3VMT and 6FTB
respectively. Crystal structure was imported and refined by a multistep process through the
protein preparation wizard of Maestro 9.3, which includes energy minimization using OPLS-
2005 force field, correct bond orders were assigned, hydrogen atoms were added and the
water molecules removed beyond 5Å from hetero atom, formal charges, amide groups of Asn
and Gln were optimized. All amino acid flips were assigned to correct geometry and
hydrogen bonds were optimized. Using PROPKA, pH was fixed and optimized to 7.5. Non-
hydrogen atoms were minimized by restrained minimization to default RMSD to 0.3Å. Using
Extra-precision (XP) docking and scoring each compound were docking into the receptor grid
of radii 30Å × 30Å × 30Å and the docking calculation were judge based on the Glide score.
Results and discussion
Biocidal activity of synthetic analog against MRSA
The synthesized analog was assessed for antibacterial property by disc diffusion
method. The piperazine and phthalimide as parental structural (ChDiPiCa) was tested against
MRSA showed biocidal effect in a dose dependent manner.The analog5have highest
significant activity compared to the standards antibiotics. The analog5 showed dose

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dependent biocidal activity in the range of 0.5 to 50 µg/mL concentration and observed after
o
12 h of MRSA plate incubation at 37 C, which reveals that the minimum inhibitory
concentration (MIC) at 26.0 µg/mL (14.5 mm) compared to the standard antibiotics
streptomycin and bacitracin at 10 µg discs14 and 10 mm respectively (Fig. 1). [37, 39] These
results were comparable to the previous research showed, as a concentration increases; the
inhibitory property also significantly increases upon due of time with respect to standards.
[38, 43] This confirms that, the synthetic piperazine derivative showed significant results due
to its structure and chemical moieties in the compound. The analogue5showed potent nature
due to its electronegative group chlorine (Cl) present in the para position of aromatic ring and
phenyl ring with substituted to the linker thioamide of piperzine moiety with promising
biological activity to govern physicochemical, pharmacokinetic and pharmacodynamic
property.
Figure 1: Antimicrobial activity of analog5 against MRSA. MRSA Pure Culture (A)The
dose dependent concentration of the analog5 (B) compared to the standard antibiotics
streptomycin and bacitracin(C).
Qualitative and quantitative effect on MRSA growth
The active analog5 has been further tested for MRSA viability upon treatment for 12
h usingSPM and growth of bacterial colonies were assessed(Fig. 2). At different

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concentrations of analog5 with MRSA showed decreased viability of the MRSA growth on
nutrient agar medium and one log reduction in the MRSA viability during 12 h of time (Fig.
2B).[37] The evaluated MRSA growth quantitatively by SPM shows, the effect of analog5 on
MRSA membrane or its cell metabolism is the reason for the viability excreted here.[39] The
MRSA colony forming unit (CFU)/mL was decreased as dose of the active analog increases
as compared to the control (Fig. 2A). Also, the quantitative measurement of the viable
bacteria showed the efficacy of the analog5 compared to the control in the present study and
it has been in accordance with the other reports of MRSA growth kinetics (40).

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Figure 2: The qualitative in vitro biocidal property of analog5against planktonic MRSA.
The qualitative biocidal effects on the CFU values of MRSA and significant role of
analog5decreased CFU/mL counts of bacteria was observed B. The typical photographs of re-
cultivated planktonic MRSA colonies on nutrient agar plates such as positive control(a),
6.5µg/mL(b), 13µg/mL(c), 26µg/mL (d) and 52µg/mL (e)of analog5and control (f).
Membrane damaging effect on MRSA
The active analog5 has been evaluated for MRSA membrane damage effect and
showed significant membrane alterations such as pores, cracks and breaks compared to
control untreated by SEM in this study (Fig. 3). The MIC of analog5 in 12 h showed the
prominent effect on the MRSA membrane which is comparable to the previous reports
carried out to understand the mechanism of synthetics against drug resistant MRSA (37). This
results also confirms that, the role of analog5 at membrane or membrane bound factors are
the responsible of the this biocidal cause excreted which leads to cellular content leakage, cell
death and adverse inflammatory conditions can be induced during MRSA infection in the
host.
A
B
Figure 3: The membrane damaging property of analog5 against MRSA. The membrane
damage effect of analog5 has been analysed using SEM and showed there is alterations on

