Biofilm inhibition and anti-Candida activity of a cationic lipo-benzamide molecule with twin-nonyl chain

Article abstract of DOI:10.1016/j.bmcl.2018.04.024

A series of cationic lipo-benzamide compounds with varying lengths of hydrocarbon chains (C2M–C18M) were evaluated for anti-Candida activity. Four compounds harbouring 8–11 hydrocarbon chains demonstrated concentration-dependent inhibition of fungal cell

Full text of DOI:10.1016/j.bmcl.2018.04.024

Accepted Manuscript
Biofilm inhibition and anti-Candida activity of a cationic lipo-benzamide mol-
ecule with twin-nonyl chain
Tushar Jain, Prathap Reddy Muktapuram, Komal Sharma, Owk Ravi, Garima
Pant, Kalyan Mitra, Surendar Reddy Bathula, Dibyendu Banerjee
PII:
S0960-894X(18)30336-6
https://doi.org/10.1016/j.bmcl.2018.04.024
BMCL 25771
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
9 February 2018
21 March 2018
11 April 2018
Please cite this article as: Jain, T., Muktapuram, P.R., Sharma, K., Ravi, O., Pant, G., Mitra, K., Bathula, S.R.,
Banerjee, D., Biofilm inhibition and anti-Candida activity of a cationic lipo-benzamide molecule with twin-nonyl
chain, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.04.024
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Bioorganic & Medicinal Chemistry Letters
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Biofilm inhibition and anti-Candida activity of a cationic lipo-benzamide
molecule with twin-nonyl chain
Tushar Jain a,b Prathap Reddy Muktapuramc, Komal Sharmac , Owk Ravib,c Garima Pant,d Kalyan Mitra,d
Surendar Reddy Bathula,*b,c Dibyendu Banerjee*a,b
aMolecular and Structural Biology Division, CSIR-Central Drug Research Institute, Lucknow-226 031, U.P., India.
bAcademy of Scientific and Innovative Research (AcSIR), Chennai, India,
cNatural Products Chemistry Division, CSIR-Indian Institute of Chemical Technology, Hyderabad -500007, India.
dElectron Microscopy Unit, Sophisticated and Analytical Instruments Facility, CSIR-Central Drug Research Institute, Lucknow-226 031, U.P, India.
ARTICLE INFO
ABSTRACT
Article history:
Received
A series of cationic lipo-benzamide compounds with varying lengths of hydrocarbon chains
(C2M-C18M) were evaluated for anti-Candida activity. Four compounds harbouring 8-11
hydrocarbon chains demonstrated concentration-dependent inhibition of fungal cell growth with
Minimum Inhibitory Concentration (MIC) of ≤ 6.2 µg ml-1. The most active compound (C9M)
inhibited growth of both Candida albicans and non-albicans strains and is equally active against
pairs of azole sensitive and resistant clinical isolates of C. albicans. Compound C9M also
inhibited different stages of Candida biofilms. Scanning Electron Microscopy (SEM) of Candida
cells after C9M treatment was also done and no significant cell lysis was observed. Hemolysis
assay was performed and only 2.5% haemolysis was observed at MIC concentration.
2009 Elsevier Ltd. All rights reserved.
Revised
Accepted
Available online
Keywords:
Candida albicans
Multi Drug Resistance
Biofilm
Cationic lipo-benzamide
Antifungal activity
Introduction
Quaternary ammonium compounds (QACs) have been known
8
to be the most useful antiseptics and disinfectants . The cationic
Fungal infections are one of the most pressing public health care
1
concerns worldwide . The human microbiota also consists of
agents supposedly react with the phospholipid components of the
cytoplasmic membrane, thereby producing membrane distortion
and a net positive charge on microbial cells 9. The positive charge
on microbial cells has often been correlated to their biocidal
action. Some polymeric QACs have been shown to induce lysis
of spheroplasts of Serratia marcescens, but not those of C.
albicans 10. The critical phenomenon determining the antifungal
effect of cationic surfactants and lipids is not cell lysis but rather
the reversal of cell surface charge from negative to positive that
leads to membrane distortion 9. QAC’s fused to varying length of
carbon chains are reported to have antimicrobial activities. It was
reported that QAC’s fused to 10-12 methylene linkers showed
growth inhibitory effect against drug-resistant bacterial strains
such as methicillin-resistant Staphylococcus aureus (MRSA) and
vancomycin-resistant enterococci (VRE) 11. Moreover, molecules
fungi that render several beneficial functions. In spite of
tremendous health benefits, some microbiota can cause mild to
severe infections, especially in conditions of immune-
2
suppression . Candida albicans is one such fungus which is a
part of commensal microflora in humans but becomes invasive
and lethal when the immune system of an individual is
3
compromised
mucosal (localized-candidiasis), as well as systemic infections
. Candida causes cutaneous, subcutaneous,
4
(Candidemia) . Candidemia or Candidiasis has been a leading
type of nosocomial infection with high morbidity and mortality
5
rates (20–40%)
Candidiasis includes polyenes, azoles and recently launched
. Conventional antifungal treatment for
echinocandins 6. However, conventional treatment options have a
narrow therapeutic index, poor bio-availability, severe side
with
a net positive charge were not only able to kill
7
.
effects and the chance of emergence of resistant strains
microorganisms in solution but also upon attachment or
adsorption onto solid surfaces 12. The organic monolayers
containing quaternary ammonium groups have been shown to
prevent deposition and growth of bacterial biofilm 13. However,
Therefore, the limitations associated with conventional treatment
necessitate the exploration of new drug candidates that can get
rid of drug-resistant and lethal systemic mycoses.

