K.K. Sharma, R. Ravi, I.K. Maurya et al.
European Journal of Medicinal Chemistry 223 (2021) 113635
Fig. 3. Stern-Volmer plots for the Trp fluorescence quenching. A) In the absence of
vesicles, B) Plots in the presence of EYPC/EYPG (7:3 w/w), and C) In the presence of
EYPC/cholesterol (10:1 w/w).
0 to 4 h (Fig. 5). The polydispersity index (Ð) for 14f sparingly in-
creases from 0.333 to 0.584, whereas for 12a it increases from 0.383
to 0.922. The experiments indicate higher dispersion, homogeneity
and uniformity within the solution for 12a in comparison to 14f.
However, inactive peptide 12a is better distributed within the so-
lution. To further understand peptide architecture, fluorescence
spectroscopic and electron microscopy techniques were employed.
2.6.2. Fluorescence spectroscopic study
Tryptophan fluorescence emission study is a useful method for
investigating the interaction of the synthetic peptides and their
assemblies [29]. One plausible interaction within the molecule
could be due to the formation of p-stacked aggregates of the
7
tryptophan residue, which were evaluated by monitoring the
fluorescence emission spectra (Fig. 5) [58e60]. Upon investigation,
initially, peptides 14f and 12a displayed a prominent emission peak
around 335 nm representing the tryptophan residue on excitation
at 280 nm.
After 24 h, 14f displayed increase in emission in fluorescence,
which is consistent with extended p-stacked aggregate formation
of tryptophan residue possibly due to the presence of ring-arylation
at the histidine residue resulting in additional p-stacked aggrega-
tion. In the case of 12a, the increase in fluorescence was less
compared to 14f due to the absence of ring-arylation.
2.6.3. Scanning electron microscopy (SEM) study
To have a microscopic insight of the peptide assembly, we
investigated the morphology of the active (14f) and control peptide
(12a). SEM study revealed that the peptide 14f upon incubation for
24 h formed unorganized aggregates (Fig. 6A and B) in comparison
to that of 12a (Fig. 6C), which was quite homogenously dispersed in
the phosphate saline buffer (PBS) solution. The SEM results and
fluorescence emission sepctroscopic study indicate that peptide 14f
forms random aggregates.
The peptide architecture studies indiactes that the synthetic
amphiphilic peptide 14f exhibits self-aggregation properties at the
pathogen surface. For better understanding, we designed other
microscopic experiments to further investigate mechanism of ac-
tion studies.
2.7. Mechanism of action study
We investigated the thorough mechanism of action by
employing various microscopic approaches. We anticipate that
determination of mechanism of action of 14f could guide in further
design of potent analogues with defined mode of action in
combating microbes. The CLSM examination was conducted to
understand the various actions of 14f on the fungal permeabiliza-
tion, intracellular localization and binding to genetic materials in
the fungal cells. The SEM study was carried out to study the effect of
active peptide on the cellular morphological changes, and the high-
resolution transmission electron microscopy (HR-TEM) study hel-
ped to observed the ultrastrucural actions of active peptide 14f on
cell surface of fungi.
2.7.1. Confocal laser scanning microscopic study
Red fluorescent propidium iodide (PI) is not permeable to intact
cell membrane, and DNA binding dye. If interacting peptide per-
meabilizes cell wall and membrane, causing in compromised fungal
membrane permeability thereby permitting PI to enter inside the
dead cells and provide red fluorescence. Under confocal micro-
scopic visualization, the uptake of PI after treatment by fungal cells
with 14f at MIC (1 h) showed red fluorescence due to per-
meabilization of cell surface (Fig. 4B), although the untreated cells
did not possess red fluorescence due to undamaged cell surface
(Fig. 4A).
These results indicate that PI entered intracellularly within the
cells after treatment with 14f as treated cells were unable to
regulate the chemical movement from its cell surface.
To study intracellular localization of 14f in C. neoformans, we
prepared FITC-labeled peptides (fluorescein isothiocyanate; Ex
480 nm) 14f and 12a. The confocal investigation of FITC-labeled 12a
revealed that it can not cross the cell surface due to the intact cell
surface as confirmed by the absence of green fluorescence (Fig. 7C).
FITC-labeled 14f permeated from cell membrane barriers via per-
meabilization and disruption after treatment with fungal cells, and
able to enter inside fungal cells by exhibiting the green fluorescence
(Fig. 7D). These results confirm the ability of 14f to cross membrane