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membrane (B) such as pores or breaks indicate that, the potential inhibition of MRSA growth
compared to the control (A) after 12 h of incubation.
Effect on MRSA membrane fatty acid content
To understand the role of synthesized analog5 on MRSA, study further evaluated at
cell fatty acid content disappearance during the bacterial growth was determined by GC-MS
by treating MIC value of analog5 compared to control, and showed significant changes in the
fatty acid profile of the MRSA. [37] This indicates that, the effect of synthetic on
biosynthetic pathway was confirmed by decreased or increased contents of the fatty acids
present in the MRSA. The saturated (Lauric acid, Myristic acid and Palmitic acid) and
unsaturated (Oleic acid, Linoleic acid and Linolenic acid) contents of treated showed
significant alterations after treatment compared to control confirms analog5 involvement in
lipid biosynthesis was deduced (Table 2). The loss of cell wall components leads to loss of
membrane integrity, which intern MRSA become susceptible to any antibiotic treatments
during pathogenic conditions. [44]
Table 2:The fatty acid profile of MRSA. The treatment of analog5 to MRSA showing
alterations in the fatty acid content confirms involvement in cell wall lipid biosynthetic
pathway.
Sl. no Methyl ester of Control (%)
Analog5 (%) MUFA/PUFA/SF
Lipid number
C6:0
1
1
2
3
4
5
6
Caproic acid
Caprillic acid
Capric acid
Lauric acid
Myristic acid
Palmitic acid
Stearic acid
13.52
1.16
19.87
Nil
SF
SF
SF
SF
SF
SF
SF
C8:0
2.37
1.07
0.53
10.65
12.72
3.02
C10:0
22.12
24.12
19.83
4.58
C12:0
C14:0
C16:0
C18:0

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7
Behenic acid
Oleic acid
0.52
Nil
2.12
2.18
Nil
SF
C22:0
C18:1 cis-9
C18:2 cis, cis9,12
C18:0
11
12
13
15
17.77
15.15
7.26
MUFA
PUFA
PUFA
MUFA
Linoleic acid
Linolenic acid
Eicosenoic acid
1.81
Nil
C20:0
The MRSA lipid fraction disassociation assessment
All living cell components protected by cell membrane composed of lipids, proteins
and carbohydrate moieties. Despite the primary functional importance of the cell, the
organization and biological role was also attributed also from the functional membrane
microdomains which are largely unknown. Here we tried to address the solution for the
human pathogen MRSA infections. The MRSA produces a largely unknown flotillin FloA,
and involved in the biosynthesis of isoprenoid membrane lipids. Apart from this, the MRSA
attracted much scientific community to address the global solution to treat and overcome the
antibiotic treatments. [45, 46, 47] The antibiotic resistance was acquired by the MRSA due to
the presence of PBP2a, in which inhibitory molecules bind the PBP active site as substrate
analogs to inhibit the PBP activity efficiently involved in the peptidoglycan synthesis during
cell division. [48, 49]The PBP2a active is inaccessible to antibiotics due to located in a deep
pocket, which licence the MRSA strain to become virulent through rapid divide and
proliferation in the journey of pathogenesis. [50, 51]
Constituents of membrane lipids of FMMs
In the present investigation, to understand the role in antibiotic resistance disruption,
the potent analogues was synthesized and studied for the time. To evidence this, MIC
concentration of the synthetic analog5 treated with MRSA confirmed the important
functional domains of the lipid membrane of MRSA compared to the untreated samples(Fig.

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3). This study shows that bacterial cells organize sophisticated programs for membrane
compartmentalization and defines an innovative strategy to overcome MRSA antibiotic
resistance and help reduce the high mortality rates caused by invasive MRSA infections.
Figure 3: Membrane functional domain disassembly in MRSA. The MRSA was used to
assess the role of potent synthesized analog5 to disrupt the functional domains of the lipid
raft in the membrane. The treated sample analog successfully confirmed the membrane
alteration and its compositional variation by LC-MS/MS for membrane DSM and DRM
fraction after the treatment.
The bacterial FMMs organization relies on the scaffold protein flotillin and
aggregation of isoprenoid lipids of yet unknown nature. [52] Staphyloxanthin is an
unphosphorylated saccharolipid of isoprenoid nature found in S. aureus membranes that gives