the activity of such compounds on fungal biofilm formation and
maturation remain elusive.
sensitive and resistant clinical isolates (Gu4/Gu5 and
DSY294/DSY296) of C. albicans and non-albicans strains
including C. glabrata, C. krusei, C. parapsilosis and C.
tropicalis. C9M were able to inhibit the planktonic growth of all
tested Candida strains with MIC values ranging from 0.7-3.1 µg
ml-1 (Table 1). Compound C9M showed dramatic anti-fungal
activity against both fluconazole-sensitive and resistant clinical
isolates of C. albicans. Fluconazole (Flu) was used as standard
anti-fungal agent.
I- R
N+
MeO
H
N
R
O
R = C2H5 to C18H38
Scheme 1: Represents general structure of lipo-benzamide.
Our present study focuses primarily on the antifungal activity of
hybrid quaternary ammonium compounds (QACs) previously
synthesized14 by our group and showed promising anticancer
activity. Lipo-benzamide compounds fused with varying lengths
of hydrocarbon chains C2M - C18M (Scheme 1) were evaluated
for antifungal activity against C. albicans. The most active
compound was tested for growth inhibition against azole-
resistant clinical isolates of C. albicans as well as non-albicans
Candida strains. The ability of such cationic lipo-benzamides to
inhibit adhesion of microbial cells to polystyrene surfaces as well
as on biofilm growth was further evaluated.
Table 1. Anti-Candida activity of cationic lipo-benzamide C9M. Fluconazole
was used as standard anti-fungal agent.
MIC (µg ml-1)a
Strain
Fluconazole
C9M
Candida albicans
ATCC90028
Figure 1. Antifungal activity of cationic lipo-benzamides. Inhibition of
planktonic growth of C. albicans ATCC 90028 was determined at varying
concentrations of cationic lipobenzamides, (A) C8M (B) C9M (C) C10M (D)
C11M. Percentage inhibition was plotted on Y-axis against the concentration
of test compound on X-axis. Percentage inhibition was determined with
respect to growth control in which no test compound was added.
4
3.1
Candida albicans
SC5314
2
1.5
Candida albicans Gu4
Candida albicans Gu5
4
3.1
3.1
128
Adhesion of C. albicans to polystyrene surfaces, prosthetic
substrate, and host tissue leads to colonization and is an
important first step leading to biofilm formation. Fungal biofilms
contain population of persister cells which shows much higher
resistance to anti-fungal drugs than planktonic cells. Quaternary
ammonium group containing C9M was able to inhibit Candida
cell adhesion to the polystyrene surface in a concentration
dependent manner. Compound C9M inhibits 90% Candida cell
adhesion on polystyrene surface at 12.5 µg ml-1 concentration
compared to growth control in which no drug was added (Fig.
2A). Fluconazole was used as control anti-fungal agent for whom
inhibition of cell adhesion was not observed at tested
concentrations upto 25 µg ml-1.
Candida albicans
DSY294
4
1.5
1.5
1.5
1.5
1.5
0.7
Candida albicans
DSY296
128
4
Candida glabrata
MTCC3814
Candida krusei
MTCC9215
64
2
Candida parapsiloisis
MTCC7043
Candida tropicalis
MTCC9038
4
a MIC - Minimum Inhibitory Concentration of test compound
that resulted in no visible growth as compared to growth control.
Results and Discussion
Synthesized cationic lipo-benzamide compounds (C2M-C18M)
were evaluated for inhibition of planktonic growth of C. albicans
strain ATCC 90028 using Broth Microdilution Assay. Four
compounds from the series, C8M, C9M, C10M and C11M
showed concentration-dependent growth inhibition with
Minimum Inhibitory Concentration (MIC) of ≤6.2 µg ml-1 (Fig.
1). Compounds with an odd number of carbon chains C9M (9
carbon chain) and C11M (11 carbon chain) were found most
active against ATCC 90028 with MIC value ≤ 3.1 µg ml-1.
Compound C9M was further tested against two pairs of azole
Figure 2. Compound C9M was tested for inhibition of different stages of C.
albicans (ATCC 90028) biofilm development, (A) Adhesion (B) Formation
and (C) Mature Biofilm, and inhibition was quantified using MTT metabolic
assay. Inhibition of adhesion to polystyrene surface and biofilm formation
was observed at 12.5µg ml-1 and 3.12µg ml-1 respectively. Mature Biofilm
was inhibited at 12.5µg ml-1 concentration.
The deposition of organic monolayers onto solid surfaces
containing quaternary ammonium groups prevents deposition and
growth of bacterial biofilms 12a. Our study reveals that C9M can