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S. aureus its typical yellow color.[53] To understand and identify the possible constituents of
lipids and those mechanisms in FMM assembly, we extracted the MRSA cell
membranes.[54] Observed the different lipid composition and their densities of compacted
FMMs in control compared to the treated one, a FMM-rich sample can be obtained by
exploiting the FMMs’ insolubility after treatment with non-ionic detergents (0.5%–1% Triton
o
X-100, 4 C) prior to phase separation.[55] This treatment generates a membrane fraction
sensitive to detergent disaggregation (detergent-sensitive membrane; DSM) and another that
is resistant to disruption with larger FMM-rich fragments (detergent-resistant membrane;
DRM). Total lipids were extracted from DRM and DSM fractions and membrane lipids
identified in untargeted lipidomics experiments using LC-MS/MS for the validation purpose
(Fig. 3, Table 3).
FMM disruption inhibits antibiotic resistance in MRSA
The well known evidence that, the FMM perturbation affects PBP2a oligomerization
and it led us to test new molecule for the first time to inhibit growth of MRSA. The present
results evidenced for the importance of membrane lipid domains in bacterial cell
organization. Our results help to explain clinical studies that detected a beneficial role of
statins in microbial infections. [56, 57, 58, 59]The cell membrane fraction, and DSM and
DRM content detected the number of membrane lipids constituents presented in the table 3,
among them LC-MS/MS detected the many lipid moieties which is has molecular weight
ranged from 200 to 600 m/z. Among them Lysophosphatidylethanolamines,
Phosphatidylglycerols, and Monoacylglycerols are majorly affected in DSM and DRM. But,
in treated cell membrane fraction of MRSA particularly altered the
Lysophosphatidylcholines, Phosphatidylserines, Sphingomyelins, Ceramide-1-phosphates,
Diacylglycerols,
Diacylated
phosphatidylinositol
monomannoside,
Diacylated
phosphatidylinositol dimannoside, Triacylated phosphatidylinositol dimannoside, and

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Tetraacylated phosphatidylinositol dimannoside lipids as compared to the untreated MRSA.
These membrane constituents may definitely impair the structural and functional role of the
MRSA can be evidenced based on the literature reports involved in disassembling the nature
of cell integrity which helps bacteria to resist many available antibiotics and hypothesized
that, the present candidates can be a impairing medicine to treat MRSA (Fig. 4). [60, 61]In
this regard, the proven hypothesis data suggest that, altering or miss loading of membrane
functional lipid domains helps to design a new type of drug like candidates to treat life
threatening infections caused by MRSA and group of ESKAPE organisms.
Figure 4: Hypothesis of present study to overcome the antibiotic resistance in MRSA.

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Table 3: Total membrane lipid assessment by LC-MS/MS in MRSA. The MRSA treated analog5 disrupted/altered the functional rafts of
membrane lipids which is confirmed by the LC-MS/MS. Note: Bold lipid names are detected in our study and unbold are lipid contents in cell.
Lipids name
Short name
Molecular
weight,
g/mol
Cell membrane
Control Analog 5 Control Analog 5 Control
DSM
DRM
Analog 5
Phosphatidylcholines
Pc
Lyso pc
Plasmeyl pc
PE
Lyso PE
Plasmenyl PE
PS
786.129
495.638
705.999
691.972
256.171
691.972
385.304
650.967
674.941
886.56
+
+
-
Lysophosphatidylcholines
Plasmenylphosphatidylcholines
Phosphatidylethanolamines
Lysophosphatidylethanolamines
Plasmenylphosphatidylethanolamines
Phosphatidylserines
Sphingomyelins
+
+
-
+
-
+
+
-
-
SM
PA
PI
PG
Phosphatidic acids
Phosphatidylinositols
Phosphatidylglycerols
Ceramide-1-phosphates
Sulfatides
Gangliosides
Monoacylglycerols
+
+
+
-
+
+
+
-
+
+
-
-
262.239
505.7
Cerp
ST
[Glycon]-Cer
MG
808.166
1546.841
266.202
594.962
881.34
+
+
+
-
Diacylglycerols
Triacylglycerols
DG
TG
Monogalactosyldiacylglycerol
Digalactosyldiacylglycerols
MGDG
DGDG
755.087
917.228
+
+
+
+
-
-
-
-
Diacylated phosphatidylinositol
monomannoside
Ac2PIM1
Ac2PIM2
Ac3PIM2
Ac4PIM2
390.234
390.234
390.234
390.234
Diacylated phosphatidylinositol
dimannoside
Triacylated phosphatidylinositol
dimannoside
Tetraacylated
phosphatidylinositol dimannoside