varying concentrations of C9M on X-axis. Complete hemolysis was observed
at 12.5µg ml-1 concentration of C9M. (B) The release of macromolecules
upon cell lysis was measured by incubating Candida cell with increasing
concentrations of C9M. OD260 and OD280 were recorded to quantify the
release of nucleic acid and proteins respectively. No significant release of
macromolecules was observed till 12.5µg ml-1 concentration of C9M.
not only inhibit Candida biofilm formation but is also able to
eradicate mature biofilms. 1x106 cells of C. albicans ATCC
90028 were allowed to grow and adhere to the polystyrene coated
wells of 96 well plates. C9M was administered at different time
points for identifying its role during biofilm formation and also
on mature biofilm. C9M was able to inhibit Candida biofilm
formation at Biofilm Inhibitory Concentration (BIC80) of 3.12 µg
ml-1, quantified using MTT assay (Fig. 2B). Microscopic
examination also revealed that biofilm formation was inhibited at
different concentrations of C9M (Fig. 3). We found that C9M
eradicates mature Candida biofilms at a Biofilm Eradicating
Concentration (BEC80) of 12.5 µg ml-1 (Fig. 2C). Fluconazole
was used as a standard anti-fungal drug and no biofilm inhibition
was observed at tested concentration.
The ability of QAC’s to inhibit the growth of microbial cells has
8
previously been attributed to cell lysis . However, in our assay,
we observed that HC90 of cationic lipo-benzamide C9M (12.5 µg
ml-1) was 4 times higher than the MIC value (3.1 µg ml-1)
observed in Candida species. To check for Candida cell lysis at
effective concentrations of C9M, we quantified the amount of
macromolecules (protein and nucleic acid) released upon
treatment with different concentrations of C9M. The increase in
the amount of these macromolecules in surrounding media gives
an indirect evidence of cell lysis. The release of proteins and
nucleic acids was quantified spectro-photometrically by
measuring OD at 280 and 260 nm respectively. In our
observation, there was no increase in OD upto a concentration of
12.5 µg ml-1 (Fig. 4B) which is about 4 folds higher than the
effective MIC value of C9M against C. albicans planktonic cells.
To further verify our findings, we checked for topological
alterations induced by test compound C9M on C. albicans cells
using Scanning Electron Microscopy (SEM). Our observations
did not reveal any significant morphological changes after C9M
treatment when compared to the control cells (Fig. 5).
Interestingly, it became evident from our study that cationic lipo-
benzamide compound C9M does not cause lysis of Candida cells
at a concentration that effectively inhibits planktonic growth.
Figure 3. Qualitative analysis of inhibition of biofilm formation by C9M
against C. albicans cells (ATCC 90028). Microscopic analysis revealed
inhibition of biofilm formation as compared to, (A) Growth control in which
no test compound (C9M) was added. (B) to (F) Biofilm inhibition with
increasing concentrations of C9M. (B) 1.56µg ml-1, (C) 3.12µg ml-1, (D)
6.25µg ml-1, (E) 12.5µg ml-1 and (F) 25µg ml-1, represents varying
concentrations of C9M.
Cytotoxicity of C9M was previously evaluated in cancerous and
non-cancerous cell lines. C9M did not exhibit cytotoxicity till 20
µmol L-1 (~10 µg ml-1) concentration in most of the cell lines
tested 14. In the present study, we evaluated the hemolytic activity
of C9M against human erythrocytes. We selected erythrocytes
(RBCs) because of their fragile cell membrane that make them
susceptible to lysis by QAC’s. Hemolytic activity of C9M was
quantified using spectrophotometer by measuring the amount of
hemoglobin released upon cell lysis. The concentration of C9M
that can lyse 90% of total RBC (HC90) was determined to be 12.5
µg ml-1 (Fig. 4A). 1% Triton X-100 was used as a control to
achieve 100% RBC lysis and buffer-treated sample was used as
negative control.
Figure 5. Representative SEM images of C. albicans cells (ATCC 90028). A)
Control C. albicans cells at 2500X and 5000X magnification. (B) C. albicans
cells (2500X-5000X magnification) after incubation for 24 h with 3.1 µg ml-1
(MIC) concentration of compound C9M.
A series of cationic lipo-benzamides containing quaternary
ammonium ion were tested and found to exhibit anti-Candida
activity. Cationic lipo-benzamides with chain lengths of 8-11
methyl units were found to inhibit the growth of C. albicans. The
most potent among them were compounds harboring 9 and 11
carbon chain lengths. The MIC of the cationic lipo-benzamide
molecule C9M was found to be quite low (0.7-3.1 µg ml-1)
against various fungal strains. Notably, the compound C9M was
equally active against fluconazole-sensitive and resistant strains
of C. albicans and against the tested non-albicans species of
Candida as well. The chain length specificity and the anti-
Figure 4. Cationic lipo-benzamide C9M does not lyses cells at the MIC
concentration. (A) Different concentrations of C9M were incubated with
RBCs. Hemoglobin released upon RBC lysis was quantified by
spectrophotometer. Percentage hemolysis was plotted on Y-axis against
a

15
.
microbial property have been corroborated by previous reports
In one such report, Locheret al., have reported nostocarbolines
Acknowledgments
linked to 10 and 12 methylene units were bactericidal against
(MRSA),
The authors acknowledge CSIR-CDRI for funds to carry out
the research. TJ, OR and PRM acknowledge CSIR, New Delhi
for their research fellowships. K.S. thanks SERB for National
Post Doctoral Fellowship PDF/2016/000319. The research is
supported by project BSC0001 from CSIR-CDRI and GAP0556
from CSIR-IICT. The authors also gratefully acknowledge SAIF,
CSIR-CDRI, Lucknow, for experimental support. The
manuscript carries a CDRI manuscript number 110/2016/DB.
Methicillin-resistant
Staphylococcus
aureus
Vancomycin-resistant Enterococci (VRE), and E. coli
16
.
However, no specific target has been identified for QAC’s till
date; rather it is assumed that their effect is global. Given their
chemical structures, it is possible that QACs interact with the
positive charge and hydrophobic regions of microbial membranes
17. The cationic agents are traditionally known to react with the
phospholipid components in the cell membrane, thereby
producing membrane distortions often leading to a complete loss
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The fungicidal action of C9M observed by us was not
associated with cell lysis since at the MIC concentration; C9M
did not show any significant RBC or Candida cell lysis. The lack
of Candida cell lysis was re-confirmed by SEM imaging and the
quantification of macromolecules (DNA and proteins) released
from Candida cells treated at MIC concentration of C9M.
Therefore, it is possible that these molecules exhibit their
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Conclusion
We have identified the anti-Candida and biofilm inhibition
potential of a cationic lipo-benzamide molecule containing
quaternary ammonium ion. More efforts will be necessary in
order to design and test QACs or similar compounds that may be
suitable for clinical usage.
Supplementary Material
Supplementary data associated with this article can be found in
the online version

Graphical Abstr
Biofilm inhibition and anti-Candida activity of a cationic
lipo-benzamide molecule with twin-nonyl chain
Leave this area blank for abstract info.
Tushar Jain a,b Prathap Reddy Muktapuramc, Komal Sharmac , Owk Ravib,c Garima Pant,d Kalyan Mitra,d
Surendar Reddy Bathula,*b,c Dibyendu Banerjee*a,b