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Molecular docking validation
In the present study, synthesized analog5was efficiently involved in the control of
MRSA growth which is confirmed in vitro antibacterial study, and involved in FMMs
disruption. To confirm this, the in silico molecular docking approach was used to give more
suitable promising way of basic understanding foundation for the drug design to treat
notorious MRSA treatment. The molecular docking was performed to evaluate the dynamics
at biological interfaces by selecting S. aureus proteins such as 3VMT and 6FTB from RCSB
Protein Data Bank which is membrane-bound transglycosylase in complex with a lipid II
analog and monofunctional glycosyltransferase in complex with moenomycin from S. aureus
respectively. The analog5 is accessed the active site of the above said proteins and confirms
that, high docking score and strong interactions such as hydrogen, vanderwaals, and
hydrophobic with possible amino acid residues (Table 4). The ligand piperazine having
phthalic anhydride, chloro groups (5e),gave the highest docking score -3.297 with 3VMT
protein and significant -3.558 with 6FTB protein compared to the standard antibiotic
streptomycin -1.009 and -7.293 respectively.
The binding site of analog5 in active site of 3VMT and 6FBT interaction 2D and 3D
were represented respectively (Fig. 5). Compound 5of protein 3MVT demonstrated hydrogen
bond,ꢀ − ꢀ stacking and ꢀ-cation interaction associations with ARG117 with compound
5and indicated other than hydrogen interactions, hydrophobhic interaction play an role with
the protein 6FTB. The involvement of the chloro, sulphur and the nitrogen groups in
electrostatic forces of attraction enhanced the docking scores of these analogues was
predicted compared to the standard streptomycin, which is interacted strongly with 3MVT
through MET 99, GLU 100, GLU 102, ARG 117, MG 302 for hydrogen interaction, and with
6FTB PO4 313 and PHE 110 showed relevant significant proposed interaction comparable

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with thepotent role of synthesized candidate analog5 antimicrobial and cell membrane lipid
constituents miss loading or disassembling to inhibit the MRSA infection was drawn and
suggested from this study.
Table 4: Molecular interaction study of analog5 and antibiotic streptomycin with 3VMT and
6FBT.
PDB id
3VMT
6FTB
Glide
Energy
(kcal/mol) (kcal/mol)
Glide
Emodel
Glide
Energy
(kcal/mol) (kcal/mol)
Glide
Emodel
Docking
Score
XP
Gscore
Docking
Score
XP
Gscore
Compounds
ChDiPiCa (5) -3.297
-3.335
-1.915
-45.215
-44.065
-57.116
-77.982
-3.558
-7.293
-3.596
-7.465
-49.97
-64.356
-57.408
-48.084
-1.099
Streptomycin

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Figure 5: Molecular docking interactive map of ligand 5 for 3VMT(A & B), for 6FTB (C & D) and antibiotic streptomycin 3VMT(E & F), for
6FTB(G & H) of binding deep inside the active site, depicting the best docking pose showing 2D and 3D respectively.

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Conclusion
The present investigation taken up with the aim to understand the role of potent
compound to treat infectious agent MRSA by disrupting the well organized cell membrane by
miss loading finest FMM such as lipid rafts to synthesize functional membrane. The
synthesized potent novel compound ChDiPiCa having biocidal property against one of the
ESKAPE group of organisms S. aureus involved in the health complications. Further, the
ChDiPiCa showed membrane damaging property validated by SEM and established the miss
loading of membrane constituents such lipid suggests the loss of cell membrane integrity and
basic organizations which causes the inhibition of MRSA cell growth and leads to death.
Also, it was validated byin silico molecular docking approaches. This confirms that, the
targeted lipid membrane and enzyme responsible for the synthesis of functional lipid
membrane was altered effectively which reduces the antibiotic resistance and susceptible to
the given drug candidate. For the future, the present result suggests that tracking the core
functional membrane microdomain miss loading, is the alternative way to reduce the
notorious MRSA infections and hot area in clinical microbiology and drug discovery
research.
Competing financial interests
The authors declare no competing financial interest.
Acknowledgments
The authors greatly thank Anand, A. P. for providing reference bacterial strain used in
the present study and SJCE, Mysore for providing instrumentation facility.
Author Contribution
The work was designed by the MHM, HSNP, CSK and PM, work carried out by HSNP,
suggestion by LM, manuscript suggestions edited, reviewed and submitted by HSNP and PM.

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